Abstract 2830: Intravital multiphoton microscopy of bone metastatic renal cell carcinoma
Authors: Maksimovic Stefan , Boscolo Nina Constanza , Barrios Sergio , Mikos Antonios , Campbell Matthew T. , Dondossola Eleonora 
Published: March 2024

Abstract
Clear cell renal carcinoma (ccRCC) represents 80% of renal neoplasms, with bone as a major site for distant spread (35-40% of patients). These secondary lesions cause a variety of skeletal-related complications, including pain, spinal cord compression, hypercalcemia, mobility issues and fractures, posing a significant negative impact on patient quality of life and survival. Furthermore, bone is well-known as a site of therapy resistance, which confers to patients treated with systemic therapies significantly worse time-to-treatment failure and progression-free and overall survival compared to those without bone metastases. Because of the complexity of the bone microenvironment and the paucity of suitable models, preclinical investigation of bone metastasis and therapy response is challenging. Preclinical models of bone metastasis that integrate tissue engineering and advanced imaging techniques can facilitate the study of the mechanism of tumor progression and therapy response. We recently developed a window model amenable to intravital multiphoton microscopy (MPM) to longitudinally monitor human ccRCC lesions in tissue engineered bone constructs (TEBCs) in immunodeficient mice. TEBCs were generated by functionalizing polymeric polycaprolactone scaffolds with bone morphogenetic protein 7. Human ccRCC lesions, after implantation into the bone cavity, were longitudinally monitored for growth and niche development, using multi-parameter recording of collagen/bone matrix (SHG), bone surface (THG), blood vessels/stromal phagocytes (fluorescent dextran), and tumor cells (GFP). However, this model lacked key components of the immune system (including B and T-cells), which are of major interest in the study of tumor progression and (immune) therapy response. Therefore, we developed a tissue-engineered preclinical model that incorporate species-specific interactions between cancer and bone stromal cells, and account for an intact immune system. To this purpose, we optimized TEBC formation in C57BL/6 fluorescent reporter mice (Sp7-mCherry, osteoblasts; Trap-td-Tomato, osteoclasts; GFP, immune cells, αSMA-RFP fibroblasts; and Flk1-GFP, blood vessels), coupled to detection by intravital or ex vivo multiphoton microscopy analysis, to monitor the evolution and fate of the TEBC. Interestingly, the bone marrow in the core of the TEBC was replaced by a desmoplastic tissue with features like the foreign body response, including foreign body giant cells, which was not present in immunodeficient mice. By applying dual color GFP/αSMA-RFP mice, which expressed red fluorescence in activated fibroblasts, we confirmed the fibrotic nature of this core. To avoid its formation, we generated scaffold-free ossicle, by direct implantation of BMP7 and VEGF in the subcutaneous tissue. To conclude, we established a protocol for generating ectopic ossicles in immunocompetent mice.
Citation Format: Stefan Maksimovic, Nina Constanza Boscolo, Sergio Barrios, Antonios Mikos, Matthew T. Campbell, Eleonora Dondossola. Intravital multiphoton microscopy of bone metastatic renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2830.