肿瘤细胞治疗研究
英文原题：Dual Targeting of FAP-Directed Nanoparticles and FRα-Specific CAR-T Cells Induces Additive Anti-Tumor Effects in Triple-Negative Breast Cancer.
这些发现支持进一步开发双靶点方法以改善TNBC的治疗效果。
三阴性乳腺癌（TNBC）是一种侵袭性恶性肿瘤，治疗选择有限。它缺乏激素受体和人表皮生长因子受体2。免疫抑制性肿瘤微环境（TME），尤其是癌相关成纤维细胞（CAFs），显著阻碍嵌合抗原受体（CAR）-T细胞治疗的成功。迫切需要克服TME介导的免疫抑制的新策略。我们在3D癌细胞-CAFs共培养异源球状体（HS）模型中评估了用成纤维细胞活化蛋白α（FAP）包被、负载8-O-methylfusarubin的纳米颗粒（称为anti-FAP@OMF-NPs）靶向CAFs，是否可增强叶酸受体α（FRα）特异性CAR-T细胞对TNBC的抗肿瘤疗效。使用免疫荧光和流式细胞术评估TNBC细胞和原代乳腺CAFs中FRα和FAP的表达。通过慢病毒转导生成anti-FRα-CAR-T细胞，并表征其活化标志物。在包含FRα高表达TNBC细胞和FAP高表达CAFs的3D-HS中，评估CAR-T细胞、anti-FAP@OMF-NPs及其联合的细胞毒性活性。荧光转染试验测量细胞活力。细胞因子微球阵列定量IFN-γ、颗粒酶A和颗粒酶B水平，以评估抗肿瘤免疫激活。与PC-B-004CAFs和正常人真皮成纤维细胞（HDFa）相比，PC-B-130CAFs和PC-B-132CAFs表现出高FAP表达。anti-FRα-CAR-T细胞选择性靶向FRα阳性TNBC细胞，同时对正常MCF-10A细胞显示最小细胞毒性。anti-FAP@OMF-NPs在表达FAP的CAFs中特异性诱导强效细胞毒性作用。与单药治疗相比，联合治疗显著增强了对MDA-MB-231/130CAF和MDA-MB-231/132CAF HSs的破坏。该联合治疗增加了抗FR-CAR-T细胞分泌IFN-、颗粒酶A和颗粒酶B。使用抗FAP@OMF-NPs靶向CAFs增强了FR特异性CAR-T细胞在TNBC中的细胞毒性效力。这种联合策略为克服TME介导的免疫抑制提供了一种有前景的方法。这些发现支持进一步开发双靶向方法以改善TNBC的治疗效果。
Triple-negative breast cancer (TNBC) is an aggressive malignancy with limited treatment options. It lacks hormone receptors and human epidermal growth factor receptor 2. The immunosuppressive tumor microenvironment (TME), particularly cancer-associated fibroblasts (CAFs), significantly hinders chimeric antigen receptor (CAR)-T cell therapy success. Novel strategies to overcome TME-mediated immunosuppression are urgently needed. We evaluated whether targeting CAFs with fibroblast activation protein alpha (FAP)-coated, 8- O -methylfusarubin-loaded nanoparticles called anti-FAP@OMF-NPs could enhance the anti-tumor efficacy of folate receptor alpha (FR )-specific CAR-T cells against TNBC in a 3D cancer cells-CAFs co-culture heterospheriod (HS) model. FR and FAP expression in TNBC cells and primary breast CAFs were assessed using immunofluorescence and flow cytometry. Anti-FR -CAR-T cells were generated via lentiviral transduction and characterised for activation markers. Cytotoxic activity of CAR-T cells, anti-FAP@OMF-NPs, and their combination was evaluated in 3D-HS comprising FR -high TNBC cells and FAP-high CAFs. A fluorescent transfection assay measured cell viability. Cytokine bead arrays quantified IFN- , granzyme A, and granzyme B levels to assess anti-tumor immune activation. PC-B-130CAFs and PC-B-132CAFs demonstrated high FAP expression compared with PC-B-004CAFs and normal human dermal fibroblast cells (HDFa). Anti-FR -CAR-T cells selectively targeted FR -positive TNBC cells whilst showing minimal cytotoxicity towards normal MCF-10A cells. Anti-FAP@OMF-NPs induced potent cytotoxic effects specifically in FAP-expressing CAFs. Combined treatment significantly enhanced the destruction of MDA-MB-231/130CAF and MDA-MB-231/132CAF HSs compared with monotherapies. This combination increased secretion of IFN- , granzyme A, and granzyme B from anti-FR -CAR-T cells. Targeting CAFs using anti-FAP@OMF-NPs enhances the cytotoxic efficacy of FR -specific CAR-T cells in TNBC. This combinatorial approach offers a promising strategy to overcome TME-mediated immunosuppression. These findings support further development of dual-targeting approaches to improve therapeutic outcomes in TNBC.
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