Multiplexed mosaic tumor models reveal natural phenotypic variations in drug response within and between populations [11 cancer cell lines]

Heterogeneity between patients poses a significant challenge in cancer drug development. Although success of a drug candidate in the clinic depends on efficacy across large numbers of diverse patients, only a few in vivo preclinical models are used for assaying a preclinical drug candidate due to the lack of scalability of traditional xenograft models. To address this limitation, we developed GENEVA, a scalable platform that enables the measurement of molecular and phenotypic responses to drug perturbations at single-cell resolution, both in 3D and in vivo. GENEVA models the genetic diversity of cancer by combining multiple patient-derived cell lines and cancer cell lines into pooled 3D cultures and xenograft models, allowing us to study drug responses across a wide range of genetic backgrounds within a single experiment. This platform integrates high-resolution transcriptomics with multiplexed single-cell profiling, providing a unique ability to uncover the interplay between cancer genotypes and drug responses. In this study, we apply GENEVA to investigate KRAS G12C inhibitors and demonstrate that mitochondrial activation is a key driver of cell death following KRAS inhibition. Additionally, we identify epithelial to mesenchymal transition (EMT) as a prominent resistance mechanism to KRAS G12C inhibition using in vivo GENEVA mice. These findings highlight the utility of GENEVA in identifying novel therapeutic targets and optimizing combination therapies, while underscoring its potential to bridge the gap between preclinical cancer models and patient outcomes by modeling the complexity of tumor heterogeneity in a scalable and robust assay. A total of 11 cancer cell lines with varying genetic backgrounds, including BRAF(V600E) and KRAS G12C mutations, were pooled together into a single experimental setup. These pooled cells were cultured in 3D conditions and then split into three treatment groups: Vehicle control (DMSO), Vemurafenib (0.8 µM) targeting BRAF(V600E) mutants, ARS-1620 (1 µM) targeting KRAS G12C mutants. After six days of treatment, cells were harvested and subjected to scRNA-seq