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Conservation Of The Fibroblast Programming Response To Solid Organ Tumors Suggests Novel Treatment Strategies Targeting The Mgp And Cd26 Surface Receptors
Michael Januszyk, MD PhD, Deshka S. Foster, MD PhD, Kathryn E. Yost, PhD, Malini Chinta, BS, Nicholas Guardino, BS, Kellen Chen, PhD, Dominic Henn, MD, Daniel Delitto, MD PhD, Derrick C. Wan, MD, Jeffrey A. Norton, MD, Howard Y. Chang, MD PhD, Geoffrey C. Gurtner, MD, Michael T. Longaker, MD.
Stanford University, Stanford, CA, USA.

PURPOSE: Cancer associated fibroblasts (CAFs) are among the most abundant cell types in desmoplastic tumors and have been found to modulate both tumor growth and spread. However, like their non-perturbed fibroblast counterparts, CAFs are characterized by significant functional and transcriptional heterogeneity, which has limited efforts to target these cells for tumor therapy.
METHODS: Using endogenous mouse breast cancer as a model system, we performed single cell RNA-seq (scRNA-seq) and ATAC-seq (scATAC-seq), in conjunction with rainbow lineage tracing and spatial transcriptomic profiling, on tumor specimens from a cohort of syngeneic mice at 4 weeks (n = 5). In silico fibroblast isolation was performed, followed by multimodal partitional analysis and spatial deconvolution. Human breast cancer core needle biopsies (n = 3) and pancreaticoduodenectomy samples (n = 3) were processed for paired scRNA/ATAC-seq analysis and integrated with our mouse data to construct consensus profiles. These were evaluated against publicly available next-gen sequencing datasets from solid organ tumors in both human and mouse using an anchor-based label transfer approach. Prospective functional studies were then applied to target subpopulations of interest using genetic knockout in mice (FAKko/ko; breast allograft; n = 5) and retrospective evaluation of pre/post checkpoint inhibitor therapy in humans (PD-1; basal cell carcinoma; n = 11).
RESULTS: Six distinct subpopulations of CAFs were identified from endogenous mouse breast tumor specimens (Panel A). Based on gene expression and chromatin profiles, we characterized three of these clusters as "mechano-responsive" and the other three "immuno-modulatory" (Panel B-D). Analysis of differentiation potential using CytoTRACE suggested that CAF heterogeneity may be the result of cell proliferation that occurs secondary to changes in tissue mechanics and ultimately determines immune responsiveness among CAF subpopulations (Panel E). Supporting this concept, using Rainbow transgenic mouse models, we determined that local fibroblasts expand poly-clonally in response to paracrine cues in the tumor microenvironment (Panel F). We found that CAF heterogeneity was recapitulated across solid tumor types in both humans and mice (Panel G-H), maintaining these two functional categories: mechano-responsive and immuno-modulatory. Moreover, these fibroblast subtypes were spatially-distinct and governed by specific changes in chromatin accessibility (Panel I). Selective disruption of underlying mechanical force or immune signaling resulted in predictable shifts in subpopulation distributions and tumor growth rates (Panel J-K). Specifically we identified the MGP and CD26 surface proteins as characteristic markers distinguishing these two fibroblast populations and found their expression to be diagnostically predictive (Panel L).
CONCLUSION: Collectively, this research elucidates the spatial dynamics of the CAF tumor microenvironment, delineates functionally-distinct CAF subpopulations, and provides a multimodal -omics framework for understanding the tumor stromal niche. The identification of subtype-specific fibroblast surface receptors may have significant diagnostic value and represent a secondary target for multi-agent cancer treatment strategies.


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