Cellular backpacks for macrophage immunotherapy
用于巨噬细胞免疫治疗的细胞背囊

Backpacks were prepared from biodegradable polymers using microcontact printing (see fig. S1) (16, 19). Each backpack contained a cell-adhesive layer, a poly(lactic-co-glycolic) acid (PLGA) layer, a polyvinyl alcohol (PVA) layer, and a second PLGA layer (Fig. 2AOpens in image viewer
利用微接触印刷技术，从可生物降解的聚合物中制备了背包（见图 S1）（16, 19）。每个背包包含一个细胞粘附层、一层聚乳酸-羟基乙酸共聚物（PLGA）、一层聚乙烯醇（PVA）以及第二层 PLGA（图 2A）。, i and ii). PVA was chosen as the interior layer due to its hydrophilicity, enabling facile incorporation of cytokine. We chose interferon-γ (IFN-γ) due to its potency in stimulating proinflammatory macrophages and its robust antitumor activity (20). PLGA was chosen to provide structural support to the PVA layer. The cell-adhesive layer was made by layer-by-layer (LBL) assembly. It comprised two sets of alternating layers of hyaluronic acid modified with aldehyde (HA-Ald) and poly(allylamine) hydrochloride (PAH). After 1.5 hours of incubation, we found that backpacks with a cell-adhesive layer bound to 86.9% of bone marrow–derived macrophages (BMDMs) from BALB/c mice, whereas backpacks without a cell-adhesive layer bound to only 63.4% of BMDMs (see fig. S2) (21). Backpacks displayed an average stiffness of 292 ± 67 MPa, an average thickness of 1.49 ± 0.14 μm, and an average width of 7.56 ± 0.37 μm, as determined by atomic force microscopy (AFM) (Fig. 2AOpens in image viewer
i 和 ii）。我们选择聚乙烯醇（PVA）作为内层，因其亲水性，便于细胞因子轻松嵌入。鉴于干扰素-γ（IFN-γ）在刺激促炎性巨噬细胞方面的强大效果及其显著的抗肿瘤活性（20），我们选用了它。聚乳酸-羟基乙酸共聚物（PLGA）则被选用以提供对 PVA 层的结构支撑。细胞粘附层通过层层组装（LBL）技术制成，由两组交替的透明质酸醛修饰层（HA-Ald）和聚（烯丙胺）盐酸盐（PAH）构成。经过 1.5 小时的孵育，我们发现带有细胞粘附层的背包装置能够结合 86.9%的 BALB/c 小鼠骨髓来源巨噬细胞（BMDMs），而没有细胞粘附层的背包装置仅能结合 63.4%的 BMDMs（见图 S2）（21）。通过原子力显微镜（AFM）测定，背包装置的平均刚度为 292 ± 67 MPa，平均厚度为 1.49 ± 0.14 μm，平均宽度为 7.56 ± 0.37 μm（图 2A）。, iii; see fig. S3).
iii；见图 S3。

We investigated the role of PVA in the interior of the backpacks on stabilizing IFN-γ. We found that increased thicknesses of PVA improved the activity of IFN-γ, despite the same loading of IFN-γ per backpack (Fig. 2BOpens in image viewer
我们研究了聚乙烯醇（PVA）在内背包内部对稳定干扰素-γ（IFN-γ）的作用。研究发现，随着 PVA 厚度的增加，尽管每个背包中 IFN-γ的载量相同，但其活性得到了提升（图 2B）。). This is likely because the PVA stabilized the IFN-γ when the second layer of PLGA dissolved in acetone was deposited. While thicker PVA layers improved the activity of IFN-γ, we fixed the thickness to 0.62 μm for the remainder of the study, as higher PVA content reduced printing efficiency (see fig. S4A). Next, we investigated the release of IFN-γ from the backpacks into serum media at 37°C over time (Fig. 2COpens in image viewer
).这可能是因为 PVA 在第二层 PLGA 溶于丙酮沉积时稳定了 IFN-γ。虽然较厚的 PVA 层提高了 IFN-γ的活性，但为了研究余下部分，我们将厚度固定为 0.62 微米，因为更高的 PVA 含量降低了打印效率（见图 S4A）。接着，我们研究了 IFN-γ从背包中在 37°C 的培养基中随时间的释放情况（图 2C）。). We found that backpacks released IFN-γ for at least 60 hours. We also found that backpacks maintained activity of IFN-γ after printing and storage for 3 months at −80°C (see fig. S5).
我们发现，背包能释放 IFN-γ至少 60 小时。此外，我们还发现，背包在打印后并在−80°C 下储存 3 个月后，仍能保持 IFN-γ的活性（见图 S5）。

Next, we evaluated the interaction of backpacks with primary BMDMs using two techniques. First, we examined the association of fluorescent backpacks with cells using flow cytometry, which included both surface-bound and phagocytosed backpacks (Fig. 2DOpens in image viewer
接下来，我们通过两种技术评估了背包与原代骨髓来源巨噬细胞（BMDMs）的相互作用。首先，我们利用流式细胞术检测了荧光背包与细胞的结合情况，包括表面结合型和被吞噬型背包（图 2D）。). We found that backpacks encapsulating IFN-γ displayed a higher affinity to BMDMs than those without, which is likely due to the enhanced activity of macrophages when stimulated by IFN-γ. Over 5 days, the association of IFN-γ backpacks reduced from 83.6 to 75.4%, whereas the association of blank backpacks reduced from 77.5 to 61.2%. Second, we examined the resistance of IFN-γ backpacks to phagocytosis compared with spheres of similar volumes using fluorescence microscopy (Fig. 2EOpens in image viewer
我们发现，封装有 IFN-γ的背包对骨髓来源巨噬细胞（BMDMs）的亲和力高于未封装者，这可能是因为 IFN-γ刺激下巨噬细胞活性增强所致。在 5 天内，IFN-γ背包的结合率从 83.6%降至 75.4%，而空白背包的结合率则从 77.5%降至 61.2%。其次，我们通过荧光显微镜比较了 IFN-γ背包与体积相似的球体对吞噬作用的抵抗能力（图 2E）。). We compared the number of surface-bound backpacks (V_BP = 49.8 μm³) to the number of surface-bound 3.3-μm spheres (V_3.3 = 18.8 μm³) and 6.2-μm spheres (V_6.2 = 124.8 μm³). Over 5 days, the proportion of backpacks that remained surface bound reduced from 89.1 to 77.3%, whereas spheres of both sizes were nearly completely internalized after 3 days (<5% remained surface-bound). Together, both sets of data suggest that the majority of cell-associated backpacks evaded phagocytosis for at least 5 days. We also imaged cells (labeled with NucBlue, blue; coumarin 6, green) displaying backpacks made from rhodamine B PLGA discs (red) using confocal microscopy (Fig. 2FOpens in image viewer
我们比较了表面结合的背包数量（V _BP = 49.8 μm ³ ）与表面结合的 3.3 微米球体（V _3.3 = 18.8 μm ³ ）和 6.2 微米球体（V _6.2 = 124.8 μm ³ ）的数量。在 5 天内，表面结合的背包比例从 89.1%下降至 77.3%，而两种尺寸的球体在 3 天后几乎完全被内化（<5%仍保持表面结合）。综合两组数据，表明大多数与细胞结合的背包至少在 5 天内逃避了吞噬作用。我们还通过共聚焦显微镜成像显示了携带罗丹明 B PLGA 圆盘（红色）背包的细胞（用 NucBlue 标记，蓝色；香豆素 6，绿色）（图 2F）。).

To assess the potency of IFN-γ backpacks to potentiate a durable shift in polarization, we evaluated the expression of several markers associated with M1 and M2 phenotypes (Fig. 3Opens in image viewer
为了评估 IFN-γ背包增强持久极化转变的效果，我们检测了与 M1 和 M2 表型相关的多个标志物的表达（图 3）。). BMDMs were cultured from murine bone marrow progenitor cells, and IFN-γ backpacks were added to cells in a ratio of 3:2, respectively. After 1.5 hours, unbound backpacks were removed, and cells were cultured for 24 to 120 hours. In addition to BMDMs with IFN-γ backpacks in standard culture conditions, we also cultured BMDMs (i) without backpacks (no IFN-γ), (ii) with blank backpacks (no IFN-γ), and (iii) without backpacks yet with an equivalent dose of free IFN-γ (16 ng/ml). We also cultured BMDMs with IFN-γ backpacks in tumor-mimicking conditions [i.e., in hypoxia (1% O₂) and 10 volume % 4T1-conditioned media]. Each day for 5 days, cells were harvested, stained, and analyzed for molecular expression by flow cytometry. Expression of each biomarker was normalized to unpolarized BMDMs.
BMDMs 来源于小鼠骨髓祖细胞，并以 3:2 的比例分别加入 IFN-γ背包装载。1.5 小时后，移除未结合的背包，细胞再培养 24 至 120 小时。除了标准培养条件下携带 IFN-γ背包的 BMDMs，我们还培养了（i）无背包的 BMDMs（无 IFN-γ），（ii）携带空白背包的 BMDMs（无 IFN-γ），以及（iii）无背包但添加了等量游离 IFN-γ（16 ng/ml）的 BMDMs。此外，我们还在模拟肿瘤环境条件下培养了携带 IFN-γ背包的 BMDMs，即在低氧（1% O ₂ ）和 10%体积比的 4T1 条件培养基中。每天持续 5 天，细胞被收获、染色并通过流式细胞术分析分子表达。每个生物标志物的表达均归一化为未极化的 BMDMs。

Macrophages carrying IFN-γ backpacks strongly exhibited traits of M1 phenotypes. We investigated the relative expression of M1 biomarkers, including inducible NO synthase (iNOS), major histocompatibility complex class II (MHCII), and CD80, because of their important role in innate immunity. iNOS is involved in the production of NO, which serves as a potent tumoricidal and antimicrobial agent (22). MHCII proteins are involved in antigen presentation to T cells to facilitate adaptive immunity (23). MHCII is expressed on macrophages with M1 and M2 phenotypes, but it is overexpressed on cells with M1 polarizations. CD80 is a costimulatory molecule used to trigger an adaptive immune response in the presence of an antigen-presenting cell (24).
携带 IFN-γ背囊的巨噬细胞显著展现出 M1 表型的特征。我们研究了 M1 标志物的相对表达情况，包括诱导型一氧化氮合酶（iNOS）、主要组织相容性复合体 II 类（MHCII）和 CD80，因为它们在先天免疫中扮演重要角色。iNOS 参与 NO 的生成，NO 作为一种强效的抗肿瘤和抗菌剂（22）。MHCII 蛋白参与向 T 细胞呈递抗原，以促进适应性免疫（23）。MHCII 在 M1 和 M2 表型的巨噬细胞上均有表达，但在 M1 极化细胞中表达量更高。CD80 是一种共刺激分子，在抗原呈递细胞存在时，用于触发适应性免疫反应（24）。

Macrophages displaying IFN-γ backpacks showed marked increases in iNOS, MHCII, and CD80 expression relative to unpolarized cells (Fig. 3AOpens in image viewer
展示 IFN-γ背包的巨噬细胞相对于未极化细胞，其 iNOS、MHCII 和 CD80 表达显著增加（图 3A）。). Here, we make several important observations. First, expression of both iNOS and MHCII in cells displaying IFN-γ backpacks was synergistic. Specifically, iNOS expression was 629.3-fold higher in the IFN-γ backpack group compared with only 2.4- and 1.3-fold higher in groups treated with blank backpacks and IFN-γ alone, respectively, after 48 hours. Similarly, MHCII expression was 6.3-folder higher in the IFN-γ backpack group compared with only 0.8- and 1.4-fold higher in groups treated with blank backpacks and IFN-γ alone, respectively, after 48 hours. While the origins of this apparent synergy needs further investigation, the differential protein expressions may arise, at least in part, from local and sustained concentration gradients of IFN-γ formed near the cells to which the backpacks are bound, thus enhancing the activity of IFN-γ. Second, the data suggest that the presence of backpacks without IFN-γ (blank backpacks) induces modest, but non-negligible phenotypic shifts toward M1 phenotypes, as evidenced by increased expressions of iNOS, MHCII, and CD80. This effect could be due to frustrated phagocytosis, whereby macrophages enhance their inflammatory phenotypes upon encountering large foreign objects (25, 26). Third, the expression of M1-related markers in BMDMs carrying IFN-γ backpacks was more durable than that of BMDMs cultured with free IFN-γ. Specifically, the relative expression of iNOS decreased by 89.1% after 5 days in cells treated with free IFN-γ, but only by 59.1% in cells with IFN-γ backpacks. Further, the relative expression of MHCII and CD80 decreased by 30.1 and 37.6%, respectively, after 5 days for cells treated with free IFN-γ; however, the relative expression of MHCII and CD80 in cells treated with IFN-γ backpacks actually increased by 95.7 and 248.4%, respectively, after 5 days. Last, no major differences in marker expression were observed between BMDMs with IFN-γ backpacks in standard culture conditions versus tumor-mimicking conditions, which we hypothesize will be critical to allow BMDMs to maintain M1 phenotypes in vivo. Overall, these data suggest that IFN-γ backpacks potentiate a shift in macrophage polarization toward M1 phenotypes that is more potent and durable than free IFN-γ.
在此，我们做出了几项重要观察。首先，在展示 IFN-γ背包的细胞中，iNOS 和 MHCII 的表达呈现协同效应。具体而言，48 小时后，与空白背包组和单独使用 IFN-γ组相比，iNOS 表达在 IFN-γ背包组中分别高出 629.3 倍、2.4 倍和 1.3 倍。同样，MHCII 表达在 IFN-γ背包组中高出 6.3 倍，而空白背包组和单独使用 IFN-γ组仅分别高出 0.8 倍和 1.4 倍。尽管这种明显协同效应的起源尚需进一步研究，但其差异蛋白表达可能至少部分源于在背包所附着细胞附近形成的局部且持续的 IFN-γ浓度梯度，从而增强了 IFN-γ的活性。其次，数据显示，虽然不含 IFN-γ的背包（空白背包）诱导了适度但不可忽视的 M1 表型转变，表现为 iNOS、MHCII 和 CD80 表达的增加。 这种效应可能是由于受挫的吞噬作用所致，即巨噬细胞在遇到大型异物时会增强其炎症表型（25, 26）。第三，携带 IFN-γ背包的 BMDMs 中 M1 相关标记的表达比自由 IFN-γ培养的 BMDMs 更为持久。具体而言，在用自由 IFN-γ处理的细胞中，5 天后 iNOS 的相对表达量下降了 89.1%，而携带 IFN-γ背包的细胞中仅下降了 59.1%。此外，自由 IFN-γ处理 5 天后，MHCII 和 CD80 的相对表达量分别下降了 30.1%和 37.6%；然而，使用 IFN-γ背包处理的细胞中，MHCII 和 CD80 的相对表达量在 5 天后分别增加了 95.7%和 248.4%。最后，在标准培养条件与模拟肿瘤条件下，携带 IFN-γ背包的 BMDMs 之间在标记物表达上未见显著差异，我们推测这对 BMDMs 在体内维持 M1 表型至关重要。总体而言，这些数据表明，IFN-γ背包能更有效地且持久地促使巨噬细胞极化向 M1 表型转变，其效果优于自由 IFN-γ。

We also investigated the expression of markers associated with M2 phenotypes: vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α), and CD206. VEGF is often overexpressed in TAMs, which serves as a source of angiogenic cytokines and proteases to promote tumor vascularization (27). HIF-1α is also overexpressed by TAMs, which suppresses T cell function and promotes tumor progression (28). CD206 is the mannose receptor, which has been linked to immunosuppression, angiogenesis, and metastasis (29). Cells displaying IFN-γ backpacks showed elevated levels of all three M2 markers relative to untreated controls; however, the magnitude of this increase was modest. The highest fold changes observed were 2.7, 3.3, and 2.6 for VEGF, HIF-1α, and CD206, respectively. These changes were less substantial than those observed for M1 markers, and the relative expression of all three M2 markers returned to values near the expression of untreated controls after 5 days.
我们还研究了与 M2 表型相关的标志物表达：血管内皮生长因子（VEGF）、缺氧诱导因子 1α（HIF-1α）和 CD206。VEGF 在肿瘤相关巨噬细胞（TAMs）中常被过度表达，作为促血管生成细胞因子和蛋白酶的来源，促进肿瘤血管化（27）。HIF-1α同样由 TAMs 过度表达，抑制 T 细胞功能并促进肿瘤进展（28）。CD206 是甘露糖受体，与免疫抑制、血管生成和转移有关（29）。表达 IFN-γ背包的细胞显示出三种 M2 标志物的水平均较未处理对照组有所升高；然而，这种增加的程度较为温和。观察到的最高倍数变化分别为 VEGF 的 2.7 倍、HIF-1α的 3.3 倍和 CD206 的 2.6 倍。这些变化不如 M1 标志物显著，且所有三种 M2 标志物的相对表达在 5 天后恢复至接近未处理对照组的水平。

Next, we sought to test our central hypothesis that macrophages carrying IFN-γ backpacks can maintain their M1 phenotypes in vivo. We chose orthotopic 4T1 breast tumors as a model immunosuppressive environment due to its association with chemotherapy resistance, tumor metastasis, and lack of tumor-specific antigens, making them challenging targets for CAR T cell therapy (10, 30, 31). To distinguish injected macrophages from TAMs, we stained BMDMs with VivoTrack 680 (see the Supplementary Materials). Macrophages were injected intratumorally. Distributions of injected cells were monitored each day for 5 days using an in vivo imaging system (IVIS) (see fig. S6). After 5 days, a second injection was administered as before. Mouse body weight, tumor growth, tumor radiance, and necrosis were monitored to the end of the study (see fig. S7). Two days after the second injection, mice were euthanized, and their tumors were extracted, digested, and tumor-associated immune cells (CD45⁺) were isolated. Dendritic cells and macrophages were stained and identified by hierarchical gating (see fig. S8). Macrophages were phenotyped as before (see table S1), except markers were used for arginase 1 (arg-1) instead of VEGF. Arg-1 affects NO synthase and down-regulates NO production (32). Expression of each marker was normalized to that of endogenous TAMs in mice treated with saline.
接下来，我们旨在验证我们的核心假设，即携带 IFN-γ背包的巨噬细胞在体内能够维持其 M1 表型。我们选择原位 4T1 乳腺癌肿瘤作为免疫抑制环境模型，因其与化疗耐药、肿瘤转移以及缺乏肿瘤特异性抗原相关，使其成为 CAR-T 细胞疗法的挑战性靶点（10, 30, 31）。为区分注射的巨噬细胞与肿瘤相关巨噬细胞（TAMs），我们对骨髓来源的巨噬细胞（BMDMs）进行了 VivoTrack 680 染色（详见补充材料）。巨噬细胞通过瘤内注射给药。利用活体成像系统（IVIS）每天监测注射细胞的分布，持续 5 天（见图 S6）。5 天后，如前所述进行第二次注射。在整个研究期间，监测小鼠体重、肿瘤生长、肿瘤发光强度及坏死情况（见图 S7）。第二次注射后两天，小鼠被安乐死，取出肿瘤并消化，分离出肿瘤相关免疫细胞（CD45+）。通过层次门控法对树突状细胞和巨噬细胞进行染色和鉴定（见图 S8）。 巨噬细胞按之前的方法进行表型鉴定（见表 S1），只是用精氨酸酶 1（arg-1）的标志物代替了 VEGF。Arg-1 影响一氧化氮合酶，并下调一氧化氮的产生（32）。每个标志物的表达量均归一化为经生理盐水处理的实验小鼠中内源性肿瘤相关巨噬细胞（TAMs）的表达水平。

Macrophages carrying IFN-γ backpacks retained M1 polarizations in solid tumors for at least 48 hours (Fig. 4AOpens in image viewer
携带 IFN-γ背包的巨噬细胞在实体肿瘤中至少保留了 48 小时的 M1 极化状态（图 4A）。). Relative to the TAMs of control mice (i.e., mice injected with saline), the expression of iNOS, MHCII, and CD80 in injected macrophages displaying IFN-γ backpacks was significantly higher than that of injected cells displaying blank backpacks or injected cells with free IFN-γ. The relative increase in MHCII and CD80 expression of cells carrying IFN-γ backpacks surpassed that of cells carrying IFN-γ backpacks in vitro (12.3- and 3.0-fold in vivo versus 6.3- and 1.3-fold in vitro for MHCII and CD80, respectively; Figs. 3AOpens in image viewer
相对于对照组小鼠的肿瘤相关巨噬细胞（即注射了生理盐水的 mice），展示 IFN-γ 背包的注射巨噬细胞中 iNOS、MHCII 和 CD80 的表达显著高于展示空白背包的注射细胞或注射自由 IFN-γ 的细胞。携带 IFN-γ 背包的细胞在 MHCII 和 CD80 表达上的相对增加，超过了体外携带 IFN-γ 背包的细胞（体内分别为 12.3 倍和 3.0 倍，而体外为 6.3 倍和 1.3 倍，分别对应 MHCII 和 CD80；图 3A）。 and 4AOpens in image viewer 和 4A). However, the relative increase in iNOS was less substantial in vivo (7.2-fold in vivo versus 629.3-fold in vitro). The reduction was due to an elevated basal expression of iNOS in the TAMs of control mice compared with untreated control cells in vitro. This observation is consistent with findings by others that iNOS expression can increase in M2-polarized TAMs (33). We also found no statistically significant differences in the relative expression of HIF-1α and CD206 between macrophages carrying IFN-γ backpacks and those carrying free backpacks or injected with free IFN-γ. Although cells carrying IFN-γ backpacks displayed significantly higher levels of Arg-1 relative to those carrying blank backpacks or injected with free IFN-γ, they did not show a significant increase in the expression of Arg-1 relative to untreated TAMs.
然而，iNOS 的相对增加在体内并不显著（体内增加 7.2 倍，而体外增加 629.3 倍）。这种减少是由于与体外未处理的控制细胞相比，对照组小鼠的肿瘤相关巨噬细胞（TAMs）中 iNOS 的基础表达水平升高所致。这一观察结果与其他研究者的发现一致，即 iNOS 表达在 M2 极化的 TAMs 中可能增加（33）。我们还发现，携带 IFN-γ背囊的巨噬细胞与携带自由背囊或注射自由 IFN-γ的巨噬细胞之间，HIF-1α和 CD206 的相对表达没有统计学上的显著差异。尽管携带 IFN-γ背囊的细胞相对于携带空白背囊或注射自由 IFN-γ的细胞显示出显著更高的 Arg-1 水平，但它们相对于未处理的 TAMs 并未表现出 Arg-1 表达的显著增加。

After demonstrating that the IFN-γ backpacks allowed macrophages to maintain their phenotypes in vivo, we sought to evaluate the phenotype of TAMs in response to adoptive transfer of macrophages carrying IFN-γ backpacks (see fig. S8 for hierarchical gating). An emergent therapeutic strategy to attack tumorous tissues is via repolarizing TAMs toward M1 phenotypes (34–39). TAMs affect cancer progression in a manner that is dependent on their polarization (40–42). Macrophages possessing M1 phenotypes have been shown to improve outcomes in cancer therapy due to their antigen-dependent and antigen-independent facets. This gives macrophages the potential to be useful in tumors that lack the tumor-specific antigens typically required for adoptive T cell therapy (43–45). This has been demonstrated by others through the delivery of nanoparticles with payloads that inhibit colony stimulating factor 1 receptor (CSF1-R) and Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP2) pathways on macrophages (46) as well as the delivery of nanoparticles encapsulating microRNA-125b (47). Still, supplying a sufficient concentration of immunomodulatory factors to repolarize TAMs while minimizing toxicity remains a major challenge.
在证实 IFN-γ背包能够使巨噬细胞在体内维持其表型后，我们试图评估接受携带 IFN-γ背包巨噬细胞过继转移后肿瘤相关巨噬细胞（TAMs）的表型变化（分层门控策略见图 S8）。一种新兴的治疗策略是通过将 TAMs 极化为 M1 表型来攻击肿瘤组织（34–39）。TAMs 对癌症进展的影响取决于其极化状态（40–42）。具有 M1 表型的巨噬细胞因其在抗原依赖性和抗原非依赖性方面的特性，已被证明能改善癌症治疗效果。这使得巨噬细胞在缺乏通常用于过继 T 细胞治疗所需的肿瘤特异性抗原的肿瘤中具有潜在应用价值（43–45）。这一点已通过其他研究得以证实，他们通过递送负载抑制集落刺激因子 1 受体（CSF1-R）和 Src 同源区 2（SH2）结构域含磷酸酶 1（SHP2）通路的纳米颗粒（46）以及递送封装微小 RNA-125b 的纳米颗粒（47）来实现。 然而，在最小化毒性的同时，提供足够浓度的免疫调节因子以重新极化肿瘤相关巨噬细胞（TAMs）仍是一个重大挑战。

We administered two intratumoral injections of macrophages with 50 ng of IFN-γ per mouse, which is 100-fold lower than the maximum total dose (MTD) administered in other studies (48). Our motivation for the comparatively low dose was to supply sufficient IFN-γ to maintain the M1 polarization of adoptively transferred macrophages while minimizing toxic side effects (49). Here, mice received two equivalent injections of saline (control) and (i) macrophages polarized ex vivo for 24 hours in IFN-γ (20 ng/ml; M1 polarized), (ii) unpolarized macrophages injected with 50 ng of free IFN-γ, and (iii) macrophages carrying IFN-γ backpacks that encapsulated 50 ng of IFN-γ (Fig. 4BOpens in image viewer
我们为每只小鼠注射了两剂含有 50 纳克 IFN-γ的巨噬细胞，这一剂量比其他研究中使用的最大总剂量（MTD）低 100 倍（48）。我们选择相对较低的剂量是为了提供足够的 IFN-γ以维持过继转移巨噬细胞的 M1 极化，同时尽量减少毒性副作用（49）。在此实验中，小鼠接受了两次等量的生理盐水注射（对照组）以及（i）在体外用 IFN-γ（20 纳克/毫升；M1 极化）极化 24 小时的巨噬细胞，（ii）未极化的巨噬细胞注射了 50 纳克游离 IFN-γ，和（iii）携带 IFN-γ背包的巨噬细胞，这些背包包裹了 50 纳克 IFN-γ（图 4B）。). Administrations were separated by 5 days.
给药间隔为 5 天。

We found that TAMs of mice treated with the IFN-γ backpack therapy were polarized toward M1 phenotypes, as evidenced by significantly increased expressions of iNOS (1.8-fold) and MHCII (1.6-fold) compared with TAMs of mice treated with saline (Fig. 4BOpens in image viewer
我们发现，接受 IFN-γ背包疗法的小鼠的肿瘤相关巨噬细胞（TAMs）向 M1 表型极化，表现为与接受生理盐水的对照组小鼠的 TAMs 相比，iNOS（增加 1.8 倍）和 MHCII（增加 1.6 倍）的表达显著增加（图 4B）。). Second, the relative increase in iNOS expression in TAMs of mice treated with the IFN-γ backpack therapy was significantly higher than in TAMs of mice treated with macrophages polarized ex vivo (1.8- versus 1.0-fold, respectively) (ii). Third, the relative increase in CD80 expression in TAMs of mice treated with the IFN-γ backpack therapy was significantly higher than in TAMs of mice treated with macrophages plus free IFN-γ (1.03- versus 0.78-fold, respectively) (iii).
其次，与体外极化的巨噬细胞治疗相比，接受 IFN-γ背包装疗法的小鼠 TAMs 中 iNOS 表达的相对增加显著更高（分别为 1.8 倍与 1.0 倍）（ii）。第三，与接受巨噬细胞联合自由 IFN-γ治疗的小鼠 TAMs 相比，IFN-γ背包装疗法处理的小鼠 TAMs 中 CD80 表达的相对增加显著更高（分别为 1.03 倍与 0.78 倍）（iii）。

We also investigated the relative expression of M2 markers in TAMs. We found that relative HIF-1α expression in TAMs of mice treated with the IFN-γ backpack therapy was significantly lower than in all other groups (Fig. 4BOpens in image viewer
我们还研究了 TAMs 中 M2 标记物的相对表达情况。结果发现，接受 IFN-γ背包疗法的小鼠 TAMs 中 HIF-1α的相对表达量显著低于其他所有组别（图 4B）。). This finding was particularly interesting, as relative HIF-1α expression in macrophages displaying IFN-γ backpacks was higher in vitro (Fig. 3BOpens in image viewer
). 这一发现尤为有趣，因为展示 IFN-γ背包的巨噬细胞在体外表现出相对较高的 HIF-1α表达（图 3B）。). TAM expression of CD206 was also significantly lower for mice treated with IFN-γ backpacks than saline. However, the group that displayed the lowest relative expression of CD206 in TAMs was in mice treated with macrophages plus free IFN-γ. No significant differences were observed in the relative expression of Arg-1. Overall, these data show that macrophages carrying IFN-γ backpacks can shift the polarization TAMs toward M1 phenotypes at a markedly reduced dose, 100-fold lower than the MTD (48). In addition, the same dose of free IFN-γ was not able to potentiate a shift in TAM polarization. Given this these findings, we sought to examine the therapeutic efficacy of macrophages with IFN-γ backpacks.
TAM 中 CD206 的表达在经 IFN-γ背包处理的小鼠中显著低于生理盐水组。然而，TAM 中 CD206 相对表达最低的组别为接受巨噬细胞加游离 IFN-γ治疗的小鼠。Arg-1 的相对表达未见显著差异。总体而言，这些数据表明，携带 IFN-γ背包的巨噬细胞能够在显著降低的剂量下，将 TAM 极化向 M1 表型转变，其剂量仅为最大耐受剂量（MTD）的百分之一（48）。此外，相同剂量的游离 IFN-γ未能促进 TAM 极化的转变。基于这些发现，我们进一步探究了携带 IFN-γ背包的巨噬细胞的治疗效果。

To evaluate the therapeutic efficacy of IFN-γ backpacks, we investigated the formation of metastases, tumor growth kinetics, and overall survival of immunocompetent BALB/c mice burdened with 4T1-Luc cells. 4T1-Luc cells were chosen because of their high luciferase expression, enabling bioluminescence imaging to visualize the formation of metastatic colonies in the chest cavities by radiance using an IVIS. We administered the same low dose of IFN-γ as before to understand the influence of the IFN-γ backpacks. After tumors became palpable (~50 mm³), mice received two equivalent injections (separated by 5 days) of (i) saline, (ii) unpolarized macrophages with 50 ng of free IFN-γ, and (iii) macrophages carrying IFN-γ backpacks encapsulating 50 ng of IFN-γ.
为评估 IFN-γ背包的治疗效果，我们研究了携带 4T1-Luc 细胞的免疫健全 BALB/c 小鼠的转移灶形成、肿瘤生长动力学及总体生存情况。选择 4T1-Luc 细胞因其高表达荧光素酶，可通过 IVIS 系统利用生物发光成像技术观察胸腔内转移灶的形成情况。我们采用与之前相同的低剂量 IFN-γ，以探究 IFN-γ背包的影响。当肿瘤可触及（约 50 mm ³ ）后，小鼠接受两次等量注射（间隔 5 天），分别为：(i) 生理盐水，(ii) 未极化巨噬细胞加 50 ng 游离 IFN-γ，以及(iii) 携带 IFN-γ背包（封装 50 ng IFN-γ）的巨噬细胞。

We found that mice treated with the IFN-γ backpack therapy had significantly fewer metastatic nodules than control mice (Fig. 5AOpens in image viewer
我们发现，接受 IFN-γ背包疗法的小鼠比对照组小鼠的转移性结节显著减少（图 5A）。). Chest cavities of mice given the IFN-γ backpack therapy showed 5.2-fold lower radiance compared with that of mice treated with saline and 4.9-fold lower radiance compared with that of mice treated with macrophages and free IFN-γ (Fig. 5BOpens in image viewer
). 接受 IFN-γ背包疗法的小鼠胸腔辐射度比接受生理盐水处理的小鼠低 5.2 倍，比接受巨噬细胞联合游离 IFN-γ处理的小鼠低 4.9 倍（图 5B）。). This suggests that, even at a low dose, IFN-γ backpacks are able to significantly inhibit the formation of metastatic colonies. To assess toxicity, peripheral blood was isolated via cardiac puncture immediately after euthanasia, and serum was analyzed for cytokines (see Methods in the Supplementary Materials). Analysis revealed that all treatments were well tolerated, and IFN-γ levels were below the limit of detection in all groups. This result was expected given that the dose of IFN-γ was 100-fold lower than the MTD used previously (48). One exception was that significantly higher IL-6 was found in the serum of mice treated with macrophages plus free IFN-γ (see fig. S9). Increased expression of IL-6 has been correlated with tumor metastasis (50); however, IL-6 is also secreted by macrophages with anti-inflammatory phenotypes (51).
这表明，即便在低剂量下，IFN-γ背包也能显著抑制转移性集落的形成。为评估毒性，实验在安乐死后立即通过心脏穿刺采集外周血，并分析血清中的细胞因子水平（详见补充材料中的方法部分）。分析结果显示，所有治疗均耐受良好，各组中 IFN-γ水平均低于检测限。鉴于所用 IFN-γ剂量比先前使用的最大耐受剂量（MTD）低 100 倍（48），这一结果在预期之中。唯一的例外是，接受巨噬细胞联合游离 IFN-γ治疗的鼠血清中 IL-6 水平显著升高（见图 S9）。IL-6 表达增加与肿瘤转移相关（50），但具有抗炎表型的巨噬细胞也会分泌 IL-6（51）。

We also assessed tumor morphology and dendritic cell infiltration. For both analyses, tumors from BALB/c mice burdened with 4T1 breast cancer (from the previous study; Fig. 4BOpens in image viewer
我们还评估了肿瘤形态和树突状细胞的浸润情况。在这两项分析中，均采用了携带 4T1 乳腺癌的 BALB/c 小鼠的肿瘤样本（来自前述研究；图 4B）。) were isolated and cut into four vertical portions. One portion was sectioned for histology, and the remaining three portions were digested and stained for phenotypic evaluation by flow cytometry. The top half of the tumor revealed large areas of digested tissue, whereas the bottom half remained largely intact (Fig. 5COpens in image viewer
）被分离并切成四个垂直部分。其中一部分用于组织学切片，其余三部分则被消化并染色，通过流式细胞术进行表型评估。肿瘤的上半部分显示出大面积的消化组织，而下半部分则基本保持完整（图 5C）。). This finding suggests that the areas of highest tumor clearance occurred in regions where the injected cells resided, as all treatments were injected toward the top each tumor. We found that mice treated with the IFN-γ backpack therapy had significantly higher infiltration of CD11c⁺ dendritic cells (Fig. 5DOpens in image viewer
这一发现表明，肿瘤清除率最高的区域是注射细胞所在的部位，因为所有治疗都向每个肿瘤的顶部注射。我们发现，接受 IFN-γ背包疗法的小鼠，其 CD11c 阳性树突状细胞的浸润显著增加（图 5D）。), as determined by the gating schema shown in fig. S8A (see the Supplementary Materials). While not studied here, we believe this could be a promising future direction of study, as higher dendritic cell populations could be used to instruct adaptive immunity as a cancer vaccine (52, 53).
), 如附图 S8A 的门控方案所确定（见补充材料）。尽管本文未对此进行研究，但我们认为这可能是一个有前景的未来研究方向，因为更高的树突状细胞群体可用于作为癌症疫苗来指导适应性免疫（52, 53）。

Last, we evaluated the progression of tumor growth and overall survival of mice treated with the IFN-γ backpack therapy (Fig. 5, E to GOpens in image viewer
最后，我们评估了接受 IFN-γ背包疗法的小鼠的肿瘤生长进程及总体生存情况（图 5，E 至 G）。). Consistent with the metastasis data, mice injected with the IFN-γ backpack therapy showed significantly smaller tumors than the two controls 14 to 23 days after the second therapeutic injection. By 37 days after inoculation, tumors of mice receiving the IFN-γ backpack therapy were 51.9 and 48.3% smaller than those of mice receiving injections of saline and macrophages with free IFN-γ, respectively. Mice receiving the IFN-γ backpack therapy showed significantly improved survival, as determined by a log-rank test. The average time of survival for mice treated with saline, macrophages with free IFN-γ, and macrophages carrying IFN-γ backpacks was 30.7, 31.7, and 35.9 days after inoculation, respectively. Together, the slowed tumor growth, smaller tumor volumes, and decrease in serum IL-6 of mice treated with the IFN-γ backpack therapy likely potentiated the reduced metastatic burdens and improved overall survival compared with controls.
与转移数据一致的是，接受 IFN-γ背包疗法注射的小鼠在第二次治疗注射后的 14 至 23 天内，肿瘤显著小于两组对照组。在接种后 37 天时，接受 IFN-γ背包疗法的小鼠肿瘤体积比接受生理盐水注射和携带游离 IFN-γ的巨噬细胞注射的小鼠分别小 51.9%和 48.3%。通过 log-rank 检验发现，接受 IFN-γ背包疗法的小鼠生存率显著提高。注射生理盐水、携带游离 IFN-γ的巨噬细胞以及携带 IFN-γ背包的巨噬细胞处理的小鼠，其平均存活时间分别为接种后 30.7 天、31.7 天和 35.9 天。综上所述，接受 IFN-γ背包疗法的小鼠肿瘤生长减缓、肿瘤体积缩小及血清 IL-6 水平降低，这些因素可能共同作用，使其转移负担减轻，整体生存率提高，优于对照组。

4T1 mammary carcinoma cells and 4T1-Fluc-Neo/eGFP-Puro cells expressing firefly luciferase were obtained from the American Type Culture Collection and Imanis Life Sciences, respectively. RPMI 1640 media, Dulbecco’s modified Eagle’s medium (DMEM) F12 media, fetal bovine serum (FBS), penicillin and streptomycin (Pen Strep), mouse IFN-γ recombinant protein, Gibco Type 1 Collagenase, SYTOX blue dead cell stain, NucBlue stain, coumarin 6 membrane dye, heparin-coated plasma preparation tubes, and UltraComp eBeads compensation beads were obtained from Thermo Fisher Scientific. Materials for backpack fabrication, including polydimethylsiloxane (PDMS), PAH, HA, PVA, PLGA, and trichloro(1H,1H,2H,2H-perfluorooctyl)silane (FDTS) were obtained from Millipore Sigma. Red blood cell lysing buffer Hybri-Max, Trypan blue, DNAse I, trypsin, and all solvents used were obtained from Millipore Sigma. Cell culture flasks, plates, and conical tubes were obtained from Corning. Mouse T helper cell type 1 (T_H1)/T_H2/T_H17 cytokine quantification kits, cell fixation/permeabilization kits, and cell strainers (40 and 70 μm) were obtained from BD Biosciences. A QuadroMACS separator, a CD45⁺ leukocyte isolation kit, and a mouse tumor dissociation kit were obtained from Miltenyi Biotec. Bambanker cell freezing media and OCT (optimum cutting temperature) compound were obtained from VWR International. Recombinant murine macrophage CSF (M-CSF) was obtained from PeproTech. Murine IFN-γ enzyme-linked immunosorbent assay (ELISA) kits were obtained from R&D Systems. Female BALB/c mice (6 to 8 weeks old) were obtained from Charles River. Information about the antibodies and their related clones and fluorophores are detailed in the Supplementary Materials (table S1).
4T1 乳腺癌细胞和表达萤火虫荧光素酶的 4T1-Fluc-Neo/eGFP-Puro 细胞分别从美国菌种保藏中心和 Imanis 生命科学公司获得。RPMI 1640 培养基、Dulbecco 改良 Eagle 培养基（DMEM）F12 培养基、胎牛血清（FBS）、青霉素和链霉素（Pen Strep）、小鼠 IFN-γ重组蛋白、Gibco I 型胶原酶、SYTOX 蓝死细胞染色剂、NucBlue 染色剂、香豆素 6 膜染料、肝素包被血浆制备管以及 UltraComp eBeads 补偿珠均购自赛默飞世尔科技公司。用于制作背包的材料，包括聚二甲基硅氧烷（PDMS）、聚乙烯亚胺（PAH）、透明质酸（HA）、聚乙烯醇（PVA）、聚乳酸-羟基乙酸共聚物（PLGA）和三氯（1H,1H,2H,2H-全氟辛基）硅烷（FDTS），均购自 Millipore Sigma。红细胞裂解缓冲液 Hybri-Max、台阶蓝、DNA 酶 I、胰蛋白酶及所有溶剂均购自 Millipore Sigma。细胞培养瓶、板和锥形管购自康宁公司。小鼠辅助性 T 细胞类型 1（T _H 1）/T _H 2/T _H 17 细胞因子定量试剂盒、细胞固定/通透试剂盒以及 40 和 70 微米细胞筛网均购自 BD 生物科学公司。 QuadroMACS 分离器、CD45 ⁺ 白细胞分离试剂盒以及小鼠肿瘤组织解离试剂盒均购自 Miltenyi Biotec 公司。Bambanker 细胞冷冻保存液和 OCT（最佳切割温度）复合物由 VWR International 提供。重组小鼠巨噬细胞集落刺激因子（M-CSF）购自 PeproTech 公司。小鼠 IFN-γ酶联免疫吸附测定（ELISA）试剂盒由 R&D Systems 提供。雌性 BALB/c 小鼠（6 至 8 周龄）来自 Charles River 公司。抗体及其相关克隆和荧光染料的详细信息见补充材料（表 S1）。

PDMS templates were prepared by soft lithography using methods similar to those described previously (16). Before use, silicon molds were fabricated by monolithic photolithography and passivated with a thin film of FDTS by vapor deposition (see Methods in the Supplementary Materials). A 10:1 weight ratio of PDMS base to cross-linker from a Sylgard 184 kit was thoroughly mixed and poured on top of the silicon molds in separate petri dishes (~20 g per mold). PDMS was degassed in a desiccator at 25°C until no visible bubbles remained. Dishes were then placed into an oven at 65°C overnight to cure the PDMS. After curing, PDMS templates were removed from the molds by cutting the petri dishes and peeling away the PDMS.
通过软光刻技术制备了 PDMS 模板，采用的方法与先前所述类似（16）。使用前，通过单片光刻技术制作硅模具，并用气相沉积法在其表面覆盖一层薄薄的 FDTS 进行钝化处理（详见补充材料中的方法）。按照 10:1 的重量比，将来自 Sylgard 184 套件的 PDMS 基础材料与交联剂充分混合，并分别倒入装有硅模具的培养皿中（每模具约 20 克）。在 25°C 下，将 PDMS 置于干燥器中脱气，直至无可见气泡残留。随后，将培养皿放入 65°C 的烘箱中过夜以固化 PDMS。固化完成后，通过切割培养皿并剥离 PDMS，将其从模具中取出。

HA (2500 kDa) was modified with aldehyde (HA-Ald) (see Methods in the Supplementary Materials). An aqueous solution of HA-Ald (2 mg/ml) was prepared in 150 mM NaCl (pH 6.8), and a aqueous solution of PAH (2 mg/ml; 17.5 kDa) in 150 mM NaCl (pH 6.8) was prepared. HA-Ald, PAH, and 150 mM NaCl (pH 6.8) solutions were separately poured into weigh boats. PDMS templates were rinsed with isopropyl alcohol and dried by a steady stream of air. Templates were then placed patterned side down in the HA-Ald solution for 15 min. Care was taken to ensure the templates were floating to maximize contact of the patterned PDMS with the solution. Templates were transferred to the NaCl solution for 2 min and were then rinsed with deionized (DI) water to remove free HA-Ald. Templates were transferred to the PAH solution for 15 min in the same fashion and then transferred to new weigh boats with the NaCl solution for 2 min. Templates were rinsed with DI water, and the entire process was repeated once more to form an LBL coating of HA-Ald/PAH/HA-Ald/PAH. Coated templates were rinsed with DI water for 30 s and dried by a stream of air. Templates were stored in petri dishes, patterned side up, at 4°C.
HA（2500 kDa）通过醛基修饰（HA-Ald）（详见补充材料中的方法）。制备了 HA-Ald 的水溶液（2 mg/ml），溶于 150 mM NaCl（pH 6.8），同时制备了 PAH 的水溶液（2 mg/ml；17.5 kDa），同样溶于 150 mM NaCl（pH 6.8）。将 HA-Ald、PAH 及 150 mM NaCl（pH 6.8）溶液分别倒入称量皿中。PDMS 模板用异丙醇冲洗后，通过稳定的气流干燥。随后，将模板图案面朝下浸入 HA-Ald 溶液中 15 分钟，确保模板浮起以最大化图案化 PDMS 与溶液的接触。之后，模板转移至 NaCl 溶液中浸泡 2 分钟，再用去离子（DI）水冲洗以去除游离的 HA-Ald。模板按相同方式转移至 PAH 溶液中浸泡 15 分钟，然后移至装有 NaCl 溶液的新称量皿中浸泡 2 分钟。模板用 DI 水冲洗，整个过程再重复一次，形成 HA-Ald/PAH/HA-Ald/PAH 的层层涂层（LBL coating）。涂层后的模板用 DI 水冲洗 30 秒，并通过气流干燥。模板存放在培养皿中，图案面朝上，置于 4°C 环境下。

An 8% w/v solution of PLGA in acetone was prepared from a 100:1 weight ratio of nonfluorescent PLGA (7 to 17 kDa; Resomer 502 H) and fluorescent PLGA (10 to 30 kDa; LG 50:50 rhodamine B; PolySciTech). PDMS templates with LBL coatings were cut into quadrants and spin coated with 225 μl of PLGA solution per quadrant at 2000 rpm for 20 s (at a 200 rpm/s ramp). Quadrants were then plasma ashed with O₂ for 60 s. A 0.5 weight % solution of PVA (146 to 186 kDa, 99 + % hydrolyzed) in phosphate-buffered saline (PBS) was prepared with IFN-γ (25 μg/ml). Immediately after plasma treatment, 100 μl of the PVA solution was evenly spread onto each quadrant by pipette. Quadrants were then placed in a desiccator under vacuum with Drierite desiccant (W.A. Hammond Drierite Co.) until dry, making a 0.6-μm-thick PVA film. A second PLGA layer was deposited using the same procedure as the first.
制备了 8%（w/v）的 PLGA 丙酮溶液，采用非荧光 PLGA（7 至 17 kDa；Resomer 502 H）与荧光 PLGA（10 至 30 kDa；LG 50:50 罗丹明 B；PolySciTech）按 100:1 重量比混合。将带有 LBL 涂层的 PDMS 模板切割成四等份，每份以 225 μl 的 PLGA 溶液在 2000 rpm 转速下旋转涂布 20 秒（以 200 rpm/s 的速率加速）。随后，各象限用氧气等离子体处理 60 秒。配制了 0.5 重量%的 PVA（146 至 186 kDa，99%以上水解）磷酸盐缓冲液（PBS）溶液，并加入 IFN-γ（25 μg/ml）。等离子处理后，立即用移液器将 100 μl 的 PVA 溶液均匀涂抹在每个象限上。随后，将象限放入装有 Drierite 干燥剂（W.A. Hammond Drierite Co.）的干燥器中，在真空条件下干燥，形成厚度为 0.6 微米的 PVA 薄膜。第二层 PLGA 的沉积采用与第一层相同的步骤进行。

PVA-coated dishes were prepared by making a 3% w/v solution of PVA (13 to 23 kDa, 87% hydrolyzed) in DI water. The solution was stirred at 80°C for several hours, and excess crystals were filtered using a 0.22-μm filter. Sterile petri dishes were coated with 2.5 ml of solution, and placed in an oven at 60° to 75°C until dry. Backpacks were printed using techniques similar to those described previously (14). Briefly, a beaker was filled with DI H₂O and heated to 65°C. The coated side of a PVA-coated dish was held ~2 cm over the beaker for 6 to 12 s. A PDMS quadrant containing backpacks was immediately pressed onto the warmed PVA dish, and consistent pressure was applied for 15 to 20 s. The quadrant was then peeled away, leaving a coating of backpacks on the dish. This was repeated until the material had fully transferred. Backpacks were then stored at −80°C until needed. To harvest backpacks, dishes were covered with 2.5 ml of PBS and were gently washed. This was repeated until the surface appeared mostly clear (typically twice). The solution was collected, passed through a 40-μm cell strainer, and centrifuged at 2500g for 5 min. Backpacks were resuspended in 5 ml of BMM− (i.e., 500 ml of DMEM F12, 50 ml of FBS, 5 ml of Pen Strep, and 25 ml of 200 mM GlutaMAX) or serum-free BMM− (i.e., BMM− sans FBS), depending on the application.
PVA 涂层培养皿的制备方法是将 PVA（分子量 13 至 23 kDa，87%水解）溶于去离子水中，配制成 3%（重量/体积）的溶液。该溶液在 80°C 下搅拌数小时，并通过 0.22 微米滤器过滤掉多余的晶体。将 2.5 毫升溶液涂覆在无菌培养皿上，然后置于 60°至 75°C 的烤箱中直至干燥。背包通过类似于先前描述的技术进行打印（14）。简而言之，将烧杯装满去离子水并加热至 65°C。将 PVA 涂层培养皿的涂层面保持在烧杯上方约 2 厘米处，持续 6 至 12 秒。立即将含有背包的 PDMS 象限压在加热的 PVA 培养皿上，并施加恒定的压力 15 至 20 秒。然后剥离象限，使培养皿上留下背包涂层。重复此过程直至材料完全转移。背包随后储存于-80°C 备用。为收获背包，将 2.5 毫升 PBS 覆盖在培养皿上并轻轻冲洗。重复此操作直至表面基本清洁（通常两次）。收集溶液，通过 40 微米细胞筛网过滤，并在 2500g 下离心 5 分钟。背包在 5 毫升 BMM-中重新悬浮。500 毫升 DMEM F12、50 毫升 FBS、5 毫升青链霉素和 25 毫升 200 mM GlutaMAX，或无血清的 BMM−（即不含 FBS 的 BMM−），视应用而定。

Progenitor cells were isolated from murine bone marrow following methods described previously (58). Briefly, 6- to 8-week-old BALB/c mice were euthanized via CO₂ inhalation. Sterile surgical scissors were used to extract the tibias, femurs, and humeri. Isolated bones were submerged in 70% ethanol, rinsed with PBS, and then transferred to a separate PBS solution. In a sterile environment, epiphyses of each bone were cut, and the bones were flushed with PBS via a syringe with a 31-gauge needle into a 50-ml collection tube. The solution was mixed thoroughly, passed through a 40-μm cell strainer, and centrifuged at 400g for 10 min at 4°C. Cells were resuspended in Bambanker (2 ml per mouse equivalent; Lymphotec Inc.) and stored in cryovials at −80°C until needed.
祖细胞通过先前描述的方法从鼠骨髓中分离出来（58）。简而言之，对 6 至 8 周龄的 BALB/c 小鼠进行 CO ₂ 吸入安乐死。使用无菌手术剪刀提取胫骨、股骨和肱骨。分离的骨骼浸泡于 70%乙醇中，用 PBS 冲洗，然后转移至另一份 PBS 溶液。在无菌环境下，切开每根骨头的骨骺，通过 31 号针头的注射器将骨骼用 PBS 冲洗入 50 毫升收集管中。溶液充分混合后，通过 40 微米细胞滤网过滤，并在 4°C 下以 400g 离心 10 分钟。细胞重悬于 Bambanker 中（每只小鼠等效 2 毫升；Lymphotec 公司），并储存于-80°C 的冷冻管中，直至需要时使用。

BMDMs were cultured from murine bone marrow progenitor cells following methods described previously (9). Briefly, frozen bone marrow was thawed and mixed with 4°C BMM− at 1:5 ratio by volume. The solution was centrifuged, the liquid was aspirated, cells were resuspended in BMM+ [i.e., BMM− with M-CSF (20 ng/ml)], and cells were counted with a hemocytometer. Approximately 4 × 10⁶ bone marrow cells were added to non–tissue culture (TC)–treated T175 flasks containing 25 ml of BMM+. Cells were incubated under standard culture conditions. Additional BMM+ (25 ml) was added to the flasks on days 3 and 7. On day 8, media were aspirated from the flasks, and cells were washed once with 10 ml of PBS. To dislodge the cells, PBS was aspirated and replaced with 10 ml of Accumax (Innovative Cell Technologies) at 4°C. Cells were incubated with the Accumax at 37°C for 10 min. The flask was then removed from the incubator and vigorously thumped several times. More Accumax (10 ml) was added to the flask, and cells were incubated for an additional 10 min and thumped again. The suspension of BMDMs was added to a 50-ml conical tube with an equal volume of BMM− and centrifuged. The supernatant was aspirated and replaced with BMM+. BMDMs were counted and plated on non-TC-treated 12-well plates at a concentration of 2.5 × 10⁵ cells per well in a volume of 1 ml per well and incubated under standard conditions for 24 hours. All centrifugation steps were performed at 400g for 10 min at 4°C.
BMDMs（骨髓来源巨噬细胞）按照先前描述的方法（9）从鼠骨髓祖细胞中培养。简言之，冷冻的骨髓被解冻并与 4°C 的 BMM−以 1:5 的体积比混合。混合液经过离心，去除上清液，细胞在 BMM+（即含 M-CSF 20 ng/ml 的 BMM−）中重悬，并使用血细胞计数器进行计数。大约 4 × 10 ⁶ 个骨髓细胞被加入到未经组织培养（TC）处理的 T175 烧瓶中，其中含有 25 ml 的 BMM+。细胞在标准培养条件下孵育。在第 3 天和第 7 天，分别向烧瓶中加入额外的 BMM+（25 ml）。第 8 天，移除培养基，细胞用 10 ml PBS 洗涤一次。为使细胞脱离烧瓶，吸去 PBS 后，加入 10 ml 4°C 的 Accumax（创新细胞技术公司）。细胞在 37°C 下与 Accumax 孵育 10 分钟。随后，烧瓶从培养箱中取出，用力拍打数次。再向烧瓶中加入 10 ml Accumax，细胞继续孵育 10 分钟并再次拍打。BMDMs 悬液被转移至 50 ml 的锥形管中，加入等体积的 BMM−后离心。 上清液被吸出并替换为 BMM+。BMDMs 计数后，以每孔 2.5 × 10¹个细胞的浓度铺于未经 TC 处理的 12 孔板上，每孔体积为 1 毫升，在标准条件下孵育 24 小时。所有离心步骤均在 4°C 下以 400g 的转速进行 10 分钟。

Backpacks were harvested and centrifuged at 2500g for 5 min at 4°C and resuspended in serum-free BMM−. Meanwhile, BMDMs cultured in 12-well plates for 24 hours were removed from the incubator, and BMM+ was exchanged with serum-free BMM− using a serial dilution technique [see Methods in the Supplementary Materials (9)]. Backpacks were counted using a hemocytometer, and 0.375 × 10⁵ backpacks were added to each well of each 12-well plate (yielding a 3:2 ratio of backpacks:cells). Plates were then centrifuged at 300g for 7.5 min to allow backpacks to gather along the bottom of the plate. Plates were then placed in a cell culture incubator for 1.5 hours to allow BMDMs to bind to backpacks. After 1.5 hours, serum-free BMM− was exchanged with BMM− via serial dilution. Plates were then incubated in either standard culturing conditions (normoxia; 74% N₂, 5% CO₂, and 21% O₂) or hypoxic conditions (94% N₂, 5% CO₂, and 1% O₂), depending on the study (see Methods in the Supplementary Materials). In cases where plates were stored in a hypoxia chamber, 100 μl of BMM− from each well was replaced with 100 μl of tumor-conditioned media, obtained from culture with 4T1 cells. In lieu of backpacks, free IFN-γ was sometimes added at a concentration of 16 ng/ml to the appropriate wells.
背包被采集并在 4°C 下以 2500g 离心 5 分钟，随后在无血清的 BMM−中重新悬浮。同时，在 12 孔板中培养 24 小时的 BMDMs 从培养箱中取出，并通过连续稀释技术将 BMM+替换为无血清的 BMM−（参见补充材料中的方法）。使用血细胞计数器对背包进行计数，并向每个 12 孔板的每孔中加入 0.375 × 10 ⁵ 个背包（形成背包与细胞 3:2 的比例）。随后，将板以 300g 离心 7.5 分钟，使背包聚集在板的底部。接着，将板放入细胞培养箱中 1.5 小时，以便 BMDMs 与背包结合。1.5 小时后，通过连续稀释法将无血清的 BMM−替换为 BMM−。然后，根据研究需要，将板在标准培养条件（常氧；74% N ₂ ，5% CO ₂ ，21% O ₂ ）或低氧条件（94% N ₂ ，5% CO ₂ ，1% O ₂ ）下培养（参见补充材料中的方法）。在板存放在低氧箱的情况下，从每孔中替换 100 μl 的 BMM−为 100 μl 的肿瘤条件培养基，该培养基来自与 4T1 细胞共培养。 作为替代，有时会在适当的孔中加入浓度为 16 纳克/毫升的游离 IFN-γ。

Serial dilution was performed to replace media in each well of the 12-well plates with Hank’s balanced salt solution (HBSS). Then, 500 μl of HBSS was aspirated from each well and replaced with 2 ml of Accumax. Plates were incubated at 37°C and 5% CO₂ for 10 to 15 min. Plates were then thumped to release BMDMs, and the respective groups were collected into separate 50-ml tubes with an equal volume BMM−. Cells were centrifuged and pellets were resuspended in 1 ml of stain buffer, comprising 1% FBS in PBS without Mg²⁺ or Ca²⁺ (pH 7.4 to 7.6). Cells were transferred into 1.5-ml Eppendorf tubes, where they were centrifuged again. Pellets were resuspended in 99 μl of stain buffer with 1 μl of Fc block and were incubated for 15 min at 4°C. After incubation, samples were diluted with 1 ml of stain buffer, centrifuged, and resuspended in 1 ml of stain buffer. Each sample was then split into two groups of 500 μl for surface marker staining and intracellular staining. For surface staining, samples were centrifuged and resuspended in an antibody mixture of anti-CD80, anti-MHCII, anti-VEGF, and stain buffer (at concentrations suggested by the manufacturer) in the dark at 4°C. After 30 min, cells were washed with 1 ml of stain buffer, centrifuged, resuspended in 300 μl, and stored in the dark at 4°C until use. For intracellular staining, samples were fixed and permeabilized following instructions from the manufacturer (BD Biosciences). Cells were centrifuged and resuspended in 100 μl of an antibody solution comprising anti-iNOS, anti-HIF-1α, anti-CD206, and Perm/Wash Buffer (at concentrations suggested by the manufacturer) in the dark at 25°C. After 30 min, cells were diluted with 1 ml of Perm/Wash Buffer, centrifuged, resuspended in 300 μl of stain buffer, and stored in the dark at 4°C until use. All centrifugation steps were performed at 350g for 5 min at 4°C. Compensation and voltage settings were determined 1 day prior using sets of compensation beads, each stained with one antibody. Up to 10,000 events were collected for each sample. Data were analyzed using FCS Express 6 Software (De Novo Software).
通过连续稀释法，将 12 孔板中各孔的培养基替换为汉克平衡盐溶液（HBSS）。随后，从每个孔中吸取 500 μl 的 HBSS，并加入 2 ml 的 Accumax。将板在 37°C 和 5% CO2 条件下孵育 10 至 15 分钟。之后轻敲板子以释放 BMDMs，并将各组分别收集到装有等量 BMM−的 50 ml 离心管中。细胞经过离心后，将沉淀物重新悬浮于 1 ml 的染色缓冲液中，该缓冲液由不含 Mg2+或 Ca2+的 PBS（pH 7.4 至 7.6）中的 1% FBS 组成。将细胞转移至 1.5 ml 的 Eppendorf 管中，再次离心。沉淀物在 99 μl 的染色缓冲液中重新悬浮，并加入 1 μl 的 Fc 阻断剂，在 4°C 下孵育 15 分钟。孵育后，样品用 1 ml 的染色缓冲液稀释，离心后重新悬浮于 1 ml 的染色缓冲液中。每个样品随后分为两组，每组 500 μl，分别用于表面标记染色和细胞内染色。对于表面染色，样品离心后在 4°C 的暗处重新悬浮于抗 CD80、抗 MHCII、抗 VEGF 和染色缓冲液（按厂家建议的浓度）的混合抗体中。 30 分钟后，细胞用 1 毫升染色缓冲液洗涤，离心，重悬于 300 微升中，并在 4°C 避光保存直至使用。对于胞内染色，样品按照制造商（BD Biosciences）的说明进行固定和通透处理。细胞离心后，在 25°C 避光条件下重悬于 100 微升抗体溶液中，该溶液包含抗 iNOS、抗 HIF-1α、抗 CD206 以及 Perm/Wash 缓冲液（浓度按制造商建议）。30 分钟后，细胞用 1 毫升 Perm/Wash 缓冲液稀释，离心，重悬于 300 微升染色缓冲液中，并在 4°C 避光保存直至使用。所有离心步骤均在 4°C 下以 350g 进行 5 分钟。补偿和电压设置在实验前一天通过分别用一种抗体染色的补偿珠组确定。每个样品最多收集 10,000 个事件。数据使用 FCS Express 6 软件（De Novo Software）进行分析。

Experiments involving animals were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Harvard University. Two orthotopic breast cancer models were used in mice, 4T1 and 4T1-Fluc-Neo/eGFP-Puro cells expressing firefly luciferase (4T1-Luc). 4T1 cells were cultured in DMEM supplemented 10% FBS and 1% Pen Strep. 4T1-Luc cells were cultured in RPMI 1640 media supplemented with 10% FBS, 1% Pen Strep, and G418 (0.1 mg/ml). Both lines were cultured in a humidified incubator maintained at 37°C and 5% CO₂. Cells were passaged twice before inoculation. Cells were released via trypsin, centrifuged, and resuspended in physiological saline. Mice were inoculated with 1 × 10⁵ 4T1 cells or 1 × 10⁶ 4T1-Luc cells (>98% cell viability) in 50 μl by subcutaneous injection into the lower left inguinal mammary fat pad of BALB/c mice 42 to 56 days in age using a 25-gauge needle. Tumor-bearing mice were randomized before treatments and monitored for tumor growth and body weight changes throughout the study. Each mouse model received two treatments, which began 14 days after inoculation in the 4T1 model (tumor volume, ~100 mm³) and 9 days after inoculation in the 4T1-Luc model (tumor volume, ~ 50 mm³). Tumor volumes were calculated using the formula: V = ½ L × W², where L and W were the longest and shortest dimensions of the tumor, respectively. Mice harboring 4T1 tumors were used for tumor-immune cell phenotyping. These mice were euthanized if L exceeded 15 mm or if body weight loss exceeded 15%. Mice in the 4T1-Luc model were enrolled in a survival study and were left alive until they succumbed to tumor burden or were euthanized with CO₂ if they became moribund.
动物实验按照哈佛大学机构动物护理和使用委员会批准的协议进行。在小鼠中使用了两种原位乳腺癌模型，分别为 4T1 和表达萤火虫荧光素酶的 4T1-Fluc-Neo/eGFP-Puro 细胞（4T1-Luc）。4T1 细胞在含 10%胎牛血清（FBS）和 1%青霉素-链霉素的 DMEM 中培养。4T1-Luc 细胞则在含 10% FBS、1%青霉素-链霉素及 G418（0.1 mg/ml）的 RPMI 1640 培养基中培养。两种细胞系均在 37°C、5% CO₂的湿润培养箱中培养，并在接种前传代两次。细胞通过胰蛋白酶消化释放，离心后重悬于生理盐水中。BALB/c 小鼠（42 至 56 日龄）通过 25 号针头皮下注射接种 1 × 10⁵个 4T1 细胞或 1 × 10⁶个 4T1-Luc 细胞（细胞存活率>98%）至左下腹股沟乳腺脂肪垫。接种后的小鼠在治疗前随机分组，并在研究期间监测肿瘤生长及体重变化。 每只小鼠模型接受两次治疗，分别在 4T1 模型中接种后 14 天（肿瘤体积约为 100 mm³）和 4T1-Luc 模型中接种后 9 天（肿瘤体积约为 50 mm³）开始。肿瘤体积通过公式计算：V = ½ L × W²，其中 L 和 W 分别为肿瘤的最长和最短尺寸。携带 4T1 肿瘤的小鼠用于肿瘤免疫细胞表型分析。若肿瘤最长尺寸超过 15 毫米或体重减轻超过 15%，则对这些小鼠实施安乐死。4T1-Luc 模型中的小鼠则纳入生存研究，直至因肿瘤负担死亡或因病情恶化通过 CO 安乐死。

Mice received two intratumoral treatment injections, occurring 14 and 18 days after inoculation in the 4T1 model and 9 and 14 days after inoculation in the 4T1-Luc model. IFN-γ was limited to 50 ng per administration (59). Mice requiring injected macrophages each received the same number of cells (i.e., 0.78 × 10⁶ macrophages per mouse per injection). Numbers were determined based on the number of macrophages necessary to deliver 50 ng worth of IFN-γ backpacks. Determinations were based on ELISA data, which revealed ~85 fg IFN-γ/backpack, and flow cytometry, which revealed ≥75% of macrophages were labeled with ≥1 backpack after the 1.5 hours incubation period. In all groups (i.e., saline, free IFN-γ, or groups with macrophages), injection volumes were 10 μl.
小鼠接受了两次瘤内治疗注射，分别在 4T1 模型中接种后第 14 天和第 18 天进行，以及在 4T1-Luc 模型中接种后第 9 天和第 14 天进行。每剂 IFN-γ剂量限制在 50 纳克（59）。需要注射巨噬细胞的小鼠每次接受相同数量的细胞（即每只小鼠每次注射 0.78 × 10^6 个巨噬细胞）。这些数量是根据输送 50 纳克 IFN-γ背包所需的巨噬细胞数量确定的。确定依据包括 ELISA 数据显示每个背包约含 85 皮克 IFN-γ，以及流式细胞术显示在 1.5 小时孵育后，≥75%的巨噬细胞被标记有≥1 个背包。所有组别（即生理盐水组、游离 IFN-γ组或含巨噬细胞组）的注射体积均为 10 微升。

Mice inoculated with 4T1 cells were treated with macrophages labeled with a near-infrared dye (VivoTrack 680, PerkinElmer). Seven days before tumor inoculations through to the end of the study, mice were fed alfalfa-free diets to reduce background fluorescence levels (Picco Rodent 5V5R 50IF irradiated pelleted, Scott Pharma Solutions). Before imaging, hair over top and near the tumor was removed using a topical formulation (Nair, Church & Dwight). Mice were imaged under anesthesia (from isoflurane) using IVIS each day after the first therapeutic administration for a total of 5 days.
接种了 4T1 细胞的小鼠接受了用近红外染料（VivoTrack 680，PerkinElmer）标记的巨噬细胞治疗。从肿瘤接种前 7 天至研究结束，小鼠被喂食不含苜蓿的饮食以降低背景荧光水平（Picco Rodent 5V5R 50IF 辐照颗粒饲料，Scott Pharma Solutions）。成像前，使用局部制剂（Nair，Church & Dwight）去除肿瘤上方及附近的毛发。小鼠在麻醉状态下（使用异氟烷）每天通过 IVIS 成像，从首次治疗开始共进行 5 天。

Lung metastases were evaluated 32 days after inoculation with 4T1-Luc. Mice were injected with 150 μl of XenoLight D-Luciferin potassium salt bioluminescence substrate (30 mg/ml) (PerkinElmer) in saline via intraperitoneal injection. Fifteen minutes after injection, mice were imaged under anesthesia (from isoflurane) using IVIS. Primary tumors were covered with strips of black paper to eliminate signal washout from the main tumors, which were brighter than the metastatic colonies in the chest cavities.
32 天后，通过接种 4T1-Luc 评估肺转移情况。小鼠经腹腔注射 150 微升的 XenoLight D-Luciferin 钾盐生物发光底物（30 毫克/毫升）（PerkinElmer），溶于生理盐水中。注射后 15 分钟，在异氟烷麻醉下对小鼠进行 IVIS 成像。为避免主肿瘤信号溢出——主肿瘤比胸腔内的转移灶更亮，主肿瘤部位覆盖了黑色纸条。

Procedures for isolating and staining tumor-associated immune cells were similar to those described previously (30). Briefly, mice were euthanized via CO₂ inhalation 2 days after administration of the second treatment. Primary tumors (from 4T1 cells) were harvested, cut into small pieces (<5 mm thick), and enzymatically degraded using a mouse tumor dissociation kit with a gentleMACS dissociator (Miltenyi Biotec). Cells were centrifuged and resuspended in ACK red cell lysis buffer supplemented with DNAse I (50 U/ml) for 5 min. Cells were again centrifuged and resuspended in PBS to quantify the remaining intact cells. Leucocytes were isolated from the general population using a CD45⁺ isolation kit, following instructions from the manufacturer (Miltenyi Biotec). For the remainder of the study, 1 × 10⁶ cells per animal were used, and all steps were performed in 100 μl of fluorescence-activated cell sorting (FACS) buffer (PBS with 3% FBS) supplemented with additional reagents as necessary. Cells were blocked for 30 min in a solution consisting of 5% rat serum, 5% mouse serum, and 1% anti-mouse CD16/32 antibody. Cells were stained with test and control antibodies (see fig. S8 and table S1) for 30 min at 25°C and for 20 min on ice in a dark enclosed space. Cells were then washed twice with ice-cold FACS buffer and resuspended in 500 μl of PBS. Following instructions from the manufacturer, cells were stained with SYTOX blue to measure cell viability at the end of all treatment steps. Stained cells were then analyzed by flow cytometry (BD LSRII). Compensation and voltage settings were determined 1 day prior to using sets of compensation beads, each stained with one antibody. Up to 100,000 events were collected for each sample. Data were analyzed using FCS Express 6 Software. All centrifugation steps were performed at 350g for 5 min.
从肿瘤相关免疫细胞的分离和染色步骤与先前描述的方法类似（30）。简而言之，在第二次治疗后 2 天，通过 CO₂吸入对小鼠实施安乐死。采集来源于 4T1 细胞的原发性肿瘤，将其切成小块（厚度<5 mm），并使用 gentleMACS 分离器（Miltenyi Biotec）配合小鼠肿瘤组织解离试剂盒进行酶解。细胞经离心后，重悬于补充有 DNA 酶 I（50 U/ml）的 ACK 红细胞裂解缓冲液中处理 5 分钟，再次离心后，用 PBS 重悬以计数剩余的完整细胞。通过 CD45 分离试剂盒，根据制造商（Miltenyi Biotec）的说明，从普通细胞群体中分离出白细胞。在本研究的其余部分中，每只动物使用 1×10⁶个细胞，所有步骤均在补充有必需附加试剂的 100 μl 荧光激活细胞分选（FACS）缓冲液（含 3%胎牛血清的 PBS）中进行。细胞在含有 5%大鼠血清、5%小鼠血清和 1%抗小鼠 CD16/32 抗体的溶液中封闭 30 分钟。随后，细胞用测试抗体和对照抗体进行染色（详见图表）。 S8 和表 S1）在 25°C 下处理 30 分钟，在暗室中冰上处理 20 分钟。随后，细胞用冰冷的 FACS 缓冲液洗涤两次，并重悬于 500 μl PBS 中。按照制造商的说明，使用 SYTOX 蓝染色以在所有处理步骤结束后检测细胞活力。染色后的细胞通过流式细胞仪（BD LSRII）进行分析。补偿和电压设置在实验前一天通过使用分别标记有一种抗体的补偿微球来确定。每个样本最多收集 100,000 个事件。数据使用 FCS Express 6 软件进行分析。所有离心步骤均在 350g 下进行 5 分钟。

Unless otherwise indicated, the data were represented as means ± SE using GraphPad (Prism 8.0). For determination of statistical significance, multiple t tests or one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests were used, as applicable. Significance was determined at the following cutoff points (*P < 0.05, **P < 0.01, ***P < 0.001). Significance from the survival time was quantified using a log-rank test.