Development of Physiologic Tissue Models to Assess Tumor Explant Response to Immune Checkpoint Blockade

项目总结 本研究项目提案的目标是开发一种体外肿瘤培养的生理模型，以 研究对免疫检查点封锁的反应性。尽管PD-1/PD-L1和CTLA4的抑制剂 免疫检查点对恶性黑色素瘤等癌症患者产生了显著而持久的反应， 预测单个患者活动的能力仍然有限，并依赖于从 固定肿瘤组织。例如，在器官模型中生长患者来源的肿瘤的能力一直是 经过过去几年的严格证明，这些系统缺乏肿瘤的关键特征。 微环境，包括血管网络和免疫细胞。因此，体外系统支持 通过概括肿瘤和免疫细胞培养的生理微环境，几乎可以肯定 对于开发能够预测患者对免疫治疗的反应的功能分析是必不可少的。 最近，我们展示了我们的三维微流控培养系统可以支持生长。 来自多种不同癌症类型的原发人类肿瘤球体，包括黑色素瘤。 重要的是，免疫图谱显示球体内的细胞也含有显著的 肿瘤相关免疫细胞的比例，包括巨噬细胞、树突状细胞和抗原 T淋巴细胞。将这些短期球体培养物暴露于免疫检查点抑制剂，如抗- PD1抗体导致免疫应答和强大的细胞因子分泌到条件 在某些情况下，还有细胞死亡的证据。 这项提案的广泛、长期目标是扩展这一初步模式，以利用 我们的实验室开发的功能将微循环网络整合到围绕 肿瘤，并利用它作为一种手段，随后引入选定的髓系细胞。初始模型和 这些扩展将经过详细的验证，以发展一种现实的生理文化 能够预测免疫治疗反应的系统。这项工作的一个独特之处是它跨越了基本的 和翻译研究，包括使用动物模型来帮助设计血管网络，以及 患者获取的样本和临床对免疫检查点封锁的反应来验证该系统。特定的 目的是：1)改进和验证现有的微流控肿瘤培养模型以评估对免疫的反应 检查点抑制疗法2)结合免疫细胞的血管流动来监测外渗和 肿瘤培养中免疫效应细胞的扩增，以及3)体外实验与患者的直接比较 对免疫检查点封锁的特定反应。通过这些互补性研究，最终目标是 开发一种最终可用于临床的健壮模型，以帮助针对免疫检查点 针对适当的患者亚组进行抑制剂治疗。这个基础平台也将在其他方面有用 设置，一旦进行了充分评估

PROJECT SUMMARY The goal of this research project proposal is to develop a physiologic model of ex vivo tumor culture to study responsiveness to immune checkpoint blockade. Although inhibitors of the PD-1/PD-L1 and CTLA4 immune checkpoints have led to remarkable and durable responses in cancers such as malignant melanoma, the ability to predict the activity for individual patients remains limited, and have relied on measurements from fixed tumor tissue. While the ability to grow patient derived tumors in organoid models, for example, has been rigorously demonstrated over the past several years, these systems lack key features of the tumor microenvironment, including a vascular network and immune cells. Thus, an ex vivo system that supports tumor and immune cell culture by recapitulating a physiologic microenvironment will almost certainly be essential to the development of functional assays that can predict patient response to immunotherapy. Recently, we demonstrated that our three-dimensional microfluidic culture system can support growth of primary human tumor spheroids derived from multiple different cancer types, including melanoma. Importantly, immune profiling of the cells within the spheroids reveals that they also contain a significant proportion of tumor associated immune cells, including macrophages, dendritic cells, and antigen experienced T lymphocytes. Exposure of these short term spheroid cultures to immune checkpoint inhibitors such as anti- PD1 antibodies results in evidence of immunologic response and robust cytokine secretion into conditioned medium, as well as evidence of cell killing in some cases. The broad, long term objective of this proposal is to extend this preliminary model to leverage the capabilities developed in our labs to incorporate a microcirculatory network in the 3D matrix surrounding the tumor and use this as a means of subsequently introducing selected myeloid cells. Both the initial model and these extensions to it will be subject to detailed validation in order to develop a realistic physiologic culture system that enables prediction of immunotherapy response. A unique aspect of this work is that it spans basic and translational research, including the use of animal models to help engineer vascularized networks, and patient-derived samples and clinical response to immune checkpoint blockade to validate the system. Specific aims are to: 1) Refine and validate an existing microfluidic tumor culture model to assess response to immune checkpoint inhibitor therapies 2) Incorporate vascular flow of immune cells to monitor extravasation and expansion of immune effector cells in tumor culture, and 3) Directly compare ex vivo experiments with patient specific response to immune checkpoint blockade. Through these complementary studies, the ultimate goal is to develop a robust model that can eventually be adapted to clinical use to help target immune checkpoint inhibitor therapies to the appropriate subgroup of patients. This basic platform will also be useful in other settings, once it has been fully evaluations