Abstract C020: Spatial and molecular landscape in clear cell renal cell carcinoma bone metastatic patients

Abstract Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, accounting for about 80% of all renal cell carcinoma cases. Of these cases, ∼40% metastasize to the bone resulting in a poor prognosis and reduced quality of life due to severe skeletal related events. The combination of antiangiogenic tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICI) has significantly improved outcomes in patients with clear cell renal cell carcinoma (ccRCC), demonstrating impressive objective response rates and a notable extension in progression-free survival. As a result, this combinatorial approach has become a standard of care for ccRCC. However, patients with bone metastases inevitably develop resistance, and the mechanisms underlying this process remain unknown. Due to (i) the complexity of the bone environment, (ii) the difficulty of processing bone tissue, and (iii) the inability of patients with symptomatic BM to be enrolled in trials due to impaired performance status, bone metastasis has limited exploration for molecular analyses. Consequently, at present, the role of the immune infiltrate in ccRCC BM progression and response to ICI in combination with TKIs remains largely unexplored. Therefore, there is an urgent need to understand the underlying mechanisms of resistance in this patient population, including the cellular determinants that drive it. To investigate the complexity of the BM ecosystem and uncover drivers of therapy resistance mechanisms, we conducted single-cell RNA sequencing and spatial biology analysis on biospecimens collected from bone metastatic ccRCC patients. 63 patients were enrolled and clinically annotation included baseline demographics, surgical history, organ metastasis and timing, distribution of bone metastasis, interventional procedures, radiation, systemic treatment and survival data. Of these patients, 52 of them (82.5%) had samples deemed eligible for integration of clinical data analysis and downstream analysis. Our single-cell RNA sequencing analysis indicated a shift in the microenvironment composition of treated patients, with an increase in the myeloid population and a decrease in the endothelial cell and lymphoid populations. Gene-set enrichment analysis (GSEA) revealed enrichment in TNF-α signaling, and IFNα and IFNγ response. Spatial analyses were performed using COMET™ multiplex immunofluorescence, evaluating 20 markers across tumor, immune and stromal compartments. Then, Visiopharm® software was used to develop an analysis pipeline for cell segmentation and phenotyping within the tumor microenvironment. This analysis showed overall reduced immune cell infiltration in patients previously treated with TKI+ICI. These findings illuminate the complexity of the bone metastatic niche. We expect that ongoing analyses of our datasets will provide critical insight into the mechanisms underlying therapy resistance. Citation Format: Andrea Ameruoso, Raymond Wang, Mohammad Moussa, Thomas J. Heyman, Jianfeng Chen, Zacharia Thomas, Christopher Alvarez-Breckenridge, Jianjun Gao, Matthew T. Campbell, Eleonora Dondossola. Spatial and molecular landscape in clear cell renal cell carcinoma bone metastatic patients abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C020.