Targeting the suppressive immune microenvironment in leptomeningeal melanoma metastases

黑色素瘤是一种高度转移性的肿瘤，有扩散到皮肤、肺部和大脑的倾向。在~5%的 例，患者还发生软脑膜黑色素瘤转移(LMM：例如黑色素瘤细胞浸润到 软脑膜、蛛网膜和脑脊液)，存活时间很短，只有8-10周。 目前还没有治疗LMM的有效方法。从药物的角度理解LMM的生物学 应对/抵抗是一个主要的未得到满足的需求。在初步研究中，我们使用了单细胞RNA-Seq(scRNA-Seq) 在LMM患者的脑脊液样本中鉴定一种新的髓系细胞群，我们认为 抑制适应性免疫反应。我们将检验LMM中的髓系细胞的假设 微环境是肿瘤进展和治疗反应的关键调节因素，而长期 LMM的治疗将需要针对黑色素瘤细胞和免疫抑制的治疗 肿瘤微环境(TME)的信号。在目标1中，我们将使用scRNA-Seq、流式细胞术和 免疫荧光检测LMM患者脑脊液免疫环境的研究 骨髓室。将进行功能研究，以1)确定脑脊液的作用 髓系细胞分化的环境2)表明滞留在脑脊液中的髓系细胞抑制T细胞 功能。然后，我们将询问来自LMM患者的Ib期临床试验的连续脑脊液样本 确定抑制PD-L1和放射治疗如何塑造脑脊液的髓系和T细胞谱系 太空。在目标2中，我们将确定髓系细胞在体内LMM进展中的作用。然后我们将在 用生长在C57/BL6小鼠软脑膜中的小鼠黑色素瘤细胞进行体内实验 全身或鞘内抑制CCR2和/或CSF1R使同系小鼠LMM对抗PD-1增敏 黑色素瘤模型。我们期望定义LMM的免疫环境，并为 可以快速评估这一最具破坏性的晚期并发症的新治疗策略 黑素瘤；黑色素瘤

Melanoma is a highly metastatic tumor, with a propensity to spread to the skin, lungs and brain. In ~5% of cases, patients also develop leptomeningeal melanoma metastases (LMM: e.g. melanoma cell infiltration into the pia mater, the arachnoid membranes and cerebrospinal fluid) which has a dismal survival of 8-10 weeks. There are no effective treatments for LMM. Understanding the biology of LMM in the context of drug response/resistance is a major unmet need. In preliminary studies, we used single cell RNA-Seq (scRNA-Seq) to identify a novel population of myeloid cells in samples of CSF from patients with LMM, which we believe suppress adaptive immune responses. We will test the hypothesis that myeloid cells in the LMM microenvironment are critical regulators of tumor progression and therapeutic response, and that the long-term management of LMM will require therapies that target both the melanoma cells and the immune-suppressive signals from the tumor microenvironment (TME). In Aim 1, we will use scRNA-Seq, flow cytometry and immunofluorescence to characterize the immune environment of CSF from patients with LMM, with a focus on the myeloid compartment. Functional studies will be undertaken to 1) determine the role of the CSF environment on myeloid cell differentiation 2) demonstrate that CSF-resident myeloid cells suppress T cell function. We will then interrogate serial CSF samples from our phase Ib clinical trial of patients with LMM to determine how inhibiting PD-L1 and radiation therapy shapes the myeloid and T cell repertoire of the CSF space. In Aim 2, we will define the role of myeloid cells in LMM progression in vivo. We will then perform in vivo experiments using mouse melanoma cells grown in the leptomeninges of C57/BL6 mice to determine if systemic or intrathecal inhibition of CCR2 and/or CSF1R sensitizes LMM to anti PD-1 in syngeneic mouse models of melanoma. We expect to define the immune environment of LMM and to develop the rationale for novel therapeutic strategies that can be rapidly evaluated in this most devastating complication of advanced melanom a.