Abstract 3184: Falcon-X 3D in vitro tumor model platform for screening patient-derived NSCLC, CRC, and RCC tumors with targeted therapy and immunotherapy

Significant challenges remain in developing oncology assays in vitro that correlate to the in vivo outcome, and in particular, including the immune compartment to effectively assay immunotherapy. Matrix-free systems such as 3D spheroids or 2D plastic plates fail to recreate critical features of the tumor microenvironment (TME) such as T cell infiltration through the extracellular matrix (ECM) stroma and desmoplastic T cell exclusion, and matrix-based systems such as basement membrane extract or collagen I suffer from batch variability and unwanted immunogenicity, as well as handling issues that impair their downstream analysis. Here, we aimed to solve these biological and technical challenges by utilizing a novel 3D hydrogel platform called Falcon-X to co-culture patient-derived xenograft (PDX) tumors of colo-rectal cancer (CRC), non-small cell lung cancer (NSCLC), or renal cell carcinoma (RCC) origin with other TME cell types such as fibroblasts and T cells in order to screen for targeted and immuno-therapy efficacy. High content confocal imaging enabled analysis of tumor size, killing, and T cell penetration. PDX tumors were cultured in a biocompatible and chemically-defined ECM hydrogel (Versagel, Hribar, 2019), layered in 96-well plates using a 3D gel patterning technology (Symphony, Hribar, 2019), and subsequently cultured in a standard cell culture medium. In some cases, fibroblasts were added to the culture in order to mimic stromal cues of the TME such as desmoplasia. For targeted therapy, compounds were added to the culture medium for 72 hours. For immunotherapy, pre-activated PBMCs were added and T cell infiltration and T-cell mediated tumor killing were quantitated in response to standard of care compounds. For CRC PDX tumors, Oxaliplatin, Cetuximab, 5-FU, Bevacizumab (anti-VEGF), and Pembrolizumab (anti-PD1) were assayed; for RCC: Sunitinib, Pazopanib, Temsirolimus, Bevacizumab, and Pembrolizumab; for NSCLC: Docetaxel, Cisplatin, Afatinib, Atezolizumab (anti-PDL1), and Pembrolizumab. Importantly, the IC50 values demonstrated a high degree of correlation to the in vivo PDX outcome. Differences in IC50s were noted with the addition of fibroblasts, suggesting active crosstalk between stromal, tumoral, and immune compartments.Together, these data reveal a novel Falcon-X 3D assay platform for culturing tumor, fibroblast, and immune cells in a 3D hydrogel and assaying critical features of the TME such as T cell infiltration and tumor killing. Such a