A Full Spectrum Fibroblast Profiling Panel Reveals Phenotypic and Metabolic Heterogeneity of Cancer-Associated Fibroblasts at the Single-Cell Level

Cancer-associated fibroblasts (CAFs) exhibit extensive transcriptional and phenotypic heterogeneity and are highly plastic. Routine approaches to resolving CAF heterogeneity at the single-cell and protein levels are lacking, making it difficult to assess changes in CAF state in response to perturbations. Here, we present an optimized 28-marker spectral flow cytometry panel that uses unsupervised clustering to resolve CAF states across in vivo and in vitro models. Using this new fibroblast profiling panel (FPP), we show that LRRC15+ CAFs in murine pancreatic ductal adenocarcinoma are phenotypically distinct from canonical SMA+ myofibroblasts, rather than representing a subset of this population. We find that marker expression is graded rather than discrete across clusters, indicating that CAF identity spans a continuum rather than fixed states. Metabolic profiling further shows that hypoxic CAFs were enriched for glucose uptake and an inflammatory phenotype. Using in vitro models, we further demonstrate how the FPP can resolve plasticity outcomes. In a 3D organoid co-culture model, direct contact between tumor cells and fibroblasts, but not organoid-derived factors alone, induces a stem-like CAF state also observed in vivo. In 2D, TGF{beta} and inflammatory/hypoxic stimuli each enrich distinct, pre-existing CAF states rather than generating new ones. These findings establish our FPP as a practical method for resolving fibroblast heterogeneity at the protein level in cancer and beyond.