Metastatic Transition of Pancreatic Ductal Cell Adenocarcinoma Is Accompanied by the Emergence of Pro-Invasive Cancer-Associated Fibroblasts

SIMPLE SUMMARY: How stromal cells control tumor progression is an active area of investigation. Cancer associated fibroblasts (CAFs) have been suggested to both limit, or promote cancer dissemination in various contexts, but a systemic understanding of stromal influence in determining cancer metastasis is lacking. This study takes a closer look at the diversity of fibroblasts that emerge in different metastatic stages of PDAC (Pancreatic Ductal Cell Adenocarcinoma), a cancer type with high stromal infiltration. Specifically, we found that the CAFs respond to the growing (but yet not metastasized) tumor by transition to a myofibroblast like stage, potentially resisting cancer invasion. As the cancer disseminates, these fibroblasts are nearly entirely replaced by highly diverse set of subpopulations with distinct functions. We have previously advanced a framework, termed Evolved Levels of Invasability (ELI), explaining the vast differences in cancer metastasis and placental invasion among mammals. We found that PDAC’s metastatic transition and stromal trespass is accompanied by the downregulation of pro-resistive and the upregulation of pro-invasability ELI signatures in CAF subpopulations. ABSTRACT: Cancer-associated fibroblasts (CAFs) are now appreciated as key regulators of cancer metastasis, particularly in cancers with high stromal content, e.g., pancreatic ductal cell carcinoma (PDAC). However, it is not yet well understood if fibroblasts are always primed to be cooperative in PDAC transition to metastasis, if they undergo transformation which ensures their cooperativity, and if such transformations are cancer-driven or intrinsic to fibroblasts. We performed a fibroblast-centric analysis of PDAC cancer, as it transitioned from the primary site to trespass stromal compartment reaching the lymph node using published single-cell RNA sequencing data by Peng et al. We have characterized the change in fibroblast response to cancer from a normal wound healing response in the initial stages to the emergence of subclasses with myofibroblast and inflammatory fibroblasts such as signatures. We have previously posited “Evolved Levels of Invasibility (ELI)”, a framework describing the evolution of stromal invasability as a selected phenotype, which explains the large and correlated reduction in stromal invasion by placental trophoblasts and cancer cells in certain mammals. Within PDAC samples, we found large changes in fibroblast subclasses at succeeding stages of PDAC progression, with the emergence of specific subclasses when cancer trespasses stroma to metastasize to proximal lymph nodes (stage IIA to IIB). Surprisingly, we found that the initial metastatic transition is accompanied by downregulation of ELI-predicted pro-resistive genes, and the emergence of a subclass of fibroblasts with ELI-predicted increased invasibility. Interestingly, this trend was also observed in stellate cells. Using a larger cohort of bulk RNAseq data from The Cancer Genome Atlas for PDAC cancers, we confirmed that genes describing this emergent fibroblast subclass are also correlated with lymph node metastasis of cancer cells. Experimental testing of selected genes characterizing pro-resistive and pro-invasive fibroblast clusters confirmed their contribution in regulating stromal invasability as a phenotype. Our data confirm that the complexity of stromal response to cancer is really a function of stage-wise emergence of distinct fibroblast clusters, characterized by distinct gene sets which confer initially a predominantly pro-resistive and then a pro-invasive property to the stroma. Stromal response therefore transitions from being tumor-limiting to a pro-metastatic state, facilitating stromal trespass and the onset of metastasis.