Investigate and inhibit microglia support of brain metastases

1摘要-项目2 2脑转移瘤是高度侵袭性、治疗抵抗性的恶性肿瘤，伴有使人衰弱的神经系统后遗症 3、严重的预后。努力了解和调节转移性脑肿瘤微环境（TME） 4具有巨大的治疗潜力。研究表明，巨噬细胞（M1或M2）的极化可能是 5有益或有害的癌症进展（1，2）。小胶质细胞（大脑的骨髓细胞）在免疫中发挥作用 6监视和介导肿瘤相关的炎症反应，包括在转移性肿瘤的背景下。 7脑肿瘤显微镜（肿瘤相关巨噬细胞和肿瘤相关小胶质细胞）。我们假设 8脑转移部分由天然小胶质细胞支持（3），这些相互作用可以是 9例为治疗获益。小胶质细胞在脑转移瘤中的作用一直难以研究 10.鉴于脑转移瘤样本相对难以获得，无法分离出人类肿瘤相关的 11小胶质细胞或监测它们在TME内的相互作用，缺乏包括或允许操纵TME的模型。 12小胶质细胞，同时仍然可靠地重演人类疾病。为了克服这些关键障碍，我们采用 （i）对新鲜人脑转移瘤进行单细胞RNA测序（scRNA-seq） 14和正常周围脑（Neuropathology Core）（4，5）;（ii）细胞的分离和动态阵列测序 15来自人脑脊液的游离信使RNA（cfRNA）（6，7）;（iii）产生人和鼠来源的 16种脑转移3D气液界面（ALI）类器官，包括肿瘤相关小胶质细胞/巨噬细胞 17（Toolkit Core）;和（iv）用循环衍生的小胶质细胞样细胞（CDMC）再增殖小胶质细胞， 18.整个大脑的效率超过90%。这四种创新方法使我们能够调查 肿瘤相关小胶质细胞在人脑转移中的作用，模型免疫细胞相互作用， 20在转移性恶性肿瘤的动物模型中操纵它们的功能。我们的多学科团队是独一无二的 21定位于测试以下三个目标：（1）测试肿瘤相关小胶质细胞在多大程度上产生肿瘤相关性小胶质细胞。 22例免疫抑制性TME支持脑转移。除了将小胶质细胞的功能与大脑 23转移局部复发和远处进展，我们将交叉参考我们的研究结果与癌细胞内在 24因子（项目1）和全身巨噬细胞反应性（项目3）;（2）模型内肿瘤相关小胶质细胞 25使用ALI类器官的脑转移性TME。我们将模拟肿瘤和肿瘤相关的小胶质细胞/巨噬细胞 26相互作用，并确定免疫治疗对小胶质细胞/巨噬细胞活化和吞噬作用的影响， 27 T细胞活化状态和细胞死亡。我们的发现将与细胞内在的扩展知识相结合， 28个因素（项目1）和脑-外周免疫相互作用（项目3）;（3）：确定效率有多高 用CDMC替代小胶质细胞抑制脑转移。我们将用“武器化”取代小胶质细胞 30 CDMC抑制癌细胞植入和疾病进展。 31

1 ABSTRACT – PROJECT 2 2 Brain metastases are highly aggressive, treatment resistant malignancies with debilitating neurological sequelae 3 and a grave prognosis. Efforts to understand and modulate the metastatic brain tumor microenvironment (TME) 4 have great therapeutic potential. Studies have suggested that polarization of macrophages (M1 or M2) may be 5 helpful or harmful in cancer progression (1, 2). Microglia, the brain’s myeloid cells, function in immune 6 surveillance and mediate the tumor-related inflammatory response, including within the context of the metastatic 7 brain tumor microniche (tumor-associated macrophages and tumor-associated microglia). We hypothesize 8 brain metastases are supported in part by native microglia (3), and that these interactions can be 9 manipulated for therapeutic benefit. The role of microglia in brain metastases has been difficult to investigate 10 given the relative inaccessibility of brain metastases samples, inability to isolate human tumor-associated 11 microglia or monitor their interactions within the TME, and lack of models that include or allow manipulation of 12 microglia while still reliably recapitulating the human disease. To overcome these critical hurdles, we employ the 13 following innovative methods: (i) single cell RNA-sequencing (scRNA-seq) on fresh, human brain metastases 14 and normal surrounding brain (Neuropathology Core)(4, 5); (ii) isolation and dynamic array sequencing of cell 15 free messenger RNA (cfRNA) from human cerebrospinal fluid (6, 7); (iii) generation of human and murine-derived 16 brain metastasis 3D air-liquid interface (ALI) organoids that include tumor-associated microglia/macrophages 17 (Toolkit Core); and (iv) repopulation of microglia with circulation derived microglia-like cells (CDMCs) with 18 greater than 90% efficiency throughout the entire brain. These four innovative methods allow us to investigate 19 the role of tumor-associated microglia in human brain metastases, model immune cell interactions, and 20 manipulate their function in an animal model of metastatic malignancy. Our multidisciplinary team is uniquely 21 positioned to test the following three aims: (1) Test the extent to which tumor-associated microglia create an 22 immunosuppressive TME supporting brain metastasis. In addition to relating microglia function to brain 23 metastasis local recurrence and distant progression, we will cross-reference our findings with cancer cell intrinsic 24 factors (Project 1) and systemic macrophage reactivity (Project 3); (2) Model tumor-associated microglia within 25 the brain metastatic TME using ALI organoids. We will model tumor and tumor-associated microglia/macrophage 26 interactions and determine the impact of immunotherapy on microglia/macrophage activation and phagocytosis, 27 T-cell activation states, and cell death. Our findings will be integrated with expanding knowledge of cell-intrinsic 28 factors (Project 1) and brain-periphery immune interactions (Project 3); (3): Determine how high efficiency 29 microglia replacement with CDMCs inhibits brain metastasis. We will replace microglia with “weaponized” 30 CDMCs to inhibit cancer cell implantation and disease progression. 31