Fibroblast response to tissue injury and neoplasia

Fibrosis contributes to 45% of all mortality in the U.S. Fibrosis occurs in the form of a scar following injury such as in the skin after a laceration or burn, in the abdominal cavity in the form of adhesions following surgery, or a stricture following intestinal resection. Other forms include chronic parenchymal fibrosis such as liver cirrhosis or idiopathic pulmonary fibrosis in the lung. Solid tumors also develop extensive fibrosis (termed desmoplasia). Fibroblasts are the cell type responsible for the deposition of fibrotic tissue in all of these circumstances, which interferes with normal organ system function. In the context of neoplasia, fibroblasts are also known to support the proliferation of transformed cells. Therapies that effectively target fibroblasts and modulate fibrosis, which could be beneficial in any of the aforementioned clinical phenomena, remained limited because the origins, heterogeneity, and behavior of fibroblasts are not yet comprehensively understood. This dissertation explores fibrosis in three distinct, but closely related settings: abdominal adhesions, wound healing, and solid tumor stroma. The Introduction provides an extensive exploration of the literature concerning similarities between solid tumor stroma and healing wounds. The focus of this review is particularly on cancer associated fibroblast (CAF) cellular heterogeneity and plasticity, and the acellular matrix components that accompany these cells. I explore how similarities and differences between healing wounds and tumor stroma continue to evolve. This in-depth analysis of the literature provides a background for the primary research presented to follow. Chapter I explores abdominal adhesions. Adhesions are fibrotic scars that form between abdominal organs following surgery or infection, which can cause bowel obstructions, chronic pain, and infertility. I systemically analyzed adhesions in mouse and human tissues, at a cellular, transcriptomic, and protein level. Using an abdominal wall transplant model, I show that adhesions derive primarily from the visceral peritoneum. Using single cell RNA-seq, I elucidate the heterogeneity among adhesion-forming fibroblasts in both mice and humans, which is more pronounced at an early timepoint. Finally, I identified that JUN pathway signaling promotes adhesion formation and results in upregulation of PDGFRA expression among adhesion fibroblasts. With JUN suppression, adhesion formation and profibrotic signaling are significantly diminished, supporting JUN as a therapeutic target to prevent adhesion formation clinically. Chapter II concerns skin wound healing. Using the Rainbow mouse, I show that tissue-resident precursor-type fibroblasts are activated following wounding and proliferate poly-clonally in a distinct radial pattern. Integrated multimodal -omics analysis including (sc)ATAC and RNA-seq of Rainbow wound fibroblasts using ArchR (Granja, 2020) reveals the highly coordinated signaling pathways that guide wound closure, which are temporally-dynamic and highly region-specific. Furthermore, as wound fibroblasts proliferate to close the wound 'gap', CytoTRACE analysis (Gulati et al., 2020) reveals that they differentiate and Rainbow fluorophore intensity decreases. We show that local tissue mechanics drive wound fibroblast proliferation and that modulation through inhibition of focal adhesion kinase (FAK) disrupts the poly-clonal proliferative response to wounding. Finally, we apply spatial transcriptomics in a model of wound healing using the 10x genomics Visium platform, which delineates fibroblast heterogeneity with unprecedented spatial detail, comprehensively defining wound healing fibroblast biology. Through integrated analysis of the single cell chromatin landscapes and gene expression states, coupled with high-resolution spatial transcriptomics, we are able, for the first time, to impute "spatial epigenomics", which elucidates the complex relationships determining cell fate during tissue repair. Taken together, these data provide a comprehensive interrogation of wound healing fibroblast phenotypes and identification of functional subpopulations with an unprecedented level of granularity. Chapter III of this dissertation explores cancer associated fibroblasts (CAFs) in breast and pancreas tumors. Using the Rainbow mouse model, I show distinct poly-clonal proliferation of CAFs in the tumor stroma using both allograft and endogenous models for breast and pancreas cancer. These data suggest that in the setting of neoplasia, similar to wound healing, tissue-resident precursor fibroblasts are activated locally and proliferate poly-clonally. Ongoing experiments for this project include obtaining scRNA-seq and scATAC-seq data for mouse and human CAFs isolated from breast and pancreas cancer specimens. I will consider the scRNA-seq data in relation to the nuclear reads from my scATAC-seq results using ArchR (Granja, 2020) to identify functionally relevant changes in chromatin accessibility and gene expression that occur with fibroblast activation, with particular focus on FAK-pathway signaling. I am also applying protocols which consider somatic mutations in mitochondrial DNA (mtDNA) variants to track cell lineage in these contexts. Using the analytic strategy described in Chapter II, I also aim to conduct spatial transcriptomics analysis using tumor specimens to spatially elucidate fibroblast heterogeneity at a gene expression and epigenetic level in this context. Finally, I am conducting in vitro and in vivo assays to determine the functional effects and explore the therapeutic potential of modulating FAK-pathway chemokines in breast and pancreatic CAFs. These experiments are expected to elucidate pathways responsible for CAF activation and potentially identify stromal targets that could be exploited therapeutically in solid tumors

Advisor: Longaker Michael T.
Chang Howard Y. (Howard Yuan-Hao)
Rankin Erinn