T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment (TME), but its complexity makes it difficult to develop the ideal model. In this study, we developed a simple co-culture assay, reproducible in any lab, but respecting the multicellular nature of the TME. Our goal is to combine in a single assay well-established techniques such as a luciferase assay for target cell viability analysis, a CD107a degranulation assay, and multicolor flow cytometry for the detection of cytokines and cytotoxicity markers. Cell suspensions of whole spleens and tumors containing splenic or tumor-infiltrating effector T cells of mice bearing Lewis lung carcinoma (LLC) or CT26 colon carcinoma tumors treated with radiation alone or in combination with immunotherapies were used for co-culture. LLC and CT26 cell lines transduced with the firefly luciferase gene were used as target cells. We demonstrated that splenocytes and tumor-infiltrating T cells derived from mice treated with combination therapy were able to kill approximately 50% of target cells after 48 h of co-culture. This effect was tumor cell-specific and dependent on CD8⁺ T cells evidenced by in vitro CD8⁺ T cell depletion. Flow cytometry demonstrated increased expression of CD107a and production of granzyme B, IFNγ, and TNFα by CD8⁺ T cells. Our co-culture assay is therefore suitable as proof of principle for in vivo therapeutic studies testing immunotherapies, and specifically to assess the involvement of cytotoxic CD8⁺ T cells in treatment response in LLC and CT26 tumor models. We also propose this assay as an ex vivo platform for high-throughput screening of immunomodulating agents to be tested in these two murine tumor models. This assay can be adapted to other tumor models after optimizations.

T 细胞免疫疗法在晚期癌症患者中显示出巨大的前景，彻底改变了治疗方法。 T 细胞的细胞毒性对其功效至关重要，因此开发测试肿瘤和 T 细胞相互作用的离体方法至关重要。人们越来越多地努力开发旨在模拟肿瘤微环境（TME）的复杂材料和平台的共培养测定，但其复杂性使得开发理想模型变得困难。在这项研究中，我们开发了一种简单的共培养测定法，可在任何实验室重复，但尊重 TME 的多细胞性质。我们的目标是在单一测定中结合成熟的技术，例如用于靶细胞活力分析的荧光素酶测定、CD107a 脱粒测定以及用于检测细胞因子和细胞毒性标记物的多色流式细胞术。将携带Lewis肺癌(LLC)或CT26结肠癌肿瘤的小鼠的整个脾脏和含有脾脏或肿瘤浸润效应T细胞的肿瘤的细胞悬浮液单独或与免疫疗法联合治疗用于共培养。使用萤火虫荧光素酶基因转导的 LLC 和 CT26 细胞系作为靶细胞。我们证明，来自接受联合治疗的小鼠的脾细胞和肿瘤浸润 T 细胞在共培养 48 小时后能够杀死大约 50% 的靶细胞。这种效应是肿瘤细胞特异性的，并且依赖于 CD8 ⁺ T 细胞，这一点通过体外CD8 ⁺ T 细胞耗竭得到证实。流式细胞术显示 CD8 ⁺ T 细胞的 CD107a 表达增加以及颗粒酶 B、IFNγ 和 TNFα 的产生增加。 因此，我们的共培养测定适合作为测试免疫疗法的体内治疗研究的原理证明，特别是评估细胞毒性 CD8 ⁺ T 细胞在 LLC 和 CT26 肿瘤模型中治疗反应中的参与情况。我们还建议将此测定作为体外平台，用于高通量筛选免疫调节剂，以在这两种小鼠肿瘤模型中进行测试。该测定经优化后可适用于其他肿瘤模型。