Illuminating the Immune Dynamics of Metastatic Renal Cell Carcinoma to Bone - PROJECT SUMMARY/ABSTRACT Clear cell renal cell carcinoma (ccRCC) accounts for ~80% of renal malignancies. Bone involvement occurs in ~40% of metastatic patients and constitutes a devastating complication, contributing significantly to morbidity, elevated mortality risk, and substantial healthcare burden. Due to both clinical and technical challenges (e.g., poor performance status that limits patient inclusion in clinical trials; difficulties in obtaining and processing bone biopsies), our understanding of the bone metastatic niche and the role of resident stromal and immune cells in the progression of ccRCC bone metastasis (BM) remains limited. This critical knowledge gap highlights the urgent need to clarify the mechanisms of resistance in preclinical models; mice represent the lowest organism to appropriately address the progression and therapy response of BM. Antiangiogenic tyrosine kinase inhibitors (TKIs) combined with immune checkpoint inhibitors (ICIs) are effective first-line therapies for patients with BM. However, resistance inevitably emerges, leading to disease progression. Understanding how TKI+ICI therapies shape the bone microenvironment and drive resistance is critical for developing more effective treatment strategies. Interestingly, chimeric antigen receptor-expressing natural killer cells (CAR-NK) recently emerged as a promising strategy against treatment-refractory ccRCC, due to their specificity, ready availability, safety profile and efficacy. A major barrier to understanding the dynamic evolution of the BM niche, immune infiltrate, and treatment response in real-time is the lack of tractable experimental models. To address this challenge, we developed a multiparametric multiphoton microscopy platform for BM (in vivo, iMPM; ex vivo, eMPM) and have successfully used it to track tumor progression and therapy response in conjunction with single-cell RNA sequencing and bioluminescence imaging. Here, we hypothesize that reciprocal interplays among ccRCC, bone stromal and immune cells foster the establishment of a distinct tumor-associated microenvironment that promotes therapy resistance and can be effectively targeted by cytotoxic cellular immunotherapy. Our relevant models will permit us to systematically characterize the dynamic nature of the ccRCC BM niche, at baseline and after therapy. Accordingly, in Aim 1 we plan to define the cellular composition, transcriptional states, and dynamics of the ccRCC BM niche by integrating longitudinal real-time multiparametric iMPM, eMPM and single cell RNA sequencing. In Aim 2 we will identify the dynamic cellular and molecular mechanisms of response (and resistance) to the standard of care TKI + ICI. In Aim 3, as a novel alternative to target ccRCC BM, we will explore CAR-NK cells and investigate outcomes. We expect our strategy to unravel the complex nature of the BM niche (including immune cells and their dynamics), at baseline and after treatment, and provide novel therapeutic strategies to overcome resistance, with the ultimate goal of enabling rational, biologically driven treatment that improves outcomes for patients afflicted with this lethal disease.
