Interrogating the response of the tumor microenvironment to combination immunotherapy using a microfluidic platform

摘要 肿瘤中细胞间相互作用的复杂性是理解肿瘤的最大障碍之一 癌症和开发有效的治疗方法。癌细胞不断地与成纤维细胞、内皮细胞和成纤维细胞相互作用。 细胞、免疫细胞、信号分子和肿瘤微环境（TME）中的细胞外基质。 宿主和癌细胞之间的相互作用是复杂的，其作用可能是肿瘤抑制性的，或 促进肿瘤的例如，巨噬细胞首先被招募来对抗癌症;然而， 细胞可以使它们支持肿瘤。因此，能够更好地理解 癌细胞与其微环境之间的相互作用可以更好地理解其机制 药物耐药性，识别新的分子靶点，并帮助解决治疗中大量未满足的需求， 癌定量技术，如我们的，整合了体外药物治疗和评估 与天然组织中的细胞和分子表型的药理学反应，应加速 新疗法的发现和发展。 然而，目前研究药物反应的工具和TME还没有跟上药物测试的需要。鉴于 几乎无限数量的潜在组合和有限的资源来实际测试它们，因此非常需要 用于测试使用人完整肿瘤组织离体预测对组合的体内反应的平台 免疫疗法以小型化、多路复用的形式，保留尽可能多的TME。 在我们的第一个R 01周期中，我们开发了一种微流体平台（称为Oncoslice）， 器官型培养物在几十种药物条件下的时空暴露，我们证明了它的效用 在异种移植物和患者GBM切片上进行细胞死亡测定。在第二个R 01周期中，我们的目标是应用我们的 评估联合免疫疗法及其与TME相互作用的肿瘤切片平台。的 该平台将应用于两种难以治疗的实体瘤类型-胰腺癌（PCa，小鼠和人） 和三阴性乳腺癌（TNBC，小鼠）。我们最近发现，CD 8 + T细胞的肿瘤内迁移 是胰腺癌中响应免疫检查点抑制剂治疗的抗肿瘤活性的必要前体 癌症使我们的断言更加可信，即发展对细胞间相互作用的微观理解， TME至关重要。PCa和TNBC两者都是极其异质的，并且对当前的免疫应答很差。 检查点抑制剂。我们平台的新发展将包括实时成像的动态变化， 免疫TME、组织和细胞因子取样、选择性转录组学和蛋白质途径分析。我们将 将切片中的表型反应与分子数据整合以建立预测统计模型。在一起， 这些方法将为免疫调节药物的发现建立一个创新平台， 提供深入了解药物的作用机制和TME在癌症治疗中的作用。

ABSTRACT The complexity of cell-cell interactions in the tumor represents one of the biggest barriers to understanding cancer and to developing effective therapeutics. Cancer cells constantly interact with fibroblast cells, endothelial cells, immune cells, signaling molecules and the extracellular matrix in the tumor microenvironment (TME). The interactions between host and cancer cells are complex, with effects that may be tumor-suppressive or tumor-promoting. For example, macrophages are first recruited to fight cancer; however, interactions with cancer cells can render them tumor-supportive. Thus, enabling a greater understanding of the complexities of the interaction between cancer cells and their microenvironment can lead to a better understanding of mechanisms of drug resistance, identification of new molecular targets and help address the large unmet needs in treating cancer. Quantitative technologies, such as ours, that integrate ex vivo drug treatments and assess pharmacological responses with cellular and molecular phenotypes in native tissues, should accelerate the discovery and development of novel therapeutics. Yet present tools to study drug responses and the TME have not kept up with drug testing needs. Given the nearly infinite number of potential combinations and limited resources to actually test them, there is a great need for testing platforms that use human, intact tumor tissue ex vivo to predict in vivo responses to combination immunotherapies in miniaturized, multiplexed formats that retain as much of the TME as possible. In our first R01 cycle, we developed a microfluidic platform (called Oncoslice) that allows for selective spatiotemporal exposure of organotypic cultures to dozens of drug conditions, and we demonstrated its utility with cell death assays on xenograft and patient GBM slices. In this second R01 cycle, our goal is to apply our Oncoslice platform to evaluate combination immunotherapies and their interaction with the TME. The platform will be applied to two difficult-to-treat solid tumor types – pancreatic cancer (PCa, mouse, and human) and triple-negative breast cancer (TNBC, mouse). Our recent finding that intratumoral migration of CD8+ T cells is a necessary precursor to anti-tumor activity in response to immune checkpoint inhibitor therapy in pancreatic cancer lends credence to our assertion that developing a micro-scale understanding of cell-cell interactions in the TME is critical. Both PCa and TNBC are extremely heterogeneous and respond poorly to current immune checkpoint inhibitors. New developments to our platform will include live imaging of dynamic changes in the immune TME, tissue and cytokine sampling, selective transcriptomics, and protein pathway profiling. We will integrate phenotypic responses in slices with molecular data to build predictive statistical models. Together, these approaches will establish an innovative platform for immuno-modulatory drug discovery designed to provide insights into the drug's mechanism of action and the TME's role in cancer treatment.