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Letter 信件 2018 年 7 月 31 日
Engineering PD-1-Presenting Platelets for Cancer Immunotherapy
工程化 PD-1 呈递血小板用于癌症免疫治疗Click to copy article link
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- Xudong Zhang 张旭东Xudong ZhangGuangdong Key Laboratory for Biomedical, Measurements and Ultrasound Imaging, Laboratory of Evolutionary Theranostics, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen 518060, ChinaDepartment of Bioengineering, California NanoSystems Institute, and Center for Minimally Invasive Therapeutics (C-MIT), University of California, Los Angeles, California 90095, United StatesJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United StatesMore by Xudong Zhang
- Jinqiang Wang 王金强Jinqiang WangDepartment of Bioengineering, California NanoSystems Institute, and Center for Minimally Invasive Therapeutics (C-MIT), University of California, Los Angeles, California 90095, United StatesJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United StatesMore by Jinqiang Wang
- Zhaowei Chen 赵伟 陈Zhaowei ChenJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United StatesMore by Zhaowei Chen
- Quanyin Hu 胡全银Quanyin HuJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United StatesMore by Quanyin Hu
- Chao Wang 王超Chao WangJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United StatesMore by Chao Wang
- Junjie Yan 闫俊杰Junjie YanJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United StatesMore by Junjie Yan
- Gianpietro Dotti 詹皮耶特罗·多蒂Gianpietro DottiLineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, United StatesMore by Gianpietro Dotti
- Peng Huang* 彭 黄*Peng Huang*E-mail: peng.huang@szu.edu.cnGuangdong Key Laboratory for Biomedical, Measurements and Ultrasound Imaging, Laboratory of Evolutionary Theranostics, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen 518060, ChinaMore by Peng Huang
- Zhen Gu* 《用于癌症免疫治疗的 PD-1 呈递血小板工程设计 | Nano Letters》Zhen Gu*E-mail: guzhen@ucla.eduDepartment of Bioengineering, California NanoSystems Institute, and Center for Minimally Invasive Therapeutics (C-MIT), University of California, Los Angeles, California 90095, United StatesJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United StatesMore by Zhen Gu
Abstract 摘要
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Radical surgery still represents the treatment choice for several malignancies. However, local and distant tumor relapses remain the major causes of treatment failure, indicating that a postsurgery consolidation treatment is necessary. Immunotherapy with checkpoint inhibitors has elicited impressive clinical responses in several types of human malignancies and may represent the ideal consolidation treatment after surgery. Here, we genetically engineered platelets from megakaryocyte (MK) progenitor cells to express the programmed cell death protein 1 (PD-1). The PD-1 platelet and its derived microparticle could accumulate within the tumor surgical wound and revert exhausted CD8+ T cells, leading to the eradication of residual tumor cells. Furthermore, when a low dose of cyclophosphamide (CP) was loaded into PD-1-expressing platelets to deplete regulatory T cells (Tregs), an increased frequency of reinvigorated CD8+ lymphocyte cells was observed within the postsurgery tumor microenvironment, directly preventing tumor relapse.
彻底的手术依然是治疗多种恶性肿瘤的首选方案。然而，局部和远处的肿瘤复发仍然是治疗失败的主要原因，表明术后巩固治疗是必要的。免疫疗法中，使用免疫检查点抑制剂在多种人类恶性肿瘤中引发了显著的临床反应，并可能成为术后理想的巩固治疗手段。在此，我们通过基因工程改造巨核细胞（MK）祖细胞来源的血小板，使其表达程序性细胞死亡蛋白 1（PD-1）。这种 PD-1 表达的血小板及其衍生的微粒能够在肿瘤手术伤口内积累，逆转耗竭的 CD8+ T 细胞，从而清除残留的肿瘤细胞。此外，当低剂量的环磷酰胺（CP）被装载到 PD-1 表达的血小板中以耗竭调节性 T 细胞（Tregs）时，术后肿瘤微环境中观察到重新激活的 CD8+淋巴细胞数量增加，直接防止了肿瘤复发。
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Copyright © 2018 American Chemical Society
版权所有 © 2018 美国化学学会
版权所有 © 2018 美国化学学会
Surgery is the main therapeutic option for most solid tumors. However, the local and distal tumor relapses frequently occur because of the incomplete resection of tumors. (1,2) Hence, there have been tremendous interests in developing effective strategies to prevent cancer relapse after surgery. (3,4) For example, tumor antigen-specific CD8+ T cells contribute to the eradication of residual tumor cells, (5) especially those that harbor neoantigens (mutant protein-derived antigens). (6−9) However, PD-L1 expression in tumors suppresses T cell responses by causing T cell exhaustion. (10) Exhausted T cells are restrained by PD-L1 ligands through the inhibitory receptors PD-1 disabling the production of immune cytokines such as interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), granzyme B, and perforin. (11,12)
手术是大多数实体肿瘤的主要治疗选择。然而，由于肿瘤切除不完全，局部和远端肿瘤复发频繁发生。（1,2）因此，开发有效策略以预防术后癌症复发引起了极大的兴趣。（3,4）例如，肿瘤抗原特异性 CD8+ T 细胞有助于清除残留的肿瘤细胞，（5）特别是那些携带新抗原（突变蛋白衍生抗原）的细胞。（6−9）然而，肿瘤中 PD-L1 的表达通过导致 T 细胞衰竭抑制了 T 细胞的反应。（10）衰竭的 T 细胞通过抑制性受体 PD-1 被 PD-L1 配体所抑制，从而无法产生免疫细胞因子，如干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）、颗粒酶 B 和穿孔素。（11,12）
手术是大多数实体肿瘤的主要治疗选择。然而，由于肿瘤切除不完全，局部和远端肿瘤复发频繁发生。（1,2）因此，开发有效策略以预防术后癌症复发引起了极大的兴趣。（3,4）例如，肿瘤抗原特异性 CD8+ T 细胞有助于清除残留的肿瘤细胞，（5）特别是那些携带新抗原（突变蛋白衍生抗原）的细胞。（6−9）然而，肿瘤中 PD-L1 的表达通过导致 T 细胞衰竭抑制了 T 细胞的反应。（10）衰竭的 T 细胞通过抑制性受体 PD-1 被 PD-L1 配体所抑制，从而无法产生免疫细胞因子，如干扰素-γ（IFN-γ）、肿瘤坏死因子-α（TNF-α）、颗粒酶 B 和穿孔素。（11,12）
Blocking the PD-1/PD-L1 axis by using checkpoint antibodies can reinvigorate exhausted T cells having led to the eradication of the tumor in ∼30% of the patients with melanoma and other types of cancers. (13−15) However, more than half of patients are not responsive or only transiently responsive to PD-1/PD-L1 blockade due to the existence of multiple immune evasion mechanisms. (16) For instance, studies indicate that there are many intrinsic and extrinsic tumor-associated mechanisms of resistance to immunotherapy that include loss of targeted tumor-associated antigens, down-regulation of major histocompatibility complex (MHC) molecules, expression of other immune checkpoint receptors, and abundance of immune suppressive cell populations (Tregs, type II macrophages, myeloid-derived suppressor cells (MDSCs)). (16) In particular, in addition to inhibiting effector T cells, Tregs compete in the consumption of interleukin-2 (IL-2) in the tumor microenvironment further impairing the proliferation of tumor infiltrated CD8+ T cells. (17,18) Moreover, activated Tregs may also directly kill T cells through perforin. (19) Thus, abundant Tregs in tumor tissue are crucial obstacles to achieve successful cancer immunotherapy. (20) Depletion of Tregs can significantly improve the response rate of PD-1/PD-L1 blockade. (14)
通过使用检查点抗体阻断 PD-1/PD-L1 轴，可以重新激活耗竭的 T 细胞，从而导致约 30%的黑色素瘤及其他类型癌症患者实现肿瘤清除。(13−15) 然而，由于存在多种免疫逃逸机制，超过半数的患者对 PD-1/PD-L1 阻断反应不佳或仅呈现短暂反应。(16) 例如，研究表明，许多内在和外在的肿瘤相关抗性机制包括靶向肿瘤相关抗原的丢失、主要组织相容性复合体（MHC）分子的下调、其他免疫检查点受体的表达以及免疫抑制细胞群体（调节性 T 细胞、II 型巨噬细胞、髓源性抑制细胞（MDSCs））的丰富。(16) 特别是，除了抑制效应 T 细胞外，调节性 T 细胞还在肿瘤微环境中竞争性消耗白细胞介素-2（IL-2），进一步损害肿瘤浸润的 CD8+ T 细胞的增殖。(17,18) 此外，激活的调节性 T 细胞可能还通过穿孔素直接杀死 T 细胞。 (19) 因此，肿瘤组织中丰富的调节性 T 细胞（Tregs）是实现成功癌症免疫治疗的关键障碍。(20) 清除 Tregs 能显著提高 PD-1/PD-L1 阻断疗法的响应率。 (14)
通过使用检查点抗体阻断 PD-1/PD-L1 轴，可以重新激活耗竭的 T 细胞，从而导致约 30%的黑色素瘤及其他类型癌症患者实现肿瘤清除。(13−15) 然而，由于存在多种免疫逃逸机制，超过半数的患者对 PD-1/PD-L1 阻断反应不佳或仅呈现短暂反应。(16) 例如，研究表明，许多内在和外在的肿瘤相关抗性机制包括靶向肿瘤相关抗原的丢失、主要组织相容性复合体（MHC）分子的下调、其他免疫检查点受体的表达以及免疫抑制细胞群体（调节性 T 细胞、II 型巨噬细胞、髓源性抑制细胞（MDSCs））的丰富。(16) 特别是，除了抑制效应 T 细胞外，调节性 T 细胞还在肿瘤微环境中竞争性消耗白细胞介素-2（IL-2），进一步损害肿瘤浸润的 CD8+ T 细胞的增殖。(17,18) 此外，激活的调节性 T 细胞可能还通过穿孔素直接杀死 T 细胞。 (19) 因此，肿瘤组织中丰富的调节性 T 细胞（Tregs）是实现成功癌症免疫治疗的关键障碍。(20) 清除 Tregs 能显著提高 PD-1/PD-L1 阻断疗法的响应率。 (14)
As the monitor of vascular damage, platelets can accumulate to the surgery wound. (21,22) By employing this property, platelets conjugated with anti-PD-L1 can accumulate within the tumor surgery wound, reinvigorating exhausted CD8+ T cells, and thus reduce postsurgical tumor recurrence and metastasis. (23) Moreover, platelet membrane is used to design formulations for wound and inflammation targeting cancer treatment. (24−27) However, since blood-originated platelets are non-nucleated and terminally differentiated cells, they cannot be expanded ex vivo and genetically manipulated to stably express transgenes, which significantly limit their clinical use as payload for cancer immunotherapy. (28) In contrast, in vitro production of platelets from megakaryocytes (MKs) can provide a large source of platelets and be genetically modified. (29,30) Herein, we genetically engineered murine MKs to stably express murine PD-1 and to produce mature platelets presenting PD-1 in vitro. We then applied these cells to target tumor cells within the surgical wound via reinvigoration of exhausted CD8+ T cells (Figure 1). In addition to PD-L1 blockade, PD-1-expressing platelets can also carry and transport cyclophosphamide, which allows the depletion of Tregs within the tumor microenvironment and further enhances the antitumor effects of CD8+ T lymphocyte cells within the surgical tumor microenvironment.
作为血管损伤的监测者，血小板能够聚集到手术伤口处。（21,22）利用这一特性，与抗 PD-L1 结合的血小板可在肿瘤手术伤口内积聚，重新激活耗竭的 CD8+ T 细胞，从而减少术后肿瘤复发与转移。（23）此外，血小板膜被用于设计针对伤口与炎症的癌症治疗制剂。（24−27）然而，由于源自血液的血小板是无核且终末分化的细胞，无法在体外扩增并进行基因操作以稳定表达转基因，这极大限制了它们作为癌症免疫治疗载体的临床应用。（28）相比之下，从巨核细胞（MKs）体外生产血小板，不仅可提供大量血小板来源，还能进行基因修饰。（29,30）在此，我们通过基因工程改造小鼠巨核细胞，使其稳定表达小鼠 PD-1，并在体外生成呈现 PD-1 的成熟血小板。随后，我们将这些细胞应用于通过重新激活耗竭的 CD8+ T 细胞来靶向手术伤口内的肿瘤细胞（图 1）。 除了 PD-L1 阻断外，表达 PD-1 的血小板还能携带并运输环磷酰胺，从而在肿瘤微环境中消耗调节性 T 细胞（Tregs），进一步增强手术肿瘤微环境中 CD8+ T 淋巴细胞的抗肿瘤效应。
作为血管损伤的监测者，血小板能够聚集到手术伤口处。（21,22）利用这一特性，与抗 PD-L1 结合的血小板可在肿瘤手术伤口内积聚，重新激活耗竭的 CD8+ T 细胞，从而减少术后肿瘤复发与转移。（23）此外，血小板膜被用于设计针对伤口与炎症的癌症治疗制剂。（24−27）然而，由于源自血液的血小板是无核且终末分化的细胞，无法在体外扩增并进行基因操作以稳定表达转基因，这极大限制了它们作为癌症免疫治疗载体的临床应用。（28）相比之下，从巨核细胞（MKs）体外生产血小板，不仅可提供大量血小板来源，还能进行基因修饰。（29,30）在此，我们通过基因工程改造小鼠巨核细胞，使其稳定表达小鼠 PD-1，并在体外生成呈现 PD-1 的成熟血小板。随后，我们将这些细胞应用于通过重新激活耗竭的 CD8+ T 细胞来靶向手术伤口内的肿瘤细胞（图 1）。 除了 PD-L1 阻断外，表达 PD-1 的血小板还能携带并运输环磷酰胺，从而在肿瘤微环境中消耗调节性 T 细胞（Tregs），进一步增强手术肿瘤微环境中 CD8+ T 淋巴细胞的抗肿瘤效应。
Figure 1 图 1
Figure 1. Schematic of the production of PD-1-expressing platelets and reinvigoration of CD8+ T cells. (A) Schematic shows L8057 cell line stably expressing murine PD-1 and production of platelets. (B) PD-1-expressing platelets target tumor cells within the surgery wound. (C) PD-L1 blockade by PD-1-expressing platelets reverts exhausted CD8+ T cells to attack tumor cells.
图 1. PD-1 表达血小板的制备及 CD8+ T 细胞的再激活示意图。(A) 示意图展示了稳定表达小鼠 PD-1 的 L8057 细胞系及血小板的生成。(B) PD-1 表达的血小板靶向手术伤口内的肿瘤细胞。(C) PD-1 表达的血小板通过阻断 PD-L1，使耗竭的 CD8+ T 细胞恢复活力并攻击肿瘤细胞。
Platelets are released from the bone marrow and lung resident MKs. (31) To produce platelets in large-scale, we treated the murine MK progenitor cells L8057 with phorbol 12-myristate 13-acetate (PMA). After stimulation, cell volume was significantly increased, accompanied by proplatelet extension (Figure S1A,B) and platelets release (Figure S1C,D). MKs with larger cell volume containing polyploid nuclei were observed, indicating MK maturation and readiness for releasing the platelets (Figure S2). To generate PD-1-expressing platelets, L8057 cell line stably expressing murine EGFP-PD-1 was established by infection with lentivirus and selection with puromycin (Figure S3). (32) Remarkably, PD-1 was expressed and localized on the cell membrane, as indicated by the colocalization of fluorescence from EGFP and the cell membrane dye Alexa Fluor 594-conjugated wheat germ agglutinin (WGA594) (Figure 2A). PD-1 expression on EGFP-PD-1 L8057 cells was confirmed by Western blot (Figure 2B). CD41a, the marker of MKs, was intensively expressed on PD-1 L8057 cell line (Figure S4A). After the stimulation with PMA, PD-1-expressing L8057 cells underwent maturation, and morphologically displayed typical peripheral nuclei and increased cytoplasmic volume (Figure S4B). CD42a, a marker of MK maturation, was expressed on the cell membrane (Figure 2C). Moreover, the platelet surface receptors glycoprotein VI (GPVI) and P-selectin were expressed in mature PD-1 L8057 cells (Figure S4C,D). Wright–Giemsa staining revealed that mature PD-1 L8057 cells contained polyploid nuclei (Figure 2D).
血小板由骨髓和肺驻留的巨核细胞（MKs）释放。（31）为了大规模生产血小板，我们用佛波醇 12-豆蔻酸 13-醋酸酯（PMA）处理了小鼠 MK 祖细胞 L8057。刺激后，细胞体积显著增大，伴随前血小板延伸（图 S1A、B）和血小板释放（图 S1C、D）。观察到含有多倍体核的较大体积的 MKs，表明 MK 成熟并准备释放血小板（图 S2）。为了生成表达 PD-1 的血小板，通过感染慢病毒并使用嘌呤霉素筛选，建立了稳定表达小鼠 EGFP-PD-1 的 L8057 细胞系（图 S3）。（32）值得注意的是，PD-1 被表达并定位于细胞膜，由 EGFP 荧光与细胞膜染料 Alexa Fluor 594 偶联的麦胚凝集素（WGA594）的共定位所指示（图 2A）。通过 Western blot 确认了 PD-1 在 EGFP-PD-1 L8057 细胞上的表达（图 2B）。作为 MKs 标志物的 CD41a 在 PD-1 L8057 细胞系上强烈表达（图 S4A）。 经 PMA 刺激后，表达 PD-1 的 L8057 细胞发生成熟，形态上显示出典型的偏心核和增大的胞质体积（图 S4B）。CD42a，作为巨核细胞（MK）成熟的标志物，在细胞膜上得以表达（图 2C）。此外，成熟 PD-1 L8057 细胞表面还表达了血小板表面受体糖蛋白 VI（GPVI）和 P-选择素（图 S4C，D）。Wright-Giemsa 染色显示，成熟的 PD-1 L8057 细胞含有多倍体核（图 2D）。
血小板由骨髓和肺驻留的巨核细胞（MKs）释放。（31）为了大规模生产血小板，我们用佛波醇 12-豆蔻酸 13-醋酸酯（PMA）处理了小鼠 MK 祖细胞 L8057。刺激后，细胞体积显著增大，伴随前血小板延伸（图 S1A、B）和血小板释放（图 S1C、D）。观察到含有多倍体核的较大体积的 MKs，表明 MK 成熟并准备释放血小板（图 S2）。为了生成表达 PD-1 的血小板，通过感染慢病毒并使用嘌呤霉素筛选，建立了稳定表达小鼠 EGFP-PD-1 的 L8057 细胞系（图 S3）。（32）值得注意的是，PD-1 被表达并定位于细胞膜，由 EGFP 荧光与细胞膜染料 Alexa Fluor 594 偶联的麦胚凝集素（WGA594）的共定位所指示（图 2A）。通过 Western blot 确认了 PD-1 在 EGFP-PD-1 L8057 细胞上的表达（图 2B）。作为 MKs 标志物的 CD41a 在 PD-1 L8057 细胞系上强烈表达（图 S4A）。 经 PMA 刺激后，表达 PD-1 的 L8057 细胞发生成熟，形态上显示出典型的偏心核和增大的胞质体积（图 S4B）。CD42a，作为巨核细胞（MK）成熟的标志物，在细胞膜上得以表达（图 2C）。此外，成熟 PD-1 L8057 细胞表面还表达了血小板表面受体糖蛋白 VI（GPVI）和 P-选择素（图 S4C，D）。Wright-Giemsa 染色显示，成熟的 PD-1 L8057 细胞含有多倍体核（图 2D）。
Figure 2 图 2
Figure 2. Production and characterization of platelets from PD-1-expressing L8057 stable cell line. (A) Confocal images present L8057 cell line stably expressing murine EGFP-PD-1 on cell membranes. WGA Alexa-Fluor 594 dye was used to stain cell membrane (scale bar: 10 μm). (B) Western blot analysis for evaluating the expression of PD-1 in L8057 cell line. L8 is short for L8057 cells. (C) EGFP-PD-1-expressing L8057 cells stimulated with 500 nM PMA for 3 days, and immunostained to detect CD42a expression. (D) L8057 cells stimulated with 500 nM PMA for 3 days, and stained with Wright–Giemsa dye (scale bar: 10 μm). (E) Evolution process of PD-1-expressing proplatelet extended from MKs (scale bar: 10 μm). (F) Morphology of PD-1 proplatelets extended from L8057 cells after 6 days of stimulation with 500 nM PMA. PD-1 proplatelets extended from L8057 cells (scale bar: 10 μm). (G) Representative confocal images of purified PD-1-expressing platelets (scale bar: 10 μm). (H) Size distribution of PD-1-expressing platelets measured by DLS. (I) CSEM image shows the morphology of PD-1-expressing platelets (scale bar: 1 μm). (J) Representative TEM image shows morphology and size of PD-1-expressing platelet (scale bar: 1 μm). (K) Number of platelets released from PD-1-expressing L8057 cells after stimulated with 500 nM PMA (n = 5). Error bar, ± SD.
图 2. 源自 PD-1 表达 L8057 稳定细胞系的血小板制备与表征。(A) 共聚焦图像展示在细胞膜上稳定表达小鼠 EGFP-PD-1 的 L8057 细胞系。WGA Alexa-Fluor 594 染料用于细胞膜染色（标尺：10 μm）。(B) 通过 Western blot 分析评估 L8057 细胞系中 PD-1 的表达情况。L8 代表 L8057 细胞。(C) 表达 EGFP-PD-1 的 L8057 细胞经 500 nM PMA 刺激 3 天后，进行免疫染色以检测 CD42a 表达。(D) L8057 细胞经 500 nM PMA 刺激 3 天后，用 Wright-Giemsa 染料染色（标尺：10 μm）。(E) PD-1 表达的前血小板从巨核细胞（MKs）延伸的演变过程（标尺：10 μm）。(F) L8057 细胞经 500 nM PMA 刺激 6 天后，PD-1 前血小板的形态。从 L8057 细胞延伸出的 PD-1 前血小板（标尺：10 μm）。(G) 纯化的 PD-1 表达血小板的代表性共聚焦图像（标尺：10 μm）。(H) 通过 DLS 测量的 PD-1 表达血小板的大小分布。(I) CSEM 图像显示 PD-1 表达血小板的形态（标尺：1 μm）。 (J) 代表性透射电子显微镜图像展示了 PD-1 表达血小板的形态与尺寸（比例尺：1 微米）。(K) PD-1 表达的 L8057 细胞在 500 nM PMA 刺激后释放的血小板数量（n = 5）。误差线表示±标准差。
Mature MKs typically reside in bone marrow and lung budding podosomes and prolong to form proplatelets. (33) Proplatelets cross through the sinusoidal endothelium and release platelets into the bloodstream. (31,33) Similarly, mature PD-1-expressing L8057 cells had budding podosomes, which prolonged to form the proplatelets (Figure 2E). Notably, the proplatelets were budded and extended from the cell membranes to form pearl-like structures (Figure 2F). The proplatelets finally disbanded and released platelets (Figure S5A,B). MK cytoplasm containing EGFP-PD-1+ membrane vesicles existed as a membrane reservoir for proplatelet formation (Figure 2F). These PD-1-expressing membrane vesicles fused to form tubular structure and budded from the cell surface (Figure 2F). Purified platelets from the culture media showed green fluorescence indicating that PD-1 was present in the platelets (Figure 2G). Binding receptors including GPVI and P-selectin were also expressed in platelets released from L8057 cells (Figure S5C). Moreover, dynamic light scattering (DLS) analysis showed that the average diameter of the platelets was around 1.5 μm and with a ζ-potential of −10 ± 2.6 mV (Figure 2H and Figure S5D). As documented by cryo-scanning electron microscopy (CSEM) and transmission electron microscopy (TEM), purified platelets showed spherical morphology (Figure 2I,J). We further quantitatively measured the platelet production from PD-1-expressing L8057 cells and found optimal platelet production at day 6 after stimulation with PMA (Figure 2K).
成熟的巨核细胞（MKs）通常定位于骨髓和肺部的发芽足体，并延长形成前血小板。（33）前血小板穿过窦状内皮，将血小板释放到血液中。（31,33）同样，成熟的 PD-1 表达 L8057 细胞也具有发芽足体，这些足体延长形成前血小板（图 2E）。值得注意的是，前血小板从细胞膜发芽并延伸，形成珍珠状结构（图 2F）。最终，前血小板解体并释放出血小板（图 S5A,B）。含有 EGFP-PD-1+膜囊泡的 MK 细胞质作为前血小板形成的膜储存库（图 2F）。这些 PD-1 表达的膜囊泡融合形成管状结构，并从细胞表面发芽（图 2F）。从培养基中纯化的血小板显示出绿色荧光，表明 PD-1 存在于血小板中（图 2G）。包括 GPVI 和 P-选择素在内的结合受体也在 L8057 细胞释放的血小板中表达（图 S5C）。此外，动态光散射（DLS）分析显示，血小板的平均直径约为 1.5 微米，ζ电位为−10 ± 2。6 mV（图 2H 和图 S5D）。通过冷冻扫描电子显微镜（CSEM）和透射电子显微镜（TEM）的记录，纯化的血小板展现出球形形态（图 2I、J）。我们进一步定量测量了来自 PD-1 表达 L8057 细胞的血小板生成，发现经过 PMA 刺激后，第 6 天达到最佳血小板生成量（图 2K）。
成熟的巨核细胞（MKs）通常定位于骨髓和肺部的发芽足体，并延长形成前血小板。（33）前血小板穿过窦状内皮，将血小板释放到血液中。（31,33）同样，成熟的 PD-1 表达 L8057 细胞也具有发芽足体，这些足体延长形成前血小板（图 2E）。值得注意的是，前血小板从细胞膜发芽并延伸，形成珍珠状结构（图 2F）。最终，前血小板解体并释放出血小板（图 S5A,B）。含有 EGFP-PD-1+膜囊泡的 MK 细胞质作为前血小板形成的膜储存库（图 2F）。这些 PD-1 表达的膜囊泡融合形成管状结构，并从细胞表面发芽（图 2F）。从培养基中纯化的血小板显示出绿色荧光，表明 PD-1 存在于血小板中（图 2G）。包括 GPVI 和 P-选择素在内的结合受体也在 L8057 细胞释放的血小板中表达（图 S5C）。此外，动态光散射（DLS）分析显示，血小板的平均直径约为 1.5 微米，ζ电位为−10 ± 2。6 mV（图 2H 和图 S5D）。通过冷冻扫描电子显微镜（CSEM）和透射电子显微镜（TEM）的记录，纯化的血小板展现出球形形态（图 2I、J）。我们进一步定量测量了来自 PD-1 表达 L8057 细胞的血小板生成，发现经过 PMA 刺激后，第 6 天达到最佳血小板生成量（图 2K）。
Platelets execute hemostasis, recruit other leukocytes for host defense responses, and release several immunoreactive molecules after adhering to vascular lesions. (34) Collagen is the primary subendothelial component for active platelet binding. As illustrated in Figure 3A,B, WGA Alexa-Fluor 594 dye-labeled free and PD-1-expressing platelets showed similar collagen adhesion ability. In contrast, blockade of the collagen receptor GPVI reduced the collagen adhesion ability of the platelets (Figure S6A). Thrombus formation by platelet aggregation is another critical event of the hemostatic response. (35) Free and PD-1-expressing platelets efficiently aggregated in response to agonistic stimulation with thrombin (Figure S6B). Platelet microparticles (PMPs) are generated from activated platelets. (36,37) To examine whether PMPs are generated from activated PD-1-expressing platelets upon stimulation, we treated the platelets with thrombin in vitro. Confocal laser scanning microscopy (CLSM), SEM, and TEM images indicated the generation of PMPs from activated platelets (Figure 3C and Figure S6C). Platelets became more dendritic and expansive after the treatment with thrombin (Figure 3C). Furthermore, DLS analysis detected the generation of small particles, substantiating the release of PMPs from activated platelets (Figure 3D).
血小板执行止血功能，招募其他白细胞参与宿主防御反应，并在附着于血管损伤处后释放多种免疫反应分子。（34）胶原蛋白是血小板主动结合的主要内皮下成分。如图 3A、B 所示，经 WGA Alexa-Fluor 594 染料标记的游离及 PD-1 表达血小板显示出相似的胶原蛋白粘附能力。相比之下，胶原蛋白受体 GPVI 的阻断降低了血小板的胶原蛋白粘附能力（图 S6A）。血小板聚集形成的血栓是止血反应的另一关键事件。（35）游离及 PD-1 表达血小板在凝血酶的激动性刺激下均能有效聚集（图 S6B）。血小板微粒（PMPs）由激活的血小板生成。（36,37）为探究 PD-1 表达血小板在刺激下是否产生 PMPs，我们在体外用凝血酶处理血小板。共聚焦激光扫描显微镜（CLSM）、扫描电镜（SEM）和透射电镜（TEM）图像显示，激活的血小板生成了 PMPs（图 3C 及图 S6C）。 血小板在经过凝血酶处理后变得更加树突状且扩展性增强（图 3C）。此外，动态光散射分析检测到了小颗粒的生成，证实了活化血小板释放 PMPs（图 3D）。
血小板执行止血功能，招募其他白细胞参与宿主防御反应，并在附着于血管损伤处后释放多种免疫反应分子。（34）胶原蛋白是血小板主动结合的主要内皮下成分。如图 3A、B 所示，经 WGA Alexa-Fluor 594 染料标记的游离及 PD-1 表达血小板显示出相似的胶原蛋白粘附能力。相比之下，胶原蛋白受体 GPVI 的阻断降低了血小板的胶原蛋白粘附能力（图 S6A）。血小板聚集形成的血栓是止血反应的另一关键事件。（35）游离及 PD-1 表达血小板在凝血酶的激动性刺激下均能有效聚集（图 S6B）。血小板微粒（PMPs）由激活的血小板生成。（36,37）为探究 PD-1 表达血小板在刺激下是否产生 PMPs，我们在体外用凝血酶处理血小板。共聚焦激光扫描显微镜（CLSM）、扫描电镜（SEM）和透射电镜（TEM）图像显示，激活的血小板生成了 PMPs（图 3C 及图 S6C）。 血小板在经过凝血酶处理后变得更加树突状且扩展性增强（图 3C）。此外，动态光散射分析检测到了小颗粒的生成，证实了活化血小板释放 PMPs（图 3D）。
Figure 3 图 3
Figure 3. In vitro and in vivo function of PD-1-expressing platelets. (A, B) Retention of platelets on collagen-coated or uncoated tissue culture slides. Green color: EGFP; Red color: WGA Alexa-Fluor 594 dye (scale bar: 50 μm). Error bar, ± SD. (C) Confocal, CSEM, and TEM images of PD-1-expressing platelets stimulated with thrombin. Platelet microparticles (PMPs) were released from the platelet (scale bar: 1 μm). (D) Measurement of the size distribution of PD-1-expressing platelets at 30 min after activation by thrombin. PMPs were produced from the platelets. (E) EGFP-PD-1-expressing platelets bound on the cell membrane of B16F10 cells. PD-1-expresing platelets or free platelets labeled with Cy5.5 were incubated with B16F10 cells for 20 h. WGA Alexa-Fluor 594 dye was used to stain the B16F10 cell membrane. The white arrows indicate the PD-1 platelets binding on the cell membrane of the cancer cells (scale bar: 10 μm). (F) B16F10 cells were transfected with DsRed-PD-L1 plasmid for 20 h, and then incubated with EGFP-PD-1 platelets for 20 h; the colocalization of EGFP-PD-1 platelets and DsRed-PD-L1 was detected (scale bar: 10 μm). (G) Cy5.5-labeled free platelets and PD-1-expressing platelets were injected through the tail-vein in mice. Fluorescence was measured at different time points (n = 3). Fluorescence intensity at 2 min as 1. Error bar, ±SD. (H) In vivo fluorescence images of free platelets and PD-1-expressing platelets in major organs and residual tumor bed. (I) Fluorescence intensity per gram of tissue in major organs and tumors (n = 3). Error bar, ± SD.
图 3. PD-1 表达血小板的体外与体内功能。(A, B) 血小板在胶原涂层或未涂层组织培养载玻片上的滞留情况。绿色：EGFP；红色：WGA Alexa-Fluor 594 染料（比例尺：50 μm）。误差棒，± 标准差。(C) PD-1 表达血小板经凝血酶刺激后的共聚焦、CSEM 及 TEM 图像。血小板释放出微粒（PMPs）（比例尺：1 μm）。(D) 经凝血酶激活 30 分钟后 PD-1 表达血小板的大小分布测量。血小板产生 PMPs。(E) EGFP-PD-1 表达血小板结合于 B16F10 细胞膜上。PD-1 表达血小板或自由标记的 Cy5.5 血小板与 B16F10 细胞共孵育 20 小时。WGA Alexa-Fluor 594 染料用于标记 B16F10 细胞膜。白色箭头指示 PD-1 血小板与癌细胞膜的结合（比例尺：10 μm）。(F) B16F10 细胞转染 DsRed-PD-L1 质粒 20 小时后，与 EGFP-PD-1 血小板共孵育 20 小时；检测 EGFP-PD-1 血小板与 DsRed-PD-L1 的共定位（比例尺：10 μm）。(G) Cy5.5-标记的自由血小板和 PD-1 表达的血小板通过小鼠尾静脉注射。在不同时间点测量荧光（n = 3）。以 2 分钟的荧光强度为 1。误差条表示±标准差。(H) 自由血小板和 PD-1 表达血小板在主要器官及残留肿瘤床中的体内荧光图像。(I) 主要器官和肿瘤中每克组织的荧光强度（n = 3）。误差条表示±标准差。
Elevation of PD-L1 expression in tumor cells causes exhaustion of T cells expressing PD-1. (10) To investigate whether PD-1 expressed platelets could bind to the surface of the melanoma cells and block PD-L1, we incubated the PD-1-expressing platelets with the B16F10 melanoma cells in vitro. We observed that PD-1-expressing platelets bound to B16F10 cells and were then internalized by the cancer cells (Figure 3E and Figure S7). In contrast, free platelets showed limited ability to bind to the B16F10 cells (Figure 3E). To examine whether the PD-L1/PD-1 interaction mediated the internalization of platelets, we added anti-PD-L1 antibody to block PD-L1 on the B16F10 cells. The confocal images showed that PD-1 platelets binding was significantly reduced when PD-L1 antibody was preincubated with the cells (Figure S7A). Furthermore, the EGFP-PD-1-expressing platelets colocalized with DsRed-PD-L1 expressed by B16F10 melanoma cells, indicating the physical interaction between PD-1 and PD-L1 (Figure 3F). To investigate the in vivo biodistribution of free and PD-1-expressing platelets, Cy5.5-labeled platelets were inoculated in mice via tail-vein injection. Free platelets showed longer blood retention than PD-1-expressing platelets (14% vs 8% at 24 h) (Figure 3G). When Cy5.5-labeled platelets were inoculated intravenously after tumor resection in B16F10 tumor-bearing mice, both free and PD-1 platelets could accumulate in the residual tumor bed (Figure 3H,I). Meanwhile the platelets intensively accumulated in the liver and spleen (Figure 3H,I). GPVI is the collagen receptor on the platelets and is responsible for the platelets targeting the wound. PD-1 platelets and free platelets showed similar binding ability on the collagen (Figure 3A). Therefore, the accumulation ability in the surgical tumors is similar between the free platelets and PD-1 platelets (Figure 3H).
PD-L1 在肿瘤细胞表面的表达上调会导致表达 PD-1 的 T 细胞功能耗竭。（10）为了探究表达 PD-1 的血小板能否与黑色素瘤细胞表面结合并阻断 PD-L1，我们在体外将表达 PD-1 的血小板与 B16F10 黑色素瘤细胞共同孵育。观察发现，表达 PD-1 的血小板能够结合到 B16F10 细胞表面，随后被癌细胞内化（图 3E 及图 S7）。相比之下，未修饰的血小板与 B16F10 细胞的结合能力有限（图 3E）。为验证 PD-L1/PD-1 相互作用是否介导了血小板的内化过程，我们在实验中加入抗 PD-L1 抗体以阻断 B16F10 细胞表面的 PD-L1。共聚焦图像显示，当预先与 PD-L1 抗体孵育后，PD-1 血小板的结合显著减少（图 S7A）。此外，EGFP 标记的 PD-1 表达血小板与 B16F10 黑色素瘤细胞表达的 DsRed-PD-L1 发生共定位，表明 PD-1 与 PD-L1 之间存在物理相互作用（图 3F）。为研究自由血小板与 PD-1 表达血小板在体内的分布情况，我们通过尾静脉注射将 Cy5.5 标记的血小板接种于小鼠体内。 自由血小板在血液中的滞留时间比表达 PD-1 的血小板更长（24 小时时分别为 14%和 8%）（图 3G）。在 B16F10 荷瘤小鼠肿瘤切除后，通过静脉注射 Cy5.5 标记的血小板，无论是自由血小板还是 PD-1 血小板，均能在残留肿瘤床中积聚（图 3H，I）。同时，血小板在肝脏和脾脏中也大量积聚（图 3H，I）。GPVI 是血小板上的胶原蛋白受体，负责引导血小板向损伤部位迁移。PD-1 血小板与自由血小板在胶原蛋白上的结合能力相似（图 3A）。因此，自由血小板与 PD-1 血小板在手术肿瘤中的积聚能力相似（图 3H）。
PD-L1 在肿瘤细胞表面的表达上调会导致表达 PD-1 的 T 细胞功能耗竭。（10）为了探究表达 PD-1 的血小板能否与黑色素瘤细胞表面结合并阻断 PD-L1，我们在体外将表达 PD-1 的血小板与 B16F10 黑色素瘤细胞共同孵育。观察发现，表达 PD-1 的血小板能够结合到 B16F10 细胞表面，随后被癌细胞内化（图 3E 及图 S7）。相比之下，未修饰的血小板与 B16F10 细胞的结合能力有限（图 3E）。为验证 PD-L1/PD-1 相互作用是否介导了血小板的内化过程，我们在实验中加入抗 PD-L1 抗体以阻断 B16F10 细胞表面的 PD-L1。共聚焦图像显示，当预先与 PD-L1 抗体孵育后，PD-1 血小板的结合显著减少（图 S7A）。此外，EGFP 标记的 PD-1 表达血小板与 B16F10 黑色素瘤细胞表达的 DsRed-PD-L1 发生共定位，表明 PD-1 与 PD-L1 之间存在物理相互作用（图 3F）。为研究自由血小板与 PD-1 表达血小板在体内的分布情况，我们通过尾静脉注射将 Cy5.5 标记的血小板接种于小鼠体内。 自由血小板在血液中的滞留时间比表达 PD-1 的血小板更长（24 小时时分别为 14%和 8%）（图 3G）。在 B16F10 荷瘤小鼠肿瘤切除后，通过静脉注射 Cy5.5 标记的血小板，无论是自由血小板还是 PD-1 血小板，均能在残留肿瘤床中积聚（图 3H，I）。同时，血小板在肝脏和脾脏中也大量积聚（图 3H，I）。GPVI 是血小板上的胶原蛋白受体，负责引导血小板向损伤部位迁移。PD-1 血小板与自由血小板在胶原蛋白上的结合能力相似（图 3A）。因此，自由血小板与 PD-1 血小板在手术肿瘤中的积聚能力相似（图 3H）。
To investigate whether PD-1-expressing platelets prevent cancer relapse after surgery, we utilized the B16F10 melanoma incomplete-tumor-resection model to mimic the postsurgical local relapse (Figure 4A). Tumor-bearing mice were injected intravenously with phosphate-buffered saline (PBS), free platelets (2 × 108), or PD-1-expressing platelets (2 × 108). After platelet infusion, tumors were resected to remove ∼90% of the tumor mass. After surgery, mice received additional treatment during the period of wound healing (Figure 4A). We observed tumor growth delay in mice treated with PD-1-expressing platelets as assessed by monitoring the tumor bioluminescence and measuring the tumor size (Figure 4B,C, and Figure S8A). In contrast, tumors rapidly progressed in mice that received free platelets or PBS (Figure 4B,C, and Figure S8A). There were 25% of mice receiving PD-1-expressing platelets that survived more than 60 days without obvious weight loss or other signs of toxicities (Figure 4D and Figure S8B). The major organs such as liver, spleen, kidney, heart, and lung were collected and assessed by immunohistochemistry to assess the systemic toxicity. There was no obvious sign of organ damage observed in the platelets treated mice (Figure S9). Remarkably, we observed increased frequency of CD8+ TILs in the tumor of mice treated with PD-1-expressing platelets (Figure 4E–G), and T cells exhibited increased expression of cytotoxic protein granzyme B (GzmB), indicating that PD-1-expressing platelets can revert T cell exhaustion within the tumor microenvironment (Figure 4H,I).
为了探究表达 PD-1 的血小板是否能预防手术后癌症复发，我们采用了 B16F10 黑色素瘤不完全肿瘤切除模型来模拟术后局部复发（图 4A）。荷瘤小鼠通过静脉注射磷酸盐缓冲液（PBS）、游离血小板（2×10^8）或表达 PD-1 的血小板（2×10^8）。在血小板输注后，切除肿瘤以移除约 90%的肿瘤组织。术后，小鼠在伤口愈合期间接受了额外治疗（图 4A）。我们通过监测肿瘤生物发光和测量肿瘤大小发现，接受 PD-1 表达血小板治疗的小鼠肿瘤生长延迟（图 4B、C 及图 S8A）。相比之下，接受游离血小板或 PBS 的小鼠肿瘤迅速进展（图 4B、C 及图 S8A）。接受 PD-1 表达血小板治疗的小鼠中有 25%存活超过 60 天，且未出现明显体重下降或其他毒性症状（图 4D 及图 S8B）。主要器官如肝脏、脾脏、肾脏、心脏和肺被收集并通过免疫组织化学方法评估全身毒性。 在经过处理的血小板治疗的小鼠中，未观察到明显的器官损伤迹象（图 S9）。值得注意的是，我们发现 PD-1 表达血小板处理的小鼠肿瘤中 CD8+肿瘤浸润淋巴细胞（TILs）的频率增加（图 4E-G），并且 T 细胞表现出细胞毒性蛋白颗粒酶 B（GzmB）表达的增强，表明 PD-1 表达的血小板能够在肿瘤微环境中逆转 T 细胞的耗竭状态（图 4H,I）。
为了探究表达 PD-1 的血小板是否能预防手术后癌症复发，我们采用了 B16F10 黑色素瘤不完全肿瘤切除模型来模拟术后局部复发（图 4A）。荷瘤小鼠通过静脉注射磷酸盐缓冲液（PBS）、游离血小板（2×10^8）或表达 PD-1 的血小板（2×10^8）。在血小板输注后，切除肿瘤以移除约 90%的肿瘤组织。术后，小鼠在伤口愈合期间接受了额外治疗（图 4A）。我们通过监测肿瘤生物发光和测量肿瘤大小发现，接受 PD-1 表达血小板治疗的小鼠肿瘤生长延迟（图 4B、C 及图 S8A）。相比之下，接受游离血小板或 PBS 的小鼠肿瘤迅速进展（图 4B、C 及图 S8A）。接受 PD-1 表达血小板治疗的小鼠中有 25%存活超过 60 天，且未出现明显体重下降或其他毒性症状（图 4D 及图 S8B）。主要器官如肝脏、脾脏、肾脏、心脏和肺被收集并通过免疫组织化学方法评估全身毒性。 在经过处理的血小板治疗的小鼠中，未观察到明显的器官损伤迹象（图 S9）。值得注意的是，我们发现 PD-1 表达血小板处理的小鼠肿瘤中 CD8+肿瘤浸润淋巴细胞（TILs）的频率增加（图 4E-G），并且 T 细胞表现出细胞毒性蛋白颗粒酶 B（GzmB）表达的增强，表明 PD-1 表达的血小板能够在肿瘤微环境中逆转 T 细胞的耗竭状态（图 4H,I）。
Figure 4 图 4
Figure 4. PD-1-expressing platelets for inhibition of tumor progression in incomplete-surgery tumor model. (A) Schematic illustration of PD-1-expressing platelets used for therapy in an incomplete-surgery tumor model. (B) In vivo bioluminescence imaging of the B16F10 tumor growth in mice treated with PBS (G1), free platelets (G2), and PD-1-expressing platelets (G3). (C) Average tumor volumes of treated mice (n = 8). Data are shown as the mean ± SEM. (D) Survival curves of mice receiving different treatments (n = 8). (E) Immunofluorescence of tumor sections showing infiltration of CD4+ and CD8+ T cells (scale bar: 100 μm). (F)Representative plots and (G) quantification of T cells in tumors analyzed by flow cytometry (gated on CD3+ T cells) (n = 3). Error bar, ± SD. (H) Representative plots and (I) quantification of GzmB in CD8+ T cells in tumors analyzed by the flow cytometry (gated on CD8+ T cells) (n = 3). Error bar, ± SD. Throughout, NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (C, G, I) One-way ANOVA with Tukey posthoc test analyses were carried out to do the analyses, or (D) by log-rank (Mantel-Cox) test.
图 4. PD-1 表达的血小板用于不完全手术肿瘤模型中抑制肿瘤进展。(A) PD-1 表达的血小板用于不完全手术肿瘤模型治疗的示意图。(B) 接受 PBS 处理（G1）、游离血小板处理（G2）及 PD-1 表达血小板处理（G3）的小鼠 B16F10 肿瘤生长的体内生物发光成像。(C) 各组处理小鼠的平均肿瘤体积（n=8），数据以均值±标准误（SEM）表示。(D) 不同治疗组小鼠的生存曲线（n=8）。(E) 肿瘤切片的免疫荧光图像，显示 CD4+和 CD8+ T 细胞的浸润（标尺：100 μm）。(F) 流式细胞术分析肿瘤内 T 细胞的代表性图谱及(G) 定量分析（以 CD3+ T 细胞为门控）（n=3）。误差棒表示±标准差（SD）。(H) 流式细胞术分析肿瘤内 CD8+ T 细胞中 GzmB 的代表性图谱及(I) 定量分析（以 CD8+ T 细胞为门控）（n=3）。误差棒表示±SD。整个实验中，NS 表示无显著性差异；*P < 0.05；**P < 0.01；***P < 0.001。(C, G, I) 采用单因素方差分析（One-way ANOVA）与 Tukey 事后检验进行分析，或(D) 采用对数秩（Mantel-Cox）检验进行分析。
Low doses of cyclophosphamides can improve immune responses in various murine tumor models and patients, which is generally attributed to selective depletion of Tregs. (38−40) To counter Tregs at the tumor site, we loaded the cyclophosphamide into the platelets. We found that platelets could internalize and release cyclophosphamide within 24 h in vitro (Figure S10). To investigate the simultaneous antitumor effect of PD-L1 blockade and cyclophosphamide-induced depletion of Tregs, we used the same B16F10 melanoma model with incomplete-tumor-resection. In this model, while cyclophosphamide and PD-1-expressing platelets showed limited results when used as single agents (Figure 5A and Figure S11A), tumor progression was significantly suppressed in mice treated with cyclophosphamide-loaded PD-1-expressing platelets (P < 0.001) (Figure 5A and Figure S11A). Treg depletion by cyclophosphamide and PD-L1 simultaneously blockade by PD-1 improved the survival of the treated mice (Figure 5B).
低剂量环磷酰胺可增强多种小鼠肿瘤模型及患者的免疫反应，这通常归因于选择性消耗调节性 T 细胞（Tregs）。（38−40）为了在肿瘤部位对抗 Tregs，我们将环磷酰胺装载到血小板中。我们发现，血小板能在体外 24 小时内内化并释放环磷酰胺（图 S10）。为了探究 PD-L1 阻断与环磷酰胺诱导的 Tregs 耗竭同时产生的抗肿瘤效果，我们采用了不完全肿瘤切除的 B16F10 黑色素瘤模型。在该模型中，单独使用环磷酰胺或表达 PD-1 的血小板效果有限（图 5A 和图 S11A），而接受装载环磷酰胺的 PD-1 表达血小板治疗的鼠肿瘤进展显著受到抑制（P < 0.001）（图 5A 和图 S11A）。环磷酰胺对 Tregs 的耗竭与 PD-1 对 PD-L1 的同时阻断改善了治疗鼠的存活率（图 5B）。
低剂量环磷酰胺可增强多种小鼠肿瘤模型及患者的免疫反应，这通常归因于选择性消耗调节性 T 细胞（Tregs）。（38−40）为了在肿瘤部位对抗 Tregs，我们将环磷酰胺装载到血小板中。我们发现，血小板能在体外 24 小时内内化并释放环磷酰胺（图 S10）。为了探究 PD-L1 阻断与环磷酰胺诱导的 Tregs 耗竭同时产生的抗肿瘤效果，我们采用了不完全肿瘤切除的 B16F10 黑色素瘤模型。在该模型中，单独使用环磷酰胺或表达 PD-1 的血小板效果有限（图 5A 和图 S11A），而接受装载环磷酰胺的 PD-1 表达血小板治疗的鼠肿瘤进展显著受到抑制（P < 0.001）（图 5A 和图 S11A）。环磷酰胺对 Tregs 的耗竭与 PD-1 对 PD-L1 的同时阻断改善了治疗鼠的存活率（图 5B）。
Figure 5 图 5
Figure 5. In vivo antitumor effect of cyclophosphamide-loaded PD-1-expressing platelets in incomplete-surgery tumor model. (A) Average tumor volumes of mice (n = 8) treated with PBS (G1), cyclophosphamide (CP) (G2), PD-1-expressing platelets (G3), CP-free platelets (G4), and CP-loaded PD-1-expressing platelets (G5). Data are shown as the mean ± SEM. ***, Compared with PBS control. (B) Survival curves of the treated mice. (C) Quantification of FoxP3 expression in CD4+ T cells within the tumors analyzed by the flow cytometry (gated on CD4+ T cells) (n = 3). (D) Representative plots and (E) quantification of Ki67 in CD8+ T cells within the tumors analyzed by the flow cytometry (gated on CD8+ T cells) (n = 3). (F) Representative plots and (G) quantification of CD8+ and CD4+ T cells within tumors analyzed by the flow cytometry (gated on CD3+ T cells) (n = 3). (H) Representative plots and (I) quantification of GzmB in CD8+ T cells within the tumors analyzed by the flow cytometry (gated on CD8+ T cells) (n = 3). (J, K) Immunofluorescence of the tumors showing CD8+ T cell infiltration (scale bar: 100 μm). Error bar of C, E, G, I, K, ± SD. Throughout, NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (A, C, E, G, I, K) Two-way ANOVA with Tukey posthoc test analyses were carried out to do the analyses or (B) by log-rank (Mantel-Cox) test (B).
图 5. 载环磷酰胺的 PD-1 表达血小板在不完全手术肿瘤模型中的体内抗肿瘤效果。(A) 接受 PBS（G1）、环磷酰胺（CP）（G2）、PD-1 表达血小板（G3）、不含 CP 的血小板（G4）及载 CP 的 PD-1 表达血小板（G5）治疗的 8 只小鼠的平均肿瘤体积。数据以均值±标准误（SEM）表示。***，与 PBS 对照组相比。(B) 治疗小鼠的生存曲线。(C) 通过流式细胞术（以 CD4+ T 细胞为门控）分析肿瘤内 CD4+ T 细胞中 FoxP3 表达的量化结果（n=3）。(D) 代表性图谱及(E) 通过流式细胞术（以 CD8+ T 细胞为门控）分析肿瘤内 CD8+ T 细胞中 Ki67 表达的量化结果（n=3）。(F) 代表性图谱及(G) 通过流式细胞术（以 CD3+ T 细胞为门控）分析肿瘤内 CD8+和 CD4+ T 细胞的量化结果（n=3）。(H) 代表性图谱及(I) 通过流式细胞术（以 CD8+ T 细胞为门控）分析肿瘤内 CD8+ T 细胞中 GzmB 表达的量化结果（n=3）。(J, K) 显示肿瘤内 CD8+ T 细胞浸润的免疫荧光图像（标尺：100 μm）。误差棒 C、E、G、I、K 为±标准差（SD）。全文标注：NS，无显著性；*P < 0.05；**P < 0.01; ***P < 0.001。（A、C、E、G、I、K）采用双因素方差分析结合 Tukey 事后检验进行分析，或（B）通过 log-rank（Mantel-Cox）检验进行分析。
We further investigated the frequencies of the CD4+ Tregs and CD8+ TILs in the tumor upon treatment. Free cyclophosphamide and cyclophosphamide-loaded platelets selectively depleted Tregs within the tumor (Figure 5C and Figure S11B) and increased the frequency of Ki67+ T cells (Figure 5D,E). Of note, despite PD-1-expressing platelets having limited effect in reducing Tregs, they still increased the frequency of Ki67+ T cells (Figure 5D,E). Remarkably, the frequency of CD8+ TILs was significantly increased in tumors collected from mice treated with cyclophosphamide-loaded PD-1-expressing platelets (Figure 5F,G), and these cells showed GzmB expression (Figure 5H,I). Immunofluorescence staining also revealed enhanced density of infiltrated CD8+ T cells in the mice treated with cyclophosphamide-loaded PD-1-expressing platelets as compared to control mice (Figure 5J,K). Mice treated with a low dose of cyclophosphamide, and cyclophosphamide-loaded platelets showed delayed hair growth in the abdomen and slighted weight loss (Figure S11A,C). These results demonstrated that the combined utilization of PD-1-expressing platelets and cyclophosphamide effectively disrupted the immune blockade of PD-L1 and depleted the Tregs, leading to the reduced tumor relapse rate after surgery.
我们进一步研究了治疗后肿瘤中 CD4+ Tregs 和 CD8+ TILs 的频率变化。游离环磷酰胺和载有环磷酰胺的血小板选择性地耗竭了肿瘤内的 Tregs（图 5C 和图 S11B），并提高了 Ki67+ T 细胞的频率（图 5D，E）。值得注意的是，尽管表达 PD-1 的血小板在减少 Tregs 方面效果有限，但它们仍然增加了 Ki67+ T 细胞的频率（图 5D，E）。值得注意的是，接受载有环磷酰胺的 PD-1 表达血小板治疗的鼠肿瘤中，CD8+ TILs 的频率显著增加（图 5F，G），这些细胞表现出 GzmB 的表达（图 5H，I）。免疫荧光染色还显示，与对照组小鼠相比，接受载有环磷酰胺的 PD-1 表达血小板治疗的小鼠中，浸润的 CD8+ T 细胞密度有所增强（图 5J，K）。接受低剂量环磷酰胺和载有环磷酰胺的血小板治疗的小鼠表现出腹部毛发生长延迟和轻微体重下降（图 S11A，C）。 这些结果表明，PD-1 表达的血小板与环磷酰胺联合使用，能有效打破 PD-L1 的免疫阻断并清除调节性 T 细胞，从而降低手术后肿瘤复发率。
我们进一步研究了治疗后肿瘤中 CD4+ Tregs 和 CD8+ TILs 的频率变化。游离环磷酰胺和载有环磷酰胺的血小板选择性地耗竭了肿瘤内的 Tregs（图 5C 和图 S11B），并提高了 Ki67+ T 细胞的频率（图 5D，E）。值得注意的是，尽管表达 PD-1 的血小板在减少 Tregs 方面效果有限，但它们仍然增加了 Ki67+ T 细胞的频率（图 5D，E）。值得注意的是，接受载有环磷酰胺的 PD-1 表达血小板治疗的鼠肿瘤中，CD8+ TILs 的频率显著增加（图 5F，G），这些细胞表现出 GzmB 的表达（图 5H，I）。免疫荧光染色还显示，与对照组小鼠相比，接受载有环磷酰胺的 PD-1 表达血小板治疗的小鼠中，浸润的 CD8+ T 细胞密度有所增强（图 5J，K）。接受低剂量环磷酰胺和载有环磷酰胺的血小板治疗的小鼠表现出腹部毛发生长延迟和轻微体重下降（图 S11A，C）。 这些结果表明，PD-1 表达的血小板与环磷酰胺联合使用，能有效打破 PD-L1 的免疫阻断并清除调节性 T 细胞，从而降低手术后肿瘤复发率。
In summary, we generated a cellular drug delivery system that leverages PD-1-presenting platelets for enhanced cancer immunotherapy after surgery. The PD-1-expressing platelets could accumulate in the surgical wound sites upon intravenous infusion, block PD-L1 on residual tumor cells, and revert exhausted CD8+ T cells to eradicate residual tumor cells. Such platelets could also function as a carrier of drugs, such as cyclophosphamide, to simultaneously disrupt the immune suppressive effects of PD-L1 and deplete Tregs, and promote the emergence of CD8+Ki67+GzmB+ lymphocytes in the surgical tumor microenvironment. This cell-mediated delivery strategy can be further exploited to deliver other checkpoint blockade inhibitors toward the tumor site as well as other immunomodulatory drugs. (41,42)
总之，我们构建了一种细胞药物递送系统，利用表达 PD-1 的血小板进行术后增强型癌症免疫治疗。静脉注射后，这些 PD-1 表达的血小板能在手术伤口部位聚集，阻断残留肿瘤细胞上的 PD-L1，使耗竭的 CD8+ T 细胞恢复功能，从而清除残留的肿瘤细胞。此外，这些血小板还能作为药物载体，如环磷酰胺，同时破坏 PD-L1 的免疫抑制作用并清除调节性 T 细胞（Tregs），促进手术肿瘤微环境中 CD8+Ki67+GzmB+淋巴细胞的出现。这种基于细胞的递送策略可进一步开发，用于向肿瘤部位递送其他检查点阻断抑制剂及其他免疫调节药物。（41,42）
总之，我们构建了一种细胞药物递送系统，利用表达 PD-1 的血小板进行术后增强型癌症免疫治疗。静脉注射后，这些 PD-1 表达的血小板能在手术伤口部位聚集，阻断残留肿瘤细胞上的 PD-L1，使耗竭的 CD8+ T 细胞恢复功能，从而清除残留的肿瘤细胞。此外，这些血小板还能作为药物载体，如环磷酰胺，同时破坏 PD-L1 的免疫抑制作用并清除调节性 T 细胞（Tregs），促进手术肿瘤微环境中 CD8+Ki67+GzmB+淋巴细胞的出现。这种基于细胞的递送策略可进一步开发，用于向肿瘤部位递送其他检查点阻断抑制剂及其他免疫调节药物。（41,42）
Chemicals and Reagents 化学品与试剂
Cyclophosphamide, thrombin, Wright–Giemsa solution, and phosphatase inhibitor cocktail were from Sigma-Aldrich. Murine PD-1 and PD-L1 antibodies were from Thermo Scientific and Sigma-Aldrich, respectively. Murine CD41a (ab63983), CD42a (ab173503), CD4, and CD8 antibodies were from Abcam. P-Selectin (sc-8419) was from Santa Cruz biotechnology. Murine GPVI (MAB6758) antibody was from R&D Systems. CD3, CD4, CD8, Ki67, and Foxp3 antibodies for FACS analysis were from Biolegend Inc. Wheat germ agglutinin (WGA) Alexa Fluor 488 and 594 dyes were from Thermo Scientific.
环磷酰胺、凝血酶、瑞氏-吉姆萨溶液及磷酸酶抑制剂混合物购自 Sigma-Aldrich。小鼠 PD-1 和 PD-L1 抗体分别来自 Thermo Scientific 和 Sigma-Aldrich。小鼠 CD41a（ab63983）、CD42a（ab173503）、CD4 和 CD8 抗体购自 Abcam。P-选择素（sc-8419）来自 Santa Cruz Biotechnology。小鼠 GPVI（MAB6758）抗体源自 R&D Systems。用于 FACS 分析的 CD3、CD4、CD8、Ki67 和 Foxp3 抗体由 Biolegend Inc.提供。麦胚凝集素（WGA）Alexa Fluor 488 和 594 染料购自 Thermo Scientific。
环磷酰胺、凝血酶、瑞氏-吉姆萨溶液及磷酸酶抑制剂混合物购自 Sigma-Aldrich。小鼠 PD-1 和 PD-L1 抗体分别来自 Thermo Scientific 和 Sigma-Aldrich。小鼠 CD41a（ab63983）、CD42a（ab173503）、CD4 和 CD8 抗体购自 Abcam。P-选择素（sc-8419）来自 Santa Cruz Biotechnology。小鼠 GPVI（MAB6758）抗体源自 R&D Systems。用于 FACS 分析的 CD3、CD4、CD8、Ki67 和 Foxp3 抗体由 Biolegend Inc.提供。麦胚凝集素（WGA）Alexa Fluor 488 和 594 染料购自 Thermo Scientific。
Cell Culture 细胞培养
HEK293T were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Mouse megakaryocyte cell line L8057 was kindly provided by professor Alan Cantor (Boston Children’s Hospital, Dana-Farber Cancer Institute) and was cultured in RPMI 1640 with 20% FBS. The murine melanoma cell line B16F10 was obtained from the American Type Culture Collection. For bioluminescent in vivo tumor imaging, B16F10-luc cells were kindly provided by Dr. Leaf Huang at UNC. B16F10 cells were cultured in DMEM supplemented with 10% FBS.
HEK293T 细胞在含 10%胎牛血清（FBS）的 Dulbecco 改良 Eagle 培养基（DMEM）中培养。小鼠巨核细胞系 L8057 由 Alan Cantor 教授（波士顿儿童医院，丹娜—法伯癌症研究所）慷慨提供，并在含 20% FBS 的 RPMI 1640 中培养。小鼠黑色素瘤细胞系 B16F10 购自美国典型培养物保藏中心。用于活体生物发光肿瘤成像的 B16F10-luc 细胞由 UNC 的 Leaf Huang 博士慷慨提供。B16F10 细胞在含 10% FBS 的 DMEM 中培养。
HEK293T 细胞在含 10%胎牛血清（FBS）的 Dulbecco 改良 Eagle 培养基（DMEM）中培养。小鼠巨核细胞系 L8057 由 Alan Cantor 教授（波士顿儿童医院，丹娜—法伯癌症研究所）慷慨提供，并在含 20% FBS 的 RPMI 1640 中培养。小鼠黑色素瘤细胞系 B16F10 购自美国典型培养物保藏中心。用于活体生物发光肿瘤成像的 B16F10-luc 细胞由 UNC 的 Leaf Huang 博士慷慨提供。B16F10 细胞在含 10% FBS 的 DMEM 中培养。
Plasmid and Stable Cell Line
质粒与稳定细胞系
Lentivirus vector encoding murine PD-1 fused at C-terminal region with GFP-tag (pLenti-C-mGFP-PD-1-puro) and Lenti-vpak packaging kit and transfection reagent were obtained from Origene. Mouse DsRed-PD-L1 plasmid was obtained from Sino biological. L8057 cells were infected with the lentivirus and incubated with 6 μg/mL polybrene. After infection, L8057 cells were cultured in RPMI 1640 with 20% FBS and with 1 μg/mL puromycin to select cell lines stably expressing murine PD-1. Established EGFP-PD-1 L8057 cells were maintained in 20% FBS complementary with 0.5–1 μg/mL puromycin.
慢病毒载体编码在小鼠 PD-1 的 C 端区域与 GFP 标签融合（pLenti-C-mGFP-PD-1-puro），以及 Lenti-vpak 包装试剂盒和转染试剂均购自 Origene。小鼠 DsRed-PD-L1 质粒由 Sino biological 提供。L8057 细胞通过慢病毒感染，并使用 6 μg/mL 聚凝胺进行孵育。感染后，L8057 细胞在含 20%胎牛血清的 RPMI 1640 培养基中培养，并添加 1 μg/mL 嘌呤霉素以筛选稳定表达小鼠 PD-1 的细胞系。建立的 EGFP-PD-1 L8057 细胞在含 20%胎牛血清的培养基中维持，并补充 0.5–1 μg/mL 嘌呤霉素。
慢病毒载体编码在小鼠 PD-1 的 C 端区域与 GFP 标签融合（pLenti-C-mGFP-PD-1-puro），以及 Lenti-vpak 包装试剂盒和转染试剂均购自 Origene。小鼠 DsRed-PD-L1 质粒由 Sino biological 提供。L8057 细胞通过慢病毒感染，并使用 6 μg/mL 聚凝胺进行孵育。感染后，L8057 细胞在含 20%胎牛血清的 RPMI 1640 培养基中培养，并添加 1 μg/mL 嘌呤霉素以筛选稳定表达小鼠 PD-1 的细胞系。建立的 EGFP-PD-1 L8057 细胞在含 20%胎牛血清的培养基中维持，并补充 0.5–1 μg/mL 嘌呤霉素。
Preparation of Platelets from L8057 Cells
L8057 细胞制备血小板
L8057 cells and PD-1-expressing L8057 cells were cultured in RPMI 1640 with 20% FBS. For maturation and differentiation, L8057 cells were stimulated with 100–500 nM PMA for 3 days. Cells were then cultured for another 6 days to produce proplatelets and platelets. To isolate platelets, the culture medium was centrifuged at 1500 rpm for 20 min to remove the cells. Supernatant was then centrifuged at 12 000 rpm for 20 min at room temperature. Platelet precipitate was resuspended in Tyrode’s buffer (134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 10 mM HEPES, pH 7.4) or PBS with 1 μM PGE1. To active platelets, 0.5 U thrombin/mL was added to the platelet suspension. PGE1 was removed prior to platelet activation.
L8057 细胞及表达 PD-1 的 L8057 细胞在含 20%胎牛血清的 RPMI 1640 培养基中培养。为促进成熟与分化，L8057 细胞经 100–500 nM PMA 刺激 3 天。随后，细胞再培养 6 天以生成前血小板及血小板。为分离血小板，培养基经 1500 rpm 离心 20 分钟去除细胞，上清液再于室温下以 12000 rpm 离心 20 分钟。所得血小板沉淀物用 Tyrode 缓冲液（134 mM NaCl，12 mM NaHCO3，2.9 mM KCl，0.34 mM Na2HPO4，1 mM MgCl2，10 mM HEPES，pH 7.4）或含 1 μM PGE1 的 PBS 重悬。为激活血小板，向血小板悬液中加入 0.5 U/mL 的凝血酶。激活前需去除 PGE1。
L8057 细胞及表达 PD-1 的 L8057 细胞在含 20%胎牛血清的 RPMI 1640 培养基中培养。为促进成熟与分化，L8057 细胞经 100–500 nM PMA 刺激 3 天。随后，细胞再培养 6 天以生成前血小板及血小板。为分离血小板，培养基经 1500 rpm 离心 20 分钟去除细胞，上清液再于室温下以 12000 rpm 离心 20 分钟。所得血小板沉淀物用 Tyrode 缓冲液（134 mM NaCl，12 mM NaHCO3，2.9 mM KCl，0.34 mM Na2HPO4，1 mM MgCl2，10 mM HEPES，pH 7.4）或含 1 μM PGE1 的 PBS 重悬。为激活血小板，向血小板悬液中加入 0.5 U/mL 的凝血酶。激活前需去除 PGE1。
Wright–Giemsa Stain 赖特-吉姆萨染色
L8057 cells stimulated with 100–500 nM PMA for 3 days were harvested and washed with PBS buffer. Cells were then fixed in absolute methanol for 5 min, stained in Wright–Giemsa stain solution for 5 min, washed with PBS buffer, and observed under microscope with 40× objective.
L8057 细胞在 100–500 nM PMA 刺激下培养 3 天后被收获，并用 PBS 缓冲液洗涤。随后，细胞在无水甲醇中固定 5 分钟，用瑞氏-吉姆萨染色液染色 5 分钟，再次用 PBS 缓冲液清洗，最后在 40 倍物镜下显微镜观察。
L8057 细胞在 100–500 nM PMA 刺激下培养 3 天后被收获，并用 PBS 缓冲液洗涤。随后，细胞在无水甲醇中固定 5 分钟，用瑞氏-吉姆萨染色液染色 5 分钟，再次用 PBS 缓冲液清洗，最后在 40 倍物镜下显微镜观察。
Cell Immunofluorescent Assay
细胞免疫荧光检测
EGFP-PD-1-expressing L8057 cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Then the cells were washed with PBS, incubated with 0.2% Triton X-100 for 5 min, and blocked with the buffer containing 3% BSA for 1 h. After that, cells were stained with CD41a, CD42a, and P-selectin antibodies overnight at 4 °C. After washing with PBS, cells were stained with Rhodamine-conjugated secondary antibody (KPL) diluted at room temperature and kept in the dark for 1 h. The nucleus was stained with DAPI for 10 min. After washing with PBS, confocal microscopy was performed on a FLUO-VIEW laser scanning confocal microscope (Zeiss) in sequential scanning mode using a 63× objective.
EGFP-PD-1 表达的 L8057 细胞用 PBS 洗涤后，以 4%多聚甲醛固定 10 分钟。随后，细胞再次用 PBS 清洗，加入 0.2% Triton X-100 孵育 5 分钟，并用含 3% BSA 的缓冲液阻断 1 小时。接着，细胞在 4°C 下与 CD41a、CD42a 及 P-选择素抗体共孵育过夜。清洗后，细胞与室温下稀释的罗丹明偶联的二抗（KPL）反应，避光孵育 1 小时。核染色使用 DAPI 进行 10 分钟。最后，经 PBS 清洗后，利用 FLUO-VIEW 激光扫描共聚焦显微镜（Zeiss）在 63×物镜下进行顺序扫描模式的共聚焦显微成像。
EGFP-PD-1 表达的 L8057 细胞用 PBS 洗涤后，以 4%多聚甲醛固定 10 分钟。随后，细胞再次用 PBS 清洗，加入 0.2% Triton X-100 孵育 5 分钟，并用含 3% BSA 的缓冲液阻断 1 小时。接着，细胞在 4°C 下与 CD41a、CD42a 及 P-选择素抗体共孵育过夜。清洗后，细胞与室温下稀释的罗丹明偶联的二抗（KPL）反应，避光孵育 1 小时。核染色使用 DAPI 进行 10 分钟。最后，经 PBS 清洗后，利用 FLUO-VIEW 激光扫描共聚焦显微镜（Zeiss）在 63×物镜下进行顺序扫描模式的共聚焦显微成像。
Western Blot 免疫印迹法
Immunoblotting analysis was performed as previously described. (43) L8057 control cells and L8057 cells stably expressing EGFP-PD1 were lysed with RIPA lysis buffer (Thermo Scientific), and cell lysates resolved on 12% SDS-PAGE. Immunoblotting was incubated with PD-1, CD41a, CD42a, P-selectin, GPVI, and β-actin antibodies, followed by enhanced chemiluminescence (ECL) detection (Thermo Scientific).
免疫印迹分析按照先前描述的方法进行。(43) L8057 对照细胞和稳定表达 EGFP-PD1 的 L8057 细胞用 RIPA 裂解缓冲液（赛默飞世尔科技）裂解，细胞裂解物通过 12% SDS-PAGE 分离。免疫印迹与 PD-1、CD41a、CD42a、P-选择素、GPVI 和β-肌动蛋白抗体孵育，随后采用增强化学发光（ECL）检测（赛默飞世尔科技）。
免疫印迹分析按照先前描述的方法进行。(43) L8057 对照细胞和稳定表达 EGFP-PD1 的 L8057 细胞用 RIPA 裂解缓冲液（赛默飞世尔科技）裂解，细胞裂解物通过 12% SDS-PAGE 分离。免疫印迹与 PD-1、CD41a、CD42a、P-选择素、GPVI 和β-肌动蛋白抗体孵育，随后采用增强化学发光（ECL）检测（赛默飞世尔科技）。
B16F10 Cell Binding Assay
B16F10 细胞结合实验
B16F10 cells were seeded in confocal wells. EGFP-PD-1-expressing platelets and free platelets (∼0.5 × 108 cell/well) labeled with cy5.5 were added to the culture medium and incubated with the B16F10 cells overnight. Wheat germ agglutinin (WGA) and Alexa Fluor 594 conjugate were added to stain the cell membrane of B16F10 for 10 min. The nucleus was stained with DAPI for 10 min. After washing with PBS, confocal microscopy was performed on a confocal microscope (Zeiss) in sequential scanning mode using a 63× objective.
B16F10 细胞被接种于共聚焦孔板中。表达 EGFP-PD-1 的血小板和未修饰的游离血小板（约 0.5 × 10^8 细胞/孔）用 Cy5.5 标记后加入培养基，并与 B16F10 细胞共同孵育过夜。随后，加入麦胚凝集素（WGA）与 Alexa Fluor 594 偶联物对 B16F10 细胞膜进行染色，染色时间为 10 分钟。细胞核则采用 DAPI 染色，同样为 10 分钟。经 PBS 洗涤后，在配备 63×物镜的共聚焦显微镜（蔡司）上，以顺序扫描模式进行共聚焦显微成像。
B16F10 细胞被接种于共聚焦孔板中。表达 EGFP-PD-1 的血小板和未修饰的游离血小板（约 0.5 × 10^8 细胞/孔）用 Cy5.5 标记后加入培养基，并与 B16F10 细胞共同孵育过夜。随后，加入麦胚凝集素（WGA）与 Alexa Fluor 594 偶联物对 B16F10 细胞膜进行染色，染色时间为 10 分钟。细胞核则采用 DAPI 染色，同样为 10 分钟。经 PBS 洗涤后，在配备 63×物镜的共聚焦显微镜（蔡司）上，以顺序扫描模式进行共聚焦显微成像。
Collagen Binding Assay 胶原蛋白结合实验
Briefly, 200 μL of the collagen solution (Murine collagen type I/III (Bio-Rad) reconstituted in 0.25% acetic acid at concentration of 2.0 mg/mL) was added to a 96-well plate and incubated overnight at 4 °C. Prior to the collagen binding study, the plate was blocked with 2% BSA and washed with PBS. Platelets were stained with WGA Alexa Fluor 594 for 30 min, washed with PBS, and added to collagen-coated or non-collagen-coated plates (∼1 × 107 cells/well) in triplicate. After 30 s of incubation, plates were washed. Retained platelets were dissolved with 100 μL of DMSO for fluorescence quantification using a TeCan Infinite M200 reader. For confocal imaging, the collagen solution was added to the confocal well and incubated overnight at 4 °C (∼1 × 108 cells/well). Wells were blocked with 2% BSA, and WGA Alexa Fluor 594-labeled platelets were incubated with collagen for 2 min, washed with PBS, and analyzed by confocal microscopy using a confocal microscope (Zeiss) in sequential scanning mode using a 63× objective.
简而言之，将 200 μL 的胶原溶液（Bio-Rad 的小鼠 I 型/III 型胶原，在 0.25%乙酸中以 2.0 mg/mL 浓度重构）加入 96 孔板中，并在 4 °C 下过夜孵育。在进行胶原结合研究之前，用 2% BSA 封闭板孔，并用 PBS 洗涤。血小板用 WGA Alexa Fluor 594 染色 30 分钟，用 PBS 洗涤后，以三重复方式加入胶原包被或未包被的板中（约 1 × 10^7 细胞/孔）。孵育 30 秒后，洗涤板子。保留的血小板用 100 μL DMSO 溶解，使用 TeCan Infinite M200 阅读器进行荧光定量。对于共聚焦成像，胶原溶液加入共聚焦孔中，在 4 °C 下过夜孵育（约 1 × 10^8 细胞/孔）。孔用 2% BSA 封闭，WGA Alexa Fluor 594 标记的血小板与胶原孵育 2 分钟，用 PBS 洗涤，并通过共聚焦显微镜（Zeiss）在顺序扫描模式下使用 63×物镜进行分析。
简而言之，将 200 μL 的胶原溶液（Bio-Rad 的小鼠 I 型/III 型胶原，在 0.25%乙酸中以 2.0 mg/mL 浓度重构）加入 96 孔板中，并在 4 °C 下过夜孵育。在进行胶原结合研究之前，用 2% BSA 封闭板孔，并用 PBS 洗涤。血小板用 WGA Alexa Fluor 594 染色 30 分钟，用 PBS 洗涤后，以三重复方式加入胶原包被或未包被的板中（约 1 × 10^7 细胞/孔）。孵育 30 秒后，洗涤板子。保留的血小板用 100 μL DMSO 溶解，使用 TeCan Infinite M200 阅读器进行荧光定量。对于共聚焦成像，胶原溶液加入共聚焦孔中，在 4 °C 下过夜孵育（约 1 × 10^8 细胞/孔）。孔用 2% BSA 封闭，WGA Alexa Fluor 594 标记的血小板与胶原孵育 2 分钟，用 PBS 洗涤，并通过共聚焦显微镜（Zeiss）在顺序扫描模式下使用 63×物镜进行分析。
Aggregation Assay 聚集试验
Aggregation of platelets was assessed using confocal imaging. Platelets were labeled with WGA Alexa Fluor 594, loaded to the confocal well, and incubated with 0.5 U/mL of thrombin for 30 min. Confocal microscopy was performed on a confocal microscope (Zeiss) in sequential scanning mode using a 63× objective.
通过共聚焦成像评估血小板的聚集情况。血小板用 WGA Alexa Fluor 594 标记，加载到共聚焦孔中，并与 0.5 U/mL 的凝血酶共同孵育 30 分钟。使用 63×物镜在共聚焦显微镜（蔡司）上以顺序扫描模式进行共聚焦显微镜检查。
通过共聚焦成像评估血小板的聚集情况。血小板用 WGA Alexa Fluor 594 标记，加载到共聚焦孔中，并与 0.5 U/mL 的凝血酶共同孵育 30 分钟。使用 63×物镜在共聚焦显微镜（蔡司）上以顺序扫描模式进行共聚焦显微镜检查。
Drug Loading and Release 药物装载与释放
To prepare cyclophosphamide-loaded platelets, 100 μg of purified platelets and 100 μg of cyclophosphamide were gently mixed in 1 mL of PBS and incubated for 2 h at 37 °C. (44,45) Platelets were then washed with PBS by centrifugation at 12 000 rpm. For electroporation shock method, 100 μg of purified platelets and 100 μg of cyclophosphamide were gently mixed in 1 mL of electroporation buffer (1.15 mM potassium phosphate pH 7.2, 25 mM potassium chloride, 21% Optiprep) at room temperature. Samples were electroporated at 300 V and 150 μF in 0.4 cm electroporation cuvettes using a MicroPulser Electro-porator (Bio-Rad). Electroporation cuvettes containing samples were then incubated for 30 min for the membrane recovery. Platelets were washed with PBS by centrifugation at 12 000 rpm. (46−48) The release of cyclophosphamide from platelets (100 μg/mL) was analyzed in PBS (pH 7.2) at different time points at 37 °C. The amount of cyclophosphamide released was quantified by using a UV–vis spectrophotometer at 205 nm. (49,50)
为制备载环磷酰胺的血小板，将 100 μg 纯化血小板与 100 μg 环磷酰胺轻柔混合于 1 mL PBS 中，并在 37 °C 下孵育 2 小时。(44,45) 随后，通过 12000 rpm 离心将血小板用 PBS 洗涤。对于电穿孔冲击法，100 μg 纯化血小板与 100 μg 环磷酰胺在室温下轻柔混合于 1 mL 电穿孔缓冲液（1.15 mM 磷酸钾 pH 7.2，25 mM 氯化钾，21% Optiprep）中。样品在 0.4 cm 电穿孔比色皿中以 300 V 和 150 μF 的条件使用 MicroPulser 电穿孔仪（Bio-Rad）进行电穿孔处理。电穿孔后的比色皿内样品再孵育 30 分钟以恢复膜结构。血小板通过 12000 rpm 离心用 PBS 洗涤。(46−48) 从血小板（100 μg/mL）中释放的环磷酰胺在 PBS（pH 7.2）中于不同时间点 37 °C 下进行分析。释放的环磷酰胺量通过在 205 nm 处使用紫外-可见分光光度计进行定量。(49,50)
为制备载环磷酰胺的血小板，将 100 μg 纯化血小板与 100 μg 环磷酰胺轻柔混合于 1 mL PBS 中，并在 37 °C 下孵育 2 小时。(44,45) 随后，通过 12000 rpm 离心将血小板用 PBS 洗涤。对于电穿孔冲击法，100 μg 纯化血小板与 100 μg 环磷酰胺在室温下轻柔混合于 1 mL 电穿孔缓冲液（1.15 mM 磷酸钾 pH 7.2，25 mM 氯化钾，21% Optiprep）中。样品在 0.4 cm 电穿孔比色皿中以 300 V 和 150 μF 的条件使用 MicroPulser 电穿孔仪（Bio-Rad）进行电穿孔处理。电穿孔后的比色皿内样品再孵育 30 分钟以恢复膜结构。血小板通过 12000 rpm 离心用 PBS 洗涤。(46−48) 从血小板（100 μg/mL）中释放的环磷酰胺在 PBS（pH 7.2）中于不同时间点 37 °C 下进行分析。释放的环磷酰胺量通过在 205 nm 处使用紫外-可见分光光度计进行定量。(49,50)
Circulation 循环
PD-1-expressing platelets and free platelets produced from L8057 cells were labeled by NHS-Cy5.5. Labeled platelets (∼2 × 108 cells) were washed with PBS and injected intravenously via tail-vein in C57BL/6 mice in 200 μL of final volume. Peripheral blood was collected at different time points after platelet injection, and fluorescence of the serum was measured.
PD-1 表达的血小板及 L8057 细胞产生的自由血小板通过 NHS-Cy5.5 进行标记。标记后的血小板（约 2 × 10^8 个细胞）用 PBS 洗涤后，以最终体积 200 μL 通过尾静脉静脉注射到 C57BL/6 小鼠体内。在血小板注射后不同时间点采集外周血，并测量血清中的荧光强度。
PD-1 表达的血小板及 L8057 细胞产生的自由血小板通过 NHS-Cy5.5 进行标记。标记后的血小板（约 2 × 10^8 个细胞）用 PBS 洗涤后，以最终体积 200 μL 通过尾静脉静脉注射到 C57BL/6 小鼠体内。在血小板注射后不同时间点采集外周血，并测量血清中的荧光强度。
Biodistribution 生物分布
Free platelets and PD-1-expressing platelets produced from L8057 cells were labeled by NHS-Cy5.5 in PBS. Following incubation overnight, Cy5.5-labeled platelets (∼2 × 108 cells/mouse) were washed with PBS and infused in melanoma tumor-bearing C57BL/6 mice. The control group was injected with PBS. After 24 h, mice were euthanized, and tumors and organs were harvested. Fluorescence imaging and average radio intensities were recorded using a Xenogen IVIS spectrum imaging system.
自由血小板及 L8057 细胞产生的 PD-1 表达型血小板在 PBS 中通过 NHS-Cy5.5 进行标记。经过一夜孵育后，Cy5.5 标记的血小板（约 2×10^8 细胞/只小鼠）用 PBS 洗涤并注入携带黑色素瘤的 C57BL/6 小鼠体内。对照组则注射 PBS。24 小时后，小鼠被安乐死，肿瘤及器官被采集。使用 Xenogen IVIS spectrum 成像系统记录荧光成像及平均放射强度。
自由血小板及 L8057 细胞产生的 PD-1 表达型血小板在 PBS 中通过 NHS-Cy5.5 进行标记。经过一夜孵育后，Cy5.5 标记的血小板（约 2×10^8 细胞/只小鼠）用 PBS 洗涤并注入携带黑色素瘤的 C57BL/6 小鼠体内。对照组则注射 PBS。24 小时后，小鼠被安乐死，肿瘤及器官被采集。使用 Xenogen IVIS spectrum 成像系统记录荧光成像及平均放射强度。
In Vivo Antitumor Effects
体内抗肿瘤效应
B16F10 luciferase-tagged B16F10 (1 × 106 cells/mouse) melanoma tumor cells were transplanted into the right flank of C57BL/6J mice. When the tumor volumes were around ∼130 mm3, the tumors were then resected, leaving about 15 mm3 (∼10%) tumor to mimic the residual tumors in the surgical bed. Briefly, animals were anesthetized in an induction chamber using isoflurane (up to 5% for induction; 1–3% for maintenance), and anesthesia was maintained via a nose cone. The tumor area was clipped and aseptically prepped. Approximately 90% of the tumor was removed using sterile instruments. The wound was closed using an Autoclip wound closing system. The mice were randomly divided into groups (n = 8). Mice were intravenously injected with PBS, free platelets (∼2 × 108 cells/mouse), PD-1 platelets (∼2 × 108 cells/mouse), cyclophosphamide (20 mg/kg), cyclophosphamide-loaded free platelets (∼2× 108 cells/mouse), or cyclophosphamide-loaded PD-1-expressing platelets (∼2 × 108 cells/mouse). Immediately after treatment, tumor resection was carried out within 10 min, one mouse by one mouse. The tumor burden was monitored via tumor bioluminescence. Images of the mice bearing tumor were taken using an IVIS Lumina imaging system (PerkinElmer). Tumor size was measured with a digital caliper. The tumor volume (mm3) was calculated as (long diameter × short diameter2)/2. Once the mice exhibit signs of impaired health or when the volume of the tumor exceeded 1.5 cm3, the mice were euthanized with CO2.
B16F10 luciferase-标记的 B16F10（每只小鼠 1×10^6 细胞）黑色素瘤肿瘤细胞被移植到 C57BL/6J 小鼠的右侧腹。当肿瘤体积达到约 130 mm³时，切除肿瘤，留下约 15 mm³（约 10%）的肿瘤以模拟手术床中的残留肿瘤。简言之，动物在诱导室中使用异氟烷麻醉（诱导时最高 5%；维持时 1-3%），并通过鼻锥维持麻醉状态。肿瘤区域被剪毛并进行无菌准备。使用无菌器械切除约 90%的肿瘤。伤口使用 Autoclip 伤口闭合系统闭合。小鼠被随机分组（n=8）。小鼠通过静脉注射 PBS、游离血小板（约 2×10^8 细胞/小鼠）、PD-1 血小板（约 2×10^8 细胞/小鼠）、环磷酰胺（20 mg/kg）、载环磷酰胺的游离血小板（约 2×10^8 细胞/小鼠）或载环磷酰胺的 PD-1 表达血小板（约 2×10^8 细胞/小鼠）。治疗后立即进行肿瘤切除，每只小鼠在 10 分钟内完成。通过肿瘤生物发光监测肿瘤负担。 使用 IVIS Lumina 成像系统（PerkinElmer）拍摄携带肿瘤的小鼠图像。肿瘤大小通过数字卡尺测量。肿瘤体积（mm³）计算公式为（长直径×短直径²）/2。一旦小鼠出现健康状况恶化迹象或肿瘤体积超过 1.5 cm³时，小鼠通过 CO₂安乐死处理。
B16F10 luciferase-标记的 B16F10（每只小鼠 1×10^6 细胞）黑色素瘤肿瘤细胞被移植到 C57BL/6J 小鼠的右侧腹。当肿瘤体积达到约 130 mm³时，切除肿瘤，留下约 15 mm³（约 10%）的肿瘤以模拟手术床中的残留肿瘤。简言之，动物在诱导室中使用异氟烷麻醉（诱导时最高 5%；维持时 1-3%），并通过鼻锥维持麻醉状态。肿瘤区域被剪毛并进行无菌准备。使用无菌器械切除约 90%的肿瘤。伤口使用 Autoclip 伤口闭合系统闭合。小鼠被随机分组（n=8）。小鼠通过静脉注射 PBS、游离血小板（约 2×10^8 细胞/小鼠）、PD-1 血小板（约 2×10^8 细胞/小鼠）、环磷酰胺（20 mg/kg）、载环磷酰胺的游离血小板（约 2×10^8 细胞/小鼠）或载环磷酰胺的 PD-1 表达血小板（约 2×10^8 细胞/小鼠）。治疗后立即进行肿瘤切除，每只小鼠在 10 分钟内完成。通过肿瘤生物发光监测肿瘤负担。 使用 IVIS Lumina 成像系统（PerkinElmer）拍摄携带肿瘤的小鼠图像。肿瘤大小通过数字卡尺测量。肿瘤体积（mm³）计算公式为（长直径×短直径²）/2。一旦小鼠出现健康状况恶化迹象或肿瘤体积超过 1.5 cm³时，小鼠通过 CO₂安乐死处理。
Tissue Immunofluorescent Assay
组织免疫荧光检测
Tumors were harvested from the mice and snap frozen in optimal cutting medium (O.C.T.). Ten micrometer sections were cut using a cryotome and mounted on slides. Frozen tumor sections were incubated in PBS for 15 min to remove the embedding medium. Specimens were blocked with the buffer containing 3% BSA, incubated with CD4 and CD8 antibodies (1:50 in 3% BSA) overnight, washed with PBS, stained with TRITC secondary antibody (KPL) diluted in 1.5% BSA at room temperature, and kept in the dark for 1 h. The nucleus was stained with DAPI and washed with PBS. Confocal microscopy was performed on a FLUO-VIEW laser scanning confocal microscope (Zeiss) in sequential scanning mode using a 40× objective.
肿瘤从实验小鼠中取出后，立即在最佳切割介质（O.C.T.）中速冻保存。使用冰冻切片机切割成 10 微米厚的切片，并固定在载玻片上。冷冻的肿瘤切片在磷酸盐缓冲液（PBS）中孵育 15 分钟以去除包埋介质。样品用含 3%牛血清白蛋白（BSA）的缓冲液进行封闭处理，随后与 CD4 和 CD8 抗体（1:50 稀释于 3% BSA 中）在 4℃下过夜孵育，再用 PBS 洗涤。接着，用室温下稀释于 1.5% BSA 中的 TRITC 二抗（KPL）进行染色，并在暗处保持 1 小时。细胞核用 DAPI 染色，并用 PBS 冲洗。使用配备 40×物镜的 FLUO-VIEW 激光扫描共聚焦显微镜（Zeiss），在顺序扫描模式下进行共聚焦显微成像。
肿瘤从实验小鼠中取出后，立即在最佳切割介质（O.C.T.）中速冻保存。使用冰冻切片机切割成 10 微米厚的切片，并固定在载玻片上。冷冻的肿瘤切片在磷酸盐缓冲液（PBS）中孵育 15 分钟以去除包埋介质。样品用含 3%牛血清白蛋白（BSA）的缓冲液进行封闭处理，随后与 CD4 和 CD8 抗体（1:50 稀释于 3% BSA 中）在 4℃下过夜孵育，再用 PBS 洗涤。接着，用室温下稀释于 1.5% BSA 中的 TRITC 二抗（KPL）进行染色，并在暗处保持 1 小时。细胞核用 DAPI 染色，并用 PBS 冲洗。使用配备 40×物镜的 FLUO-VIEW 激光扫描共聚焦显微镜（Zeiss），在顺序扫描模式下进行共聚焦显微成像。
Statistical Analysis 统计分析
All results are expressed as the mean ± SD or the mean ± SEM as indicated. Biological replicates were used in all experiments unless otherwise stated. One-way or two-way analysis of variance (ANOVA) and Tukey posthoc tests were used when more than two groups were compared (multiple comparisons). Survival benefit was determined using a log-rank test. All statistical analyses were performed using the IBM SPSS statistics 19. The threshold for statistical significance was P < 0.05.
所有结果均以均值±标准差或均值±标准误表示，具体视情况而定。除非另有说明，所有实验均使用生物学重复。当比较两组以上时，采用单因素或双因素方差分析（ANOVA）及 Tukey 事后检验（多重比较）。生存获益通过 log-rank 检验确定。所有统计分析均使用 IBM SPSS 统计 19 软件进行。统计显著性阈值为 P < 0.05。
所有结果均以均值±标准差或均值±标准误表示，具体视情况而定。除非另有说明，所有实验均使用生物学重复。当比较两组以上时，采用单因素或双因素方差分析（ANOVA）及 Tukey 事后检验（多重比较）。生存获益通过 log-rank 检验确定。所有统计分析均使用 IBM SPSS 统计 19 软件进行。统计显著性阈值为 P < 0.05。
Supporting Information 支持信息
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b02321.
支持信息可免费在 ACS Publications 网站上获取，DOI: 10.1021/acs.nanolett.8b02321。
In vitro process of platelet releasing from L8057 cells, establishment of L8057 cell line stably expressing EGFP-PD-1, activity of platelets released from L8057 cells, in vitro binding and uptake of platelets by tumor cells, tumor size and body weight of mice, histological images for H&E staining, in vitro loading and release of cyclophosphamide by PD-1-expressing platelets, and the bioluminescence imaging of B16F10 tumor growth in mice after partial tumor resection and receiving the treatments (PDF)
体外从 L8057 细胞释放血小板的过程，建立稳定表达 EGFP-PD-1 的 L8057 细胞系，L8057 细胞释放血小板的活性，血小板与肿瘤细胞的体外结合与摄取，小鼠的肿瘤大小与体重，H&E 染色组织学图像，PD-1 表达血小板体外装载与释放环磷酰胺，以及部分肿瘤切除后接受治疗的小鼠 B16F10 肿瘤生长的生物发光成像（PDF）
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Author Information 作者信息
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- Peng Huang - Guangdong Key Laboratory for Biomedical, Measurements and Ultrasound Imaging, Laboratory of Evolutionary Theranostics, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen 518060, China;
http://orcid.org/0000-0003-3651-7813;
黄鹏 - 广东省生物医学测量与超声成像重点实验室，进化治疗学实验室，生物医学工程学院，健康科学中心，深圳大学，深圳 518060，中国； http://orcid.org/0000-0003-3651-7813；电子邮箱：peng.huang@szu.edu.cn - Zhen Gu - Department of Bioengineering, California NanoSystems Institute, and Center for Minimally Invasive Therapeutics (C-MIT), University of California, Los Angeles, California 90095, United States; Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United States;http://orcid.org/0000-0003-2947-4456;
Zhen Gu - 生物工程系，加州纳米系统研究所，微创治疗中心（C-MIT），加州大学洛杉矶分校，加利福尼亚州，邮编 90095，美国；联合生物医学工程系，北卡罗来纳大学教堂山分校与北卡罗来纳州立大学，罗利市，北卡罗来纳州，邮编 27695，美国； http://orcid.org/0000-0003-2947-4456；电子邮箱：guzhen@ucla.edu
- Zhaowei Chen - Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United States;http://orcid.org/0000-0001-9007-5513
赵伟 陈 - 联合生物医学工程系，北卡罗来纳大学教堂山分校与北卡罗来纳州立大学，罗利，北卡罗来纳州 27695，美国； http://orcid.org/0000-0001-9007-5513 - Junjie Yan - Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, North Carolina 27695, United States;http://orcid.org/0000-0001-8016-2277
闫俊杰 - 联合生物医学工程系，北卡罗来纳大学教堂山分校与北卡罗来纳州立大学，罗利，北卡罗来纳州 27695，美国； http://orcid.org/0000-0001-8016-2277
X.Z. and J.W. contributed equally to this work. X.Z., P.H.. and Z.G. designed the study. X.Z., Z.C., Q.H., C.W., J.W., and J.Y. performed the experiments. X.Z. interpreted the data. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
X.Z. 和 J.W. 对本研究贡献相同。X.Z.、P.H. 和 Z.G. 设计了研究。X.Z.、Z.C.、Q.H.、C.W.、J.W. 和 J.Y. 进行了实验。X.Z. 解读了数据。论文由所有作者共同撰写。所有作者均已批准了稿件的最终版本。
Acknowledgments 致谢
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This work was supported by grants from the Alfred P. Sloan Foundation (Sloan Research Fellowship), and a pilot grant from the University of North Carolina (UNC) Cancer Center, the National Natural Science Foundation of China (31771036, 51573096, 51703132, 51728301), the Basic Research Program of Shenzhen (JCYJ20170412111100742, JCYJ20160422091238319), Fok Ying-Tong Education Foundation for Young Teachers in the Higher Education Institutions of China (161032), and China Podtdoctoral Science Foundation (2017M612742). We acknowledge Professor Alan Cantor at Boston Children’s Hospital and Dana-Farber Cancer Institute for providing the mouse megakaryocyte cell line L8057 cells, and Professor Leaf Huang at University of North Carolina at Chapel Hill for providing B16F10-luc cells.
本研究得到了阿尔弗雷德·P·斯隆基金会（斯隆研究奖学金）、北卡罗来纳大学（UNC）癌症中心试点资助、国家自然科学基金（31771036、51573096、51703132、51728301）、深圳市基础研究计划（JCYJ20170412111100742、JCYJ20160422091238319）、中国高等学校青年教师霍英东教育基金会（161032）以及中国博士后科学基金会（2017M612742）的资助。我们感谢波士顿儿童医院和达纳-法伯癌症研究所的 Alan Cantor 教授提供小鼠巨核细胞系 L8057 细胞，以及北卡罗来纳大学教堂山分校的 Leaf Huang 教授提供 B16F10-luc 细胞。
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- 4Kanwar, S. S.; Poolla, A.; Majumdar, A. P. World J. Gastrointest. Pathophysiol. 2012, 3, 1– 9, DOI: 10.4291/wjgp.v3.i1.1Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC383msFentA%253D%253D&md5=3c7eaa049a7b7089a803f822f08dbb16Regulation of colon cancer recurrence and development of therapeutic strategiesKanwar Shailender Singh; Poolla Anuradha; Majumdar Adhip PnWorld journal of gastrointestinal pathophysiology (2012), 3 (1), 1-9 ISSN:.Recurrence of colon cancer still remains a major issue which affects nearly 50% of patients treated by conventional therapeutics. Although the underlying causative factor(s) is not fully understood, development of drug-resistance has been associated with induction of cancer stem or stem-like cells (CSCs) which constitute a small sub-population of tumor cells known to be highly resistant to chemotherapy. In fact, the discovery of CSCs in a variety of tumors (including colon cancer) has changed the view of carcinogenesis and therapeutic strategies. Emerging reports have indicated that to improve patient outcomes, conventional anticancer therapies should be replaced with specific approaches targeting CSCs. Thus, therapeutic strategies that specifically target CSCs are being sought to reduce the risk of relapse and metastasis. In order to specifically target colon CSCs (while sparing somatic intestinal stem cells), it is critical to identify unique deregulated pathways responsible for self-renewal of CSCs and colon cancer recurrence. Colon CSCs present a unique opportunity to better understand the biology of solid tumors. Thus, a better understanding of the clinical signs and symptoms of colon cancer patients (undergoing surgery or chemotherapy) during perioperative periods, along with the underlying regulatory events affecting the stem/progenitor cell self-renewal and differentiation of colon epithelial cells, is of immense importance. In this review we discuss the implication of clinical factors and the emerging role of CSCs during recurrence of colon cancer along with the development of new therapeutic strategies involving the use of natural agents.
- 5Disis, M. L.; Stanton, S. E. Clin. Cancer Res. 2013, 19, 6398– 6403, DOI: 10.1158/1078-0432.CCR-13-0734Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVOitb7E&md5=1bf5983c54196d826179f2793d6c55fdCan Immunity to Breast Cancer Eliminate Residual Micrometastases?Disis, Mary L.; Stanton, Sasha E.Clinical Cancer Research (2013), 19 (23), 6398-6403CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)A review. An effective immune response has the potential for breast cancer sterilization with marked redn. in the potential for disease relapse. Adaptive type I immune cells uniquely have the capability of (i) cytotoxic T-cell activation and proliferation until all antigen expressing cells are eradicated, (ii) traversing endothelial barriers to penetrate tumor deposits wherever they occur, and (iii) immunol. memory, which allows the persistence of destructive immunity over the years it may take for breast cancer micrometastases to become clin. evident. Numerous recent investigations suggest that some breast cancers stimulate the type of immunity that results in a decreased risk of relapse. Moreover, the endogenous type I tumor microenvironment or type I immunity induced by drugs or biol. agents may improve response to std. therapies, further lowering the probability of disease recurrence. Clin Cancer Res; 19(23); 6398-403. ©2013 AACR.
- 6Schumacher, T. N.; Schreiber, R. D. Science 2015, 348, 69– 74, DOI: 10.1126/science.aaa4971
- 7Balachandran, V. P.; Luksza, M.; Zhao, J. N.; Makarov, V.; Moral, J. A.; Remark, R.; Herbst, B.; Askan, G.; Bhanot, U.; Senbabaoglu, Y.; Wells, D. K. Nature 2017, 551 (7681), 512– 516Google ScholarThere is no corresponding record for this reference.
- 8Ott, P. A.; Hu, Z.; Keskin, D. B.; Shukla, S. A.; Sun, J.; Bozym, D. J.; Zhang, W.; Luoma, A.; Giobbie-Hurder, A.; Peter, L. Nature 2017, 547, 217– 221, DOI: 10.1038/nature22991Google ScholarThere is no corresponding record for this reference.
- 9Gubin, M. M.; Zhang, X.; Schuster, H.; Caron, E.; Ward, J. P.; Noguchi, T.; Ivanova, Y.; Hundal, J.; Arthur, C. D.; Krebber, W. J. Nature 2014, 515 (7528), 577– 81, DOI: 10.1038/nature13988Google ScholarThere is no corresponding record for this reference.
- 10Sharma, P.; Allison, J. P. Cell 2015, 161, 205– 214, DOI: 10.1016/j.cell.2015.03.030Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmsVWqsbc%253D&md5=a9cc49380f787e851564e3e5ac9b418fImmune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative PotentialSharma, Padmanee; Allison, James P.Cell (Cambridge, MA, United States) (2015), 161 (2), 205-214CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Research in two fronts has enabled the development of therapies that provide significant benefit to cancer patients. One area stems from a detailed knowledge of mutations that activate or inactivate signaling pathways that drive cancer development. This work triggered the development of targeted therapies that lead to clin. responses in the majority of patients bearing the targeted mutation, although responses are often of limited duration. In the second front are the advances in mol. immunol. that unveiled the complexity of the mechanisms regulating cellular immune responses. These developments led to the successful targeting of immune checkpoints to unleash anti-tumor T cell responses, resulting in durable long-lasting responses but only in a fraction of patients. In this Review, we discuss the evolution of research in these two areas and propose that intercrossing them and increasing funding to guide research of combination of agents represent a path forward for the development of curative therapies for the majority of cancer patients.
- 11Boussiotis, V. A. N. Engl. J. Med. 2016, 375, 1767– 1778, DOI: 10.1056/NEJMra1514296Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvF2ksrbI&md5=856e935a0b34db47b57190db05218c24Molecular and biochemical aspects of the PD-1 checkpoint pathwayBoussiotis, Vassiliki A.New England Journal of Medicine (2016), 375 (18), 1767-1778CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)The pathway consisting of the receptor programmed cell death 1 (PD-1; also called CD279) and its ligands, PD-L1 (B7-H1 or CD274) and PD-L2 (B7-DC or CD273), plays a vital role in the maintenance of peripheral tolerance (i.e., mechanisms that maintain the quiescence of autoreactive T cells that have already matured and escaped the mechanisms of central tolerance during development in the thymus). Tumors and pathogens that cause chronic infections can exploit this pathway to escape T-cell-mediated tumor-specific and pathogen-specific immunity. Therapies with antibodies targeting PD-1 and its ligands have been shown to be assocd. with remarkable response rates in various cancers and, together with antibodies targeting CTLA-4, have revolutionized cancer treatment. (See the Supplementary Appendix, available with the full text of this article at NEJM.org, for a list of the protein abbreviations used in this review.) In addn. to the clin. success, ongoing work is currently revealing the mol. mechanisms targeted by PD-1. Here, I provide a brief overview of the mol. and biochem. events that are regulated by PD-1 ligation and their implications for mechanisms intrinsic and extrinsic to the cell that det. the fate and function of T cells.
- 12Zou, W.; Wolchok, J. D.; Chen, L. Sci. Transl. Med. 2016, 8, 328rv4, DOI: 10.1126/scitranslmed.aad7118Google ScholarThere is no corresponding record for this reference.
- 13Sharma, P.; Allison, J. P. Science 2015, 348, 56– 61, DOI: 10.1126/science.aaa8172Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXls1Wmurg%253D&md5=d63ae85b9c651ec64a3b5b002c609e35The future of immune checkpoint therapySharma, Padmanee; Allison, James P.Science (Washington, DC, United States) (2015), 348 (6230), 56-61CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clin. advances and provided a new weapon against cancer. This therapy has elicited durable clin. responses and, in a fraction of patients, long-term remissions where patients exhibit no clin. signs of cancer for many years. The way forward for this class of novel agents lies in our ability to understand human immune responses in the tumor microenvironment. This will provide valuable information regarding the dynamic nature of the immune response and regulation of addnl. pathways that will need to be targeted through combination therapies to provide survival benefit for greater nos. of patients.
- 14Hoos, A. Nat. Rev. Drug Discovery 2016, 15, 235– 247, DOI: 10.1038/nrd.2015.35Google ScholarThere is no corresponding record for this reference.
- 15Chen, Q.; Xu, L.; Liang, C.; Wang, C.; Peng, R.; Liu, Z. Nat. Commun. 2016, 7, 13193, DOI: 10.1038/ncomms13193Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslGrtrzN&md5=82551fdfc13ac97849f58618cbd90f7ePhotothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapyChen, Qian; Xu, Ligeng; Liang, Chao; Wang, Chao; Peng, Rui; Liu, ZhuangNature Communications (2016), 7 (), 13193CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)A therapeutic strategy that can eliminate primary tumors, inhibit metastases, and prevent tumor relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clin. approved components can be used for near-IR laser-triggered photothermal ablation of primary tumors, generating tumor-assocd. antigens, which in the presence of R837-contg. nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunol. responses will be able to attack remaining tumor cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumor models. Furthermore, such strategy offers a strong immunol. memory effect, which can provide protection against tumor rechallenging post elimination of their initial tumors.
- 16Sharma, P.; Hu-Lieskovan, S.; Wargo, J. A.; Ribas, A. Cell 2017, 168, 707– 723, DOI: 10.1016/j.cell.2017.01.017Google ScholarThere is no corresponding record for this reference.
- 17von Boehmer, H. Nat. Immunol. 2005, 6, 338– 44, DOI: 10.1038/ni1180Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisVyitb4%253D&md5=5a70a3f463538e8a6e9e534868fe7eabMechanisms of suppression by suppressor T cellsvon Boehmer, HaraldNature Immunology (2005), 6 (4), 338-344CODEN: NIAMCZ; ISSN:1529-2908. (Nature Publishing Group)A review. Mechanisms of immunosuppression by CD4+CD25+ suppressor T cells have been addressed using many in vitro and in vivo conditions. However, those studies have not yielded a single mode of action. This review will discuss the mechanisms of suppression, which include the local secretion of cytokines such as TGF-β and direct cell contact through binding of cell surface mols. such as CTLA-4 on suppressor T cells to CD80 and CD86 mols. on effector T cells. Suppression requires the appropriate colocalization of suppressor and effector T cells in different tissue and may involve the interference with T cell receptor signaling that triggers transcription factors important in regulating effector cell function.
- 18Zou, W. Nat. Rev. Immunol. 2006, 6, 295– 307, DOI: 10.1038/nri1806Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XivVyru7k%253D&md5=c08b6710a4fc3c59fd3a1349d5802ad7Regulatory T cells, tumour immunity and immunotherapyZou, WeipingNature Reviews Immunology (2006), 6 (4), 295-307CODEN: NRIABX; ISSN:1474-1733. (Nature Publishing Group)A review. Tumors express a range of antigens, including self-antigens. Regulatory T cells are crucial for maintaining T-cell tolerance to self-antigens. Regulatory T cells are thought to dampen T-cell immunity to tumor-assocd. antigens and to be the main obstacle tempering successful immunotherapy and active vaccination. In this Review, I consider the nature and characteristics of regulatory T cells in the tumor microenvironment and their potential multiple suppressive mechanisms. Strategies for therapeutic targeting of regulatory T cells and the effect of regulatory T cells on current immunotherapeutic and vaccine regimens are discussed.
- 19Grossman, W. J.; Verbsky, J. W.; Barchet, W.; Colonna, M.; Atkinson, J. P.; Ley, T. J. Immunity 2004, 21, 589– 601, DOI: 10.1016/j.immuni.2004.09.002Google ScholarThere is no corresponding record for this reference.
- 20Maj, T.; Wang, W.; Crespo, J.; Zhang, H.; Wei, S.; Zhao, L.; Vatan, L.; Shao, I.; Szeliga, W.; Lyssiotis, C.; Liu, J. R.; Kryczek, I.; Zou, W. Nat. Immunol. 2017, 18, 1332– 1341, DOI: 10.1038/ni.3868Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslejtLrO&md5=ece2a07be77ac266d2384e64ce9622bbOxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumorMaj, Tomasz; Wang, Wei; Crespo, Joel; Zhang, Hongjuan; Wang, Weimin; Wei, Shuang; Zhao, Lili; Vatan, Linda; Shao, Irene; Szeliga, Wojciech; Lyssiotis, Costas; Liu, J. Rebecca; Kryczek, Ilona; Zou, WeipingNature Immunology (2017), 18 (12), 1332-1341CODEN: NIAMCZ; ISSN:1529-2908. (Nature Research)Live regulatory T cells (Treg cells) suppress antitumor immunity, but how Treg cells behave in the metabolically abnormal tumor microenvironment remains unknown. Here we show that tumor Treg cells undergo apoptosis, and such apoptotic Treg cells abolish spontaneous and PD-L1-blockade-mediated antitumor T cell immunity. Biochem. and functional analyses show that adenosine, but not typical suppressive factors such as PD-L1, CTLA-4, TGF-β, IL-35, and IL-10, contributes to apoptotic Treg-cell-mediated immunosuppression. Mechanistically, apoptotic Treg cells release and convert a large amt. of ATP to adenosine via CD39 and CD73, and mediate immunosuppression via the adenosine and A2A pathways. Apoptosis in Treg cells is attributed to their weak NRF2-assocd. antioxidant system and high vulnerability to free oxygen species in the tumor microenvironment. Thus, the data support a model wherein tumor Treg cells sustain and amplify their suppressor capacity through inadvertent death via oxidative stress. This work highlights the oxidative pathway as a metabolic checkpoint that controls Treg cell behavior and affects the efficacy of therapeutics targeting cancer checkpoints.
- 21Golebiewska, E. M.; Poole, A. W. Blood Rev. 2015, 29, 153– 162, DOI: 10.1016/j.blre.2014.10.003Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFSnu73P&md5=5546fb06f1ff5d85fe6bcae2d31a0ac4Platelet secretion: From haemostasis to wound healing and beyondGolebiewska, Ewelina M.; Poole, Alastair W.Blood Reviews (2015), 29 (3), 153-162CODEN: BLOREB; ISSN:0268-960X. (Elsevier Ltd.)Upon activation, platelets secrete more than 300 active substances from their intracellular granules. Platelet dense granule components, such as ADP and polyphosphates, contribute to haemostasis and coagulation, but also play a role in cancer metastasis. α-Granules contain multiple cytokines, mitogens, pro- and anti-inflammatory factors and other bioactive mols. that are essential regulators in the complex microenvironment of the growing thrombus but also contribute to a no. of disease processes. Our understanding of the mol. mechanisms of secretion and the genetic regulation of granule biogenesis still remains incomplete. In this review we summarise our current understanding of the roles of platelet secretion in health and disease, and discuss some of the hypotheses that may explain how platelets may control the release of its many secreted components in a context-specific manner, to allow platelets to play multiple roles in health and disease.
- 22Nurden, A. T.; Nurden, P.; Sanchez, M.; Andia, I.; Anitua, E. Front Biosci. 2008, 13, 3532– 3548Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1czksVymsQ%253D%253D&md5=bc9408067264404e8f17e44fcbfaffe8Platelets and wound healingNurden Alan T; Nurden Paquita; Sanchez Mikel; Andia Isabel; Anitua EduardoFrontiers in bioscience : a journal and virtual library (2008), 13 (), 3532-48 ISSN:1093-9946.Platelets help prevent blood loss at sites of vascular injury. To do this, they adhere, aggregate and form a procoagulant surface favoring thrombin generation and fibrin formation. In addition, platelets express and release substances that promote tissue repair and influence processes such as angiogenesis, inflammation and the immune response. They contain large secretable pools of biologically active proteins, while newly synthesized active metabolites are also released. Although anucleate, activated platelets possess a spliceosome and can synthesize tissue factor and interleukin-1beta. The binding of secreted proteins within a developing fibrin mesh or to the extracellular matrix can create chemotactic gradients favoring the recruitment of stem cells, stimulating cell migration and differentiation, and promoting repair. The therapeutic use of platelets in a fibrin clot has a positive influence in clinical situations requiring rapid healing. Dental implant surgery, orthopaedic surgery, muscle and tendon repair, skin ulcers, hole repair in eye surgery and cardiac surgery are situations where the use of autologous platelets accelerates healing. We now review the ways in which platelets participate in these processes.
- 23Wang, C.; Sun, W.; Ye, Y.; Hu, Q.; Bomba, H. N.; Gu, Z. Nat. Biomed. Eng. 2017, 1, 0011, DOI: 10.1038/s41551-016-0011Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1ahur%252FO&md5=f19b7813302dd9569c51e471417ed119In situ activation of platelets with checkpoint inhibitors for post-surgical cancer immunotherapyWang, Chao; Sun, Wujin; Ye, Yanqi; Hu, Quanyin; Bomba, Hunter N.; Gu, ZhenNature Biomedical Engineering (2017), 1 (2), 0011CODEN: NBEAB3; ISSN:2157-846X. (Nature Research)Cancer recurrence after surgical resection remains a significant challenge in cancer therapy. Platelets, which accumulate in wound sites and interact with circulating tumor cells (CTCs), can however trigger inflammation and repair processes in the remaining tumor microenvironment. Inspired by this intrinsic ability of platelets and the clin. success of immune checkpoint inhibitors, here we show that conjugating anti-PDL1 (engineered monoclonal antibodies against programmed-death ligand 1) to the surface of platelets can reduce post-surgical tumor recurrence and metastasis. Using mice bearing partially removed primary melanomas (B16-F10) or triple-neg. breast carcinomas (4T1), we found that anti-PDL1 was effectively released on platelet activation by platelet-derived microparticles, and that the administration of platelet-bound anti-PDL1 significantly prolonged overall mouse survival after surgery by reducing the risk of cancer regrowth and metastatic spread. Our findings suggest that engineered platelets can facilitate the delivery of the immunotherapeutic anti-PDL1 to the surgical bed and target CTCs in the bloodstream, thereby potentially improving the objective response rate.
- 24Hu, C. M.; Fang, R. H.; Wang, K. C.; Luk, B. T.; Thamphiwatana, S.; Dehaini, D.; Nguyen, P.; Angsantikul, P.; Wen, C. H.; Kroll, A. V. Nature 2015, 526, 118– 121, DOI: 10.1038/nature15373Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFersL3L&md5=b3ac12cfaaec406bc86e7b816f843a80Nanoparticle biointerfacing by platelet membrane cloakingHu, Che-Ming J.; Fang, Ronnie H.; Wang, Kuei-Chun; Luk, Brian T.; Thamphiwatana, Soracha; Dehaini, Diana; Nguyen, Phu; Angsantikul, Pavimol; Wen, Cindy H.; Kroll, Ashley V.; Carpenter, Cody; Ramesh, Manikantan; Qu, Vivian; Patel, Sherrina H.; Zhu, Jie; Shi, William; Hofman, Florence M.; Chen, Thomas C.; Gao, Weiwei; Zhang, Kang; Chien, Shu; Zhang, LiangfangNature (London, United Kingdom) (2015), 526 (7571), 118-121CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Development of functional nanoparticles can be encumbered by unanticipated material properties and biol. events, which can affect nanoparticle effectiveness in complex, physiol. relevant systems. Despite the advances in bottom-up nanoengineering and surface chem., reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the prepn. of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens assocd. with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an exptl. rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, resp., show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.
- 25Anselmo, A. C.; Modery-Pawlowski, C. L.; Menegatti, S.; Kumar, S.; Vogus, D. R.; Tian, L. L.; Chen, M.; Squires, T. M.; Sen Gupta, A.; Mitragotri, S. ACS Nano 2014, 8, 11243– 11253, DOI: 10.1021/nn503732mGoogle ScholarThere is no corresponding record for this reference.
- 26Hu, Q.; Sun, W.; Qian, C.; Wang, C.; Bomba, H. N.; Gu, Z. Adv. Mater. 2015, 27, 7043– 7050, DOI: 10.1002/adma.201503323Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFyqsrnM&md5=fb41127552a9ab172439df259a7c2cbbAnticancer Platelet-Mimicking NanovehiclesHu, Quanyin; Sun, Wujin; Qian, Chengen; Wang, Chao; Bomba, Hunter N.; Gu, ZhenAdvanced Materials (Weinheim, Germany) (2015), 27 (44), 7043-7050CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)Human plasma membranes were isolated and made into nanocapsules that contained doxorubicin. TRAIL was also added to provide antitumor activity. The nanocapsules were targeted to human breast cancer cells by platelet P-selectin binding to CD44 and TRAIL binding to death receptors. Tumor apoptosis was demonstrated.
- 27Li, S.; Jiang, Q.; Liu, S.; Zhang, Y.; Tian, Y.; Song, C.; Wang, J.; Zou, Y.; Anderson, G. J.; Han, J. Y.; Chang, Y.; Liu, Y.; Zhang, C.; Chen, L.; Zhou, G.; Nie, G.; Yan, H.; Ding, B.; Zhao, Y. Nat. Biotechnol. 2018, 36, 258– 264, DOI: 10.1038/nbt.4071Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFCiurY%253D&md5=b780d2a8958409aeee721f6d96b3aee0A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivoLi, Suping; Jiang, Qiao; Liu, Shaoli; Zhang, Yinlong; Tian, Yanhua; Song, Chen; Wang, Jing; Zou, Yiguo; Anderson, Gregory J.; Han, Jing-Yan; Chang, Yung; Liu, Yan; Zhang, Chen; Chen, Liang; Zhou, Guangbiao; Nie, Guangjun; Yan, Hao; Ding, Baoquan; Zhao, YuliangNature Biotechnology (2018), 36 (3), 258-264CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Nanoscale robots have potential as intelligent drug delivery systems that respond to mol. triggers. Using DNA origami we constructed an autonomous DNA robot programmed to transport payloads and present them specifically in tumors. Our nanorobot is functionalized on the outside with a DNA aptamer that binds nucleolin, a protein specifically expressed on tumor-assocd. endothelial cells, and the blood coagulation protease thrombin within its inner cavity. The nucleolin-targeting aptamer serves both as a targeting domain and as a mol. trigger for the mech. opening of the DNA nanorobot. The thrombin inside is thus exposed and activates coagulation at the tumor site. Using tumor-bearing mouse models, we demonstrate that i.v. injected DNA nanorobots deliver thrombin specifically to tumor-assocd. blood vessels and induce intravascular thrombosis, resulting in tumor necrosis and inhibition of tumor growth. The nanorobot proved safe and immunol. inert in mice and Bama miniature pigs. Our data show that DNA nanorobots represent a promising strategy for precise drug delivery in cancer therapy.
- 28Stroncek, D. F.; Rebulla, P. Lancet 2007, 370, 427– 438, DOI: 10.1016/S0140-6736(07)61198-2Google ScholarThere is no corresponding record for this reference.
- 29Moreau, T.; Evans, A. L.; Vasquez, L.; Tijssen, M. R.; Yan, Y.; Trotter, M. W.; Howard, D.; Colzani, M.; Arumugam, M.; Wu, W. H. Nat. Commun. 2016, 7, 11208, DOI: 10.1038/ncomms11208Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xls1Kmtb8%253D&md5=f0a2623373828c01d2fe868e5a10ac9dLarge-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programmingMoreau, Thomas; Evans, Amanda L.; Vasquez, Louella; Tijssen, Marloes R.; Yan, Ying; Trotter, Matthew W.; Howard, Daniel; Colzani, Maria; Arumugam, Meera; Wu, Wing Han; Dalby, Amanda; Lampela, Riina; Bouet, Guenaelle; Hobbs, Catherine M.; Pask, Dean C.; Payne, Holly; Ponomaryov, Tatyana; Brill, Alexander; Soranzo, Nicole; Ouwehand, Willem H.; Pedersen, Roger A.; Ghevaert, CedricNature Communications (2016), 7 (), 11208CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)The prodn. of megakaryocytes (MKs)-the precursors of blood platelets-from human pluripotent stem cells (hPSCs) offers exciting clin. opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chem. defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 105 mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufg. practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro prodn. of platelets for transfusion and basic research in MK and platelet biol.
- 30Li, J.; Sharkey, C. C.; Wun, B.; Liesveld, J. L.; King, M. R. J. Controlled Release 2016, 228, 38– 47, DOI: 10.1016/j.jconrel.2016.02.036Google ScholarThere is no corresponding record for this reference.
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- 47El-Andaloussi, S.; Lee, Y.; Lakhal-Littleton, S.; Li, J.; Seow, Y.; Gardiner, C.; Alvarez-Erviti, L.; Sargent, I. L.; Wood, M. J. Nat. Protoc. 2012, 7, 2112– 2126, DOI: 10.1038/nprot.2012.131Google ScholarThere is no corresponding record for this reference.
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Abstract 摘要
Figure 1 图 1
Figure 1. Schematic of the production of PD-1-expressing platelets and reinvigoration of CD8+ T cells. (A) Schematic shows L8057 cell line stably expressing murine PD-1 and production of platelets. (B) PD-1-expressing platelets target tumor cells within the surgery wound. (C) PD-L1 blockade by PD-1-expressing platelets reverts exhausted CD8+ T cells to attack tumor cells.
图 1. PD-1 表达血小板的制备及 CD8+ T 细胞的再激活示意图。(A) 示意图展示了稳定表达小鼠 PD-1 的 L8057 细胞系及血小板的生成。(B) PD-1 表达的血小板靶向手术伤口内的肿瘤细胞。(C) PD-1 表达的血小板通过阻断 PD-L1，使耗竭的 CD8+ T 细胞恢复活力并攻击肿瘤细胞。Figure 2
Figure 2. Production and characterization of platelets from PD-1-expressing L8057 stable cell line. (A) Confocal images present L8057 cell line stably expressing murine EGFP-PD-1 on cell membranes. WGA Alexa-Fluor 594 dye was used to stain cell membrane (scale bar: 10 μm). (B) Western blot analysis for evaluating the expression of PD-1 in L8057 cell line. L8 is short for L8057 cells. (C) EGFP-PD-1-expressing L8057 cells stimulated with 500 nM PMA for 3 days, and immunostained to detect CD42a expression. (D) L8057 cells stimulated with 500 nM PMA for 3 days, and stained with Wright–Giemsa dye (scale bar: 10 μm). (E) Evolution process of PD-1-expressing proplatelet extended from MKs (scale bar: 10 μm). (F) Morphology of PD-1 proplatelets extended from L8057 cells after 6 days of stimulation with 500 nM PMA. PD-1 proplatelets extended from L8057 cells (scale bar: 10 μm). (G) Representative confocal images of purified PD-1-expressing platelets (scale bar: 10 μm). (H) Size distribution of PD-1-expressing platelets measured by DLS. (I) CSEM image shows the morphology of PD-1-expressing platelets (scale bar: 1 μm). (J) Representative TEM image shows morphology and size of PD-1-expressing platelet (scale bar: 1 μm). (K) Number of platelets released from PD-1-expressing L8057 cells after stimulated with 500 nM PMA (n = 5). Error bar, ± SD.
Figure 3
Figure 3. In vitro and in vivo function of PD-1-expressing platelets. (A, B) Retention of platelets on collagen-coated or uncoated tissue culture slides. Green color: EGFP; Red color: WGA Alexa-Fluor 594 dye (scale bar: 50 μm). Error bar, ± SD. (C) Confocal, CSEM, and TEM images of PD-1-expressing platelets stimulated with thrombin. Platelet microparticles (PMPs) were released from the platelet (scale bar: 1 μm). (D) Measurement of the size distribution of PD-1-expressing platelets at 30 min after activation by thrombin. PMPs were produced from the platelets. (E) EGFP-PD-1-expressing platelets bound on the cell membrane of B16F10 cells. PD-1-expresing platelets or free platelets labeled with Cy5.5 were incubated with B16F10 cells for 20 h. WGA Alexa-Fluor 594 dye was used to stain the B16F10 cell membrane. The white arrows indicate the PD-1 platelets binding on the cell membrane of the cancer cells (scale bar: 10 μm). (F) B16F10 cells were transfected with DsRed-PD-L1 plasmid for 20 h, and then incubated with EGFP-PD-1 platelets for 20 h; the colocalization of EGFP-PD-1 platelets and DsRed-PD-L1 was detected (scale bar: 10 μm). (G) Cy5.5-labeled free platelets and PD-1-expressing platelets were injected through the tail-vein in mice. Fluorescence was measured at different time points (n = 3). Fluorescence intensity at 2 min as 1. Error bar, ±SD. (H) In vivo fluorescence images of free platelets and PD-1-expressing platelets in major organs and residual tumor bed. (I) Fluorescence intensity per gram of tissue in major organs and tumors (n = 3). Error bar, ± SD.
Figure 4
Figure 4. PD-1-expressing platelets for inhibition of tumor progression in incomplete-surgery tumor model. (A) Schematic illustration of PD-1-expressing platelets used for therapy in an incomplete-surgery tumor model. (B) In vivo bioluminescence imaging of the B16F10 tumor growth in mice treated with PBS (G1), free platelets (G2), and PD-1-expressing platelets (G3). (C) Average tumor volumes of treated mice (n = 8). Data are shown as the mean ± SEM. (D) Survival curves of mice receiving different treatments (n = 8). (E) Immunofluorescence of tumor sections showing infiltration of CD4+ and CD8+ T cells (scale bar: 100 μm). (F)Representative plots and (G) quantification of T cells in tumors analyzed by flow cytometry (gated on CD3+ T cells) (n = 3). Error bar, ± SD. (H) Representative plots and (I) quantification of GzmB in CD8+ T cells in tumors analyzed by the flow cytometry (gated on CD8+ T cells) (n = 3). Error bar, ± SD. Throughout, NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (C, G, I) One-way ANOVA with Tukey posthoc test analyses were carried out to do the analyses, or (D) by log-rank (Mantel-Cox) test.
Figure 5
Figure 5. In vivo antitumor effect of cyclophosphamide-loaded PD-1-expressing platelets in incomplete-surgery tumor model. (A) Average tumor volumes of mice (n = 8) treated with PBS (G1), cyclophosphamide (CP) (G2), PD-1-expressing platelets (G3), CP-free platelets (G4), and CP-loaded PD-1-expressing platelets (G5). Data are shown as the mean ± SEM. ***, Compared with PBS control. (B) Survival curves of the treated mice. (C) Quantification of FoxP3 expression in CD4+ T cells within the tumors analyzed by the flow cytometry (gated on CD4+ T cells) (n = 3). (D) Representative plots and (E) quantification of Ki67 in CD8+ T cells within the tumors analyzed by the flow cytometry (gated on CD8+ T cells) (n = 3). (F) Representative plots and (G) quantification of CD8+ and CD4+ T cells within tumors analyzed by the flow cytometry (gated on CD3+ T cells) (n = 3). (H) Representative plots and (I) quantification of GzmB in CD8+ T cells within the tumors analyzed by the flow cytometry (gated on CD8+ T cells) (n = 3). (J, K) Immunofluorescence of the tumors showing CD8+ T cell infiltration (scale bar: 100 μm). Error bar of C, E, G, I, K, ± SD. Throughout, NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. (A, C, E, G, I, K) Two-way ANOVA with Tukey posthoc test analyses were carried out to do the analyses or (B) by log-rank (Mantel-Cox) test (B).
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- 4Kanwar, S. S.; Poolla, A.; Majumdar, A. P. World J. Gastrointest. Pathophysiol. 2012, 3, 1– 9, DOI: 10.4291/wjgp.v3.i1.14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC383msFentA%253D%253D&md5=3c7eaa049a7b7089a803f822f08dbb16Regulation of colon cancer recurrence and development of therapeutic strategiesKanwar Shailender Singh; Poolla Anuradha; Majumdar Adhip PnWorld journal of gastrointestinal pathophysiology (2012), 3 (1), 1-9 ISSN:.Recurrence of colon cancer still remains a major issue which affects nearly 50% of patients treated by conventional therapeutics. Although the underlying causative factor(s) is not fully understood, development of drug-resistance has been associated with induction of cancer stem or stem-like cells (CSCs) which constitute a small sub-population of tumor cells known to be highly resistant to chemotherapy. In fact, the discovery of CSCs in a variety of tumors (including colon cancer) has changed the view of carcinogenesis and therapeutic strategies. Emerging reports have indicated that to improve patient outcomes, conventional anticancer therapies should be replaced with specific approaches targeting CSCs. Thus, therapeutic strategies that specifically target CSCs are being sought to reduce the risk of relapse and metastasis. In order to specifically target colon CSCs (while sparing somatic intestinal stem cells), it is critical to identify unique deregulated pathways responsible for self-renewal of CSCs and colon cancer recurrence. Colon CSCs present a unique opportunity to better understand the biology of solid tumors. Thus, a better understanding of the clinical signs and symptoms of colon cancer patients (undergoing surgery or chemotherapy) during perioperative periods, along with the underlying regulatory events affecting the stem/progenitor cell self-renewal and differentiation of colon epithelial cells, is of immense importance. In this review we discuss the implication of clinical factors and the emerging role of CSCs during recurrence of colon cancer along with the development of new therapeutic strategies involving the use of natural agents.
- 5Disis, M. L.; Stanton, S. E. Clin. Cancer Res. 2013, 19, 6398– 6403, DOI: 10.1158/1078-0432.CCR-13-07345https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvVOitb7E&md5=1bf5983c54196d826179f2793d6c55fdCan Immunity to Breast Cancer Eliminate Residual Micrometastases?Disis, Mary L.; Stanton, Sasha E.Clinical Cancer Research (2013), 19 (23), 6398-6403CODEN: CCREF4; ISSN:1078-0432. (American Association for Cancer Research)A review. An effective immune response has the potential for breast cancer sterilization with marked redn. in the potential for disease relapse. Adaptive type I immune cells uniquely have the capability of (i) cytotoxic T-cell activation and proliferation until all antigen expressing cells are eradicated, (ii) traversing endothelial barriers to penetrate tumor deposits wherever they occur, and (iii) immunol. memory, which allows the persistence of destructive immunity over the years it may take for breast cancer micrometastases to become clin. evident. Numerous recent investigations suggest that some breast cancers stimulate the type of immunity that results in a decreased risk of relapse. Moreover, the endogenous type I tumor microenvironment or type I immunity induced by drugs or biol. agents may improve response to std. therapies, further lowering the probability of disease recurrence. Clin Cancer Res; 19(23); 6398-403. ©2013 AACR.
- 6Schumacher, T. N.; Schreiber, R. D. Science 2015, 348, 69– 74, DOI: 10.1126/science.aaa4971There is no corresponding record for this reference.
- 7Balachandran, V. P.; Luksza, M.; Zhao, J. N.; Makarov, V.; Moral, J. A.; Remark, R.; Herbst, B.; Askan, G.; Bhanot, U.; Senbabaoglu, Y.; Wells, D. K. Nature 2017, 551 (7681), 512– 516There is no corresponding record for this reference.
- 8Ott, P. A.; Hu, Z.; Keskin, D. B.; Shukla, S. A.; Sun, J.; Bozym, D. J.; Zhang, W.; Luoma, A.; Giobbie-Hurder, A.; Peter, L. Nature 2017, 547, 217– 221, DOI: 10.1038/nature22991There is no corresponding record for this reference.
- 9Gubin, M. M.; Zhang, X.; Schuster, H.; Caron, E.; Ward, J. P.; Noguchi, T.; Ivanova, Y.; Hundal, J.; Arthur, C. D.; Krebber, W. J. Nature 2014, 515 (7528), 577– 81, DOI: 10.1038/nature13988There is no corresponding record for this reference.
- 10Sharma, P.; Allison, J. P. Cell 2015, 161, 205– 214, DOI: 10.1016/j.cell.2015.03.03010https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXmsVWqsbc%253D&md5=a9cc49380f787e851564e3e5ac9b418fImmune Checkpoint Targeting in Cancer Therapy: Toward Combination Strategies with Curative PotentialSharma, Padmanee; Allison, James P.Cell (Cambridge, MA, United States) (2015), 161 (2), 205-214CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A review. Research in two fronts has enabled the development of therapies that provide significant benefit to cancer patients. One area stems from a detailed knowledge of mutations that activate or inactivate signaling pathways that drive cancer development. This work triggered the development of targeted therapies that lead to clin. responses in the majority of patients bearing the targeted mutation, although responses are often of limited duration. In the second front are the advances in mol. immunol. that unveiled the complexity of the mechanisms regulating cellular immune responses. These developments led to the successful targeting of immune checkpoints to unleash anti-tumor T cell responses, resulting in durable long-lasting responses but only in a fraction of patients. In this Review, we discuss the evolution of research in these two areas and propose that intercrossing them and increasing funding to guide research of combination of agents represent a path forward for the development of curative therapies for the majority of cancer patients.
- 11Boussiotis, V. A. N. Engl. J. Med. 2016, 375, 1767– 1778, DOI: 10.1056/NEJMra151429611https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhvF2ksrbI&md5=856e935a0b34db47b57190db05218c24Molecular and biochemical aspects of the PD-1 checkpoint pathwayBoussiotis, Vassiliki A.New England Journal of Medicine (2016), 375 (18), 1767-1778CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)The pathway consisting of the receptor programmed cell death 1 (PD-1; also called CD279) and its ligands, PD-L1 (B7-H1 or CD274) and PD-L2 (B7-DC or CD273), plays a vital role in the maintenance of peripheral tolerance (i.e., mechanisms that maintain the quiescence of autoreactive T cells that have already matured and escaped the mechanisms of central tolerance during development in the thymus). Tumors and pathogens that cause chronic infections can exploit this pathway to escape T-cell-mediated tumor-specific and pathogen-specific immunity. Therapies with antibodies targeting PD-1 and its ligands have been shown to be assocd. with remarkable response rates in various cancers and, together with antibodies targeting CTLA-4, have revolutionized cancer treatment. (See the Supplementary Appendix, available with the full text of this article at NEJM.org, for a list of the protein abbreviations used in this review.) In addn. to the clin. success, ongoing work is currently revealing the mol. mechanisms targeted by PD-1. Here, I provide a brief overview of the mol. and biochem. events that are regulated by PD-1 ligation and their implications for mechanisms intrinsic and extrinsic to the cell that det. the fate and function of T cells.
- 12Zou, W.; Wolchok, J. D.; Chen, L. Sci. Transl. Med. 2016, 8, 328rv4, DOI: 10.1126/scitranslmed.aad7118There is no corresponding record for this reference.
- 13Sharma, P.; Allison, J. P. Science 2015, 348, 56– 61, DOI: 10.1126/science.aaa817213https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXls1Wmurg%253D&md5=d63ae85b9c651ec64a3b5b002c609e35The future of immune checkpoint therapySharma, Padmanee; Allison, James P.Science (Washington, DC, United States) (2015), 348 (6230), 56-61CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)A review. Immune checkpoint therapy, which targets regulatory pathways in T cells to enhance antitumor immune responses, has led to important clin. advances and provided a new weapon against cancer. This therapy has elicited durable clin. responses and, in a fraction of patients, long-term remissions where patients exhibit no clin. signs of cancer for many years. The way forward for this class of novel agents lies in our ability to understand human immune responses in the tumor microenvironment. This will provide valuable information regarding the dynamic nature of the immune response and regulation of addnl. pathways that will need to be targeted through combination therapies to provide survival benefit for greater nos. of patients.
- 14Hoos, A. Nat. Rev. Drug Discovery 2016, 15, 235– 247, DOI: 10.1038/nrd.2015.35There is no corresponding record for this reference.
- 15Chen, Q.; Xu, L.; Liang, C.; Wang, C.; Peng, R.; Liu, Z. Nat. Commun. 2016, 7, 13193, DOI: 10.1038/ncomms1319315https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhslGrtrzN&md5=82551fdfc13ac97849f58618cbd90f7ePhotothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapyChen, Qian; Xu, Ligeng; Liang, Chao; Wang, Chao; Peng, Rui; Liu, ZhuangNature Communications (2016), 7 (), 13193CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)A therapeutic strategy that can eliminate primary tumors, inhibit metastases, and prevent tumor relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clin. approved components can be used for near-IR laser-triggered photothermal ablation of primary tumors, generating tumor-assocd. antigens, which in the presence of R837-contg. nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunol. responses will be able to attack remaining tumor cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumor models. Furthermore, such strategy offers a strong immunol. memory effect, which can provide protection against tumor rechallenging post elimination of their initial tumors.
- 16Sharma, P.; Hu-Lieskovan, S.; Wargo, J. A.; Ribas, A. Cell 2017, 168, 707– 723, DOI: 10.1016/j.cell.2017.01.017There is no corresponding record for this reference.
- 17von Boehmer, H. Nat. Immunol. 2005, 6, 338– 44, DOI: 10.1038/ni118017https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisVyitb4%253D&md5=5a70a3f463538e8a6e9e534868fe7eabMechanisms of suppression by suppressor T cellsvon Boehmer, HaraldNature Immunology (2005), 6 (4), 338-344CODEN: NIAMCZ; ISSN:1529-2908. (Nature Publishing Group)A review. Mechanisms of immunosuppression by CD4+CD25+ suppressor T cells have been addressed using many in vitro and in vivo conditions. However, those studies have not yielded a single mode of action. This review will discuss the mechanisms of suppression, which include the local secretion of cytokines such as TGF-β and direct cell contact through binding of cell surface mols. such as CTLA-4 on suppressor T cells to CD80 and CD86 mols. on effector T cells. Suppression requires the appropriate colocalization of suppressor and effector T cells in different tissue and may involve the interference with T cell receptor signaling that triggers transcription factors important in regulating effector cell function.
- 18Zou, W. Nat. Rev. Immunol. 2006, 6, 295– 307, DOI: 10.1038/nri180618https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28XivVyru7k%253D&md5=c08b6710a4fc3c59fd3a1349d5802ad7Regulatory T cells, tumour immunity and immunotherapyZou, WeipingNature Reviews Immunology (2006), 6 (4), 295-307CODEN: NRIABX; ISSN:1474-1733. (Nature Publishing Group)A review. Tumors express a range of antigens, including self-antigens. Regulatory T cells are crucial for maintaining T-cell tolerance to self-antigens. Regulatory T cells are thought to dampen T-cell immunity to tumor-assocd. antigens and to be the main obstacle tempering successful immunotherapy and active vaccination. In this Review, I consider the nature and characteristics of regulatory T cells in the tumor microenvironment and their potential multiple suppressive mechanisms. Strategies for therapeutic targeting of regulatory T cells and the effect of regulatory T cells on current immunotherapeutic and vaccine regimens are discussed.
- 19Grossman, W. J.; Verbsky, J. W.; Barchet, W.; Colonna, M.; Atkinson, J. P.; Ley, T. J. Immunity 2004, 21, 589– 601, DOI: 10.1016/j.immuni.2004.09.002There is no corresponding record for this reference.
- 20Maj, T.; Wang, W.; Crespo, J.; Zhang, H.; Wei, S.; Zhao, L.; Vatan, L.; Shao, I.; Szeliga, W.; Lyssiotis, C.; Liu, J. R.; Kryczek, I.; Zou, W. Nat. Immunol. 2017, 18, 1332– 1341, DOI: 10.1038/ni.386820https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhslejtLrO&md5=ece2a07be77ac266d2384e64ce9622bbOxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumorMaj, Tomasz; Wang, Wei; Crespo, Joel; Zhang, Hongjuan; Wang, Weimin; Wei, Shuang; Zhao, Lili; Vatan, Linda; Shao, Irene; Szeliga, Wojciech; Lyssiotis, Costas; Liu, J. Rebecca; Kryczek, Ilona; Zou, WeipingNature Immunology (2017), 18 (12), 1332-1341CODEN: NIAMCZ; ISSN:1529-2908. (Nature Research)Live regulatory T cells (Treg cells) suppress antitumor immunity, but how Treg cells behave in the metabolically abnormal tumor microenvironment remains unknown. Here we show that tumor Treg cells undergo apoptosis, and such apoptotic Treg cells abolish spontaneous and PD-L1-blockade-mediated antitumor T cell immunity. Biochem. and functional analyses show that adenosine, but not typical suppressive factors such as PD-L1, CTLA-4, TGF-β, IL-35, and IL-10, contributes to apoptotic Treg-cell-mediated immunosuppression. Mechanistically, apoptotic Treg cells release and convert a large amt. of ATP to adenosine via CD39 and CD73, and mediate immunosuppression via the adenosine and A2A pathways. Apoptosis in Treg cells is attributed to their weak NRF2-assocd. antioxidant system and high vulnerability to free oxygen species in the tumor microenvironment. Thus, the data support a model wherein tumor Treg cells sustain and amplify their suppressor capacity through inadvertent death via oxidative stress. This work highlights the oxidative pathway as a metabolic checkpoint that controls Treg cell behavior and affects the efficacy of therapeutics targeting cancer checkpoints.
- 21Golebiewska, E. M.; Poole, A. W. Blood Rev. 2015, 29, 153– 162, DOI: 10.1016/j.blre.2014.10.00321https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFSnu73P&md5=5546fb06f1ff5d85fe6bcae2d31a0ac4Platelet secretion: From haemostasis to wound healing and beyondGolebiewska, Ewelina M.; Poole, Alastair W.Blood Reviews (2015), 29 (3), 153-162CODEN: BLOREB; ISSN:0268-960X. (Elsevier Ltd.)Upon activation, platelets secrete more than 300 active substances from their intracellular granules. Platelet dense granule components, such as ADP and polyphosphates, contribute to haemostasis and coagulation, but also play a role in cancer metastasis. α-Granules contain multiple cytokines, mitogens, pro- and anti-inflammatory factors and other bioactive mols. that are essential regulators in the complex microenvironment of the growing thrombus but also contribute to a no. of disease processes. Our understanding of the mol. mechanisms of secretion and the genetic regulation of granule biogenesis still remains incomplete. In this review we summarise our current understanding of the roles of platelet secretion in health and disease, and discuss some of the hypotheses that may explain how platelets may control the release of its many secreted components in a context-specific manner, to allow platelets to play multiple roles in health and disease.
- 22Nurden, A. T.; Nurden, P.; Sanchez, M.; Andia, I.; Anitua, E. Front Biosci. 2008, 13, 3532– 354822https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD1czksVymsQ%253D%253D&md5=bc9408067264404e8f17e44fcbfaffe8Platelets and wound healingNurden Alan T; Nurden Paquita; Sanchez Mikel; Andia Isabel; Anitua EduardoFrontiers in bioscience : a journal and virtual library (2008), 13 (), 3532-48 ISSN:1093-9946.Platelets help prevent blood loss at sites of vascular injury. To do this, they adhere, aggregate and form a procoagulant surface favoring thrombin generation and fibrin formation. In addition, platelets express and release substances that promote tissue repair and influence processes such as angiogenesis, inflammation and the immune response. They contain large secretable pools of biologically active proteins, while newly synthesized active metabolites are also released. Although anucleate, activated platelets possess a spliceosome and can synthesize tissue factor and interleukin-1beta. The binding of secreted proteins within a developing fibrin mesh or to the extracellular matrix can create chemotactic gradients favoring the recruitment of stem cells, stimulating cell migration and differentiation, and promoting repair. The therapeutic use of platelets in a fibrin clot has a positive influence in clinical situations requiring rapid healing. Dental implant surgery, orthopaedic surgery, muscle and tendon repair, skin ulcers, hole repair in eye surgery and cardiac surgery are situations where the use of autologous platelets accelerates healing. We now review the ways in which platelets participate in these processes.
- 23Wang, C.; Sun, W.; Ye, Y.; Hu, Q.; Bomba, H. N.; Gu, Z. Nat. Biomed. Eng. 2017, 1, 0011, DOI: 10.1038/s41551-016-001123https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXit1ahur%252FO&md5=f19b7813302dd9569c51e471417ed119In situ activation of platelets with checkpoint inhibitors for post-surgical cancer immunotherapyWang, Chao; Sun, Wujin; Ye, Yanqi; Hu, Quanyin; Bomba, Hunter N.; Gu, ZhenNature Biomedical Engineering (2017), 1 (2), 0011CODEN: NBEAB3; ISSN:2157-846X. (Nature Research)Cancer recurrence after surgical resection remains a significant challenge in cancer therapy. Platelets, which accumulate in wound sites and interact with circulating tumor cells (CTCs), can however trigger inflammation and repair processes in the remaining tumor microenvironment. Inspired by this intrinsic ability of platelets and the clin. success of immune checkpoint inhibitors, here we show that conjugating anti-PDL1 (engineered monoclonal antibodies against programmed-death ligand 1) to the surface of platelets can reduce post-surgical tumor recurrence and metastasis. Using mice bearing partially removed primary melanomas (B16-F10) or triple-neg. breast carcinomas (4T1), we found that anti-PDL1 was effectively released on platelet activation by platelet-derived microparticles, and that the administration of platelet-bound anti-PDL1 significantly prolonged overall mouse survival after surgery by reducing the risk of cancer regrowth and metastatic spread. Our findings suggest that engineered platelets can facilitate the delivery of the immunotherapeutic anti-PDL1 to the surgical bed and target CTCs in the bloodstream, thereby potentially improving the objective response rate.
- 24Hu, C. M.; Fang, R. H.; Wang, K. C.; Luk, B. T.; Thamphiwatana, S.; Dehaini, D.; Nguyen, P.; Angsantikul, P.; Wen, C. H.; Kroll, A. V. Nature 2015, 526, 118– 121, DOI: 10.1038/nature1537324https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFersL3L&md5=b3ac12cfaaec406bc86e7b816f843a80Nanoparticle biointerfacing by platelet membrane cloakingHu, Che-Ming J.; Fang, Ronnie H.; Wang, Kuei-Chun; Luk, Brian T.; Thamphiwatana, Soracha; Dehaini, Diana; Nguyen, Phu; Angsantikul, Pavimol; Wen, Cindy H.; Kroll, Ashley V.; Carpenter, Cody; Ramesh, Manikantan; Qu, Vivian; Patel, Sherrina H.; Zhu, Jie; Shi, William; Hofman, Florence M.; Chen, Thomas C.; Gao, Weiwei; Zhang, Kang; Chien, Shu; Zhang, LiangfangNature (London, United Kingdom) (2015), 526 (7571), 118-121CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Development of functional nanoparticles can be encumbered by unanticipated material properties and biol. events, which can affect nanoparticle effectiveness in complex, physiol. relevant systems. Despite the advances in bottom-up nanoengineering and surface chem., reductionist functionalization approaches remain inadequate in replicating the complex interfaces present in nature and cannot avoid exposure of foreign materials. Here we report on the prepn. of polymeric nanoparticles enclosed in the plasma membrane of human platelets, which are a unique population of cellular fragments that adhere to a variety of disease-relevant substrates. The resulting nanoparticles possess a right-side-out unilamellar membrane coating functionalized with immunomodulatory and adhesion antigens assocd. with platelets. Compared to uncoated particles, the platelet membrane-cloaked nanoparticles have reduced cellular uptake by macrophage-like cells and lack particle-induced complement activation in autologous human plasma. The cloaked nanoparticles also display platelet-mimicking properties such as selective adhesion to damaged human and rodent vasculatures as well as enhanced binding to platelet-adhering pathogens. In an exptl. rat model of coronary restenosis and a mouse model of systemic bacterial infection, docetaxel and vancomycin, resp., show enhanced therapeutic efficacy when delivered by the platelet-mimetic nanoparticles. The multifaceted biointerfacing enabled by the platelet membrane cloaking method provides a new approach in developing functional nanoparticles for disease-targeted delivery.
- 25Anselmo, A. C.; Modery-Pawlowski, C. L.; Menegatti, S.; Kumar, S.; Vogus, D. R.; Tian, L. L.; Chen, M.; Squires, T. M.; Sen Gupta, A.; Mitragotri, S. ACS Nano 2014, 8, 11243– 11253, DOI: 10.1021/nn503732mThere is no corresponding record for this reference.
- 26Hu, Q.; Sun, W.; Qian, C.; Wang, C.; Bomba, H. N.; Gu, Z. Adv. Mater. 2015, 27, 7043– 7050, DOI: 10.1002/adma.20150332326https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFyqsrnM&md5=fb41127552a9ab172439df259a7c2cbbAnticancer Platelet-Mimicking NanovehiclesHu, Quanyin; Sun, Wujin; Qian, Chengen; Wang, Chao; Bomba, Hunter N.; Gu, ZhenAdvanced Materials (Weinheim, Germany) (2015), 27 (44), 7043-7050CODEN: ADVMEW; ISSN:0935-9648. (Wiley-VCH Verlag GmbH & Co. KGaA)Human plasma membranes were isolated and made into nanocapsules that contained doxorubicin. TRAIL was also added to provide antitumor activity. The nanocapsules were targeted to human breast cancer cells by platelet P-selectin binding to CD44 and TRAIL binding to death receptors. Tumor apoptosis was demonstrated.
- 27Li, S.; Jiang, Q.; Liu, S.; Zhang, Y.; Tian, Y.; Song, C.; Wang, J.; Zou, Y.; Anderson, G. J.; Han, J. Y.; Chang, Y.; Liu, Y.; Zhang, C.; Chen, L.; Zhou, G.; Nie, G.; Yan, H.; Ding, B.; Zhao, Y. Nat. Biotechnol. 2018, 36, 258– 264, DOI: 10.1038/nbt.407127https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXisFCiurY%253D&md5=b780d2a8958409aeee721f6d96b3aee0A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivoLi, Suping; Jiang, Qiao; Liu, Shaoli; Zhang, Yinlong; Tian, Yanhua; Song, Chen; Wang, Jing; Zou, Yiguo; Anderson, Gregory J.; Han, Jing-Yan; Chang, Yung; Liu, Yan; Zhang, Chen; Chen, Liang; Zhou, Guangbiao; Nie, Guangjun; Yan, Hao; Ding, Baoquan; Zhao, YuliangNature Biotechnology (2018), 36 (3), 258-264CODEN: NABIF9; ISSN:1087-0156. (Nature Research)Nanoscale robots have potential as intelligent drug delivery systems that respond to mol. triggers. Using DNA origami we constructed an autonomous DNA robot programmed to transport payloads and present them specifically in tumors. Our nanorobot is functionalized on the outside with a DNA aptamer that binds nucleolin, a protein specifically expressed on tumor-assocd. endothelial cells, and the blood coagulation protease thrombin within its inner cavity. The nucleolin-targeting aptamer serves both as a targeting domain and as a mol. trigger for the mech. opening of the DNA nanorobot. The thrombin inside is thus exposed and activates coagulation at the tumor site. Using tumor-bearing mouse models, we demonstrate that i.v. injected DNA nanorobots deliver thrombin specifically to tumor-assocd. blood vessels and induce intravascular thrombosis, resulting in tumor necrosis and inhibition of tumor growth. The nanorobot proved safe and immunol. inert in mice and Bama miniature pigs. Our data show that DNA nanorobots represent a promising strategy for precise drug delivery in cancer therapy.
- 28Stroncek, D. F.; Rebulla, P. Lancet 2007, 370, 427– 438, DOI: 10.1016/S0140-6736(07)61198-2There is no corresponding record for this reference.
- 29Moreau, T.; Evans, A. L.; Vasquez, L.; Tijssen, M. R.; Yan, Y.; Trotter, M. W.; Howard, D.; Colzani, M.; Arumugam, M.; Wu, W. H. Nat. Commun. 2016, 7, 11208, DOI: 10.1038/ncomms1120829https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xls1Kmtb8%253D&md5=f0a2623373828c01d2fe868e5a10ac9dLarge-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programmingMoreau, Thomas; Evans, Amanda L.; Vasquez, Louella; Tijssen, Marloes R.; Yan, Ying; Trotter, Matthew W.; Howard, Daniel; Colzani, Maria; Arumugam, Meera; Wu, Wing Han; Dalby, Amanda; Lampela, Riina; Bouet, Guenaelle; Hobbs, Catherine M.; Pask, Dean C.; Payne, Holly; Ponomaryov, Tatyana; Brill, Alexander; Soranzo, Nicole; Ouwehand, Willem H.; Pedersen, Roger A.; Ghevaert, CedricNature Communications (2016), 7 (), 11208CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)The prodn. of megakaryocytes (MKs)-the precursors of blood platelets-from human pluripotent stem cells (hPSCs) offers exciting clin. opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chem. defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 105 mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufg. practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro prodn. of platelets for transfusion and basic research in MK and platelet biol.
- 30Li, J.; Sharkey, C. C.; Wun, B.; Liesveld, J. L.; King, M. R. J. Controlled Release 2016, 228, 38– 47, DOI: 10.1016/j.jconrel.2016.02.036There is no corresponding record for this reference.
- 31Lefrancais, E.; Ortiz-Munoz, G.; Caudrillier, A.; Mallavia, B.; Liu, F.; Sayah, D. M.; Thornton, E. E.; Headley, M. B.; David, T.; Coughlin, S. R. Nature 2017, 544 (7648), 105– 109, DOI: 10.1038/nature21706There is no corresponding record for this reference.
- 32Zhang, X.; Wang, C.; Wang, J.; Hu, Q.; Langworthy, B.; Ye, Y.; Sun, W.; Lin, J.; Wang, T.; Fine, J.; Cheng, H.; Dotti, G.; Huang, P.; Gu, Z. Adv. Mater. 2018, 30, 1707112, DOI: 10.1002/adma.201707112There is no corresponding record for this reference.
- 33Machlus, K. R.; Italiano, J. E., Jr. J. Cell Biol. 2013, 201, 785– 796, DOI: 10.1083/jcb.201304054There is no corresponding record for this reference.
- 34Ruggeri, Z. M.; Mendolicchio, G. L. Circ. Res. 2007, 100, 1673– 1685, DOI: 10.1161/01.RES.0000267878.97021.ab34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXmslygt7c%253D&md5=91f340e290373377e8a8b543f7e3f133Adhesion Mechanisms in Platelet FunctionRuggeri, Zaverio M.; Mendolicchio, G. LoredanaCirculation Research (2007), 100 (12), 1673-1685CODEN: CIRUAL; ISSN:0009-7330. (Lippincott Williams & Wilkins)Platelet adhesion is an essential function in response to vascular injury and is generally viewed as the first step during which single platelets bind through specific membrane receptors to cellular and extracellular matrix constituents of the vessel wall and tissues. This response initiates thrombus formation that arrests hemorrhage and permits wound healing. Pathol. conditions that cause vascular alterations and blood flow disturbances may turn this beneficial process into a disease mechanism that results in arterial occlusion, most frequently in atherosclerotic vessels of the heart and brain. Besides their relevant role in hemostasis and thrombosis, platelet adhesive properties are central to a variety of pathophysiol. processes that extend from inflammation to immune-mediated host defense and pathogenic mechanisms as well as cancer metastasis. All of these activities depend on the ability of platelets to circulate in blood as sentinels of vascular integrity, adhere where alterations are detected, and signal the abnormality to other platelets and blood cells. In this respect, therefore, platelet adhesion to vascular wall structures, to one another (aggregation), or to other blood cells, represent different aspects of the same fundamental biol. process. Detailed studies by many investigators over the past several years have been aimed to dissect the complexity of these functions, and the results obtained now permit an attempt to integrate all the available information into a picture that highlights the balanced diversity and synergy of distinct platelet adhesive interactions.
- 35Semple, J. W.; Italiano, J. E., Jr.; Freedman, J. Nat. Rev. Immunol. 2011, 11, 264– 274, DOI: 10.1038/nri295635https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjvVOhu7s%253D&md5=dac67170a4368952bc133aacc5b1bdbdPlatelets and the immune continuumSemple, John W.; Italiano, Joseph E.; Freedman, JohnNature Reviews Immunology (2011), 11 (4), 264-274CODEN: NRIABX; ISSN:1474-1733. (Nature Publishing Group)A review. Platelets are anucleate cells that are crucial mediators of haemostasis. Most immunologists probably don't think about platelets every day, and may even consider these cells to be 'nuisances' in certain in vitro studies. However, it is becoming increasingly clear that platelets have inflammatory functions and can influence both innate and adaptive immune responses. Here, we discuss the mechanisms by which platelets contribute to immunity: these small cells are more immunol. savvy than we once thought.
- 36Siljander, P. R. Thromb. Res. 2011, 127, S30– S33, DOI: 10.1016/S0049-3848(10)70152-336https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhs1GksA%253D%253D&md5=8a9f0eee648e1a4ce1e4929e5f0ab038Platelet-derived microparticles - an updated perspectiveSiljander, Pia R. M.Thrombosis Research (2011), 127 (Suppl. 2), S30-S33CODEN: THBRAA; ISSN:0049-3848. (Elsevier Ltd.)A review. Platelet-derived microparticles (PMP) are a heterogeneous population of vesicles (<1mm) generated from the plasma membrane upon platelet activation by various stimuli. They are a discrete population differing from the exosomes which originate from the intracellular multivesicular bodies. PMP also differ from the microparticles derived from megakaryocytes despite the presence of several identical surface markers on the latter. The mol. properties and the functional roles of the PMP are beginning to be elucidated by the rapidly evolving research interest, but novel questions are simultaneously raised. This updated perspective discusses the most recent highlights in the PMP research in context with the methodol. problems and the paradoxical role of the PMP in health and disease.
- 37Mause, S. F.; von Hundelshausen, P.; Zernecke, A.; Koenen, R. R.; Weber, C. Arterioscler., Thromb., Vasc. Biol. 2005, 25, 1512– 1518, DOI: 10.1161/01.ATV.0000170133.43608.3737https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXltlSitb0%253D&md5=a60e3f963f73b8bdebb85cbd87a1e55aPlatelet Microparticles: A Transcellular Delivery System for RANTES Promoting Monocyte Recruitment on EndotheliumMause, Sebastian F.; von Hundelshausen, Philipp; Zernecke, Alma; Koenen, Rory R.; Weber, ChristianArteriosclerosis, Thrombosis, and Vascular Biology (2005), 25 (7), 1512-1518CODEN: ATVBFA; ISSN:1079-5642. (Lippincott Williams & Wilkins)Objective: Platelet activation mediates multiple cellular responses, including secretion of chemokines such as RANTES (CCL5), and formation of platelet microparticles (PMPs). We studied the role of PMPs in delivering RANTES and promoting monocyte recruitment. Methods and Results: Here we show that PMPs contain substantial amts. of RANTES and deposit RANTES on activated endothelium or murine atherosclerotic carotid arteries. RANTES deposition is facilitated by flow conditions and more efficient than that conferred by PMP supernatants. Interactions of PMPs with activated endothelium in flow were mostly characterized by rolling. RANTES deposition showed a diffuse distribution pattern and was rarely colocalized with firmly adherent PMPs, substantiating that RANTES deposition occurs during transient interactions. Importantly, preperfusion with PMPs enhanced monocyte arrest on activated endothelium or atherosclerotic carotid arteries, which could be inhibited by a blocking antibody or a RANTES receptor antagonist. Blockade or deficiency of PMP-expressed adhesion receptors demonstrated differential requirement of P-selectin, glycoprotein Ib (GPIb), GPIIb/IIIa, and junctional adhesion mol.-A for PMP interactions with endothelium, PMP-dependent RANTES deposition, and subsequent monocyte arrest. Conclusion: Circulating PMPs may serve as a finely tuned transcellular delivery system for RANTES, triggering monocyte arrest to inflamed and atherosclerotic endothelium, introducing a novel mechanism for platelet-dependent monocyte recruitment in inflammation and atherosclerosis.
- 38Rollinghoff, M.; Starzinski-Powitz, A.; Pfizenmaier, K.; Wagner, H. J. Exp. Med. 1977, 145, 455– 459, DOI: 10.1084/jem.145.2.455There is no corresponding record for this reference.
- 39Yoshida, S.; Nomoto, K.; Himeno, K.; Takeya, K. Clin. Exp. Immunol. 1979, 38 (2), 211– 21739https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADyaL3c7htFOgtA%253D%253D&md5=ceee5b85ffd7b15cfad133e147e71833Immune response to syngeneic or autologous testicular cells in mice. I. Augmented delayed footpad reaction in cyclophosphamide-treated miceYoshida S; Nomoto K; Himeno K; Takeya KClinical and experimental immunology (1979), 38 (2), 211-7 ISSN:0009-9104.A delayed footpad reaction against syngeneic or autologous testicular cells was detected in mice of inbred C57BL/6, AKR and C3H/He strains. The reaction was only provoked to a measurable level if the immunization was preceded by treatment with cyclophosphamide (CY). Footpad reaction was strongest on day 6 after immunization and was detected in both male and female mice. It was found that the reaction was elicited not only with the immunizing antigen, but also with allogeneic or xenogeneic testicular antigen in mice immunized with syngeneic testicular cells.
- 40Berd, D.; Mastrangelo, M. J. Cancer Res. 1988, 48 (6), 1671– 1675There is no corresponding record for this reference.
- 41Chen, Q.; Wang, C.; Chen, G.; Hu, Q.; Gu, Z. Adv. Healthcare Mater. 2018, 1800424, DOI: 10.1002/adhm.201800424There is no corresponding record for this reference.
- 42Wang, C.; Wang, J.; Zhang, X.; Yu, S.; Wen, D.; Hu, Q.; Ye, Y.; Bomba, H.; Hu, X.; Liu, Z.; Dotti, G.; Gu, Z. Sci. Transl. Med. 2018, 10, 429, DOI: 10.1126/scitranslmed.aan3682There is no corresponding record for this reference.
- 43Zhang, X.; Dong, Y.; Zeng, X.; Liang, X.; Li, X.; Tao, W.; Chen, H.; Jiang, Y.; Mei, L.; Feng, S. S. Biomaterials 2014, 35 (6), 1932– 43, DOI: 10.1016/j.biomaterials.2013.10.034There is no corresponding record for this reference.
- 44Jang, S. C.; Kim, O. Y.; Yoon, C. M.; Choi, D. S.; Roh, T. Y.; Park, J.; Nilsson, J.; Lotvall, J.; Kim, Y. K.; Gho, Y. S. ACS Nano 2013, 7, 7698– 7710, DOI: 10.1021/nn402232gThere is no corresponding record for this reference.
- 45Wen, D.; Peng, Y.; Liu, D.; Weizmann, Y.; Mahato, R. I. J. Controlled Release 2016, 238, 166– 175, DOI: 10.1016/j.jconrel.2016.07.044There is no corresponding record for this reference.
- 46Alvarez-Erviti, L.; Seow, Y.; Yin, H.; Betts, C.; Lakhal, S.; Wood, M. J. Nat. Biotechnol. 2011, 29, 341– 345, DOI: 10.1038/nbt.180746https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXjsVKqsLw%253D&md5=b86a2f2c2338e8dfe6d4cf2f1e788296Delivery of siRNA to the mouse brain by systemic injection of targeted exosomesAlvarez-Erviti, Lydia; Seow, Yiqi; Yin, HaiFang; Betts, Corinne; Lakhal, Samira; Wood, Matthew J. A.Nature Biotechnology (2011), 29 (4), 341-345CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)To realize the therapeutic potential of RNA drugs, efficient, tissue-specific and nonimmunogenic delivery technologies must be developed. Here we show that exosomes-endogenous nano-vesicles that transport RNAs and proteins-can deliver short interfering (si)RNA to the brain in mice. To reduce immunogenicity, we used self-derived dendritic cells for exosome prodn. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide. Purified exosomes were loaded with exogenous siRNA by electroporation. I.v. injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not obsd. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease, in wild-type mice.
- 47El-Andaloussi, S.; Lee, Y.; Lakhal-Littleton, S.; Li, J.; Seow, Y.; Gardiner, C.; Alvarez-Erviti, L.; Sargent, I. L.; Wood, M. J. Nat. Protoc. 2012, 7, 2112– 2126, DOI: 10.1038/nprot.2012.131There is no corresponding record for this reference.
- 48Tian, Y.; Li, S.; Song, J.; Ji, T.; Zhu, M.; Anderson, G. J.; Wei, J.; Nie, G. Biomaterials 2014, 35, 2383– 2390, DOI: 10.1016/j.biomaterials.2013.11.08348https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFGrsbzJ&md5=02b3a5d88c448c81b2d57c70b011698cA doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapyTian, Yanhua; Li, Suping; Song, Jian; Ji, Tianjiao; Zhu, Motao; Anderson, Gregory J.; Wei, Jingyan; Nie, GuangjunBiomaterials (2014), 35 (7), 2383-2390CODEN: BIMADU; ISSN:0142-9612. (Elsevier Ltd.)Targeted drug delivery vehicles with low immunogenicity and toxicity are needed for cancer therapy. Here we show that exosomes, endogenous nano-sized membrane vesicles secreted by most cell types, can deliver chemotherapeutics such as doxorubicin (Dox) to tumor tissue in BALB/c nude mice. To reduce immunogenicity and toxicity, mouse immature dendritic cells (imDCs) were used for exosome prodn. Tumor targeting was facilitated by engineering the imDCs to express a well-characterized exosomal membrane protein (Lamp2b) fused to αv integrin-specific iRGD peptide (CRGDKGPDC). Purified exosomes from imDCs were loaded with Dox via electroporation, with an encapsulation efficiency of up to 20%. IRGD exosomes showed highly efficient targeting and Dox delivery to αv integrin-pos. breast cancer cells in vitro as demonstrated by confocal imaging and flow cytometry. I.v. injected targeted exosomes delivered Dox specifically to tumor tissues, leading to inhibition of tumor growth without overt toxicity. Our results suggest that exosomes modified by targeting ligands can be used therapeutically for the delivery of Dox to tumors, thus having great potential value for clin. applications.
- 49Gulfam, M.; Kim, J. E.; Lee, J. M.; Ku, B.; Chung, B. H.; Chung, B. G. Langmuir 2012, 28, 8216– 8223, DOI: 10.1021/la300691nThere is no corresponding record for this reference.
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Supporting Information
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b02321.
In vitro process of platelet releasing from L8057 cells, establishment of L8057 cell line stably expressing EGFP-PD-1, activity of platelets released from L8057 cells, in vitro binding and uptake of platelets by tumor cells, tumor size and body weight of mice, histological images for H&E staining, in vitro loading and release of cyclophosphamide by PD-1-expressing platelets, and the bioluminescence imaging of B16F10 tumor growth in mice after partial tumor resection and receiving the treatments (PDF)
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