Treatment of cancers has largely benefited from the development of immunotherapy. In particular, Chimeric Antigen Receptor (CAR) redirected T cells have demonstrated impressive efficacy against B-cell malignancies and continuous efforts are made to adapt this new therapy to solid tumors, where the immunosuppressive tumor microenvironment is a barrier for delivery. CAR T-cell validation relies on in vitro functional assays using monolayer or suspension cells and in vivo xenograft models in immunodeficient animals. However, the efficacy of CAR therapies remains difficult to predict with these systems, in particular when challenged against 3D organized solid tumors with highly intricate microenvironment. An increasing number of reports have now included an additional step in the development process in which redirected T cells are tested against tumor spheres. Here, we report a method to produce 3D structures, or cysts, out of a colorectal cancer cell line, Caco-2, which has the ability to form polarized spheroids as a validation tool for adoptive cell therapy in general. We used CD19CAR T cells to explore this method and we show that it can be adapted to various platforms including high resolution microscopy, bioluminescence assays and high-throughput live cell imaging systems. We developed an affordable, reliable and practical method to produce cysts to validate therapeutic CAR T cells. The integration of this additional layer between in vitro and in vivo studies could be an important tool in the pre-clinical workflow of cell-based immunotherapy.

中文翻译：

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结直肠囊肿可作为CAR治疗的验证工具。

癌症的治疗在很大程度上得益于免疫疗法的发展。尤其是，嵌合抗原受体（CAR）重定向的T细胞在对抗B细胞恶性肿瘤方面已显示出令人印象深刻的功效，并不断努力使这种新疗法适应实体瘤，其中免疫抑制性肿瘤微环境是传递的障碍。CAR T细胞验证依赖于免疫缺陷动物中使用单层或悬浮细胞的体外功能测定以及体内异种移植模型。但是，使用这些系统仍难以预测CAR疗法的功效，尤其是在针对具有高度复杂的微环境的3D组织实体瘤的挑战时。现在越来越多的报告在开发过程中包括了另外的步骤，其中针对肿瘤球对重定向的T细胞进行了测试。在这里，我们报告了一种从结直肠癌细胞系Caco-2中产生3D结构或囊肿的方法，该方法具有形成极化球体的能力，通常作为过继细胞疗法的验证工具。我们使用CD19CAR T细胞来探索这种方法，我们证明它可以适应各种平台，包括高分辨率显微镜，生物发光测定和高通量活细胞成像系统。我们开发了一种经济实惠，可靠且实用的方法来生产囊肿，以验证治疗性CAR T细胞。在体外和体内研究之间，这一附加层的整合可能是基于细胞的免疫疗法临床前工作流程中的重要工具。