Abstract B021: Optimizing combination immunotherapy regimens in kidney cancer using ex-vivo multicellular 3D immune-microtumors and multiplex immunoassays
Authors: Hare Roseanna , Peerani Eleonora , Nan Keqian , Maximchik Polina , Hudson Jessica , Ient Jonathan , Bah Nourdine , Kearney Jay , Iori Francesco , Williams Matthew , Tran Maxine , Ranatunga Duleek 
Published: February 2025

Abstract
Renal cell carcinoma (RCC) presents a challenging treatment landscape in oncology due to its varied therapeutic approaches and the lack of reliable, predictive clinical decision-making tools. Our study focuses on developing a functional precision medicine test to predict responses to combination immunotherapy/receptor tyrosine kinase (TKI) regimens in RCC by optimizing a 3D immune-microtumor platform. Using RCC resections and matched bloods, we isolated tumor cells and peripheral blood mononuclear cells (PBMCs) as single cells. Cells were labeled with fluorescent dyes to enable tracking of tumor, CD8+ and CD8- PBMC single-cell behaviors. Immunofluorescence (IF), RNAseq and flow cytometry (FCm) were used to deconvolute cell subpopulations within the tumor, while cytokine production was analyzed pre and post-encapsulation into 3D immune-microtumor cocultures using a Luminex assay. Our functional 3D assay assessed live cell behaviors such as cell viability, immune cell infiltration and killing (N=20). In a 20 patient cohort, IF analysis confirmed that cell-type diversity, including tumor, endothelial, immune cells, and cancer-associated fibroblasts, was preserved within 3D immune-microtumors from day 0 to day 4 of culture. Dose-determination of immune checkpoint inhibitors (ICIs) was conducted on primary PBMCs. A targeted 8-plex Luminex panel allowed the evaluation of ICI exposure time across five drugs (pembrolizumab, ipilimumab, nivolumab, avelumab, atezolizumab) and PBMC activation conditions (none; CD3; CD3/CD28) based on maximal differential cytokine release versus day 0 control, when measuring granzyme B, IFN-γ, TNF-α, IL-12, and IL-17. Dose-response assays were then conducted in the full 3D immune-microtumor setup (N=10). The functional assay revealed changes in tumor cell viability and immune cell infiltration upon ICI treatment in a dose-dependent fashion, alongside molecular readouts from PBMC FCm for checkpoint marker expression (incl. PD-1/PD-L1), tumor IF (incl. PD-L1), and ELISA for IFN-γ and granzyme B. Post optimization, the 3D immune-microtumor model was used to evaluate clinical responses to combination therapies for RCC (ICI + TKI) in our trial (PEAR-TREE N=20 ISRCTN10001405). Pembrolizumab alone led to a 26% rise in PBMC infiltration, a 14% increase in CD8+ cell infiltration, and a 15% rise in tumor cell death relative to untreated controls (N=5). When combined with either lenvatinib or axitinib, pembrolizumab further reduced tumor cell viability by 10% and 19%, respectively. Patient responses displayed intra-patient variability. In conclusion, this 3D immune-microtumor model provides an ex-vivo system for understanding individual patient responses to ICIs, supporting personalized treatment strategies in RCC. By testing FDA-approved treatments in this model and through our metastatic RCC clinical trial (NCT06264479) we are advancing a functional precision medicine approach that may shape future individualized cancer care.
Citation Format: Roseanna Hare, Eleonora Peerani, Keqian Nan, Polina Maximchik, Jessica Hudson, Jonathan Ient, Nourdine Bah, Jay Kearney, Francesco Iori, Matthew Williams, Maxine Tran, Duleek Ranatunga. Optimizing combination immunotherapy regimens in kidney cancer using ex-vivo multicellular 3D immune-microtumors and multiplex immunoassays [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B021.