Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells
利用携带纳米颗粒的 T 细胞实现化疗对播散性肿瘤的主动靶向
Authors Info & Affiliations
黄邦妮, 吴伯特·D·亚伯拉罕, 郑一然, 桑德拉·C·布斯塔曼特·洛佩斯, 萨曼莎·S·卢奥, 达雷尔·J·欧文 作者信息与单位
黄邦妮, 吴伯特·D·亚伯拉罕, 郑一然, 桑德拉·C·布斯塔曼特·洛佩斯, 萨曼莎·S·卢奥, 达雷尔·J·欧文 作者信息与单位
Science Translational Medicine
科学转化医学
科学转化医学
10 Jun 2015 2015 年 6 月 10 日
Vol 7, Issue 291 第 7 卷，第 291 期
p. 291ra94 第 291 页，RA94 部分
T cell backpacks carry chemo to tumors
T 细胞“背包”携带化疗药物直达肿瘤
Getting drugs into tumors is no small feat, especially when they are disseminated throughout the body or harbored in the lymph nodes—often considered a “sanctuary” for cancer cells. Huang et al. devised a clever drug delivery system for such tumors that capitalizes on the natural movement of immune cells throughout the body. The authors first expanded T cells ex vivo under conditions that would make them home to lymphoid tissues. Then, nanocapsules loaded with a common chemotherapeutic were attached to the lymphocytes. The modified cells were infused into a mouse model of Burkitt’s lymphoma, where they carried their toxic backpacks to multiple lymphoid organs. The animals experienced not only reduced tumor burden but also prolonged survival compared with systemic therapy with the same drug. Because T cells are easily obtained from blood and are already being used in the clinic for cancer therapy, this “pharmacyte” strategy could be tailored to home to different organs, carrying unique cargos, in an effort to eradicate hard-to-reach tumors.
将药物送入肿瘤绝非易事，尤其是在肿瘤扩散至全身或藏匿于淋巴结内时，淋巴结常被视为癌细胞的“避难所”。Huang 等人设计了一种巧妙的药物递送系统，利用免疫细胞在体内的自然运动来针对此类肿瘤。研究者首先在体外扩增 T 细胞，使其在特定条件下归巢至淋巴组织，随后将装载常见化疗药物的纳米胶囊附着于这些淋巴细胞上。改造后的细胞被注入伯基特淋巴瘤小鼠模型体内，它们携带毒性“背包”抵达多个淋巴器官。与全身使用相同药物的治疗相比，这些小鼠不仅肿瘤负荷减轻，且生存期延长。由于 T 细胞易于从血液中获取，且已在临床上用于癌症治疗，这种“药理细胞”策略可针对不同器官进行定制，携带特异性药物，旨在消灭难以触及的肿瘤。
将药物送入肿瘤绝非易事，尤其是在肿瘤扩散至全身或藏匿于淋巴结内时，淋巴结常被视为癌细胞的“避难所”。Huang 等人设计了一种巧妙的药物递送系统，利用免疫细胞在体内的自然运动来针对此类肿瘤。研究者首先在体外扩增 T 细胞，使其在特定条件下归巢至淋巴组织，随后将装载常见化疗药物的纳米胶囊附着于这些淋巴细胞上。改造后的细胞被注入伯基特淋巴瘤小鼠模型体内，它们携带毒性“背包”抵达多个淋巴器官。与全身使用相同药物的治疗相比，这些小鼠不仅肿瘤负荷减轻，且生存期延长。由于 T 细胞易于从血液中获取，且已在临床上用于癌症治疗，这种“药理细胞”策略可针对不同器官进行定制，携带特异性药物，旨在消灭难以触及的肿瘤。
Abstract 摘要
Tumor cells disseminate into compartments that are poorly accessible from circulation, which necessitates high doses of systemic chemotherapy. However, the effectiveness of many drugs, such as the potent topoisomerase I poison SN-38, is hampered by poor pharmacokinetics. To deliver SN-38 to lymphoma tumors in vivo, we took advantage of the fact that healthy lymphocytes can be programmed to phenocopy the biodistribution of the tumor cells. In a murine model of disseminated lymphoma, we expanded autologous polyclonal T cells ex vivo under conditions that retained homing receptors mirroring lymphoma cells, and functionalized these T cells to carry SN-38–loaded nanocapsules on their surfaces. Nanocapsule-functionalized T cells were resistant to SN-38 but mediated efficient killing of lymphoma cells in vitro. Upon adoptive transfer into tumor-bearing mice, these T cells served as active vectors to deliver the chemotherapeutic into tumor-bearing lymphoid organs. Cell-mediated delivery concentrated SN-38 in lymph nodes at levels 90-fold greater than free drug systemically administered at 10-fold higher doses. The live T cell delivery approach reduced tumor burden significantly after 2 weeks of treatment and enhanced survival under conditions where free SN-38 and SN-38–loaded nanocapsules alone were ineffective. These results suggest that tissue-homing lymphocytes can serve as specific targeting agents to deliver nanoparticles into sites difficult to access from the circulation, and thus improve the therapeutic index of chemotherapeutic drugs with unfavorable pharmacokinetics.
肿瘤细胞扩散至血液循环难以有效到达的区域，这要求高剂量的全身化疗。然而，许多药物如强力拓扑异构酶 I 抑制剂 SN-38，其疗效受限于不良的药代动力学特性。为了将 SN-38 有效递送至体内淋巴瘤肿瘤，我们利用了健康淋巴细胞可被编程以模仿肿瘤细胞生物分布的特性。在一种播散性淋巴瘤的小鼠模型中，我们在体外扩增了自体多克隆 T 细胞，维持了与淋巴瘤细胞相似的归巢受体，并对其进行功能化改造，使其表面携带装载 SN-38 的纳米胶囊。纳米胶囊功能化的 T 细胞对 SN-38 具有抗性，但在体外能有效杀伤淋巴瘤细胞。通过过继转移至荷瘤小鼠体内，这些 T 细胞作为活性载体将化疗药物递送至肿瘤浸润的淋巴组织。细胞介导的递送方式使 SN-38 在淋巴结中的浓度比全身给予高 10 倍剂量的游离药物高出 90 倍。 活体 T 细胞递送方法在治疗 2 周后显著降低了肿瘤负荷，并在单独使用游离 SN-38 和装载 SN-38 的纳米胶囊无效的情况下增强了生存率。这些结果表明，组织归巢淋巴细胞可以作为特异性靶向载体，将纳米颗粒递送到血液循环难以触及的部位，从而提高具有不利药代动力学的化疗药物的治疗指数。
肿瘤细胞扩散至血液循环难以有效到达的区域，这要求高剂量的全身化疗。然而，许多药物如强力拓扑异构酶 I 抑制剂 SN-38，其疗效受限于不良的药代动力学特性。为了将 SN-38 有效递送至体内淋巴瘤肿瘤，我们利用了健康淋巴细胞可被编程以模仿肿瘤细胞生物分布的特性。在一种播散性淋巴瘤的小鼠模型中，我们在体外扩增了自体多克隆 T 细胞，维持了与淋巴瘤细胞相似的归巢受体，并对其进行功能化改造，使其表面携带装载 SN-38 的纳米胶囊。纳米胶囊功能化的 T 细胞对 SN-38 具有抗性，但在体外能有效杀伤淋巴瘤细胞。通过过继转移至荷瘤小鼠体内，这些 T 细胞作为活性载体将化疗药物递送至肿瘤浸润的淋巴组织。细胞介导的递送方式使 SN-38 在淋巴结中的浓度比全身给予高 10 倍剂量的游离药物高出 90 倍。 活体 T 细胞递送方法在治疗 2 周后显著降低了肿瘤负荷，并在单独使用游离 SN-38 和装载 SN-38 的纳米胶囊无效的情况下增强了生存率。这些结果表明，组织归巢淋巴细胞可以作为特异性靶向载体，将纳米颗粒递送到血液循环难以触及的部位，从而提高具有不利药代动力学的化疗药物的治疗指数。
INTRODUCTION 引言
Lymphomas are a heterogeneous family of hematological cancers that are generally sensitive to a wide variety of cytotoxic drugs. However, survival rates vary between subtypes, and their disseminated nature poses a challenge for therapy—tumor cell detection in an increased number of lymph nodes and extranodal sites is directly associated with more advanced staging and poorer prognosis (1). Being derived from immune cells that normally recirculate among lymphoid organs, lymphomas seed tumors in multiple organs and surgery cannot prevent the spread of disease. One route by which lymphomas evade chemotherapy in circulation is through entry into lymph nodes by transmigration across the high endothelial venules. Lymph nodes are a common tissue site for metastasis of many tumors, but systemic administration of some cytotoxic drugs has been shown to generate barely detectable levels of drug in the lymph nodes of cancer patients (2).
淋巴瘤是一组异质性的血液系统恶性肿瘤，通常对多种细胞毒性药物敏感。然而，不同亚型的生存率存在差异，且其播散性特征对治疗构成挑战——淋巴结和淋巴结外部位的肿瘤细胞检出数量增加直接与疾病分期更晚和预后更差相关（1）。由于淋巴瘤起源于通常在淋巴器官间再循环的免疫细胞，它们会在多个器官中形成肿瘤，而手术无法阻止疾病的扩散。淋巴瘤逃避化疗循环系统的一种途径是通过跨高内皮小静脉迁移进入淋巴结。淋巴结是许多肿瘤转移的常见组织部位，但系统性给予某些细胞毒性药物后，在癌症患者淋巴结中产生的药物浓度几乎无法检测到（2）。
淋巴瘤是一组异质性的血液系统恶性肿瘤，通常对多种细胞毒性药物敏感。然而，不同亚型的生存率存在差异，且其播散性特征对治疗构成挑战——淋巴结和淋巴结外部位的肿瘤细胞检出数量增加直接与疾病分期更晚和预后更差相关（1）。由于淋巴瘤起源于通常在淋巴器官间再循环的免疫细胞，它们会在多个器官中形成肿瘤，而手术无法阻止疾病的扩散。淋巴瘤逃避化疗循环系统的一种途径是通过跨高内皮小静脉迁移进入淋巴结。淋巴结是许多肿瘤转移的常见组织部位，但系统性给予某些细胞毒性药物后，在癌症患者淋巴结中产生的药物浓度几乎无法检测到（2）。
A second issue is permeation of drug throughout a tumor mass. For example, in breast cancer patients given intravenous doxorubicin, the drug was found to permeate only tumor cells in close proximity to blood vessels (3). Pharmacological studies have highlighted many obstacles to drug accumulation in tumors, including rapid metabolism or excretion and the physical barrier of the tumor stroma (4, 5). Administered by traditional intravenous routes, toxic systemic doses may be required to overcome these bottlenecks and achieve therapeutically relevant drug concentrations in the tumor. In addition, some tissue compartments, such as the lymph nodes, may serve as tumor sanctuaries, out of reach of normally effective drugs.
第二个问题是药物在整个肿瘤块中的渗透。例如，在给予静脉注射多柔比星的乳腺癌患者中，发现药物仅能渗透到靠近血管的肿瘤细胞（3）。药理学研究揭示了药物在肿瘤中积累的诸多障碍，包括快速代谢或排泄以及肿瘤基质的物理屏障（4, 5）。通过传统的静脉途径给药，可能需要毒性全身剂量才能克服这些瓶颈，在肿瘤中达到具有治疗意义的药物浓度。此外，某些组织腔室，如淋巴结，可能作为肿瘤的避难所，使常规有效药物难以触及。
第二个问题是药物在整个肿瘤块中的渗透。例如，在给予静脉注射多柔比星的乳腺癌患者中，发现药物仅能渗透到靠近血管的肿瘤细胞（3）。药理学研究揭示了药物在肿瘤中积累的诸多障碍，包括快速代谢或排泄以及肿瘤基质的物理屏障（4, 5）。通过传统的静脉途径给药，可能需要毒性全身剂量才能克服这些瓶颈，在肿瘤中达到具有治疗意义的药物浓度。此外，某些组织腔室，如淋巴结，可能作为肿瘤的避难所，使常规有效药物难以触及。
To overcome these issues, nanoparticles have been engineered to regulate the time scale of drug circulation in vivo, as well as to improve accumulation of drug payloads in tumors (6–8). Modification of particle material properties, surface chemistry, and microenvironment responsiveness can facilitate particle escape from scavenging by the reticuloendothelial system, as well as extend payload retention and, thus, increase the time between doses. Nanoparticle drug delivery has shown the best efficacy in preclinical models of solid tumors with leaky vasculature, where the enhanced permeation and retention (EPR) effect is most active. However, nanoparticles often become trapped in the matrix just outside tumor vessels, and thus fail to reach tumor cells distal from the vasculature. In addition, the EPR effect is heterogeneous and may be completely lacking in some tumors (9), including lymphoma subtypes that do not exhibit abnormal angiogenesis (10, 11).
为解决这些问题，纳米颗粒被设计用于调控药物在体内的循环时间，并增强药物在肿瘤中的积累（6-8）。通过调整颗粒的材料特性、表面化学性质及对微环境的响应性，可以促进颗粒逃避免疫系统的吞噬，延长药物载荷的保留时间，从而增加给药间隔。在具有渗漏血管的实体瘤临床前模型中，纳米颗粒药物递送显示出最佳疗效，此时增强的渗透与保留（EPR）效应最为活跃。然而，纳米颗粒常常滞留在肿瘤血管外基质中，无法到达远离血管的肿瘤细胞。此外，EPR 效应具有异质性，在某些肿瘤中可能完全缺失（9），包括不显示异常血管生成的淋巴瘤亚型（10, 11）。
为解决这些问题，纳米颗粒被设计用于调控药物在体内的循环时间，并增强药物在肿瘤中的积累（6-8）。通过调整颗粒的材料特性、表面化学性质及对微环境的响应性，可以促进颗粒逃避免疫系统的吞噬，延长药物载荷的保留时间，从而增加给药间隔。在具有渗漏血管的实体瘤临床前模型中，纳米颗粒药物递送显示出最佳疗效，此时增强的渗透与保留（EPR）效应最为活跃。然而，纳米颗粒常常滞留在肿瘤血管外基质中，无法到达远离血管的肿瘤细胞。此外，EPR 效应具有异质性，在某些肿瘤中可能完全缺失（9），包括不显示异常血管生成的淋巴瘤亚型（10, 11）。
We hypothesized that the efficacy of chemotherapy could be enhanced while reducing off-target toxicity if autologous lymphocytes were used as carriers to target drug-loaded nanoparticles to the lymphoid tissue sites where lymphomas home. Because the normal function of lymphocytes is to migrate throughout lymphoid tissues in search of antigen, we reasoned that polyclonal T cells, which express lymph node–homing receptors but do not specifically recognize tumor cell antigens, could serve as effective chaperones for targeting of chemotherapy drugs to tumor-ridden lymphoid organs. By homing into these tumor sanctuary sites and distributing throughout the tissue, each nanoparticle-carrying cell would serve as a local micro-depot of drug to dose surrounding tumor cells. The use of polyclonal T cell carriers would be attractive for clinical implementation, because tumor antigen–specific T cells can only be isolated from a subset of cancer patients (12), and by contrast, large numbers of T cells (250 to 500 million) can be obtained from a single leukapheresis and quickly expanded as much as 5000-fold using established clinical procedures in adoptive cell therapy (13).
我们假设，如果使用自体淋巴细胞作为载体，将载药纳米颗粒靶向至淋巴瘤归巢的淋巴组织部位，可以增强化疗的疗效并减少脱靶毒性。由于淋巴细胞的正常功能是在淋巴组织中迁移以寻找抗原，我们推论，表达淋巴结归巢受体但不特异性识别肿瘤细胞抗原的多克隆 T 细胞，可以作为有效的向导，将化疗药物靶向至肿瘤侵袭的淋巴器官。通过归巢到这些肿瘤庇护所并分布于组织中，每个载有纳米颗粒的细胞将作为局部药物微储库，对周围的肿瘤细胞进行剂量传递。使用多克隆 T 细胞作为载体在临床实施上具有吸引力，因为肿瘤抗原特异性 T 细胞只能从部分癌症患者中分离得到（12），而相比之下，单次白细胞分离术即可获得大量 T 细胞（2.5 亿至 5 亿），并可通过已建立的过继细胞治疗临床程序迅速扩增至 5000 倍（13）。
我们假设，如果使用自体淋巴细胞作为载体，将载药纳米颗粒靶向至淋巴瘤归巢的淋巴组织部位，可以增强化疗的疗效并减少脱靶毒性。由于淋巴细胞的正常功能是在淋巴组织中迁移以寻找抗原，我们推论，表达淋巴结归巢受体但不特异性识别肿瘤细胞抗原的多克隆 T 细胞，可以作为有效的向导，将化疗药物靶向至肿瘤侵袭的淋巴器官。通过归巢到这些肿瘤庇护所并分布于组织中，每个载有纳米颗粒的细胞将作为局部药物微储库，对周围的肿瘤细胞进行剂量传递。使用多克隆 T 细胞作为载体在临床实施上具有吸引力，因为肿瘤抗原特异性 T 细胞只能从部分癌症患者中分离得到（12），而相比之下，单次白细胞分离术即可获得大量 T 细胞（2.5 亿至 5 亿），并可通过已建立的过继细胞治疗临床程序迅速扩增至 5000 倍（13）。
To test this concept in a murine model of Burkitt’s lymphoma, we prepared controlled-release lipid nanocapsules (NCs) loaded with the potent topoisomerase I poison SN-38. These NCs were covalently conjugated to the plasma membrane of in vitro–activated primary T cells expanded under conditions promoting retention of CD62L and CCR7 receptor expression required for lymph node homing. T cells conjugated to SN-38–releasing NCs were used as live vectors to transport NCs systemically into lymphoid organs where lymphoma cells are enriched. SN-38 NC–functionalized T cells rapidly reduced tumor burden in multiple anatomical sites and significantly extend survival compared to systemic drug therapy in an aggressive transplanted Eμ-myc Arf−/− lymphoma model. These results suggest that autologous lymphocytes with engineered tissue-specific homing receptors can serve as effective chaperones for drug delivery to systemic cancer sites.
为了在小鼠伯基特淋巴瘤模型中验证这一概念，我们制备了装载强效拓扑异构酶 I 抑制剂 SN-38 的控释脂质纳米胶囊（NCs），并将这些 NCs 共价结合到体外激活的、经条件培养扩增以保留 CD62L 和 CCR7 受体表达（这些受体对于淋巴结归巢至关重要）的初始 T 细胞的细胞膜上。携带 SN-38 释放型 NCs 的 T 细胞作为活性载体，将 NCs 系统性地输送到富含淋巴瘤细胞的淋巴器官中。与全身性药物治疗相比，SN-38 NC 功能化的 T 细胞在侵袭性移植的 Eμ-myc Arf −/− 淋巴瘤模型中迅速减轻了多个解剖部位的肿瘤负荷，并显著延长了生存期。这些结果表明，经过工程改造携带组织特异性归巢受体的自体淋巴细胞可作为向全身性癌症部位递送药物的有效载体。
为了在小鼠伯基特淋巴瘤模型中验证这一概念，我们制备了装载强效拓扑异构酶 I 抑制剂 SN-38 的控释脂质纳米胶囊（NCs），并将这些 NCs 共价结合到体外激活的、经条件培养扩增以保留 CD62L 和 CCR7 受体表达（这些受体对于淋巴结归巢至关重要）的初始 T 细胞的细胞膜上。携带 SN-38 释放型 NCs 的 T 细胞作为活性载体，将 NCs 系统性地输送到富含淋巴瘤细胞的淋巴器官中。与全身性药物治疗相比，SN-38 NC 功能化的 T 细胞在侵袭性移植的 Eμ-myc Arf −/− 淋巴瘤模型中迅速减轻了多个解剖部位的肿瘤负荷，并显著延长了生存期。这些结果表明，经过工程改造携带组织特异性归巢受体的自体淋巴细胞可作为向全身性癌症部位递送药物的有效载体。
RESULTS 结果
Poor uptake of systemically administered SN-38 in sites of lymphoma dissemination
系统性给药的 SN-38 在淋巴瘤扩散部位的摄取较差
To model Burkitt’s lymphoma, we used malignant B cells isolated from double-transgenic Eμ-myc Arf−/− mice, in which the Eμ enhancer drives c-myc oncogene overexpression in B cells and the Arf tumor suppressor gene is deleted, thus inactivating the tumor suppressor p53 (14). To track tumor distribution and growth kinetics in multiple tissues, Eμ-myc Arf−/− cells were transduced with dual green fluorescent protein (GFP) and firefly luciferase reporters (hereafter, Eμ-myc cells) (15). Consistent with previous studies (14), when transplanted into wild-type recipients, Eμ-myc cells established systemic disseminated disease, accumulating in peripheral lymph nodes, bone marrow, and spleen (Fig. 1AOpens in image viewer
为了模拟伯基特淋巴瘤，我们使用了从双转基因 Eμ-myc Arf −/− 小鼠中分离出的恶性 B 细胞，在这些小鼠中，Eμ增强子驱动 c-myc 原癌基因在 B 细胞中的过度表达，同时 Arf 抑癌基因被删除，从而使 p53 抑癌蛋白失活（14）。为了追踪肿瘤在多种组织中的分布及生长动力学，Eμ-myc Arf −/− 细胞被转导了双绿色荧光蛋白（GFP）和萤火虫荧光素酶报告基因（以下简称 Eμ-myc 细胞）（15）。与先前研究一致（14），当移植到野生型受体中时，Eμ-myc 细胞建立了全身性播散性疾病，在周围淋巴结、骨髓和脾脏中积累（图 1A）。). This pattern of tumor cell dissemination was accompanied by the expression of a suite of chemokine and adhesion receptors associated with normal lymphocyte trafficking, including the lymph node–homing receptors CD62L and CCR7, bone marrow–homing receptors CXCR4 and α4, β1, and β2 integrins, and the mucosal homing receptor α4β7 (fig. S1).
这种肿瘤细胞播散模式伴随着一系列与正常淋巴细胞迁移相关的趋化因子和粘附受体的表达，包括淋巴结归巢受体 CD62L 和 CCR7，骨髓归巢受体 CXCR4 及α4β1、β2 整合素，以及黏膜归巢受体α4β7（图 S1）。
为了模拟伯基特淋巴瘤，我们使用了从双转基因 Eμ-myc Arf −/− 小鼠中分离出的恶性 B 细胞，在这些小鼠中，Eμ增强子驱动 c-myc 原癌基因在 B 细胞中的过度表达，同时 Arf 抑癌基因被删除，从而使 p53 抑癌蛋白失活（14）。为了追踪肿瘤在多种组织中的分布及生长动力学，Eμ-myc Arf −/− 细胞被转导了双绿色荧光蛋白（GFP）和萤火虫荧光素酶报告基因（以下简称 Eμ-myc 细胞）（15）。与先前研究一致（14），当移植到野生型受体中时，Eμ-myc 细胞建立了全身性播散性疾病，在周围淋巴结、骨髓和脾脏中积累（图 1A）。). This pattern of tumor cell dissemination was accompanied by the expression of a suite of chemokine and adhesion receptors associated with normal lymphocyte trafficking, including the lymph node–homing receptors CD62L and CCR7, bone marrow–homing receptors CXCR4 and α4, β1, and β2 integrins, and the mucosal homing receptor α4β7 (fig. S1).
这种肿瘤细胞播散模式伴随着一系列与正常淋巴细胞迁移相关的趋化因子和粘附受体的表达，包括淋巴结归巢受体 CD62L 和 CCR7，骨髓归巢受体 CXCR4 及α4β1、β2 整合素，以及黏膜归巢受体α4β7（图 S1）。
Fig. 1. SN-38 is a potent cytotoxic agent against Eμ-myc lymphoma cells but fails to access sites of lymphoma dissemination in vivo.
图 1. SN-38 是一种对 Eμ-myc 淋巴瘤细胞具有强效细胞毒性的药物，但在体内无法到达淋巴瘤扩散的部位。
图 1. SN-38 是一种对 Eμ-myc 淋巴瘤细胞具有强效细胞毒性的药物，但在体内无法到达淋巴瘤扩散的部位。
C57BL/6J recipients were injected intravenously with 1 × 106 luciferase- and GFP-expressing Eμ-myc cells. (A) On day 21 after inoculation, lymphoma biodistribution was imaged by IVIS in intact animals, followed by flow cytometry to detect GFP+ tumor cells (green gates). Images are representative ventral and side views of the same animal. LN, lymph node; PE, phycoerythrin. (B) On day 17 after inoculation, free SN-38 (10 mg/kg) was injected intravenously into tumor-bearing mice, and tissue drug concentrations were measured by high-performance liquid chromatography (HPLC) over time. Data are means ± SEM (n = 3 per group). (C) On day 17 after inoculation, tumor-bearing mice were intravenously injected with empty fluorescent liposomes (36.3 mg/kg lipid). Tissues were collected 24 hours after injection for histology. (D) On day 17 after inoculation, tumor-bearing mice were intravenously injected with SN-38–containing liposomes (1 mg/kg SN-38). Tissues were collected 24 hours after injection for HPLC analysis. All data are representative of one of two independent experiments (n = 3 animals per group). Dashed lines in (B) and (D) denote limit of detection (0.5 ng/g tissue) for SN-38 by HPLC.
C57BL/6J 小鼠接受者通过静脉注射了 1 × 10^6 表达荧光素酶和 GFP 的 Eμ-myc 细胞。(A) 接种后第 21 天，通过 IVIS 在完整动物体内成像观察淋巴瘤的生物分布，随后进行流式细胞术检测 GFP 标记的肿瘤细胞（绿色门）。图像为同一动物的腹面和侧面代表性视图。LN，淋巴结；PE，藻红蛋白。(B) 接种后第 17 天，向荷瘤小鼠静脉注射游离 SN-38（10 mg/kg），并随时间通过高效液相色谱法（HPLC）测定组织药物浓度。数据为平均值±标准误差（每组 n=3）。(C) 接种后第 17 天，向荷瘤小鼠静脉注射空荧光脂质体（36.3 mg/kg 脂质）。注射后 24 小时采集组织进行组织学检查。(D) 接种后第 17 天，向荷瘤小鼠静脉注射含 SN-38 的脂质体（1 mg/kg SN-38）。注射后 24 小时采集组织进行 HPLC 分析。所有数据均为两次独立实验中的一次的代表性结果（每组 n=3 只动物）。(B)和(D)中的虚线表示检测限（0.通过高效液相色谱法（HPLC）测定，SN-38 在组织中的浓度为 5 纳克/克。
C57BL/6J 小鼠接受者通过静脉注射了 1 × 10^6 表达荧光素酶和 GFP 的 Eμ-myc 细胞。(A) 接种后第 21 天，通过 IVIS 在完整动物体内成像观察淋巴瘤的生物分布，随后进行流式细胞术检测 GFP 标记的肿瘤细胞（绿色门）。图像为同一动物的腹面和侧面代表性视图。LN，淋巴结；PE，藻红蛋白。(B) 接种后第 17 天，向荷瘤小鼠静脉注射游离 SN-38（10 mg/kg），并随时间通过高效液相色谱法（HPLC）测定组织药物浓度。数据为平均值±标准误差（每组 n=3）。(C) 接种后第 17 天，向荷瘤小鼠静脉注射空荧光脂质体（36.3 mg/kg 脂质）。注射后 24 小时采集组织进行组织学检查。(D) 接种后第 17 天，向荷瘤小鼠静脉注射含 SN-38 的脂质体（1 mg/kg SN-38）。注射后 24 小时采集组织进行 HPLC 分析。所有数据均为两次独立实验中的一次的代表性结果（每组 n=3 只动物）。(B)和(D)中的虚线表示检测限（0.通过高效液相色谱法（HPLC）测定，SN-38 在组织中的浓度为 5 纳克/克。
As an advanced chemotherapy strategy for treatment of lymphoma, we focused on SN-38, the active form of the camptothecin derivative irinotecan. Although 1000-fold more potent than irinotecan (16), SN-38 exhibits poor pharmacokinetics in vivo, with a 7-min half-life in the bloodstream and rapid clearance through the liver (17). Consistent with these previous reports, after intravenous injection, we found that most of the drug was in the liver within 1 hour (Fig. 1BOpens in image viewer
作为一种针对淋巴瘤的高级化疗策略，我们重点关注了 SN-38，它是喜树碱衍生物伊立替康的活性形式。尽管 SN-38 的效力比伊立替康高出 1000 倍（16），但其在体内的药代动力学特性较差，血液中的半衰期仅为 7 分钟，并迅速通过肝脏清除（17）。与之前的报告一致，我们在静脉注射后发现，大部分药物在 1 小时内就集中于肝脏（图 1B）。). SN-38 showed some accumulation in the spleen and bone marrow at 10 hours after injection, but was rapidly cleared from these compartments. Less than 0.05% of the injected dose was detected in the lymph nodes at any time point (Fig. 1BOpens in image viewer
SN-38 在注射后 10 小时在脾脏和骨髓中显示出一定程度的积累，但很快从这些部位清除。在任何时间点，淋巴结中检测到的剂量均不足注射量的 0.05%（图 1B）。).
作为一种针对淋巴瘤的高级化疗策略，我们重点关注了 SN-38，它是喜树碱衍生物伊立替康的活性形式。尽管 SN-38 的效力比伊立替康高出 1000 倍（16），但其在体内的药代动力学特性较差，血液中的半衰期仅为 7 分钟，并迅速通过肝脏清除（17）。与之前的报告一致，我们在静脉注射后发现，大部分药物在 1 小时内就集中于肝脏（图 1B）。). SN-38 showed some accumulation in the spleen and bone marrow at 10 hours after injection, but was rapidly cleared from these compartments. Less than 0.05% of the injected dose was detected in the lymph nodes at any time point (Fig. 1BOpens in image viewer
SN-38 在注射后 10 小时在脾脏和骨髓中显示出一定程度的积累，但很快从这些部位清除。在任何时间点，淋巴结中检测到的剂量均不足注射量的 0.05%（图 1B）。).
A common strategy to alter the biodistribution of chemotherapy agents is encapsulation in nanoparticles (18). After intravenous administration of fluorescently labeled stealth liposomes (120 ± 24 nm in diameter) to Eμ-myc tumor-bearing mice, some liposomes were found in the liver and spleen at 24 hours. By contrast, no particles were detected in the lymph nodes (Fig. 1COpens in image viewer
一种常见的改变化疗药物生物分布的策略是将它们封装在纳米颗粒中（18）。在向 Eμ-myc 荷瘤小鼠静脉注射荧光标记的隐形脂质体（直径 120 ± 24 nm）后，24 小时时在肝脏和脾脏中发现了部分脂质体。相比之下，淋巴结中未检测到颗粒（图 1C）。). Systemic injection of stealth liposomes carrying SN-38 (136 ± 11 nm diameter, 4.8 ± 1.5 μg of SN-38 per micromole of lipid) into tumor-bearing mice resulted in high levels of SN-38 in the blood, spleen, and liver, but accumulation of drug in lymph nodes was 10-fold lower than in these organs and indistinguishable from the result obtained with systemic free drug administration (Fig. 1DOpens in image viewer
). 将携带 SN-38 的隐形脂质体（直径 136 ± 11 nm，每微摩尔脂质含 4.8 ± 1.5 μg SN-38）系统性注射入荷瘤小鼠后，血液、脾脏和肝脏中 SN-38 水平较高，但淋巴结中的药物积聚量比这些器官低 10 倍，且与系统性注射游离药物的结果无明显差异（图 1D）。). Thus, free drug or liposome formulations that promote accumulation of chemotherapy agents in tumors by the EPR effect failed to access tumor-ridden lymph nodes efficiently, and thus, these tissues may serve as a survival niche for lymphoma cells in the face of chemotherapy.
因此，通过 EPR 效应促进化疗药物在肿瘤中积累的游离药物或脂质体制剂，未能有效进入肿瘤浸润的淋巴结，从而这些组织可能在化疗面前成为淋巴瘤细胞的生存龛。
一种常见的改变化疗药物生物分布的策略是将它们封装在纳米颗粒中（18）。在向 Eμ-myc 荷瘤小鼠静脉注射荧光标记的隐形脂质体（直径 120 ± 24 nm）后，24 小时时在肝脏和脾脏中发现了部分脂质体。相比之下，淋巴结中未检测到颗粒（图 1C）。). Systemic injection of stealth liposomes carrying SN-38 (136 ± 11 nm diameter, 4.8 ± 1.5 μg of SN-38 per micromole of lipid) into tumor-bearing mice resulted in high levels of SN-38 in the blood, spleen, and liver, but accumulation of drug in lymph nodes was 10-fold lower than in these organs and indistinguishable from the result obtained with systemic free drug administration (Fig. 1DOpens in image viewer
). 将携带 SN-38 的隐形脂质体（直径 136 ± 11 nm，每微摩尔脂质含 4.8 ± 1.5 μg SN-38）系统性注射入荷瘤小鼠后，血液、脾脏和肝脏中 SN-38 水平较高，但淋巴结中的药物积聚量比这些器官低 10 倍，且与系统性注射游离药物的结果无明显差异（图 1D）。). Thus, free drug or liposome formulations that promote accumulation of chemotherapy agents in tumors by the EPR effect failed to access tumor-ridden lymph nodes efficiently, and thus, these tissues may serve as a survival niche for lymphoma cells in the face of chemotherapy.
因此，通过 EPR 效应促进化疗药物在肿瘤中积累的游离药物或脂质体制剂，未能有效进入肿瘤浸润的淋巴结，从而这些组织可能在化疗面前成为淋巴瘤细胞的生存龛。
Generation of T cells as chaperones for SN-38 delivery
生成作为 SN-38 递送载体的 T 细胞
We recently described a strategy to enhance the functionality of tumor-specific T cells via conjugation of cytokine-releasing or small-molecule drug–releasing nanoparticles to the plasma membrane of adoptively transferred lymphocytes; adjuvant drugs released from cell-bound particles provided autocrine stimulation to the carrier T cells to support their antitumor activity in vivo (19, 20). The failure of free or liposome-formulated SN-38 to effectively reach lymphoid organs led us to test whether a similar “pharmacyte” strategy could be used for paracrine delivery of chemotherapy to tumor cells, using the intrinsic tissue-homing pattern of lymphocytes rather than specific antigen recognition as a means to deliver drugs to sites of lymphoma dissemination (Fig. 2AOpens in image viewer
我们最近提出了一种策略，通过将释放细胞因子或小分子药物的纳米颗粒共价结合到过继转移的淋巴细胞的质膜上，来增强肿瘤特异性 T 细胞的功能；细胞结合颗粒释放的辅助药物提供了自分泌刺激，支持载体 T 细胞在体内的抗肿瘤活性（19, 20）。由于游离或脂质体包裹的 SN-38 未能有效到达淋巴器官，我们测试了类似的“药细胞”策略是否可用于通过淋巴细胞固有的组织归巢模式进行化疗的旁分泌传递，而非依赖特异性抗原识别，以此将药物送达淋巴瘤扩散的部位（图 2A）。). For this approach to succeed, several conditions needed to be met: (i) the tropism of the carrier cell needed to match as closely as possible the tissue distribution of the target tumor cells; (ii) the chaperone T cell needed to be resistant to SN-38 to avoid death of the carrier cell before arrival in target tissues; and (iii) the lymphocytes needed to carry a dosage of SN-38 NCs sufficient to kill lymphoma cells, which were expected to be in excess of the chaperone T cells.
为使这一方法成功，需满足以下几个条件：(i) 载体细胞的趋向性需尽可能与目标肿瘤细胞的组织分布相匹配；(ii) 伴随 T 细胞需对 SN-38 具有抗性，以免在到达目标组织前载体细胞死亡；(iii) 淋巴细胞需携带足够剂量的 SN-38 纳米颗粒，以杀灭预期的淋巴瘤细胞，且该剂量应超过伴随 T 细胞的数量。
我们最近提出了一种策略，通过将释放细胞因子或小分子药物的纳米颗粒共价结合到过继转移的淋巴细胞的质膜上，来增强肿瘤特异性 T 细胞的功能；细胞结合颗粒释放的辅助药物提供了自分泌刺激，支持载体 T 细胞在体内的抗肿瘤活性（19, 20）。由于游离或脂质体包裹的 SN-38 未能有效到达淋巴器官，我们测试了类似的“药细胞”策略是否可用于通过淋巴细胞固有的组织归巢模式进行化疗的旁分泌传递，而非依赖特异性抗原识别，以此将药物送达淋巴瘤扩散的部位（图 2A）。). For this approach to succeed, several conditions needed to be met: (i) the tropism of the carrier cell needed to match as closely as possible the tissue distribution of the target tumor cells; (ii) the chaperone T cell needed to be resistant to SN-38 to avoid death of the carrier cell before arrival in target tissues; and (iii) the lymphocytes needed to carry a dosage of SN-38 NCs sufficient to kill lymphoma cells, which were expected to be in excess of the chaperone T cells.
为使这一方法成功，需满足以下几个条件：(i) 载体细胞的趋向性需尽可能与目标肿瘤细胞的组织分布相匹配；(ii) 伴随 T 细胞需对 SN-38 具有抗性，以免在到达目标组织前载体细胞死亡；(iii) 淋巴细胞需携带足够剂量的 SN-38 纳米颗粒，以杀灭预期的淋巴瘤细胞，且该剂量应超过伴随 T 细胞的数量。
Fig. 2. IL-2/rapamycin–expanded T cells express homing receptors to traffic to lymphoma sites and are resistant to SN-38 toxicity.
图 2. 经 IL-2/雷帕霉素扩增的 T 细胞表达归巢受体，能够迁移至淋巴瘤部位，并对 SN-38 毒性具有抗性。
图 2. 经 IL-2/雷帕霉素扩增的 T 细胞表达归巢受体，能够迁移至淋巴瘤部位，并对 SN-38 毒性具有抗性。
(A) Schematic of T cell functionalization and cell-mediated delivery of SN-38 NCs into tumors. (B and C) Polyclonal T cells from C57BL/6J mice were primed with concanavalin A and IL-7 for 2 days, then expanded in IL-2 with or without rapamycin for 2 days, and analyzed for expression of tissue-homing receptors by flow cytometry. Shown are representative staining histograms (B) and quantification markers (C) of n = 7 replicate cultures from two independent experiments. Data are means ± SEM. P value was determined by t test. MFI, median fluorescence intensity. (D) Eμ-myc cells or IL-2/rapamycin–expanded T cells were cultured in vitro with SN-38 at indicated doses, and viability was assessed by flow cytometry after 24 hours. Data are means ± SEM of pooled four to eight replicate cultures from four independent experiments. (E) IL-2– or IL-2/rapamycin–cultured T cells were taken directly from culture or incubated with SN-38 (20 ng/ml) for 12 hours, then stained for intracellular Bcl-2 expression, and analyzed by flow cytometry. Data are representative plots for n = 3 cultures per condition from one of three independent experiments. P values were determined by two-way analysis of variance (ANOVA) with Bonferroni post-test. (F) IL-2– or IL-2/rapamycin–cultured Thy1.1+ T cells (40 × 106) were adoptively transferred into C57BL/6J mice (n = 6 to 7 per group). The number of Thy1.1+ T cells in blood, spleen, and lymph nodes was enumerated by flow cytometry 2 days after transfer. Data are means ± SEM from one of two independent experiments. P values were determined by t test.
(A) T 细胞功能化及细胞介导的 SN-38 纳米颗粒（NCs）向肿瘤递送的示意图。(B 和 C) 来自 C57BL/6J 小鼠的多克隆 T 细胞经刀豆蛋白 A 和 IL-7 预处理 2 天，随后在 IL-2 中或联合雷帕霉素扩增 2 天，并通过流式细胞术分析其组织归巢受体的表达。图中展示了代表性的染色直方图（B）和定量标记（C），数据来自两次独立实验中的 n=7 个重复培养物。数据为平均值±标准误差（SEM）。P 值通过 t 检验确定。MFI，中位荧光强度。(D) Eμ-myc 细胞或 IL-2/雷帕霉素扩增的 T 细胞在体外与指示剂量的 SN-38 共培养 24 小时后，通过流式细胞术评估其存活率。数据为四至八个重复培养物合并后的平均值±SEM，来自四次独立实验。(E) IL-2 或 IL-2/雷帕霉素培养的 T 细胞直接从培养中取出或与 SN-38（20 ng/ml）共孵育 12 小时，随后进行 Bcl-2 胞内表达的染色，并通过流式细胞术分析。数据为代表性图谱，每种条件下 n=3 个培养物，来自三次独立实验中的一次。 P 值通过双因素方差分析（ANOVA）结合 Bonferroni 事后检验确定。(F) IL-2 或 IL-2/雷帕霉素培养的 Thy1.1 + T 细胞（40 × 10 6 ）被过继转移至 C57BL/6J 小鼠（每组 n=6 至 7）。转移后 2 天，通过流式细胞术计数血液、脾脏和淋巴结中的 Thy1.1 + T 细胞数量。数据为两次独立实验中一次的平均值±标准误（SEM）。P 值通过 t 检验确定。
(A) T 细胞功能化及细胞介导的 SN-38 纳米颗粒（NCs）向肿瘤递送的示意图。(B 和 C) 来自 C57BL/6J 小鼠的多克隆 T 细胞经刀豆蛋白 A 和 IL-7 预处理 2 天，随后在 IL-2 中或联合雷帕霉素扩增 2 天，并通过流式细胞术分析其组织归巢受体的表达。图中展示了代表性的染色直方图（B）和定量标记（C），数据来自两次独立实验中的 n=7 个重复培养物。数据为平均值±标准误差（SEM）。P 值通过 t 检验确定。MFI，中位荧光强度。(D) Eμ-myc 细胞或 IL-2/雷帕霉素扩增的 T 细胞在体外与指示剂量的 SN-38 共培养 24 小时后，通过流式细胞术评估其存活率。数据为四至八个重复培养物合并后的平均值±SEM，来自四次独立实验。(E) IL-2 或 IL-2/雷帕霉素培养的 T 细胞直接从培养中取出或与 SN-38（20 ng/ml）共孵育 12 小时，随后进行 Bcl-2 胞内表达的染色，并通过流式细胞术分析。数据为代表性图谱，每种条件下 n=3 个培养物，来自三次独立实验中的一次。 P 值通过双因素方差分析（ANOVA）结合 Bonferroni 事后检验确定。(F) IL-2 或 IL-2/雷帕霉素培养的 Thy1.1 + T 细胞（40 × 10 6 ）被过继转移至 C57BL/6J 小鼠（每组 n=6 至 7）。转移后 2 天，通过流式细胞术计数血液、脾脏和淋巴结中的 Thy1.1 + T 细胞数量。数据为两次独立实验中一次的平均值±标准误（SEM）。P 值通过 t 检验确定。
To generate large populations of lymphocytes capable of targeting SN-38 to lymphoid organs, we first established an ex vivo T cell priming protocol that allowed robust expansion of primary T cells while retaining key homing receptors required for lymphoid tissue trafficking. Both mouse and human T cells can be rapidly expanded to large numbers in vitro by polyclonal T cell receptor (TCR) triggering followed by culture in interleukin-2 (IL-2). However, after TCR stimulation, CD62L is rapidly shed/down-regulated, resulting in decreased T cell homing to lymph nodes, mediated in part by mammalian target of rapamycin (mTOR) signaling (21). To counteract these effects, we expanded primary T cells isolated from C57BL/6J mice in the presence of IL-2 and the mTOR inhibitor rapamycin, which has been shown to preserve CD62L and CCR7 expression during IL-2–induced growth and proliferation of T cells (21). As expected, IL-2 expanded both CD4+ and CD8+ T cells with an activated CD25+CD44+CD69+ phenotype (fig. S2, A and B), regardless of whether rapamycin was present. However, only T cells cotreated with rapamycin retained high levels of CD62L (Fig. 2Opens in image viewer
为了生成能够将 SN-38 靶向淋巴器官的大量淋巴细胞群体，我们首先建立了一种体外 T 细胞预处理方案，该方案能够在保持淋巴组织归巢所需关键归巢受体的同时，促进初始 T 细胞的稳健扩增。小鼠和人类 T 细胞均可在体外通过多克隆 T 细胞受体（TCR）激活后，在白细胞介素-2（IL-2）环境中迅速扩增至大量。然而，TCR 刺激后，CD62L 会迅速脱落/下调，导致 T 细胞向淋巴结的归巢能力下降，部分原因是由哺乳动物雷帕霉素靶蛋白（mTOR）信号传导介导的（21）。为了对抗这些效应，我们将从 C57BL/6J 小鼠中分离出的初始 T 细胞在 IL-2 和 mTOR 抑制剂雷帕霉素的存在下进行扩增，研究表明，这能在 IL-2 诱导的 T 细胞生长和增殖过程中维持 CD62L 和 CCR7 的表达（21）。正如预期，IL-2 扩增了 CD4+和 CD8+ T 细胞，这些细胞表现出激活的 CD25+ CD44+ CD69+表型（图 S2，A 和 B），无论是否存在雷帕霉素。然而，只有与雷帕霉素共同处理的 T 细胞保留了高水平的 CD62L（图 2）。, B and C). IL-2/rapamycin–treated T cells also expressed the integrins α4β7, β1, and β2 and the chemokine receptor CXCR4 (fig. S2C), thus imitating the homing receptor repertoire of Eμ-myc cells.
B 和 C）。IL-2/雷帕霉素处理的 T 细胞同样表达了整合素 α 4 β 7 、β 1 和 β 2 以及趋化因子受体 CXCR4（图 S2C），从而模仿了 Eμ-myc 细胞的归巢受体谱系。
为了生成能够将 SN-38 靶向淋巴器官的大量淋巴细胞群体，我们首先建立了一种体外 T 细胞预处理方案，该方案能够在保持淋巴组织归巢所需关键归巢受体的同时，促进初始 T 细胞的稳健扩增。小鼠和人类 T 细胞均可在体外通过多克隆 T 细胞受体（TCR）激活后，在白细胞介素-2（IL-2）环境中迅速扩增至大量。然而，TCR 刺激后，CD62L 会迅速脱落/下调，导致 T 细胞向淋巴结的归巢能力下降，部分原因是由哺乳动物雷帕霉素靶蛋白（mTOR）信号传导介导的（21）。为了对抗这些效应，我们将从 C57BL/6J 小鼠中分离出的初始 T 细胞在 IL-2 和 mTOR 抑制剂雷帕霉素的存在下进行扩增，研究表明，这能在 IL-2 诱导的 T 细胞生长和增殖过程中维持 CD62L 和 CCR7 的表达（21）。正如预期，IL-2 扩增了 CD4+和 CD8+ T 细胞，这些细胞表现出激活的 CD25+ CD44+ CD69+表型（图 S2，A 和 B），无论是否存在雷帕霉素。然而，只有与雷帕霉素共同处理的 T 细胞保留了高水平的 CD62L（图 2）。, B and C). IL-2/rapamycin–treated T cells also expressed the integrins α4β7, β1, and β2 and the chemokine receptor CXCR4 (fig. S2C), thus imitating the homing receptor repertoire of Eμ-myc cells.
B 和 C）。IL-2/雷帕霉素处理的 T 细胞同样表达了整合素 α 4 β 7 、β 1 和 β 2 以及趋化因子受体 CXCR4（图 S2C），从而模仿了 Eμ-myc 细胞的归巢受体谱系。
Eμ-myc cells were sensitive to SN-38–induced apoptosis in vitro at concentrations as low as 2 ng/ml and were essentially eradicated at 10 ng/ml (Fig. 2DOpens in image viewer
Eμ-myc 细胞在体外对低至 2 ng/ml 的 SN-38 诱导的凋亡敏感，并在 10 ng/ml 浓度下基本被根除（图 2D）。). In contrast, IL-2/rapamycin–expanded T cells were minimally affected over the same concentration range. This selective activity of SN-38 toward Eμ-myc cells is consistent with previous reports of tumor cells having increased sensitivity to topoisomerase I poisons (22). These results suggest a therapeutic window in which T cells could carry therapeutic doses of SN-38 without undergoing apoptosis themselves.
相比之下，白介素-2/雷帕霉素扩增的 T 细胞在相同浓度范围内受到的影响极小。SN-38 对 Eμ-myc 细胞的选择性活性与先前报道的肿瘤细胞对拓扑异构酶 I 抑制剂的敏感性增加相一致（22）。这些结果表明，存在一个治疗窗口，在此窗口内，T 细胞能够携带治疗剂量的 SN-38 而不发生自身凋亡。
Eμ-myc 细胞在体外对低至 2 ng/ml 的 SN-38 诱导的凋亡敏感，并在 10 ng/ml 浓度下基本被根除（图 2D）。). In contrast, IL-2/rapamycin–expanded T cells were minimally affected over the same concentration range. This selective activity of SN-38 toward Eμ-myc cells is consistent with previous reports of tumor cells having increased sensitivity to topoisomerase I poisons (22). These results suggest a therapeutic window in which T cells could carry therapeutic doses of SN-38 without undergoing apoptosis themselves.
相比之下，白介素-2/雷帕霉素扩增的 T 细胞在相同浓度范围内受到的影响极小。SN-38 对 Eμ-myc 细胞的选择性活性与先前报道的肿瘤细胞对拓扑异构酶 I 抑制剂的敏感性增加相一致（22）。这些结果表明，存在一个治疗窗口，在此窗口内，T 细胞能够携带治疗剂量的 SN-38 而不发生自身凋亡。
Both sustained TCR signaling and IL-2 withdrawal promote apoptosis in T cells (23); rapamycin counteracts this by increasing levels of the antiapoptotic protein Bcl-2 (24). Consistent with these reports, IL-2/rapamycin–treated T cells had higher Bcl-2 expression, as compared to T cells expanded only in IL-2, and this expression difference was maintained in the presence of SN-38 (Fig. 2EOpens in image viewer
TCR 信号的持续激活和 IL-2 的撤除均会促进 T 细胞凋亡（23）；而雷帕霉素则通过提高抗凋亡蛋白 Bcl-2 的水平来对抗这一现象（24）。与这些报道一致，相较于仅在 IL-2 中扩增的 T 细胞，经过 IL-2/雷帕霉素处理的 T 细胞表现出更高的 Bcl-2 表达水平，并且在 SN-38 存在的情况下，这种表达差异依然得以维持（图 2E）。), suggesting that IL-2/rapamycin T cells would preferentially survive in vivo. Indeed, when we transferred IL-2– or IL-2/rapamycin–expanded T cells into naïve hosts and analyzed the biodistribution of the transferred cells 2 days later, we observed between 10- and 100-fold more viable IL-2/rapamycin T cells in the blood, spleen, and lymph nodes compared to IL-2 T cells (Fig. 2FOpens in image viewer
),表明 IL-2/雷帕霉素 T 细胞在体内具有优先存活的优势。事实上，当我们向未接触过抗原的宿主中转移 IL-2 或 IL-2/雷帕霉素扩增的 T 细胞，并在两天后分析转移细胞的生物分布时，我们观察到在血液、脾脏和淋巴结中，IL-2/雷帕霉素 T 细胞的存活数量比 IL-2 T 细胞多出 10 到 100 倍（图 2F）。). Together, these data demonstrate that combined IL-2/rapamycin “programming” yields expanded T cells that are resistant to SN-38 treatment, exhibit lymphoid tissue homing, and maintain effective survival in vivo.
综上所述，这些数据表明，联合使用 IL-2/雷帕霉素“编程”可产生扩增的 T 细胞，这些细胞对 SN-38 治疗具有抗性，表现出淋巴组织归巢能力，并在体内维持有效的生存。
TCR 信号的持续激活和 IL-2 的撤除均会促进 T 细胞凋亡（23）；而雷帕霉素则通过提高抗凋亡蛋白 Bcl-2 的水平来对抗这一现象（24）。与这些报道一致，相较于仅在 IL-2 中扩增的 T 细胞，经过 IL-2/雷帕霉素处理的 T 细胞表现出更高的 Bcl-2 表达水平，并且在 SN-38 存在的情况下，这种表达差异依然得以维持（图 2E）。), suggesting that IL-2/rapamycin T cells would preferentially survive in vivo. Indeed, when we transferred IL-2– or IL-2/rapamycin–expanded T cells into naïve hosts and analyzed the biodistribution of the transferred cells 2 days later, we observed between 10- and 100-fold more viable IL-2/rapamycin T cells in the blood, spleen, and lymph nodes compared to IL-2 T cells (Fig. 2FOpens in image viewer
),表明 IL-2/雷帕霉素 T 细胞在体内具有优先存活的优势。事实上，当我们向未接触过抗原的宿主中转移 IL-2 或 IL-2/雷帕霉素扩增的 T 细胞，并在两天后分析转移细胞的生物分布时，我们观察到在血液、脾脏和淋巴结中，IL-2/雷帕霉素 T 细胞的存活数量比 IL-2 T 细胞多出 10 到 100 倍（图 2F）。). Together, these data demonstrate that combined IL-2/rapamycin “programming” yields expanded T cells that are resistant to SN-38 treatment, exhibit lymphoid tissue homing, and maintain effective survival in vivo.
综上所述，这些数据表明，联合使用 IL-2/雷帕霉素“编程”可产生扩增的 T 细胞，这些细胞对 SN-38 治疗具有抗性，表现出淋巴组织归巢能力，并在体内维持有效的生存。
To enable T cell–mediated delivery of highly hydrophobic SN-38 over a period of days, we entrapped the drug in multilamellar lipid NCs (25) designed to covalently react with T cell surface thiols (Fig. 2AOpens in image viewer
为了实现 T 细胞介导的、在数天内持续递送高疏水性药物 SN-38，我们将该药物包裹于多层脂质纳米颗粒（NCs）中，这些纳米颗粒设计成能与 T 细胞表面的硫醇发生共价反应（图 2A）。). NCs were formed by a variation on the synthesis of interbilayer-crosslinked multilamellar vesicles we previously described: In brief, a co-solution of phosphatidylglycerol lipid, maleimide-headgroup lipid, and SN-38 formed precursor vesicles, which were fused together, and each liposome wall was covalently crosslinked to others to form multilamellar lipid capsules (Fig. 3AOpens in image viewer
NCs 是通过我们之前描述的层间交联多层囊泡合成方法的变体制备而成：简而言之，磷脂酰甘油脂、马来酰亚胺头基脂和 SN-38 的共溶液形成前体囊泡，这些囊泡融合在一起，并且每个脂质体壁通过共价交联与其他脂质体壁连接，形成多层脂质胶囊（图 3A）。 and fig. S3, A and B). The resulting SN-38 NCs had a mean diameter of 340 ± 12 nm and entrapped 14.3 μg of SN-38 per milligram of lipid, which was completely released over 3 days in vitro (Fig. 3BOpens in image viewer
并图 S3，A 和 B）。制得的 SN-38 纳米脂质体（NCs）平均直径为 340 ± 12 nm，每毫克脂质可包封 14.3 微克的 SN-38，该药物在体外 3 天内完全释放（图 3B）。).
为了实现 T 细胞介导的、在数天内持续递送高疏水性药物 SN-38，我们将该药物包裹于多层脂质纳米颗粒（NCs）中，这些纳米颗粒设计成能与 T 细胞表面的硫醇发生共价反应（图 2A）。). NCs were formed by a variation on the synthesis of interbilayer-crosslinked multilamellar vesicles we previously described: In brief, a co-solution of phosphatidylglycerol lipid, maleimide-headgroup lipid, and SN-38 formed precursor vesicles, which were fused together, and each liposome wall was covalently crosslinked to others to form multilamellar lipid capsules (Fig. 3AOpens in image viewer
NCs 是通过我们之前描述的层间交联多层囊泡合成方法的变体制备而成：简而言之，磷脂酰甘油脂、马来酰亚胺头基脂和 SN-38 的共溶液形成前体囊泡，这些囊泡融合在一起，并且每个脂质体壁通过共价交联与其他脂质体壁连接，形成多层脂质胶囊（图 3A）。 and fig. S3, A and B). The resulting SN-38 NCs had a mean diameter of 340 ± 12 nm and entrapped 14.3 μg of SN-38 per milligram of lipid, which was completely released over 3 days in vitro (Fig. 3BOpens in image viewer
并图 S3，A 和 B）。制得的 SN-38 纳米脂质体（NCs）平均直径为 340 ± 12 nm，每毫克脂质可包封 14.3 微克的 SN-38，该药物在体外 3 天内完全释放（图 3B）。).
Fig. 3. T cells conjugated with SN-38 NCs kill bystander lymphoma cells but not the T cells themselves.
图 3. 与 SN-38 纳米颗粒结合的 T 细胞能够杀死旁观性淋巴瘤细胞，但不会损伤 T 细胞本身。
图 3. 与 SN-38 纳米颗粒结合的 T 细胞能够杀死旁观性淋巴瘤细胞，但不会损伤 T 细胞本身。
(A) Cryo–electron microscopy image of SN-38 NCs. (B) Kinetics of SN-38 release from NCs at 37 °C in 10% serum. Data are means ± SEM (n = 8). (C) T cells stained with carboxyfluorescein diacetate succinimidyl ester (blue) and conjugated to fluorescently labeled NCs (pink) were imaged by confocal microscopy. (D) T cells were conjugated to fluorescent DiD NCs either lacking maleimide-lipid (control NCs) or containing maleimide-headgroup lipids (maleimide NCs), washed, and then analyzed by flow cytometry. (E) T cells were conjugated with SN-38 NCs over a range of NC/cell ratios and then lysed to measure the final conjugated amount of SN-38. (F and G) Eμ-myc cells were cocultured with unmodified T cells, empty (“blank”) NC–conjugated T cells, or SN-38 NC–conjugated T cells at indicated T cell/lymphoma cell ratios. Viability of Eμ-myc cells (F) and T cells (G) was measured by flow cytometry 24 hours later. Data are means ± SEM (n = 3 to 8 samples per group). ns, not significant, two-way ANOVA with Bonferroni post-test. All data are representative of two to three independent experiments.
(A) SN-38 纳米晶体的冷冻电镜图像。(B) SN-38 纳米晶体在 37°C、10%血清条件下释放动力学。数据为平均值±标准误差（n=8）。(C) 用羧基荧光素二乙酸琥珀酰亚胺酯染色的 T 细胞（蓝色）与荧光标记的纳米晶体（粉色）结合后，通过共聚焦显微镜成像。(D) T 细胞分别与不含马来酰亚胺脂质（对照纳米晶体）或含马来酰亚胺头基脂质（马来酰亚胺纳米晶体）的荧光 DiD 纳米晶体结合，洗涤后通过流式细胞术分析。(E) T 细胞在不同纳米晶体/细胞比例下与 SN-38 纳米晶体结合，随后裂解以测量最终结合的 SN-38 量。(F 和 G) Eμ-myc 细胞分别与未修饰的 T 细胞、空载（“空白”）纳米晶体结合的 T 细胞或 SN-38 纳米晶体结合的 T 细胞按指示的 T 细胞/淋巴瘤细胞比例共培养。24 小时后通过流式细胞术测量 Eμ-myc 细胞（F）和 T 细胞（G）的存活率。数据为平均值±标准误差（每组 n=3 至 8 个样本）。ns 表示无显著性差异，采用双因素方差分析及 Bonferroni 事后检验。所有数据均代表两到三次独立实验。
(A) SN-38 纳米晶体的冷冻电镜图像。(B) SN-38 纳米晶体在 37°C、10%血清条件下释放动力学。数据为平均值±标准误差（n=8）。(C) 用羧基荧光素二乙酸琥珀酰亚胺酯染色的 T 细胞（蓝色）与荧光标记的纳米晶体（粉色）结合后，通过共聚焦显微镜成像。(D) T 细胞分别与不含马来酰亚胺脂质（对照纳米晶体）或含马来酰亚胺头基脂质（马来酰亚胺纳米晶体）的荧光 DiD 纳米晶体结合，洗涤后通过流式细胞术分析。(E) T 细胞在不同纳米晶体/细胞比例下与 SN-38 纳米晶体结合，随后裂解以测量最终结合的 SN-38 量。(F 和 G) Eμ-myc 细胞分别与未修饰的 T 细胞、空载（“空白”）纳米晶体结合的 T 细胞或 SN-38 纳米晶体结合的 T 细胞按指示的 T 细胞/淋巴瘤细胞比例共培养。24 小时后通过流式细胞术测量 Eμ-myc 细胞（F）和 T 细胞（G）的存活率。数据为平均值±标准误差（每组 n=3 至 8 个样本）。ns 表示无显著性差异，采用双因素方差分析及 Bonferroni 事后检验。所有数据均代表两到三次独立实验。
After crosslinking, sufficient maleimide groups remained on the particle surfaces to allow conjugation of NCs to T cell surface proteins; residual maleimide groups were quenched with polyethylene glycol (PEG)–thiol (Fig. 2AOpens in image viewer
交联后，纳米颗粒表面保留了足够的马来酰亚胺基团，可用于与 T 细胞表面蛋白的共轭结合；剩余的马来酰亚胺基团通过聚乙二醇（PEG）-硫醇进行淬灭（图 2A）。). SN-38 NCs were then stably conjugated to the surfaces of T cells and retained after washing (Fig. 3COpens in image viewer
SN-38 纳米颗粒随后被稳定地连接到 T 细胞表面，并在洗涤后仍保持附着（图 3C）。), whereas maleimide-free (control) NCs showed minimal nonspecific binding to T cells (Fig. 3DOpens in image viewer
),而未含马来酰亚胺（对照组）的纳米颗粒（NCs）对 T 细胞表现出极少的非特异性结合（图 3D）。). Titration of the NC/cell ratio showed that T cells could be readily coupled with NCs carrying up to ~0.4 pg of SN-38 per cell (Fig. 3EOpens in image viewer
滴定 NC 与细胞的比例显示，T 细胞可以轻易地与携带高达每细胞约 0.4 皮克 SN-38 的 NCs 结合（图 3E）。).
交联后，纳米颗粒表面保留了足够的马来酰亚胺基团，可用于与 T 细胞表面蛋白的共轭结合；剩余的马来酰亚胺基团通过聚乙二醇（PEG）-硫醇进行淬灭（图 2A）。). SN-38 NCs were then stably conjugated to the surfaces of T cells and retained after washing (Fig. 3COpens in image viewer
SN-38 纳米颗粒随后被稳定地连接到 T 细胞表面，并在洗涤后仍保持附着（图 3C）。), whereas maleimide-free (control) NCs showed minimal nonspecific binding to T cells (Fig. 3DOpens in image viewer
),而未含马来酰亚胺（对照组）的纳米颗粒（NCs）对 T 细胞表现出极少的非特异性结合（图 3D）。). Titration of the NC/cell ratio showed that T cells could be readily coupled with NCs carrying up to ~0.4 pg of SN-38 per cell (Fig. 3EOpens in image viewer
滴定 NC 与细胞的比例显示，T 细胞可以轻易地与携带高达每细胞约 0.4 皮克 SN-38 的 NCs 结合（图 3E）。).
In vitro killing of lymphoma cells by NC-functionalized T cells
体外通过 NC 功能化 T 细胞杀伤淋巴瘤细胞
To test the capacity of SN-38–carrying T cells to deliver drug in trans to lymphoma cells, we cultured unmodified cells, T cells conjugated with empty NCs, or T cells conjugated with SN-38–loaded NCs (SN-38 NC-T cells, 0.2 pg of SN-38 per cell) for 24 hours with Eμ-myc cells, and viability was assessed by flow cytometry. Coculture of Eμ-myc cells with unmodified T cells or T cells decorated with empty NCs (blank NC-T) did not affect tumor cell viability, but SN-38 NC-T cells elicited tumor cell killing at ratios as low as 1 SN-38 NC-T cell per 20 lymphoma cells (Fig. 3FOpens in image viewer
为了测试 SN-38 携带型 T 细胞在体外向淋巴瘤细胞递送药物的能力，我们将未修饰的 T 细胞、与空纳米颗粒（NCs）结合的 T 细胞或与装载 SN-38 的 NCs 结合的 T 细胞（SN-38 NC-T 细胞，每细胞 0.2 皮克 SN-38）与 Eμ-myc 细胞共培养 24 小时，并通过流式细胞术评估细胞活力。结果显示，Eμ-myc 细胞与未修饰的 T 细胞或与空 NCs 结合的 T 细胞（空白 NC-T）共培养并未影响肿瘤细胞的存活，但 SN-38 NC-T 细胞在 T 细胞与淋巴瘤细胞比例低至 1:20 时，仍能诱导肿瘤细胞的杀伤作用（图 3F）。). In this same coculture assay, SN-38 NC-T cells showed viability comparable to T cells that were unmodified or conjugated with empty NCs, demonstrating that T cells remained resistant to SN-38 even when drug-loaded capsules were directly conjugated to their plasma membranes (Fig. 3GOpens in image viewer
). 在此共培养实验中，SN-38 NC-T 细胞的存活率与未修饰或仅与空纳米胶囊（NCs）结合的 T 细胞相当，表明即使药物负载的胶囊直接结合到 T 细胞的质膜上，T 细胞仍对 SN-38 保持耐受性（图 3G）。). Thus, particle-decorated T cells are capable of carrying doses of SN-38 that are therapeutically relevant for clearing surrounding lymphoma cells, without causing acute toxicity to the carrier cell.
因此，经过粒子修饰的 T 细胞能够携带治疗剂量的 SN-38，有效清除周围的淋巴瘤细胞，同时不会对携带细胞造成急性毒性。
为了测试 SN-38 携带型 T 细胞在体外向淋巴瘤细胞递送药物的能力，我们将未修饰的 T 细胞、与空纳米颗粒（NCs）结合的 T 细胞或与装载 SN-38 的 NCs 结合的 T 细胞（SN-38 NC-T 细胞，每细胞 0.2 皮克 SN-38）与 Eμ-myc 细胞共培养 24 小时，并通过流式细胞术评估细胞活力。结果显示，Eμ-myc 细胞与未修饰的 T 细胞或与空 NCs 结合的 T 细胞（空白 NC-T）共培养并未影响肿瘤细胞的存活，但 SN-38 NC-T 细胞在 T 细胞与淋巴瘤细胞比例低至 1:20 时，仍能诱导肿瘤细胞的杀伤作用（图 3F）。). In this same coculture assay, SN-38 NC-T cells showed viability comparable to T cells that were unmodified or conjugated with empty NCs, demonstrating that T cells remained resistant to SN-38 even when drug-loaded capsules were directly conjugated to their plasma membranes (Fig. 3GOpens in image viewer
). 在此共培养实验中，SN-38 NC-T 细胞的存活率与未修饰或仅与空纳米胶囊（NCs）结合的 T 细胞相当，表明即使药物负载的胶囊直接结合到 T 细胞的质膜上，T 细胞仍对 SN-38 保持耐受性（图 3G）。). Thus, particle-decorated T cells are capable of carrying doses of SN-38 that are therapeutically relevant for clearing surrounding lymphoma cells, without causing acute toxicity to the carrier cell.
因此，经过粒子修饰的 T 细胞能够携带治疗剂量的 SN-38，有效清除周围的淋巴瘤细胞，同时不会对携带细胞造成急性毒性。
Delivery of SN-38 to lymphoid organs in vivo by nanoparticle-decorated T cells
通过纳米颗粒修饰的 T 细胞体内递送 SN-38 至淋巴器官
We next tested whether T cells could transport drug-loaded nanoparticles into lymphoma-infiltrated organs in vivo. After injection into mice bearing established Eμ-myc tumors, luciferase+Thy1.1+ T cells conjugated with SN-38 NCs trafficked to the spleen, lymph nodes, and bone marrow (Fig. 4AOpens in image viewer
接下来，我们测试了 T 细胞是否能在体内将载药纳米颗粒运输到淋巴瘤浸润的器官中。将携带 Eμ-myc 肿瘤的小鼠注射了与 SN-38 纳米颗粒（NCs）结合的荧光素酶 + Thy1.1 + T 细胞后，这些 T 细胞迁移到了脾脏、淋巴结和骨髓（图 4A）。). Flow cytometry analysis showed substantial NC-T cell accumulation in each of these lymphoid organs by 20 hours, reaching peak levels by ~40 hours with kinetics similar to unmodified T cells, suggesting that NC conjugation did not impair the survival or trafficking of the transferred T cells (Fig. 4BOpens in image viewer
). 流式细胞术分析显示，20 小时内各淋巴器官中均有大量 NC-T 细胞积聚，并在约 40 小时达到峰值，其动力学与未修饰的 T 细胞相似，表明 NC 偶联并未损害转移 T 细胞的存活或迁移能力（图 4B）。). Twenty-four hours after transfer, NC-T cells dispersed throughout the lymph node, in proximity to Eμ-myc cells (Fig. 4COpens in image viewer
). 转移后 24 小时，NC-T 细胞遍布于淋巴结中，靠近 Eμ-myc 细胞（图 4C）。). When conjugated with fluorescent particles, transferred T cells recovered from lymph nodes were uniformly positive for NC fluorescence, demonstrating retention of their particle cargo during homing and a lack of particle transfer to other cells in the lymph node (Fig. 4DOpens in image viewer
).当与荧光颗粒结合后，从淋巴结中回收的转移 T 细胞均一地显示出 NC 荧光阳性，表明在归巢过程中颗粒货物得以保留，且未发生颗粒向淋巴结内其他细胞的转移（图 4D）。).
接下来，我们测试了 T 细胞是否能在体内将载药纳米颗粒运输到淋巴瘤浸润的器官中。将携带 Eμ-myc 肿瘤的小鼠注射了与 SN-38 纳米颗粒（NCs）结合的荧光素酶 + Thy1.1 + T 细胞后，这些 T 细胞迁移到了脾脏、淋巴结和骨髓（图 4A）。). Flow cytometry analysis showed substantial NC-T cell accumulation in each of these lymphoid organs by 20 hours, reaching peak levels by ~40 hours with kinetics similar to unmodified T cells, suggesting that NC conjugation did not impair the survival or trafficking of the transferred T cells (Fig. 4BOpens in image viewer
). 流式细胞术分析显示，20 小时内各淋巴器官中均有大量 NC-T 细胞积聚，并在约 40 小时达到峰值，其动力学与未修饰的 T 细胞相似，表明 NC 偶联并未损害转移 T 细胞的存活或迁移能力（图 4B）。). Twenty-four hours after transfer, NC-T cells dispersed throughout the lymph node, in proximity to Eμ-myc cells (Fig. 4COpens in image viewer
). 转移后 24 小时，NC-T 细胞遍布于淋巴结中，靠近 Eμ-myc 细胞（图 4C）。). When conjugated with fluorescent particles, transferred T cells recovered from lymph nodes were uniformly positive for NC fluorescence, demonstrating retention of their particle cargo during homing and a lack of particle transfer to other cells in the lymph node (Fig. 4DOpens in image viewer
).当与荧光颗粒结合后，从淋巴结中回收的转移 T 细胞均一地显示出 NC 荧光阳性，表明在归巢过程中颗粒货物得以保留，且未发生颗粒向淋巴结内其他细胞的转移（图 4D）。).
Fig. 4. SN-38 NC–conjugated T cells traffic into lymphoma-bearing lymph nodes in vivo and sustain elevated intranodal SN-38 levels over time.
图 4. 携带 SN-38 NC 的 T 细胞在体内迁移至淋巴瘤负荷的淋巴结，并随时间维持较高的淋巴结内 SN-38 水平。
图 4. 携带 SN-38 NC 的 T 细胞在体内迁移至淋巴瘤负荷的淋巴结，并随时间维持较高的淋巴结内 SN-38 水平。
C57BL/6J mice (n = 3 to 5 per group) were injected intravenously with 1 × 106 Eμ-myc cells and, 17 days later, received intravenous injection of 2 × 108 luciferase+ SN-38–carrying NC-T cells (1 mg/kg equivalent SN-38), control unmodified T cells, or an equivalent dose of free NCs. (A) T cell biodistribution was assessed by whole-animal bioluminescence 38 hours after transfer. Images are ventral views of three different animals and side view of one representative animal. (B) Kinetics of T cell and SN-38 NC-T cell accumulation in tissues assessed by flow cytometry. Data are means ± SEM (n = 4 to 12 animals per group per time point), pooled from five independent experiments. **P < 0.01 versus T cells alone, by two-way ANOVA with Bonferroni post-test. (C) Mice (n = 4 per group) were injected with SN-38 NC–conjugated fluorescently labeled T cells, and tumor (Eμ-myc)–bearing lymph nodes were harvested at 24 hours after injection for histological analysis. Representative image from one of two experiments. (D) Retention of fluorescently labeled SN-38 NCs by T cells (Thy1.1+) in tumor-bearing lymph nodes was measured by flow cytometry 15 hours after transfer. Plot is from one of two representative experiments. (E) Animals were treated with free SN-38 NCs, SN-38 NC-T cells, or free SN-38 at a dose 10-fold greater than that in the NCs. Tumor-bearing lymph nodes were harvested at various times after treatment for HPLC analysis. Data are means ± SEM (n = 3 to 5 animals per group per time point, pooled from four independent experiments). Dashed line denotes limit of SN-38 detection (0.5 ng/g tissue). *P < 0.05, ****P < 0.0001 versus SN-38 NCs, by two-way ANOVA with Bonferroni post-test.
C57BL/6J 小鼠（每组 3 至 5 只）通过静脉注射 1 × 10^6 Eμ-myc 细胞，17 天后，再静脉注射 2 × 10^7 携带荧光素酶标记的 SN-38 纳米颗粒的 T 细胞（相当于 1 mg/kg SN-38 剂量）、未修饰的对照 T 细胞或等量的游离纳米颗粒。(A) 通过全身生物发光成像评估 T 细胞的生物分布，成像于转移后 38 小时进行。图像为三只不同小鼠的腹面视图及一只代表性小鼠的侧面视图。(B) 通过流式细胞术评估 T 细胞及 SN-38 纳米颗粒 T 细胞在组织中的累积动力学。数据为平均值±标准误差（每个时间点每组 4 至 12 只小鼠），汇总自五次独立实验。**P < 0.01，与单独 T 细胞相比，采用双因素方差分析及 Bonferroni 后验测试。(C) 小鼠（每组 4 只）注射了荧光标记的 SN-38 纳米颗粒偶联 T 细胞，并在注射后 24 小时采集肿瘤（Eμ-myc）负荷的淋巴结进行组织学分析。图为两次实验中的一次代表性图像。(D) 通过流式细胞术测量转移后 15 小时，荧光标记的 SN-38 纳米颗粒在肿瘤负荷淋巴结中被 Thy1.1^+^ T 细胞的保留情况。 图来自两个代表性实验之一。(E) 动物分别接受游离 SN-38 NCs、SN-38 NC-T 细胞或剂量高于 NCs 中 10 倍的游离 SN-38 处理。治疗后不同时间点采集肿瘤负荷的淋巴结进行 HPLC 分析。数据为平均值±标准误（n = 每组每时间点 3 至 5 只动物，来自四项独立实验的数据汇总）。虚线表示 SN-38 检测限（0.5 ng/g 组织）。*P < 0.05，****P < 0.0001，与 SN-38 NCs 相比，采用两因素方差分析及 Bonferroni 事后检验。
C57BL/6J 小鼠（每组 3 至 5 只）通过静脉注射 1 × 10^6 Eμ-myc 细胞，17 天后，再静脉注射 2 × 10^7 携带荧光素酶标记的 SN-38 纳米颗粒的 T 细胞（相当于 1 mg/kg SN-38 剂量）、未修饰的对照 T 细胞或等量的游离纳米颗粒。(A) 通过全身生物发光成像评估 T 细胞的生物分布，成像于转移后 38 小时进行。图像为三只不同小鼠的腹面视图及一只代表性小鼠的侧面视图。(B) 通过流式细胞术评估 T 细胞及 SN-38 纳米颗粒 T 细胞在组织中的累积动力学。数据为平均值±标准误差（每个时间点每组 4 至 12 只小鼠），汇总自五次独立实验。**P < 0.01，与单独 T 细胞相比，采用双因素方差分析及 Bonferroni 后验测试。(C) 小鼠（每组 4 只）注射了荧光标记的 SN-38 纳米颗粒偶联 T 细胞，并在注射后 24 小时采集肿瘤（Eμ-myc）负荷的淋巴结进行组织学分析。图为两次实验中的一次代表性图像。(D) 通过流式细胞术测量转移后 15 小时，荧光标记的 SN-38 纳米颗粒在肿瘤负荷淋巴结中被 Thy1.1^+^ T 细胞的保留情况。 图来自两个代表性实验之一。(E) 动物分别接受游离 SN-38 NCs、SN-38 NC-T 细胞或剂量高于 NCs 中 10 倍的游离 SN-38 处理。治疗后不同时间点采集肿瘤负荷的淋巴结进行 HPLC 分析。数据为平均值±标准误（n = 每组每时间点 3 至 5 只动物，来自四项独立实验的数据汇总）。虚线表示 SN-38 检测限（0.5 ng/g 组织）。*P < 0.05，****P < 0.0001，与 SN-38 NCs 相比，采用两因素方差分析及 Bonferroni 事后检验。
To determine the impact of T cell–mediated NC transport on tissue levels of the chemotherapy cargo, we measured SN-38 concentrations in tumor-bearing lymph nodes. Consistent with the T cell/NC biodistribution, SN-38 accumulation was greatly increased in tumor-bearing lymph nodes when delivered via T cell–bound NCs, reaching concentrations 63-fold greater than free NCs at 20 hours and remaining at high levels for at least 4 days (Fig. 4EOpens in image viewer
为了评估 T 细胞介导的纳米载体（NC）运输对化疗药物在组织中分布的影响，我们测量了携带肿瘤的淋巴结中 SN-38 的浓度。与 T 细胞/NC 的生物分布一致，当通过 T 细胞结合的 NC 递送时，携带肿瘤的淋巴结中 SN-38 的积累显著增加，20 小时时达到的浓度是自由 NC 的 63 倍，并且在至少 4 天内保持在较高水平（图 4E）。).
为了评估 T 细胞介导的纳米载体（NC）运输对化疗药物在组织中分布的影响，我们测量了携带肿瘤的淋巴结中 SN-38 的浓度。与 T 细胞/NC 的生物分布一致，当通过 T 细胞结合的 NC 递送时，携带肿瘤的淋巴结中 SN-38 的积累显著增加，20 小时时达到的浓度是自由 NC 的 63 倍，并且在至少 4 天内保持在较高水平（图 4E）。).
Enhanced efficacy of SN-38 chemotherapy by T cell–mediated drug delivery
通过 T 细胞介导的药物递送增强 SN-38 化疗的疗效
We next tested the therapeutic efficacy of pharmacyte-mediated drug delivery against these aggressive disseminated lymphoma tumors. Five days after tumor inoculation, mice were treated with SN-38 as free drug intravenously, SN-38 NCs, or SN-38 NC-T cells (four doses total given every 3 days, SN-38 at 1 mg/kg per dose for all groups). Mice treated with saline or free SN-38 demonstrated high Eμ-myc tumor burdens in the blood, bone marrow, spleen, and lymph nodes (Fig. 5AOpens in image viewer
接下来，我们测试了药学介导的药物递送系统对这些高度扩散的淋巴瘤肿瘤的治疗效果。在肿瘤接种后的第五天，小鼠接受静脉注射的游离 SN-38、SN-38 纳米颗粒（NCs）或 SN-38 纳米颗粒-T 细胞复合物（NC-T 细胞）治疗，总共四次给药，每隔三天一次，所有组别每次剂量均为 1 mg/kg 的 SN-38。接受生理盐水或游离 SN-38 治疗的小鼠表现出血液、骨髓、脾脏和淋巴结中高负荷的 Eμ-myc 肿瘤（图 5A）。). Imaging revealed that free SN-38 at this dose did not suppress tumor growth at any point during the therapy (Fig. 5BOpens in image viewer
). 影像学显示，在该剂量下，游离的 SN-38 在整个治疗过程中并未抑制肿瘤生长（图 5B）。). We also confirmed that transfer of IL-2/rapamycin–treated T cells alone without NC conjugation had no effect on tumor progression (fig. S4). By contrast, mice treated with SN-38–loaded NCs had a significant reduction of tumor burden in all of these compartments, with the total tumor burden reduced by 5.1-fold (Fig. 5Opens in image viewer
我们也证实了，仅转移经 IL-2/雷帕霉素处理的 T 细胞而不进行纳米颗粒（NC）连接，对肿瘤进展无影响（图 S4）。相比之下，接受 SN-38 负载纳米颗粒（NC）处理的小鼠，在所有这些部位的肿瘤负荷均显著减少，总肿瘤负荷降低了 5.1 倍（图 5）。, A and B). However, animals treated with SN-38 NC-T cells showed the most marked tumor eradication, exhibiting a 60-fold reduction in tumor burden on day 16 relative to the free drug or untreated animals (Fig. 5Opens in image viewer
, A 和 B）。然而，接受 SN-38 NC-T 细胞治疗的动物显示出最显著的肿瘤消除效果，在第 16 天时肿瘤负担相较于自由药物或未治疗动物减少了 60 倍（图 5）。, A and B). These results confirm that the lymph nodes are an important growth niche for Eμ-myc cells and that increasing the local SN-38 concentration by T cell delivery suppressed tumor growth.
, A 和 B）。这些结果证实淋巴结是 Eμ-myc 细胞的重要生长微环境，而通过 T 细胞递送提高局部 SN-38 浓度则抑制了肿瘤生长。
接下来，我们测试了药学介导的药物递送系统对这些高度扩散的淋巴瘤肿瘤的治疗效果。在肿瘤接种后的第五天，小鼠接受静脉注射的游离 SN-38、SN-38 纳米颗粒（NCs）或 SN-38 纳米颗粒-T 细胞复合物（NC-T 细胞）治疗，总共四次给药，每隔三天一次，所有组别每次剂量均为 1 mg/kg 的 SN-38。接受生理盐水或游离 SN-38 治疗的小鼠表现出血液、骨髓、脾脏和淋巴结中高负荷的 Eμ-myc 肿瘤（图 5A）。). Imaging revealed that free SN-38 at this dose did not suppress tumor growth at any point during the therapy (Fig. 5BOpens in image viewer
). 影像学显示，在该剂量下，游离的 SN-38 在整个治疗过程中并未抑制肿瘤生长（图 5B）。). We also confirmed that transfer of IL-2/rapamycin–treated T cells alone without NC conjugation had no effect on tumor progression (fig. S4). By contrast, mice treated with SN-38–loaded NCs had a significant reduction of tumor burden in all of these compartments, with the total tumor burden reduced by 5.1-fold (Fig. 5Opens in image viewer
我们也证实了，仅转移经 IL-2/雷帕霉素处理的 T 细胞而不进行纳米颗粒（NC）连接，对肿瘤进展无影响（图 S4）。相比之下，接受 SN-38 负载纳米颗粒（NC）处理的小鼠，在所有这些部位的肿瘤负荷均显著减少，总肿瘤负荷降低了 5.1 倍（图 5）。, A and B). However, animals treated with SN-38 NC-T cells showed the most marked tumor eradication, exhibiting a 60-fold reduction in tumor burden on day 16 relative to the free drug or untreated animals (Fig. 5Opens in image viewer
, A 和 B）。然而，接受 SN-38 NC-T 细胞治疗的动物显示出最显著的肿瘤消除效果，在第 16 天时肿瘤负担相较于自由药物或未治疗动物减少了 60 倍（图 5）。, A and B). These results confirm that the lymph nodes are an important growth niche for Eμ-myc cells and that increasing the local SN-38 concentration by T cell delivery suppressed tumor growth.
, A 和 B）。这些结果证实淋巴结是 Eμ-myc 细胞的重要生长微环境，而通过 T 细胞递送提高局部 SN-38 浓度则抑制了肿瘤生长。
Fig. 5. T cell–mediated delivery of NCs improves the therapeutic efficacy of SN-38 without toxicity.
图 5. T 细胞介导的纳米载体递送提高了 SN-38 的治疗效果且无毒性。
图 5. T 细胞介导的纳米载体递送提高了 SN-38 的治疗效果且无毒性。
(A and B) Albino C57BL/6J mice (n = 5 animals per group) were inoculated with 1 × 106 Eμ-myc cells on day 0 and then received four treatments of free SN-38, free SN-38 NCs, or 2 × 108 NC-T cells (1 mg/kg SN-38 in each group), as indicated [arrows on (B)]. Shown are representative results from one of two independent experiments. (A) Bioluminescence images of tumor burden on day 16. (B) Tumor burden as assessed by Eμ-myc bioluminescence (normalized to signal at the start of therapy) over time. *P < 0.01 by two-way ANOVA with Bonferroni post-test on day 16. (C and D) Albino C57BL/6J mice (n = 5 to 6 animals per group) were inoculated as in (A) and then received free SN-38 at 1 or 10 mg/kg, free SN-38 NCs at 1 mg/kg, or 2 × 108 or 5 × 107 NC-T cells (1 or 0.25 mg/kg SN-38, respectively) every 3 days, starting on day 5, for a total of seven treatments. Shown are representative results from one of two independent experiments. (C) Normalized total body bioluminescence measured on day 15. **P < 0.01, by t test. (D) Overall survival. ***P < 0.001 by log-rank test. (E and F) Animals were inoculated and treated as in (C). (E) Weights of animals normalized individually to the pretherapy weight. (F) On day 28, serum was collected for alanine aminotransferase (ALT) and blood urea nitrogen (BUN) measurement; dashed lines indicate reference healthy ranges. Data shown are means ± SEM (n = 4 to 6 animals per group). Shown are representative results from one of three independent experiments.
(A 和 B) 白化 C57BL/6J 小鼠（每组 n = 5 只动物）在第 0 天接种 1 × 10^6 Eμ-myc 细胞，随后分别接受四次自由 SN-38、自由 SN-38 NCs 或 2 × 10^7 NC-T 细胞（每组 1 mg/kg SN-38）治疗，如所示[箭头在 (B) 中]。图中展示了两次独立实验中一次的代表性结果。(A) 第 16 天的肿瘤负担生物发光图像。(B) 通过 Eμ-myc 生物发光评估的肿瘤负担（以治疗开始时的信号为基准进行归一化）随时间变化。*P < 0.01，通过双因素方差分析（ANOVA）及 Bonferroni 事后检验，第 16 天。(C 和 D) 白化 C57BL/6J 小鼠（每组 n = 5 至 6 只动物）按 (A) 所述接种，随后从第 5 天开始，每 3 天接受一次 1 或 10 mg/kg 的自由 SN-38、1 mg/kg 的自由 SN-38 NCs，或 2 × 10^8 或 5 × 10^9 NC-T 细胞（分别为 1 或 0.25 mg/kg SN-38），共进行七次治疗。图中展示了两次独立实验中一次的代表性结果。(C) 第 15 天测得的归一化全身生物发光。**P < 0.01，通过 t 检验。(D) 总体生存率。***P < 0.001，通过对数秩检验。(E 和 F) 动物按 (C) 所述接种并治疗。 (E) 各动物体重相对于治疗前体重进行个体化标准化。(F) 第 28 天收集血清用于检测丙氨酸氨基转移酶（ALT）和血尿素氮（BUN）；虚线表示参考健康范围。数据显示为平均值±标准误（每组 n = 4 至 6 只动物）。图中展示的是三次独立实验中的一次代表性结果。
(A 和 B) 白化 C57BL/6J 小鼠（每组 n = 5 只动物）在第 0 天接种 1 × 10^6 Eμ-myc 细胞，随后分别接受四次自由 SN-38、自由 SN-38 NCs 或 2 × 10^7 NC-T 细胞（每组 1 mg/kg SN-38）治疗，如所示[箭头在 (B) 中]。图中展示了两次独立实验中一次的代表性结果。(A) 第 16 天的肿瘤负担生物发光图像。(B) 通过 Eμ-myc 生物发光评估的肿瘤负担（以治疗开始时的信号为基准进行归一化）随时间变化。*P < 0.01，通过双因素方差分析（ANOVA）及 Bonferroni 事后检验，第 16 天。(C 和 D) 白化 C57BL/6J 小鼠（每组 n = 5 至 6 只动物）按 (A) 所述接种，随后从第 5 天开始，每 3 天接受一次 1 或 10 mg/kg 的自由 SN-38、1 mg/kg 的自由 SN-38 NCs，或 2 × 10^8 或 5 × 10^9 NC-T 细胞（分别为 1 或 0.25 mg/kg SN-38），共进行七次治疗。图中展示了两次独立实验中一次的代表性结果。(C) 第 15 天测得的归一化全身生物发光。**P < 0.01，通过 t 检验。(D) 总体生存率。***P < 0.001，通过对数秩检验。(E 和 F) 动物按 (C) 所述接种并治疗。 (E) 各动物体重相对于治疗前体重进行个体化标准化。(F) 第 28 天收集血清用于检测丙氨酸氨基转移酶（ALT）和血尿素氮（BUN）；虚线表示参考健康范围。数据显示为平均值±标准误（每组 n = 4 至 6 只动物）。图中展示的是三次独立实验中的一次代表性结果。
To evaluate the efficacy of the therapy within the context of a wider dose range, we carried out survival studies comparing the same four treatments as well as two new treatments: a 10-fold higher dose of free SN-38 (10 mg/kg) and a one-quarter dose of 50 × 106 NC-T cells per dose (corresponding to an SN-38 dose of 0.25 mg/kg). Each group was treated seven times, once every 3 days, and then followed for overall survival. On day 15, tumor burdens were measured by whole-body imaging (Fig. 5COpens in image viewer
为了评估在更广泛剂量范围内的治疗效果，我们进行了生存研究，比较了相同的四种治疗方案以及两种新的治疗方案：一种是 10 倍剂量的游离 SN-38（10 mg/kg），另一种是每剂使用 50 × 10 6 NC-T 细胞的四分之一剂量（相当于 SN-38 剂量为 0.25 mg/kg）。每组治疗七次，每隔三天一次，随后监测总生存期。在第 15 天，通过全身成像测量肿瘤负荷（图 5C）。). As before, free SN-38 at 1 mg/kg did not affect tumor burden, but increasing the dose of free SN-38 10-fold still did not achieve efficacy comparable to drug-carrying T cells. The one-quarter cell dose NC-T cell treatment (one-fourth the drug dose as the free SN-38 group) had about the same tumor burden at this time point as the 10× free SN-38 group, implying that T cell–mediated drug delivery enhanced the potency of SN-38 by at least 40-fold (Fig. 5COpens in image viewer
）。与之前一样，1 mg/kg 的游离 SN-38 对肿瘤负荷没有影响，但将游离 SN-38 的剂量增加 10 倍仍未能达到与药物载运 T 细胞相当的疗效。四分之一细胞剂量（即游离 SN-38 组药物剂量的四分之一）的 NC-T 细胞治疗在此时间点上的肿瘤负荷与 10 倍剂量游离 SN-38 组大致相同，这表明 T 细胞介导的药物递送至少增强了 SN-38 的效力达 40 倍（图 5C）。).
为了评估在更广泛剂量范围内的治疗效果，我们进行了生存研究，比较了相同的四种治疗方案以及两种新的治疗方案：一种是 10 倍剂量的游离 SN-38（10 mg/kg），另一种是每剂使用 50 × 10 6 NC-T 细胞的四分之一剂量（相当于 SN-38 剂量为 0.25 mg/kg）。每组治疗七次，每隔三天一次，随后监测总生存期。在第 15 天，通过全身成像测量肿瘤负荷（图 5C）。). As before, free SN-38 at 1 mg/kg did not affect tumor burden, but increasing the dose of free SN-38 10-fold still did not achieve efficacy comparable to drug-carrying T cells. The one-quarter cell dose NC-T cell treatment (one-fourth the drug dose as the free SN-38 group) had about the same tumor burden at this time point as the 10× free SN-38 group, implying that T cell–mediated drug delivery enhanced the potency of SN-38 by at least 40-fold (Fig. 5COpens in image viewer
）。与之前一样，1 mg/kg 的游离 SN-38 对肿瘤负荷没有影响，但将游离 SN-38 的剂量增加 10 倍仍未能达到与药物载运 T 细胞相当的疗效。四分之一细胞剂量（即游离 SN-38 组药物剂量的四分之一）的 NC-T 细胞治疗在此时间点上的肿瘤负荷与 10 倍剂量游离 SN-38 组大致相同，这表明 T 细胞介导的药物递送至少增强了 SN-38 的效力达 40 倍（图 5C）。).
Despite the early slowing of tumor growth achieved by treatment with 10× free SN-38 or free NCs, tumor growth control decayed over the course of therapy (as seen for free NCs in Fig. 5BOpens in image viewer
尽管使用 10 倍剂量的游离 SN-38 或游离 NCs 治疗初期能够减缓肿瘤生长，但随着治疗的进行，肿瘤生长控制逐渐减弱（如图 5B 中所示的游离 NCs 情况）。), and the median survival times of these groups were not significantly different from untreated controls (~24 days; Fig. 5DOpens in image viewer
这些组的平均生存时间与未治疗的对照组（约 24 天；图 5D）相比没有显著差异。). Increasing the free drug dose 10× only increased the survival of one of six animals. By contrast, animals with lymphoma receiving SN-38 NC-T cells showed significant increases in life span, with median survival extended to 35 days. Infusion of a fourfold lower dose of SN-38 NC-T cells also modestly extended survival of 50% of the cohort, although the overall median survival time was not statistically different than the controls (Fig. 5DOpens in image viewer
).将游离药物剂量增加 10 倍仅使六只动物中的一只存活时间延长。相比之下，接受 SN-38 NC-T 细胞的淋巴瘤动物显示出显著的生命延长，中位生存期延长至 35 天。注射四分之一剂量的 SN-38 NC-T 细胞也适度延长了 50%实验组的生存期，尽管总体中位生存时间与对照组相比在统计学上并无显著差异（图 5D）。).
尽管使用 10 倍剂量的游离 SN-38 或游离 NCs 治疗初期能够减缓肿瘤生长，但随着治疗的进行，肿瘤生长控制逐渐减弱（如图 5B 中所示的游离 NCs 情况）。), and the median survival times of these groups were not significantly different from untreated controls (~24 days; Fig. 5DOpens in image viewer
这些组的平均生存时间与未治疗的对照组（约 24 天；图 5D）相比没有显著差异。). Increasing the free drug dose 10× only increased the survival of one of six animals. By contrast, animals with lymphoma receiving SN-38 NC-T cells showed significant increases in life span, with median survival extended to 35 days. Infusion of a fourfold lower dose of SN-38 NC-T cells also modestly extended survival of 50% of the cohort, although the overall median survival time was not statistically different than the controls (Fig. 5DOpens in image viewer
).将游离药物剂量增加 10 倍仅使六只动物中的一只存活时间延长。相比之下，接受 SN-38 NC-T 细胞的淋巴瘤动物显示出显著的生命延长，中位生存期延长至 35 天。注射四分之一剂量的 SN-38 NC-T 细胞也适度延长了 50%实验组的生存期，尽管总体中位生存时间与对照组相比在统计学上并无显著差异（图 5D）。).
To assess possible toxicities from SN-38 NC-T cell therapy, we tracked animal weights and liver enzymes, but saw no weight loss at any time point in any group and found that ALT, BUN, and other serum measurements fell in the range of healthy animals (Fig. 5EOpens in image viewer
为了评估 SN-38 NC-T 细胞疗法可能产生的毒性，我们监测了动物体重和肝酶，但未观察到任何时间点任何组别的体重下降，并发现 ALT、BUN 及其他血清指标均处于健康动物的正常范围内（图 5E）。 and fig. S5). Thus, NC-T cell therapy significantly improved the efficacy of SN-38 without increasing the risk of adverse side effects.
因此，NC-T 细胞疗法显著提升了 SN-38 的疗效，同时并未增加不良副作用的风险。
为了评估 SN-38 NC-T 细胞疗法可能产生的毒性，我们监测了动物体重和肝酶，但未观察到任何时间点任何组别的体重下降，并发现 ALT、BUN 及其他血清指标均处于健康动物的正常范围内（图 5E）。 and fig. S5). Thus, NC-T cell therapy significantly improved the efficacy of SN-38 without increasing the risk of adverse side effects.
因此，NC-T 细胞疗法显著提升了 SN-38 的疗效，同时并未增加不良副作用的风险。
DISCUSSION 讨论
SN-38 is representative of a large class of potent chemotherapy drugs with limited in vivo efficacy owing to very poor pharmacokinetics and toxicity. Nanoparticle formulation has been pursued as a strategy to overcome these issues, and several SN-38 formulations have been developed—including liposomes, polylactic-co-glycolic acid nanoparticles, and micelles—some of which are in clinical trials for colorectal and other solid cancers (26). However, nanoparticle delivery faces its own challenges, because tumor accumulation is dependent on the presence of a leaky tumor vasculature (the EPR effect), and particles often become trapped perivascularly in tumors. Autochthonous Eμ-myc Arf+/+ tumors developing in lymph nodes show higher densities of blood vessels and lymphatic vessels compared to normal nodes, but these vessels are not permissively leaky to systemically injected dyes (27). Similar observations of increased angiogenesis have been made in patient samples but are inconsistent across lymphoma subtypes (10, 11). Within the transplanted Eμ-myc Arf−/− model used in our studies, we did not observe an EPR effect in tumor-ridden lymph nodes with “stealth” liposomes (Fig. 1Opens in image viewer
SN-38 代表了一类强效化疗药物，但由于其极差的药代动力学特性和毒性，体内疗效有限。纳米颗粒制剂已被探索作为克服这些问题的策略，并开发了几种 SN-38 制剂，包括脂质体、聚乳酸-羟基乙酸共聚物纳米颗粒和胶束，其中一些正在进行结直肠癌及其他实体瘤的临床试验（26）。然而，纳米颗粒递送本身也面临挑战，因为肿瘤的积累依赖于肿瘤血管的渗漏性（EPR 效应），且颗粒常常在肿瘤内皮周围滞留。在淋巴结中自发形成的 Eμ-myc Arf +/+ 肿瘤相比正常淋巴结显示出更高密度的血管和淋巴管，但这些血管对系统性注射的染料并不表现出明显的渗漏性（27）。类似地，在患者样本中也观察到血管生成增加的现象，但在不同淋巴瘤亚型间表现不一致（10, 11）。在我们研究中使用的 Eμ-myc Arf −/− 移植模型中，未观察到携带“隐形”脂质体的肿瘤浸润淋巴结出现 EPR 效应（图 1）。) or with SN-38 NCs (Fig. 4Opens in image viewer
）或使用 SN-38 纳米颗粒（图 4）). Thus, nanoparticle-based delivery of SN-38, or similar drugs, would be insufficient to reach lymphoid tissue–homing lymphomas or leukemias.
因此，基于纳米颗粒的 SN-38 或类似药物的递送方式，将不足以使药物到达归巢于淋巴组织的淋巴瘤或白血病。
SN-38 代表了一类强效化疗药物，但由于其极差的药代动力学特性和毒性，体内疗效有限。纳米颗粒制剂已被探索作为克服这些问题的策略，并开发了几种 SN-38 制剂，包括脂质体、聚乳酸-羟基乙酸共聚物纳米颗粒和胶束，其中一些正在进行结直肠癌及其他实体瘤的临床试验（26）。然而，纳米颗粒递送本身也面临挑战，因为肿瘤的积累依赖于肿瘤血管的渗漏性（EPR 效应），且颗粒常常在肿瘤内皮周围滞留。在淋巴结中自发形成的 Eμ-myc Arf +/+ 肿瘤相比正常淋巴结显示出更高密度的血管和淋巴管，但这些血管对系统性注射的染料并不表现出明显的渗漏性（27）。类似地，在患者样本中也观察到血管生成增加的现象，但在不同淋巴瘤亚型间表现不一致（10, 11）。在我们研究中使用的 Eμ-myc Arf −/− 移植模型中，未观察到携带“隐形”脂质体的肿瘤浸润淋巴结出现 EPR 效应（图 1）。) or with SN-38 NCs (Fig. 4Opens in image viewer
）或使用 SN-38 纳米颗粒（图 4）). Thus, nanoparticle-based delivery of SN-38, or similar drugs, would be insufficient to reach lymphoid tissue–homing lymphomas or leukemias.
因此，基于纳米颗粒的 SN-38 或类似药物的递送方式，将不足以使药物到达归巢于淋巴组织的淋巴瘤或白血病。
To overcome these hurdles, here, we demonstrated a strategy for active targeting of chemotherapy to disseminated tumors, using lymphocytes as living chaperones to deliver drug-loaded NCs to tumor sites. By expanding T cells under conditions that maintained chemokine and adhesion receptors necessary for lymphoid tissue homing, we were able to generate large numbers of lymphocyte chaperones with highly specific tumor-targeting tropism. These T cell chaperones were resistant to SN-38, even with high doses of drug-loaded particles bound directly to the cell membrane. Although T cell homing occurs over days, even low numbers of NC-T cells entering tumors at early times after transfer could initiate therapeutic responses, as seen from the high concentration [20-fold above the EC90 (90% effective concentration)] of SN-38 in tumor-bearing lymph nodes at 20 hours after transfer (Fig. 4Opens in image viewer
为了克服这些障碍，本文展示了一种主动靶向化疗药物至播散性肿瘤的策略，利用淋巴细胞作为活体向导，将载药纳米颗粒（NCs）递送至肿瘤部位。通过在维持趋化因子和粘附受体表达的条件下扩增 T 细胞，这些受体对于淋巴组织归巢至关重要，我们成功制备了大量具有高度特异性肿瘤靶向倾向的淋巴细胞向导。这些 T 细胞向导即使在高剂量载药颗粒直接结合到细胞膜的情况下，仍对 SN-38 具有抗性。尽管 T 细胞归巢需要数天时间，但在转移后早期进入肿瘤的少量 NC-T 细胞即可启动治疗反应，正如在转移后 20 小时肿瘤负荷的淋巴结中观察到的高浓度 SN-38（达到 90%有效浓度 EC 90 的 20 倍）所示（图 4）。). The substantially lower efficacy of free SN-38 NCs, which effectively accumulated in one lymphoma residence site (the spleen) but not lymph nodes, suggests that increased lymph node delivery is key to the efficacy of this approach. This enhanced delivery of SN-38 to tumors yielded therapeutic benefits at modest doses of SN-38—doses that had no effect in free drug form and limited efficacy in NCs only. We demonstrated a 12-day extension of survival using T cell–mediated SN-38 NC delivery at an SN-38 cumulative dose of only 7 mg/kg. This efficacy was far greater than the free SN-38 dose of 70 mg/kg because of the poor pharmacokinetics of the free drug. However, even with irinotecan, the water-soluble, U.S. Food and Drug Administration (FDA)–approved analog of SN-38, a cumulative dose of 100 mg/kg was required to extend survival by a maximum of 9 days in this model (28). Doxorubicin, another cytotoxic chemotherapeutic, achieved a 14- to 15-day survival extension at 10 mg/kg (28), but this is the maximal lifetime tolerated dose and causes potentially lethal myocardial damage. By contrast, we saw no evidence for toxicity with SN-38 NC-T cells, suggesting that the therapeutic window for higher levels of drug dosing with this approach is much wider than for traditional chemotherapy.
). 自由 SN-38 纳米晶体（NCs）的显著较低疗效表明，尽管其在某一淋巴瘤寄居部位（脾脏）有效积累，但未能进入淋巴结，这提示增加淋巴结递送是该方法疗效的关键。通过增强 SN-38 向肿瘤的递送，在适中的 SN-38 剂量下即获得了治疗效果——这些剂量在自由药物形式下无效，且仅在纳米晶体形式下具有有限疗效。我们展示了使用 T 细胞介导的 SN-38 纳米晶体递送，在仅 7 mg/kg 的累积 SN-38 剂量下，生存期延长了 12 天。这一疗效远超 70 mg/kg 自由 SN-38 剂量的效果，原因在于自由药物的药代动力学较差。然而，即便是伊立替康（irinotecan），作为 SN-38 的水溶性、美国食品药品监督管理局（FDA）批准的类似物，在此模型中也需要 100 mg/kg 的累积剂量才能使生存期最多延长 9 天（28）。另一种细胞毒性化疗药物阿霉素（doxorubicin）在 10 mg/kg 剂量下实现了 14 至 15 天的生存期延长（28），但这已是其终生耐受的最大剂量，并可能导致潜在致命的心肌损伤。 相比之下，我们未观察到 SN-38 NC-T 细胞存在毒性，这表明采用此方法进行更高剂量药物治疗的安全范围远大于传统化疗。
为了克服这些障碍，本文展示了一种主动靶向化疗药物至播散性肿瘤的策略，利用淋巴细胞作为活体向导，将载药纳米颗粒（NCs）递送至肿瘤部位。通过在维持趋化因子和粘附受体表达的条件下扩增 T 细胞，这些受体对于淋巴组织归巢至关重要，我们成功制备了大量具有高度特异性肿瘤靶向倾向的淋巴细胞向导。这些 T 细胞向导即使在高剂量载药颗粒直接结合到细胞膜的情况下，仍对 SN-38 具有抗性。尽管 T 细胞归巢需要数天时间，但在转移后早期进入肿瘤的少量 NC-T 细胞即可启动治疗反应，正如在转移后 20 小时肿瘤负荷的淋巴结中观察到的高浓度 SN-38（达到 90%有效浓度 EC 90 的 20 倍）所示（图 4）。). The substantially lower efficacy of free SN-38 NCs, which effectively accumulated in one lymphoma residence site (the spleen) but not lymph nodes, suggests that increased lymph node delivery is key to the efficacy of this approach. This enhanced delivery of SN-38 to tumors yielded therapeutic benefits at modest doses of SN-38—doses that had no effect in free drug form and limited efficacy in NCs only. We demonstrated a 12-day extension of survival using T cell–mediated SN-38 NC delivery at an SN-38 cumulative dose of only 7 mg/kg. This efficacy was far greater than the free SN-38 dose of 70 mg/kg because of the poor pharmacokinetics of the free drug. However, even with irinotecan, the water-soluble, U.S. Food and Drug Administration (FDA)–approved analog of SN-38, a cumulative dose of 100 mg/kg was required to extend survival by a maximum of 9 days in this model (28). Doxorubicin, another cytotoxic chemotherapeutic, achieved a 14- to 15-day survival extension at 10 mg/kg (28), but this is the maximal lifetime tolerated dose and causes potentially lethal myocardial damage. By contrast, we saw no evidence for toxicity with SN-38 NC-T cells, suggesting that the therapeutic window for higher levels of drug dosing with this approach is much wider than for traditional chemotherapy.
). 自由 SN-38 纳米晶体（NCs）的显著较低疗效表明，尽管其在某一淋巴瘤寄居部位（脾脏）有效积累，但未能进入淋巴结，这提示增加淋巴结递送是该方法疗效的关键。通过增强 SN-38 向肿瘤的递送，在适中的 SN-38 剂量下即获得了治疗效果——这些剂量在自由药物形式下无效，且仅在纳米晶体形式下具有有限疗效。我们展示了使用 T 细胞介导的 SN-38 纳米晶体递送，在仅 7 mg/kg 的累积 SN-38 剂量下，生存期延长了 12 天。这一疗效远超 70 mg/kg 自由 SN-38 剂量的效果，原因在于自由药物的药代动力学较差。然而，即便是伊立替康（irinotecan），作为 SN-38 的水溶性、美国食品药品监督管理局（FDA）批准的类似物，在此模型中也需要 100 mg/kg 的累积剂量才能使生存期最多延长 9 天（28）。另一种细胞毒性化疗药物阿霉素（doxorubicin）在 10 mg/kg 剂量下实现了 14 至 15 天的生存期延长（28），但这已是其终生耐受的最大剂量，并可能导致潜在致命的心肌损伤。 相比之下，我们未观察到 SN-38 NC-T 细胞存在毒性，这表明采用此方法进行更高剂量药物治疗的安全范围远大于传统化疗。
The intrinsic trafficking ability of host cells to infiltrate disease sites and act as self-directed vectors for therapeutics is being explored in a number of contexts, most commonly using phagocytic cells as drug carriers (29, 30). Monocytes, macrophages, and mesenchymal stem cells readily phagocytose nanoparticles and microparticles, and particle-loaded cells have been used as transporters for gold nanoshells for photothermal tumor ablation (31), chemotherapy- or imaging agent–loaded particles for tumor treatment (32, 33), and antiretroviral drugs for treatment of HIV infection (34). A limitation of such approaches is that they can only be applied to cell-permeable drug cargos or agents that provide a function from within the carrier cell. By contrast, the plasma membrane–conjugation approach described here allows particles to deliver cargos, such as biologics, that are not membrane-permeable and must access cell surface receptors of nearby target cells (19).
宿主细胞固有的穿透疾病部位并作为自我导向的药物递送载体的能力，正在多个领域中被探索，最常见的是利用吞噬细胞作为药物载体（29, 30）。单核细胞、巨噬细胞和间充质干细胞能够轻易吞噬纳米颗粒和微粒，而载有颗粒的细胞已被用作金纳米壳的运输工具，用于光热肿瘤消融（31），以及携带化疗药物或成像剂的颗粒用于肿瘤治疗（32, 33），还有抗逆转录病毒药物用于治疗 HIV 感染（34）。这类方法的一个局限性在于，它们仅适用于细胞可渗透的药物载荷或能在载体细胞内部发挥功能的药物。相比之下，本文所述的质膜结合方法使得颗粒能够递送生物制剂等非膜渗透性货物，这些货物必须接触到邻近靶细胞的细胞表面受体（19）。
宿主细胞固有的穿透疾病部位并作为自我导向的药物递送载体的能力，正在多个领域中被探索，最常见的是利用吞噬细胞作为药物载体（29, 30）。单核细胞、巨噬细胞和间充质干细胞能够轻易吞噬纳米颗粒和微粒，而载有颗粒的细胞已被用作金纳米壳的运输工具，用于光热肿瘤消融（31），以及携带化疗药物或成像剂的颗粒用于肿瘤治疗（32, 33），还有抗逆转录病毒药物用于治疗 HIV 感染（34）。这类方法的一个局限性在于，它们仅适用于细胞可渗透的药物载荷或能在载体细胞内部发挥功能的药物。相比之下，本文所述的质膜结合方法使得颗粒能够递送生物制剂等非膜渗透性货物，这些货物必须接触到邻近靶细胞的细胞表面受体（19）。
We used polyclonal T cells as nanoparticle carriers, using lymphocytes that express homing receptors to traffic to the lymphoid organs where tumor cells reside, but which do not target tumor antigens explicitly. The pronounced therapeutic effects shown here using non–antigen-specific cells demonstrate the utility of organ-specific targeting in this disease model (as opposed to tumor cell–specific targeting). The effectiveness of polyclonal T cells facilitates clinical implementation because isolation of endogenous tumor antigen–specific T cells is only possible for a fraction of cancer patients (12). However, in diseases such as melanoma where tumor-specific T cells are more readily obtained or cancers where genetically engineered artificial antigen receptors can be safely introduced [for example, leukemias (35)], our approach could be combined with tumor antigen–specific T cells.
我们采用多克隆 T 细胞作为纳米颗粒载体，利用表达归巢受体的淋巴细胞前往肿瘤细胞所在的淋巴器官，但这些细胞并不特异性地针对肿瘤抗原。在此展示的非抗原特异性细胞所表现出的显著治疗效果，证明了在此疾病模型中器官特异性靶向的实用性（与肿瘤细胞特异性靶向相对）。多克隆 T 细胞的有效性促进了临床应用，因为只有部分癌症患者能够分离出内源性肿瘤抗原特异性 T 细胞（12）。然而，在黑色素瘤等肿瘤特异性 T 细胞更易获得的疾病中，或是在可安全引入基因工程人工抗原受体的癌症中（例如白血病，35），我们的方法可以与肿瘤抗原特异性 T 细胞相结合。
我们采用多克隆 T 细胞作为纳米颗粒载体，利用表达归巢受体的淋巴细胞前往肿瘤细胞所在的淋巴器官，但这些细胞并不特异性地针对肿瘤抗原。在此展示的非抗原特异性细胞所表现出的显著治疗效果，证明了在此疾病模型中器官特异性靶向的实用性（与肿瘤细胞特异性靶向相对）。多克隆 T 细胞的有效性促进了临床应用，因为只有部分癌症患者能够分离出内源性肿瘤抗原特异性 T 细胞（12）。然而，在黑色素瘤等肿瘤特异性 T 细胞更易获得的疾病中，或是在可安全引入基因工程人工抗原受体的癌症中（例如白血病，35），我们的方法可以与肿瘤抗原特异性 T 细胞相结合。
Although significant tumor cell killing was seen at a T cell/tumor cell ratio of 1:20 in vitro, complete eradication required ratios of at least ~1:10. Owing to in vivo confounding factors, such as lymph flow, secreted factors, and stromal cells that augment Eμ-myc tumor growth and survival (36, 37), we aimed to transfer enough T cells to reach at least 1:5 T cell/tumor ratios in the nodes. Although the cell numbers needed to reach this density of pharmacytes in lymph nodes is high, it is within the range of cell doses that have been used in clinical trials of adoptive T cell therapy for cancer. Using the FDA guidelines for determining dose conversions between species (38), the equivalent of our 2 × 108 cell dosing for a human patient would be ~4.6 × 1010 lymphocytes. Early adoptive T cell therapy clinical trials treated melanoma patients with >2 × 1011 total tumor-infiltrating lymphocytes in the first course alone, with some patients receiving up to five total courses (39). More recent trials have used up to 1.5 × 1011 to 1.6 × 1011 total cells per patient (40, 41). These numbers reflect the large quantity of T cells required to successfully eliminate large tumor burdens even when relying on antigen-specific recognition for tumor elimination, and suggest that the number of SN-38 NC–conjugated T cells required for human patients would be both technologically and clinically reasonable to achieve. This cell dosing could be substantially reduced with the development of NCs loaded more efficiently with drug cargo, but our objective here was to demonstrate proof of concept in a cell dose that has been used in patients.
虽然在体外 T 细胞与肿瘤细胞比例为 1:20 时观察到了显著的肿瘤细胞杀伤效果，但完全清除肿瘤需要至少约 1:10 的比例。由于体内存在淋巴流动、分泌因子及基质细胞等干扰因素，这些因素会促进 Eμ-myc 肿瘤的生长和存活（36, 37），我们的目标是转移足够数量的 T 细胞，以在淋巴结中达到至少 1:5 的 T 细胞与肿瘤比例。尽管达到淋巴结中如此密度的效应细胞所需的细胞数量较高，但这一剂量仍处于癌症适应性 T 细胞疗法临床试验所用细胞剂量的范围内。根据美国食品药品监督管理局（FDA）关于确定物种间剂量转换的指南（38），我们用于小鼠的 2 × 10^6 细胞剂量相当于人类患者约 4.6 × 10^7 淋巴细胞。早期的适应性 T 细胞疗法临床试验在首次疗程中单独为黑色素瘤患者输注了超过 2 × 10^8 的肿瘤浸润淋巴细胞，部分患者甚至接受了多达五个疗程的治疗（39）。近期的试验则每名患者使用高达 1.5 × 10^9 至 1.6 × 10^10 的总细胞量（40, 41）。 这些数据反映了即使在依赖抗原特异性识别来消除肿瘤的情况下，成功清除大量肿瘤负荷所需的 T 细胞数量仍然庞大，并表明对于人类患者而言，所需的 SN-38 NC-偶联 T 细胞数量在技术和临床上都是可实现的目标。随着更高效载药纳米颗粒（NCs）的研发，这一细胞剂量有望显著减少，但本研究旨在通过已在患者中使用的细胞剂量来验证概念的可行性。
虽然在体外 T 细胞与肿瘤细胞比例为 1:20 时观察到了显著的肿瘤细胞杀伤效果，但完全清除肿瘤需要至少约 1:10 的比例。由于体内存在淋巴流动、分泌因子及基质细胞等干扰因素，这些因素会促进 Eμ-myc 肿瘤的生长和存活（36, 37），我们的目标是转移足够数量的 T 细胞，以在淋巴结中达到至少 1:5 的 T 细胞与肿瘤比例。尽管达到淋巴结中如此密度的效应细胞所需的细胞数量较高，但这一剂量仍处于癌症适应性 T 细胞疗法临床试验所用细胞剂量的范围内。根据美国食品药品监督管理局（FDA）关于确定物种间剂量转换的指南（38），我们用于小鼠的 2 × 10^6 细胞剂量相当于人类患者约 4.6 × 10^7 淋巴细胞。早期的适应性 T 细胞疗法临床试验在首次疗程中单独为黑色素瘤患者输注了超过 2 × 10^8 的肿瘤浸润淋巴细胞，部分患者甚至接受了多达五个疗程的治疗（39）。近期的试验则每名患者使用高达 1.5 × 10^9 至 1.6 × 10^10 的总细胞量（40, 41）。 这些数据反映了即使在依赖抗原特异性识别来消除肿瘤的情况下，成功清除大量肿瘤负荷所需的 T 细胞数量仍然庞大，并表明对于人类患者而言，所需的 SN-38 NC-偶联 T 细胞数量在技术和临床上都是可实现的目标。随着更高效载药纳米颗粒（NCs）的研发，这一细胞剂量有望显著减少，但本研究旨在通过已在患者中使用的细胞剂量来验证概念的可行性。
We also expect that our approach would have synergy with traditional chemotherapy dosing, using the pharmacyte approach to eradicate residual disease in lymphoid tissue sanctuaries rather than as a sole treatment regimen; this is an area for future study. Further efficacy with this strategy in patients might also be expected because human lymphomas exhibit a broad range of phenotypes. For reference, the Eμ-myc tumors used here with constitutive expression of Myc and deletion of Arf model the most aggressive subtypes of these cancers (42).
我们还期望，我们的方法能与传统化疗剂量方案协同作用，利用药理学途径来清除淋巴组织庇护所中的残留疾病，而非作为单一治疗方案；这是未来研究的一个领域。鉴于人类淋巴瘤表现出广泛的表型，采用此策略在患者中进一步提高疗效也是可预期的。作为参考，此处使用的 Eμ-myc 肿瘤模型具有 Myc 的组成性表达和 Arf 的缺失，模拟了这些癌症中最具侵袭性的亚型（42）。
我们还期望，我们的方法能与传统化疗剂量方案协同作用，利用药理学途径来清除淋巴组织庇护所中的残留疾病，而非作为单一治疗方案；这是未来研究的一个领域。鉴于人类淋巴瘤表现出广泛的表型，采用此策略在患者中进一步提高疗效也是可预期的。作为参考，此处使用的 Eμ-myc 肿瘤模型具有 Myc 的组成性表达和 Arf 的缺失，模拟了这些癌症中最具侵袭性的亚型（42）。
The fundamental approach for cell-mediated drug delivery demonstrated here should be generalizable to many types of cellular carriers. Lymphocytes as drug carriers offer a number of advantages. First, clinical protocols for T cell expansion are well established in the adoptive therapy field, and autologous lymphocytes are easily obtained from blood. Second, lymphocytes exhibit substantial plasticity in their expression of tissue-homing markers. T cells can be induced under conditions of inflammation or disease to enter nearly every tissue, and the receptors required for T cell trafficking to the lungs, skin, gut, and brain, as well as the molecular stimuli required to induce expression of these homing markers have been characterized (43) and could potentially be used to generate lymphocyte carriers to deliver drugs to each of these organs.
此处展示的基于细胞介导药物递送的基本方法应可推广至多种细胞载体。作为药物载体的淋巴细胞具有诸多优势。首先，在过继疗法领域，T 细胞扩增的临床方案已相当成熟，且自体淋巴细胞易于从血液中获取。其次，淋巴细胞在组织归巢标记的表达上表现出显著的可塑性。在炎症或疾病条件下，T 细胞可被诱导进入几乎所有组织，用于 T 细胞向肺、皮肤、肠道和大脑迁移所需的受体，以及诱导这些归巢标记表达所需的分子刺激因素已被阐明（43），并可能用于生成针对各器官靶向递送药物的淋巴细胞载体。
此处展示的基于细胞介导药物递送的基本方法应可推广至多种细胞载体。作为药物载体的淋巴细胞具有诸多优势。首先，在过继疗法领域，T 细胞扩增的临床方案已相当成熟，且自体淋巴细胞易于从血液中获取。其次，淋巴细胞在组织归巢标记的表达上表现出显著的可塑性。在炎症或疾病条件下，T 细胞可被诱导进入几乎所有组织，用于 T 细胞向肺、皮肤、肠道和大脑迁移所需的受体，以及诱导这些归巢标记表达所需的分子刺激因素已被阐明（43），并可能用于生成针对各器官靶向递送药物的淋巴细胞载体。
In summary, we have developed here a strategy for active targeting of drugs to disease sites, using autologous lymphocytes as “Trojan horses” to deliver drug-loaded NCs. A limitation of this approach is the total payload of drug that can be loaded per cell and the number of cells that can be transferred, but as shown here, the greatly enhanced efficacy of drug delivery to tumor sites allows even modest total doses of drug to be highly efficacious. Further, ongoing advances in nanoparticle design can facilitate loading of very high payloads of drug (44–46). We envision that this approach could be particularly relevant as a means to eliminate residual disease in tissue sanctuaries that are difficult to reach at safe doses and by traditional nanomedicine.
总之，我们在此开发了一种利用自体淋巴细胞作为“特洛伊木马”将载药纳米颗粒主动靶向疾病部位的策略。该方法的一个局限性是每个细胞可装载的药物总负荷量以及可转移的细胞数量，但如本文所示，药物向肿瘤部位递送的显著增强效果使得即使较小总剂量的药物也能高度有效。此外，纳米颗粒设计的持续进展有望实现极高负荷的药物装载（44-46）。我们预见，这种方法在消除难以安全剂量触及且传统纳米医学难以攻克的组织庇护所中的残留疾病方面，将具有特别重要的意义。
总之，我们在此开发了一种利用自体淋巴细胞作为“特洛伊木马”将载药纳米颗粒主动靶向疾病部位的策略。该方法的一个局限性是每个细胞可装载的药物总负荷量以及可转移的细胞数量，但如本文所示，药物向肿瘤部位递送的显著增强效果使得即使较小总剂量的药物也能高度有效。此外，纳米颗粒设计的持续进展有望实现极高负荷的药物装载（44-46）。我们预见，这种方法在消除难以安全剂量触及且传统纳米医学难以攻克的组织庇护所中的残留疾病方面，将具有特别重要的意义。
MATERIALS AND METHODS 材料与方法
Study design 研究设计
The hypothesis was that SN-38–carrying nanoparticles attached to T cells would show greater uptake in tumor tissues and enhanced antitumor efficacy compared to free drug or free nanoparticle treatments. All experiments were performed independently at least twice. In vivo therapy studies were designed to evaluate the impact of active versus passive delivery of a chemotherapeutic agent to disseminated tumor sites in a syngeneic mouse model of lymphoma, and were executed with at least five animals per group. Before treatment, cumulative tumor burden was measured by whole-body radiance, and animals were randomized to minimize variances between groups. The radiance value of each animal was normalized by its pretreatment radiance. Representative data are shown for tumor therapy experiments owing to variation in tumor growth kinetics between inoculations. Pooled data are shown for NC-T cell biodistribution experiments to obtain sufficient replicates for each time point and condition. Data analyses were not blinded. Outliers were not excluded.
假设是，与游离药物或游离纳米颗粒治疗相比，附着于 T 细胞的 SN-38 载纳米颗粒在肿瘤组织中的摄取量更大，且抗肿瘤效果增强。所有实验均至少独立进行两次。体内治疗研究旨在评估在同基因小鼠淋巴瘤模型中，主动与被动递送化疗药物至播散性肿瘤部位的影响，每组至少使用五只动物进行实验。治疗前，通过全身发光度测量累积肿瘤负荷，并将动物随机分组以尽量减少各组间的差异。每只动物的发光值以其治疗前的发光值进行归一化处理。由于不同接种之间的肿瘤生长动力学存在差异，肿瘤治疗实验的代表性数据得以展示。为了在每个时间点和条件下获得足够的重复次数，NC-T 细胞生物分布实验的数据进行了合并展示。数据分析未进行盲法处理，未排除异常值。
假设是，与游离药物或游离纳米颗粒治疗相比，附着于 T 细胞的 SN-38 载纳米颗粒在肿瘤组织中的摄取量更大，且抗肿瘤效果增强。所有实验均至少独立进行两次。体内治疗研究旨在评估在同基因小鼠淋巴瘤模型中，主动与被动递送化疗药物至播散性肿瘤部位的影响，每组至少使用五只动物进行实验。治疗前，通过全身发光度测量累积肿瘤负荷，并将动物随机分组以尽量减少各组间的差异。每只动物的发光值以其治疗前的发光值进行归一化处理。由于不同接种之间的肿瘤生长动力学存在差异，肿瘤治疗实验的代表性数据得以展示。为了在每个时间点和条件下获得足够的重复次数，NC-T 细胞生物分布实验的数据进行了合并展示。数据分析未进行盲法处理，未排除异常值。
T cell–NC conjugation T 细胞-纳米颗粒结合
Live T cells were purified by Ficoll gradient, washed in phosphate-buffered saline (PBS), and resuspended in serum-free unsupplemented RPMI 1640 at 50 × 106/ml. NCs were added and incubated with gentle mixing at 4 °C for 30 min. Cells were washed by pelleting and resuspending in 50 ml of PBS twice. Cells were then resuspended in serum-free RPMI with PEG2000-SH (1 mg/ml) and incubated with gentle mixing at 4 °C for 30 min. Cells were washed in PBS and used immediately. The amount of SN-38 NC conjugated to T cells was quantified by lysing T cell pellets in 0.1 M NaOH + 0.5% Triton X-100, pelleting at 21,000g, and reading the SN-38 fluorescence in the supernatant.
活 T 细胞通过 Ficoll 梯度法纯化，用磷酸盐缓冲液（PBS）洗涤，并在无血清、无补充的 RPMI 1640 培养基中以 50 × 10⁶/ml 的浓度重悬。随后加入纳米载体（NCs），在 4°C 下轻轻搅拌孵育 30 分钟。细胞通过离心和重悬于 50ml PBS 中洗涤两次。接着，细胞在含有 PEG2000-SH（1 mg/ml）的无血清 RPMI 中重悬，同样在 4°C 下轻轻搅拌孵育 30 分钟。之后，细胞用 PBS 洗涤并立即使用。通过在 0.1 M NaOH + 0.5% Triton X-100 中裂解 T 细胞沉淀，21,000g 离心后测定上清液中 SN-38 荧光强度，定量 T 细胞上结合的 SN-38 纳米载体量。
活 T 细胞通过 Ficoll 梯度法纯化，用磷酸盐缓冲液（PBS）洗涤，并在无血清、无补充的 RPMI 1640 培养基中以 50 × 10⁶/ml 的浓度重悬。随后加入纳米载体（NCs），在 4°C 下轻轻搅拌孵育 30 分钟。细胞通过离心和重悬于 50ml PBS 中洗涤两次。接着，细胞在含有 PEG2000-SH（1 mg/ml）的无血清 RPMI 中重悬，同样在 4°C 下轻轻搅拌孵育 30 分钟。之后，细胞用 PBS 洗涤并立即使用。通过在 0.1 M NaOH + 0.5% Triton X-100 中裂解 T 细胞沉淀，21,000g 离心后测定上清液中 SN-38 荧光强度，定量 T 细胞上结合的 SN-38 纳米载体量。
In vivo tumor experiments
体内肿瘤实验
Animals were cared for following federal, state, and local guidelines. To inoculate tumors, 1 × 106 Eμ-myc cells were injected via the tail vein. For luciferase+GFP+ Eμ-myc Arf−/− cells, tumor burden was assessed by whole-animal bioluminescent imaging (Xenogen Spectrum 200). Animals were injected subcutaneously with d-luciferin (150 mg/kg) 10 min before imaging. At the terminal time point, blood, spleen, lymph nodes (cervical, axillary, brachial, inguinal, mesenteric, iliac), bone marrow (from femurs and tibia), and liver were collected. For flow cytometry analysis, tissues were mechanically dissociated into single-cell suspensions, and Eμ-myc cells were gated on GFP.
根据联邦、州和地方的指导原则对动物进行护理。为了接种肿瘤，通过尾静脉注射 1 × 10^6 个 Eμ-myc 细胞。对于表达荧光素酶^1^ GFP^2^ Eμ-myc Arf^3^的细胞，通过全动物生物发光成像（Xenogen Spectrum 200）评估肿瘤负荷。在成像前 10 分钟，动物皮下注射 d-荧光素（150 mg/kg）。在终末时间点，采集血液、脾脏、淋巴结（颈部、腋窝、臂部、腹股沟、肠系膜、髂骨）、骨髓（来自股骨和胫骨）和肝脏。对于流式细胞术分析，组织被机械分离成单细胞悬液，并通过 GFP 对 Eμ-myc 细胞进行门控分析。
根据联邦、州和地方的指导原则对动物进行护理。为了接种肿瘤，通过尾静脉注射 1 × 10^6 个 Eμ-myc 细胞。对于表达荧光素酶^1^ GFP^2^ Eμ-myc Arf^3^的细胞，通过全动物生物发光成像（Xenogen Spectrum 200）评估肿瘤负荷。在成像前 10 分钟，动物皮下注射 d-荧光素（150 mg/kg）。在终末时间点，采集血液、脾脏、淋巴结（颈部、腋窝、臂部、腹股沟、肠系膜、髂骨）、骨髓（来自股骨和胫骨）和肝脏。对于流式细胞术分析，组织被机械分离成单细胞悬液，并通过 GFP 对 Eμ-myc 细胞进行门控分析。
Statistical analysis 统计分析
Statistical analyses were performed using GraphPad Prism 5.0. All plots show mean ± SEM. Statistical significance threshold was set at P ≤ 0.05. All tests assumed normal distribution and were two-sided.
统计分析采用 GraphPad Prism 5.0 软件进行。所有图表展示均值±标准误（SEM）。统计显著性阈值设定为 P ≤ 0.05。所有检验均假设数据呈正态分布，且为双侧检验。
统计分析采用 GraphPad Prism 5.0 软件进行。所有图表展示均值±标准误（SEM）。统计显著性阈值设定为 P ≤ 0.05。所有检验均假设数据呈正态分布，且为双侧检验。
Acknowledgments 致谢
Histology samples were sectioned by the Koch Institute Swanson Biotechnology Center. Eμ-myc Arf−/− cells were a gift of M. T. Hemann. Funding: This work was supported in part by the Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute, the NIH (CA140476 and CA172164), and the Department of Defense (W81XWH-10-1-0290). D.J.I. is an investigator of the Howard Hughes Medical Institute. Author contributions: B.H. performed all experiments and statistical analyses. W.D.A., Y.Z., and S.C.B.L. contributed to animal experiments. S.S.L. contributed to nanoparticle development and characterization. B.H. and D.J.I. designed the experiments and wrote the paper. Competing interests: A patent related to the technology described here is pending. Data and materials availability: No material used in this study was obtained from external parties under material transfer agreement.
组织学样本由科赫研究所斯旺森生物技术中心切片。Eμ-myc Arf −/− 细胞由 M. T. Hemann 赠送。经费支持：本研究部分由国家癌症研究所的科赫研究所支持（核心）资助金 P30-CA14051、NIH（CA140476 和 CA172164）以及国防部（W81XWH-10-1-0290）资助。D.J.I.是霍华德·休斯医学研究所的研究员。作者贡献：B.H.完成所有实验和统计分析。W.D.A.、Y.Z.和 S.C.B.L.参与动物实验。S.S.L.参与纳米颗粒的开发与表征。B.H.和 D.J.I.设计实验并撰写论文。竞争利益：与本文所述技术相关的专利正在申请中。数据和材料可用性：本研究中使用的材料未通过材料转移协议从外部获取。
组织学样本由科赫研究所斯旺森生物技术中心切片。Eμ-myc Arf −/− 细胞由 M. T. Hemann 赠送。经费支持：本研究部分由国家癌症研究所的科赫研究所支持（核心）资助金 P30-CA14051、NIH（CA140476 和 CA172164）以及国防部（W81XWH-10-1-0290）资助。D.J.I.是霍华德·休斯医学研究所的研究员。作者贡献：B.H.完成所有实验和统计分析。W.D.A.、Y.Z.和 S.C.B.L.参与动物实验。S.S.L.参与纳米颗粒的开发与表征。B.H.和 D.J.I.设计实验并撰写论文。竞争利益：与本文所述技术相关的专利正在申请中。数据和材料可用性：本研究中使用的材料未通过材料转移协议从外部获取。
Supplementary Material 补充材料
Summary 摘要
Materials and Methods 材料与方法
Fig. S1. Eμ-myc lymphoma cells express homing markers for peripheral lymph nodes, gut, and bone marrow.
图 S1. Eμ-myc 淋巴瘤细胞表达归巢标志物，可定向至外周淋巴结、肠道及骨髓。
图 S1. Eμ-myc 淋巴瘤细胞表达归巢标志物，可定向至外周淋巴结、肠道及骨髓。
Fig. S2. T cells expanded in IL-2 with or without rapamycin express similar activation and subset markers.
图 S2. 在有或无雷帕霉素的情况下，通过 IL-2 扩增的 T 细胞表达相似的激活和亚群标志物。
图 S2. 在有或无雷帕霉素的情况下，通过 IL-2 扩增的 T 细胞表达相似的激活和亚群标志物。
Fig. S3. NCs are synthesized by fusion and covalent stabilization of SN-38–containing liposomes.
图 S3. 通过融合和共价稳定 SN-38 脂质体合成纳米载体。
图 S3. 通过融合和共价稳定 SN-38 脂质体合成纳米载体。
Fig. S4. T cell transfer alone has no impact on lymphoma progression.
图 S4. 单纯 T 细胞转移对淋巴瘤进展无影响。
图 S4. 单纯 T 细胞转移对淋巴瘤进展无影响。
Fig. S5. Animals treated with SN-38 therapies show no systemic toxicity.
图 S5. 接受 SN-38 疗法的动物未表现出系统性毒性。
图 S5. 接受 SN-38 疗法的动物未表现出系统性毒性。
Reference (47) 参考文献 (47)
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Science Translational Medicine
Volume 7 | Issue 291
June 2015
June 2015
Copyright
Copyright © 2015, American Association for the Advancement of Science.
Submission history
Received: 22 December 2014
Accepted: 20 April 2015
Acknowledgments
Histology samples were sectioned by the Koch Institute Swanson Biotechnology Center. Eμ-myc Arf−/− cells were a gift of M. T. Hemann. Funding: This work was supported in part by the Koch Institute Support (core) grant P30-CA14051 from the National Cancer Institute, the NIH (CA140476 and CA172164), and the Department of Defense (W81XWH-10-1-0290). D.J.I. is an investigator of the Howard Hughes Medical Institute. Author contributions: B.H. performed all experiments and statistical analyses. W.D.A., Y.Z., and S.C.B.L. contributed to animal experiments. S.S.L. contributed to nanoparticle development and characterization. B.H. and D.J.I. designed the experiments and wrote the paper. Competing interests: A patent related to the technology described here is pending. Data and materials availability: No material used in this study was obtained from external parties under material transfer agreement.
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Figures
Fig. 1. SN-38 is a potent cytotoxic agent against Eμ-myc lymphoma cells but fails to access sites of lymphoma dissemination in vivo.
C57BL/6J recipients were injected intravenously with 1 × 106 luciferase- and GFP-expressing Eμ-myc cells. (A) On day 21 after inoculation, lymphoma biodistribution was imaged by IVIS in intact animals, followed by flow cytometry to detect GFP+ tumor cells (green gates). Images are representative ventral and side views of the same animal. LN, lymph node; PE, phycoerythrin. (B) On day 17 after inoculation, free SN-38 (10 mg/kg) was injected intravenously into tumor-bearing mice, and tissue drug concentrations were measured by high-performance liquid chromatography (HPLC) over time. Data are means ± SEM (n = 3 per group). (C) On day 17 after inoculation, tumor-bearing mice were intravenously injected with empty fluorescent liposomes (36.3 mg/kg lipid). Tissues were collected 24 hours after injection for histology. (D) On day 17 after inoculation, tumor-bearing mice were intravenously injected with SN-38–containing liposomes (1 mg/kg SN-38). Tissues were collected 24 hours after injection for HPLC analysis. All data are representative of one of two independent experiments (n = 3 animals per group). Dashed lines in (B) and (D) denote limit of detection (0.5 ng/g tissue) for SN-38 by HPLC.
Fig. 2. IL-2/rapamycin–expanded T cells express homing receptors to traffic to lymphoma sites and are resistant to SN-38 toxicity.
(A) Schematic of T cell functionalization and cell-mediated delivery of SN-38 NCs into tumors. (B and C) Polyclonal T cells from C57BL/6J mice were primed with concanavalin A and IL-7 for 2 days, then expanded in IL-2 with or without rapamycin for 2 days, and analyzed for expression of tissue-homing receptors by flow cytometry. Shown are representative staining histograms (B) and quantification markers (C) of n = 7 replicate cultures from two independent experiments. Data are means ± SEM. P value was determined by t test. MFI, median fluorescence intensity. (D) Eμ-myc cells or IL-2/rapamycin–expanded T cells were cultured in vitro with SN-38 at indicated doses, and viability was assessed by flow cytometry after 24 hours. Data are means ± SEM of pooled four to eight replicate cultures from four independent experiments. (E) IL-2– or IL-2/rapamycin–cultured T cells were taken directly from culture or incubated with SN-38 (20 ng/ml) for 12 hours, then stained for intracellular Bcl-2 expression, and analyzed by flow cytometry. Data are representative plots for n = 3 cultures per condition from one of three independent experiments. P values were determined by two-way analysis of variance (ANOVA) with Bonferroni post-test. (F) IL-2– or IL-2/rapamycin–cultured Thy1.1+ T cells (40 × 106) were adoptively transferred into C57BL/6J mice (n = 6 to 7 per group). The number of Thy1.1+ T cells in blood, spleen, and lymph nodes was enumerated by flow cytometry 2 days after transfer. Data are means ± SEM from one of two independent experiments. P values were determined by t test.
Fig. 3. T cells conjugated with SN-38 NCs kill bystander lymphoma cells but not the T cells themselves.
(A) Cryo–electron microscopy image of SN-38 NCs. (B) Kinetics of SN-38 release from NCs at 37 °C in 10% serum. Data are means ± SEM (n = 8). (C) T cells stained with carboxyfluorescein diacetate succinimidyl ester (blue) and conjugated to fluorescently labeled NCs (pink) were imaged by confocal microscopy. (D) T cells were conjugated to fluorescent DiD NCs either lacking maleimide-lipid (control NCs) or containing maleimide-headgroup lipids (maleimide NCs), washed, and then analyzed by flow cytometry. (E) T cells were conjugated with SN-38 NCs over a range of NC/cell ratios and then lysed to measure the final conjugated amount of SN-38. (F and G) Eμ-myc cells were cocultured with unmodified T cells, empty (“blank”) NC–conjugated T cells, or SN-38 NC–conjugated T cells at indicated T cell/lymphoma cell ratios. Viability of Eμ-myc cells (F) and T cells (G) was measured by flow cytometry 24 hours later. Data are means ± SEM (n = 3 to 8 samples per group). ns, not significant, two-way ANOVA with Bonferroni post-test. All data are representative of two to three independent experiments.
Fig. 4. SN-38 NC–conjugated T cells traffic into lymphoma-bearing lymph nodes in vivo and sustain elevated intranodal SN-38 levels over time.
C57BL/6J mice (n = 3 to 5 per group) were injected intravenously with 1 × 106 Eμ-myc cells and, 17 days later, received intravenous injection of 2 × 108 luciferase+ SN-38–carrying NC-T cells (1 mg/kg equivalent SN-38), control unmodified T cells, or an equivalent dose of free NCs. (A) T cell biodistribution was assessed by whole-animal bioluminescence 38 hours after transfer. Images are ventral views of three different animals and side view of one representative animal. (B) Kinetics of T cell and SN-38 NC-T cell accumulation in tissues assessed by flow cytometry. Data are means ± SEM (n = 4 to 12 animals per group per time point), pooled from five independent experiments. **P < 0.01 versus T cells alone, by two-way ANOVA with Bonferroni post-test. (C) Mice (n = 4 per group) were injected with SN-38 NC–conjugated fluorescently labeled T cells, and tumor (Eμ-myc)–bearing lymph nodes were harvested at 24 hours after injection for histological analysis. Representative image from one of two experiments. (D) Retention of fluorescently labeled SN-38 NCs by T cells (Thy1.1+) in tumor-bearing lymph nodes was measured by flow cytometry 15 hours after transfer. Plot is from one of two representative experiments. (E) Animals were treated with free SN-38 NCs, SN-38 NC-T cells, or free SN-38 at a dose 10-fold greater than that in the NCs. Tumor-bearing lymph nodes were harvested at various times after treatment for HPLC analysis. Data are means ± SEM (n = 3 to 5 animals per group per time point, pooled from four independent experiments). Dashed line denotes limit of SN-38 detection (0.5 ng/g tissue). *P < 0.05, ****P < 0.0001 versus SN-38 NCs, by two-way ANOVA with Bonferroni post-test.
Fig. 5. T cell–mediated delivery of NCs improves the therapeutic efficacy of SN-38 without toxicity.
(A and B) Albino C57BL/6J mice (n = 5 animals per group) were inoculated with 1 × 106 Eμ-myc cells on day 0 and then received four treatments of free SN-38, free SN-38 NCs, or 2 × 108 NC-T cells (1 mg/kg SN-38 in each group), as indicated [arrows on (B)]. Shown are representative results from one of two independent experiments. (A) Bioluminescence images of tumor burden on day 16. (B) Tumor burden as assessed by Eμ-myc bioluminescence (normalized to signal at the start of therapy) over time. *P < 0.01 by two-way ANOVA with Bonferroni post-test on day 16. (C and D) Albino C57BL/6J mice (n = 5 to 6 animals per group) were inoculated as in (A) and then received free SN-38 at 1 or 10 mg/kg, free SN-38 NCs at 1 mg/kg, or 2 × 108 or 5 × 107 NC-T cells (1 or 0.25 mg/kg SN-38, respectively) every 3 days, starting on day 5, for a total of seven treatments. Shown are representative results from one of two independent experiments. (C) Normalized total body bioluminescence measured on day 15. **P < 0.01, by t test. (D) Overall survival. ***P < 0.001 by log-rank test. (E and F) Animals were inoculated and treated as in (C). (E) Weights of animals normalized individually to the pretherapy weight. (F) On day 28, serum was collected for alanine aminotransferase (ALT) and blood urea nitrogen (BUN) measurement; dashed lines indicate reference healthy ranges. Data shown are means ± SEM (n = 4 to 6 animals per group). Shown are representative results from one of three independent experiments.
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