Tumour-stroma signaling drives a reversible program with neural-like features in cancer-associated fibroblasts

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, in part due to a dense and highly plastic tumor microenvironment dominated by cancer-associated fibroblasts (CAFs). In previous work, we identified a CAF population expressing a neural-associated NES+SOX4+NOTCH1+ program that increased with disease progression. The signals driving this state and the mechanisms regulating it remained unclear. Here, we investigated how tumor-derived signals activate this program and whether it represents a stable fibroblast identity or a reversible state. Using spatial transcriptomic data spanning normal pancreas to invasive carcinoma, we identified EGF, FGF2 and BDNF as candidate tumor-to-fibroblast signals associated with disease progression. Functional experiments in patient-derived CAFs and human fibroblast cell lines showed that EGF and FGF2 are sufficient to induce the NES+SOX4+NOTCH1+ program. Growth factor withdrawal reversed the NES+SOX4+NOTCH1+ program at both the protein and transcriptional levels, indicating that this state is dynamic and environment dependent. Single-cell transcriptomic analysis further revealed a multi-phase response involving early loss of fibroblast-associated features, transient activation of neural-associated genes, and a later stress-associated state. Regulatory analyses identified ETS-family transcription factors as key regulators of the program, while phosphoproteomic profiling and inhibitor studies showed that MAPK/ERK signaling plays a central role in its transcriptional activation. Finally, pan-cancer single-cell analysis showed that this state is enriched in CAFs and increases from normal to tumor tissue across multiple cancer types. Together, these findings define a reversible, growth factor-driven CAF program and identify signaling mechanisms that may provide opportunities to selectively modulate tumor-supportive fibroblast states.