Abstract 2574: Cancer associated fibroblasts with high expression of immunoglobulin superfamily containing leucine-rich repeat show tumor inhibitory capabilities in brain metastasis

Abstract Brain metastasis (BM) remains mostly incurable with a dismal mean 2-year survival of less than 2%. The tumor microenvironment (TME) of BM is complex and includes cancer associated fibroblasts (CAFs), which we have shown can either support or inhibit tumor growth, pointing to their heterogeneity. To examine the CAF heterogeneity in the BM TME, we performed a cell multiome sequencing analysis (scMultiome-seq) of BM patient tissues and identified 4 diverse CAF subpopulations in the TME. Among these was a subpopulation of CAFs that expressed high levels of tumor inhibitory CAF marker immunoglobulin superfamily containing leucine-rich repeat (ISLR). High expression of ISLR was also observed in CAFs we have previously reported as tumor inhibitory in an in vivo model of BM. We thus hypothesized that ISLR-CAFs could be tumor inhibitory in the BM TME. Further analysis of our scMultiome-seq data to infer cell-cell communication revealed the anti-proliferative IGFBP3-TMEM219 pathway as a potential communication mechanism between ISLR-CAFs and BM tumor cells. In the present study, we thus aimed to investigate the role of ISLR in BM CAFs and to study the significance of the IGFBP3-TMEM219 pathway in the tumor-inhibitory effects of ISLR-CAFs in vitro. To do so, patient-derived BM tumor (CM04) and CAF (CM01, CM02, CM03 and CM08) cell lines previously established in our lab were used. BM tumor cells were treated with CAF-derived conditioned media (CM) and cell viability was assessed using the CellTiter-Glo assay. The expression of ISLR, IGFBP3 and TMEM219, along with the activation of caspase-8 signaling linked to the anti-proliferative effects of the IGFBP3-TMEM219 pathway, was analyzed via western blotting. We observed high expression of ISLR in CM03 and CM08 CAFs, as compared to CM01 and CM02 CAFs, which expressed low to no ISLR. CM04 tumor cells showed high expression of TMEM219 compared to CAFs, while CM03 and CM08 CAFs expressed the highest levels of IGFBP3. Both ISLR and IGFBP3 were also detected in CM derived from CM03 and CM08 CAFs. We observed that CM03 and CM08 CAF-derived CM significantly inhibited CM04 tumor cell viability in vitro. In addition, activation of the caspase 8 signaling was detected in CM04 tumor cells treated with CM08 CAF-derived CM. Next, ISLR knock-down was performed in CAFs using lentiviral shRNA particles to confirm a role for ISLR in CAF-mediated tumor inhibition. ISLR knock-down in CM08 CAFs decreased expression of IGFBP3, suggesting a role for ISLR in regulating IGFBP3 expression in CAFs. Altogether the present data revealed that CAFs with high ISLR expression could inhibit tumor growth via the IGFBP3-TMEM219 pathway in the BM TME. Further in-depth understanding of the roles of ISLR-CAFs in the BM TME could help develop strategies that selectively target tumor supportive CAFs while protecting tumor inhibitory ones. Citation Format: Thomas Simon, Aspiara Roosmin, David N. Buckley, Ben Y. Tew, Gerald C. Gooden, Gabriel Zada, Bodour Salhia. Cancer associated fibroblasts with high expression of immunoglobulin superfamily containing leucine-rich repeat show tumor inhibitory capabilities in brain metastasis [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 2574.