Abstract 3750: Spatial crosstalk modeling of the tumor microenvironment uncovers CCR5-mediated glia-to-glia signaling as a key regulator of brain metastases

Abstract Purpose: Therapies targeting tumor cells and their microenvironment have demonstrated efficacy across various cancers, yet limited impact on brain metastases due to challenges with blood-brain barrier (BBB) penetration, the brain's immune privilege, and its glia composition. Here, we performed a detailed spatiotemporal analysis of glial cells in the brain metastasis microenvironment to characterize tumor-glial versus glia-to-glia communication. Methods: Astrocytes, microglia, and oligodendrocytes were isolated from mouse brains on days 3, 14, and 28 post-tumor injection using Miltenyi Biotech MicroBead Kits. RNA sequencing was performed on extracted RNA (>100 ng) from ≥200, 000 cells per sample, generating 25-60 million single-end 50-bp reads on an Illumina HiSeq 2000 platform. Spatial transcriptomics on late-stage brain metastases using the 10X Genomics Visium platform, followed by spatial analysis via our cellular crosstalk modeling tool, Spatial Single Cell Crosstalk (S2C2), to elucidate heterogenous tumor-glia signaling. Results: Spatial crosstalk analysis revealed astrocytes, microglia, and oligodendrocytes converging on Ras and Gap junction signaling pathways in tumor cells to promote macrometastasis. Notably, glial autocrine/paracrine signaling was distinct substantially from tumor-to-glia interactions, with unique ligand-receptor pairs characterizing glial communication. Beyond common pathways like PI3K-AKT and MAPK, we identified unique pathways of glia-glia crosstalk, including OSM-OSMR, CCL4-CCR5, CXCL16-CXCR6, IL1A/B-IL1R, and TNF-TNFR, as key contributors to brain metastasis progression. CCR5 inhibition with maraviroc, an FDA-approved antiviral drug, significantly reduced brain metastasis progression in preclinical models. In late-stage treatment groups, maraviroc decreased tumor burden by 62% compared to vehicle controls (p=0.004, Wilcoxon rank-sum test, n=10 per group), without inducing direct cytotoxicity in tumor cells. This highlights the critical role of CCR5-mediated glial signaling in sustaining a supportive tumor microenvironment. Conclusions: Our findings highlight CCR5-mediated glia-to-glia signaling as a key regulator of brain metastases. Unlike traditional therapies, this strategy disrupts the supportive glial niche that facilitates tumor growth. Maraviroc’s efficacy in reducing brain tumor burden highlights a promising approach focused on modifying glial interactions within the brain metastasis microenvironment, shifting the therapeutic focus from tumor cells to their supportive cellular environment. Citation Format: Ju Young Ahn, Jianting Sheng, Xin Wang, Matthew Vasquez, Wenjuan Dong, Hong Zhao, Stephen Wong. Spatial crosstalk modeling of the tumor microenvironment uncovers CCR5-mediated glia-to-glia signaling as a key regulator of brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3750.