由肿瘤浸润淋巴细胞（TIL）介导的过继性免疫疗法已对多种实体瘤显示出明确的疗效。然而，基于TIL体外扩增的传统方法效率低下，无法达到治疗目的所需的细胞计数和高肿瘤杀伤活性。本研究探讨了3D肿瘤球体对TILs体外激活和扩增的影响，旨在为TILs的扩增提供一种新的方法。我们从肺癌患者的手术样本中获取 TIL 和原代肿瘤细胞，然后比较肺癌细胞系 NCI-H1975 和在 2D 和 3D 条件下培养的原代肺癌细胞对 TIL 的激活、扩增和抗肿瘤活性的影响。 TIL。此外，我们将程序性细胞死亡蛋白 1 (PD-1) 抗体添加到 3D 环境中原代肿瘤细胞和 TIL 的共培养中，以评估该抗体对 TIL 的影响。结果显示，与2D培养的肿瘤细胞相比，3D培养的H1975细胞显着增强了TIL的扩增，将TIL中CD3/CD8细胞的比例提高至61.6%。 3D原发肿瘤模型还提高了TIL中CD3/CD8细胞的比例（45.5%、54.4%），诱导肿瘤上皮细胞凋亡，并降低了共培养后肿瘤细胞的总体存活率（16.7%）。 PD-1抗体进一步提高了3D肿瘤球体介导的TIL的体外扩增能力，导致CD3/CD8细胞比例达到50.9%和57.0%，显着增强抗肿瘤活性（将总体肿瘤存活率降低至9.36%） ）。综上所述，3D肿瘤球体的使用显着促进了TILs的扩增并提高了TILs的抗肿瘤效果，而PD-1抗体的使用进一步促进了TILs的扩增和杀伤效果。

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.