Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a ‘super engager’ behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

将针对血癌的细胞免疫疗法的成功扩展到实体瘤领域将需要改进的体外模型，以揭示分子水平的治疗作用模式。在这里，我们描述了一个名为 BEHAV3D 的系统，该系统开发用于通过成像和转录组学研究免疫细胞和患者癌症类器官的动态相互作用。我们将 BEHAV3D 应用于实时跟踪 >150,000 个工程化 T 细胞，这些细胞与源自患者的实体瘤类器官一起培养，确定了一个包含具有强大连续杀伤能力的 T 细胞的“超级参与者”行为集群。在其他 T 细胞概念中，我们还研究癌症代谢组传感工程 T 细胞 (TEG) 并检测行为特异性基因特征，其中包括一组 27 个基因，这些基因没有先前描述的由超级接合杀手 TEG 表达的 T 细胞功能。我们进一步表明，I 型干扰素可以引发抗性类器官用于 TEG 介导的杀伤。BEHAV3D 是一种很有前途的工具，可用于表征细胞免疫疗法的行为表型异质性，并可能支持优化个性化实体瘤靶向细胞疗法。