Neural organoid models of the immunological microenvironment of glioblastoma for drug discovery applications

项目概要/摘要 胶质母细胞瘤（GBM）是成人中最常见的原发性脑肿瘤， 存活率和基本不变的护理标准。虽然有许多挑战， 开发更好的GBM治疗，主要挑战之一是免疫抑制 在GBM肿瘤中常见的环境。这种免疫抑制性质导致 不适合对GBM细胞产生免疫应答的肿瘤， 免疫疗法无效。为了解决这个问题，可以询问 GBM癌细胞和小胶质细胞之间的复杂相互作用 巨噬细胞对于靶点鉴定、新治疗剂的筛选以及 行动模式研究。肿瘤相关的小胶质细胞和巨噬细胞是特别感兴趣的 这是由于它们在形成GBM肿瘤的免疫环境中的主要作用。人类 类器官技术非常适合模拟人类复杂的多细胞相互作用 组织样环境。Stem Pharm的水凝胶神经类器官允许掺入 非神经细胞群，如小胶质细胞和巨噬细胞，在可重复的96孔板中 适合筛选应用程序的格式。因此，本提案中的工作将得到发展， 通过掺入小胶质细胞验证人体外胶质母细胞瘤类器官模型， 巨噬细胞和患者来源的GBM细胞在我们的神经类器官中。具体目标：（1） 表征并入GBM的类器官，评估GBM存活、侵袭和增殖; 并表征对GBM的细胞类型特异性转录应答， 肿瘤和公开可用的数据集。2)显示免疫抑制激活 神经类器官内的小胶质细胞和巨噬细胞响应于浸润的GBM细胞。 多重细胞因子组、共刺激和检查点分子表达以及直接的 使用外周血单核细胞衍生的T细胞的免疫抑制测定将用于 评估小胶质细胞和巨噬细胞免疫抑制。最后，用三小治疗 将在类器官内评估已知调节巨噬细胞活化的分子， 证明了调节小胶质细胞和巨噬细胞对GBM细胞应答的能力。 这些特定目标的成功完成将导致稳健的体外类器官模型， 询问GBM侵袭和随后的小胶质细胞和巨噬细胞的新能力 免疫抑制这将为制药合作伙伴提供研究治疗的能力， 以前由于这种免疫抑制环境而失败，并测试新的治疗方法 接近。II期研究将扩大可用的患者来源样本的数量， 更好地捕捉GBM肿瘤的多样性和异质性，探索性别连锁差异， 评估CAR-T和联合疗法在GBM模型中的有效性， 我们的目标是为这种毁灭性疾病的患者提供更好的治疗选择。

Project Summary/Abstract Glioblastoma (GBM) is the most prevalent primary brain tumor in adults with extremely poor survival rates and largely unchanged standard of care. While there are many challenges to developing better GBM treatments, one of the major challenges is the immune-suppressive environment commonly found within GBM tumors. This immune-suppressive nature results in a tumor that is not suitable for mounting an immune response to GBM cells, rendering emerging immunotherapies ineffective. To address this issue, suitable models that can interrogate the complex interactions between GBM cancer cells and microglia and peripherally derived macrophages would be invaluable for target identification, screening of novel therapeutics and for mode of action studies. Tumor-associated microglia and macrophages are of particular interest due to their primary role in shaping the immunological environment of GBM tumors. Human organoid technology is well-suited for modeling complex, multicellular interactions in a human tissue-like environment. Stem Pharm’s hydrogel-enabled neural organoids allow for incorporation of non-neural populations such as microglia and macrophages in a reproducible, 96-well plate format amenable to screening applications. Therefore, work in this proposal will develop and validate a human in vitro glioblastoma organoid model through incorporation of microglia, macrophages, and patient derived GBM cells in our neural organoids. Specific aims will 1) characterize organoids incorporating GBM, evaluate GBM survival, invasion, and proliferation; and characterize cell-type specific transcriptional responses to GBM and compare them to parent tumors and publicly available data sets. 2) demonstrate immunosuppressive activation of microglia and macrophages within the neural organoid in response to infiltrating GBM cells. Multiplex cytokine panels, co-stimulatory and checkpoint molecule expression, and a direct immunosuppression assay with peripheral blood mononuclear cell-derived T-cells will be used to evaluate microglia and macrophage immunosuppression. Finally, treatment with three small molecules known to modulate macrophage activation will be assessed within the organoids to demonstrate the ability to regulate the microglial and macrophage response to GBM cells. Successful completion of these specific aims will result in a robust in vitro organoid model with novel capabilities to interrogate GBM invasion and subsequent microglia and macrophage immunosuppression. This will provide pharma partners with the ability to study therapies that previously failed due to this immunosuppressive environment, and test new therapeutic approaches. Phase II studies will expand the number of available patient-derived samples to better capture the diversity and heterogeneity of GBM tumors, explore sex-linked differences, and evaluate the effectiveness of CAR-Ts and combination therapies within the GBM model with the goal of bringing better treatment options to patients for this devastating condition.