<i>In vitro</i> models of cancer‐associated fibroblast heterogeneity uncover subtype‐specific effects of <scp>CRISPR</scp> perturbations

Cancer-associated fibroblasts (CAFs) are sought after as potential therapeutic targets due to their pro- and antitumorigenic functions, which are attributed to specializations in CAF subtypes. A precise targeting of specific subtypes would be required to design therapies that effectively modulate CAF phenotypes, necessitating translatable model systems to support target discovery efforts. However, not only is our knowledge of CAF heterogeneity in solid tumors lacking, particularly in pancreatic tumors, but the translatability of CAF models has also not been rigorously evaluated. Here, we develop a coculturing model with primary CAFs and immortalized tumor cell lines that can reliably represent CAF phenotypes observed in tumors, with correlations to immuno-resistant and immunomodulatory phenotypes. Using single-cell transcriptomics, we characterize the CAF subtype heterogeneity in the in vitro CAF cell lines isolated from pancreatic cancer patients and investigate the impact of perturbing potential stromal genes on different CAF subtypes. We also infer the continuum of state changes underlying the interconvertibility of CAF subtypes. Finally, we use immortalized CAF cell lines to perform single-cell CRISPR perturbations of stromal targets, revealing the subtype-specific effects of perturbations and the impact of model-type selection on the translatability of insights.