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

项目摘要 本研究项目提案的目标是开发一种离体肿瘤培养的生理模型， 研究对免疫检查点封锁的反应性。尽管PD-1/PD-L1和CTLA 4的抑制剂 免疫检查点已经在癌症如恶性黑素瘤中导致显著和持久的应答， 预测个体患者活动的能力仍然有限，并且依赖于来自 固定的肿瘤组织。虽然在类器官模型中生长患者来源的肿瘤的能力，例如， 在过去几年中，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，一种离体系统， 通过重现生理微环境进行肿瘤和免疫细胞培养， 这对于开发可以预测患者对免疫疗法的反应的功能测定至关重要。 最近，我们证明了我们的三维微流控培养系统可以支持生长 来源于多种不同癌症类型的原发性人类肿瘤球体，包括黑素瘤。 重要的是，球状体内细胞的免疫谱显示，它们还含有显著的 肿瘤相关免疫细胞（包括巨噬细胞、树突状细胞和抗原）的比例 T淋巴细胞。将这些短期球状体培养物暴露于免疫检查点抑制剂如抗- PD 1抗体导致免疫应答的证据和向条件性细胞内的稳健细胞因子分泌 培养基，以及在某些情况下细胞杀伤的证据。 本提案的广泛、长期目标是扩展这一初步模式，以利用 在我们的实验室中开发的功能，将微循环网络纳入3D矩阵， 肿瘤，并将其用作随后引入选定的骨髓细胞的手段。初始模型和 为了发展一种现实的生理文化，对它的这些扩展将进行详细的验证 该系统能够预测免疫治疗反应。这项工作的一个独特之处在于它跨越了基本的 和转化研究，包括使用动物模型来帮助设计血管化网络，以及 患者来源的样品和对免疫检查点阻断的临床反应，以验证系统。具体 目的是：1）完善和验证现有的微流控肿瘤培养模型，以评估免疫应答 检查点抑制剂疗法2）纳入免疫细胞的血管流动以监测外渗， 在肿瘤培养物中扩增免疫效应细胞，和3）直接比较与患者的离体实验 对免疫检查点阻断的特异性反应。通过这些补充研究，最终目标是 开发一个强大的模型，最终可以适应临床使用，以帮助靶向免疫检查点 抑制剂治疗适当的患者亚组。这一基本平台也将在其他领域发挥作用。 设置，一旦它已经完全评估

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