肿瘤细胞治疗研究
英文原题：Enhanced efficacy of dual chimeric antigen receptor-T cells targeting programmed death-ligand 1 and cancer-associated fibroblasts in colorectal cancer in vitro.
Enhanced efficacy of dual chimeric antigen receptor-T cells targeting programmed death-ligand 1 and cancer-associated fibroblasts in colorectal cancer in vitro.
本研究成功开发了靶向 PD-L1 和 CAF 的双靶点 CAR-T 细胞，并证明其在 CRC 治疗中具有优越的抗肿瘤活性和免疫调节作用。
目的：结直肠癌（CRC）治疗面临免疫逃逸和肿瘤微环境（TME）抑制等重大挑战。嵌合抗原受体（CAR）T细胞疗法在血液系统恶性肿瘤中显示潜力，但TME的不利影响限制了其治疗实体瘤的效果。本文探讨同时靶向程序性死亡配体1（PD-L1）和癌症相关成纤维细胞（CAF）的双特异性CAR-T细胞治疗CRC的潜力。材料与方法：使用GV400慢病毒载体构建靶向PD-L1和CAF的双靶点CAR-T细胞；制备编码程序性死亡蛋白1（PD-1）/纳米抗体（Nb）及成纤维细胞活化蛋白（FAP）/Nb的慢病毒载体，并通过293T细胞三质粒系统制备CAR-T细胞。分离人外周血单个核细胞（PBMC），用这些载体转导后扩增。采用ELISA、蛋白质印迹、流式细胞术、TUNEL及CCK-8检测对CAR-T细胞进行功能表征；使用Transwell小室评估FAP-PD-1/Nb CAR-T细胞向肿瘤细胞迁移及侵袭细胞外基质的能力。结果：研究者构建了包含PD-L1和CAF纳米抗体、可持续分泌PD-1/Nb的双靶点CAR-T细胞。蛋白质印迹证实PD-1/Nb和FAP-PD-1/Nb CAR-T细胞表达PD-1/Nb，未处理组（UTD）则无表达（P<.01）。经FAP阳性靶细胞刺激后，与其他组相比，FAP-PD-1/Nb CAR-T细胞CD25和CD69表达显著升高（P<.01）。TUNEL、流式细胞术和CCK-8检测显示，该细胞对FAP阳性HCT116细胞具有更强细胞毒作用并更有效抑制增殖（P<.01）。ELISA显示IFN-γ和TNF-α增加、IL-10降低（P<.01），提示细胞因子调节和抗肿瘤免疫增强。与单靶点CAR-T及UTD相比，FAP-PD-1/Nb CAR-T细胞穿透Matrigel基质和侵袭能力显著增强（P<.01）。安全性检测证实其对正常PBMC细胞毒作用极低。结论：本研究成功开发出靶向PD-L1和CAF的双靶点CAR-T细胞，并证实其在CRC治疗中具有更强抗肿瘤活性及免疫调节作用，建立了一种应用CAR-T技术治疗CRC的新策略。
OBJECTIVE: Colorectal cancer (CRC) presents significant treatment challenges, including immune evasion and tumor microenvironment (TME) suppression. Chimeric antigen receptor (CAR) T-cell therapy has shown promise in hematologic malignancies, but its effectiveness against solid tumors is hampered by the detrimental effects of the TME. This article aims to explore the potential of bispecific CAR T cells targeting programmed death-ligand 1 (PD-L1) and cancer-associated fibroblasts (CAFs) in CRC treatment. MATERIAL AND METHODS: Dual-targeted CAR-T cells against PD-L1 and CAF were engineered using the GV400 lentiviral vector. Programmed death-1 (PD-1)/nanobody (Nb) and fibroblast activation protein (FAP)/Nb-encoding lentiviral vectors were generated, and CAR T cells were produced through a three-plasmid system in 293T cells. Human peripheral blood mononuclear cells (PBMCs) were separated, transduced with these vectors, and then expanded. Functional characterization of CAR-T cells was performed through enzyme-linked immunosorbent assay (ELISA), Western blot analysis, flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays, and cell counting kit-8 (CCK-8) assay. Migration and invasion assays were conducted using Transwell chambers to assess the ability of FAP-PD-1/Nb CAR-T cells to migrate toward tumor cells and invade the extracellular matrix. RESULTS: We developed dual-targeted CAR-T cells incorporating PD-L1 and CAF Nbs, which continuously secreted PD-1/Nb. Western blot confirmed PD-1/Nb expression in PD-1/Nb and FAP-PD-1/Nb CAR-T cells, with no expression in the untreated (UTD) group ( P < 0.01). Flow cytometry showed a significantly higher cluster of differentiation (CD)25 and CD69 expression in FAP-PD-1/Nb CAR-T cells upon stimulation with FAP-positive target cells compared with the other groups ( P < 0.01). TUNEL, flow cytometry, and CCK-8 assays revealed that FAP-PD-1/Nb CAR-T cells exhibited superior cytotoxicity and proliferation inhibition against FAP-positive HCT116 cells ( P < 0.01). ELISA demonstrated increased interferon-gamma and tumor necrosis factor-alpha levels and reduced interleukin-10 ( P < 0.01), suggesting enhanced cytokine modulation and antitumor immunity. Compared with single-target CAR-T cells and UTD, FAP-PD-1/Nb CAR-T cells showed notably enhanced Matrigel penetration and invasion ( P < 0.01). Safety tests confirmed minimal cytotoxicity to normal PBMCs, indicating favorable safety. CONCLUSION: This study successfully developed dual-targeted CAR-T cells against PD-L1 and CAF and demonstrated their superior antitumor activity and immunomodulatory effects on CRC treatment. This novel therapeutic strategy was established using CAR T-cell technology for the treatment of CRC.
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