肿瘤球对肿瘤浸润淋巴细胞的体外激活扩增及抗肿瘤的作用

3D tumor spheroids promote activation, expansion, and anti-tumor effects of tumor-infiltrating lymphocytes in vitro

摘要:

肿瘤浸润淋巴细胞(tumor-infiltrating lymphocytes, TILs)介导的过继性免疫治疗已在多种实体瘤中取得良好疗效。然而，传统TILs体外扩增方法效率低，难以达到治疗级别的细胞数量和高肿瘤杀伤活性要求。为了研究3D肿瘤模型对TILs体外激活扩增及肿瘤杀伤活性的影响，为TILs体外扩增提供新策略，本研究从肺癌患者手术样本中获取TILs和原代肿瘤细胞，对比观察2D及3D培养条件下肺癌细胞系NCI-H1975及原代肺癌细胞对TILs激活扩增及抗肿瘤作用的影响；向3D培养原代肿瘤+TILs中添加程序性死亡受体1 (programmed cell death protein 1, PD-1)抗体，验证PD-1抗体对该体系中TILs浸润杀伤肿瘤细胞的影响。结果表明，相比于2D培养，3D培养的H1975肺癌细胞系可使TILs在一定时间内显著扩增，并提升TILs中CD3⁺/CD8⁺细胞的比例(61.6%)；3D培养的原代肿瘤球也可刺激增加CD3⁺/CD8⁺ TILs细胞的比例(45.5%, 54.4%)，诱导肿瘤上皮细胞的凋亡，使其存活率降至16.7%；进一步研究表明，PD-1抗体的引入促进3D原代肿瘤球共培养介导的TILs的增殖、提高CD3⁺/CD8⁺细胞的比例(50.9%, 57.0%)，并显著提高其抗肿瘤效力(肿瘤上皮细胞整体存活率降至9.36%)。综上所述，3D肿瘤球显著增强TILs细胞的体外激活增殖及抗肿瘤能力，且PD-1抗体进一步促进3D肿瘤球介导的TILs细胞增殖及其杀伤肿瘤的效力。

Abstract:

The adoptive immunotherapy mediated by tumor-infiltrating lymphocytes (TILs) has shown definite efficacy against various solid tumors. However, the inefficiency of the conventional method based on in vitro expansion of TILs fails to achieve the cell count and high tumor-killing activity required for therapeutic purposes. This study investigated the effect of 3D tumor spheroids on the activation and expansion of TILs in vitro, aiming to provide a novel approach for the expansion of TILs. We procured TILs and primary tumor cells from surgical samples of lung cancer patients and then compared the impacts of lung cancer cell line NCI-H1975 and primary lung cancer cells cultured under 2D and 3D conditions on the activation, expansion, and anti-tumor activity of TILs. Furthermore, we added the programmed cell death protein 1 (PD-1) antibody into the co-culture of primary tumor cells and TILs within a 3D environment to assess the effects of the antibody on TILs. The results showed that compared with 2D cultured tumor cells, the 3D cultured H1975 cells significantly enhanced the expansion of TILs, increasing the proportion of CD3⁺/CD8⁺ cells in TILs to 61.6%. The 3D primary tumor model also enhanced the proportion of CD3⁺/CD8⁺ cells in TILs (45.5%, 54.4%), induced apoptosis of tumor epithelial cells and decreased the overall tumor cells survival rate (16.7%) after co-culture. PD-1 antibodies further improved the in vitro expansion capacity of TILs mediated by 3D tumor spheroids, resulting in the proportions of 50.9% and 57.0% for CD3⁺/CD8⁺ cells and enhancing the antitumor activity significantly (reducing the overall tumor survival rate to 9.36%). In summary, the use of 3D tumor spheroids significantly promoted the expansion and improved the anti-tumor effect of TILs, and the use of the PD-1 antibody further promoted the expansion and tumor-killing effect of TILs.