Regulation of Early Breast Cancer Invasion by Cancer-Associated Fibroblasts - Project Summary / Abstract Ductal carcinoma in situ (DCIS) is a non-deadly, pre-invasive lesion, which, if left untreated, can progress to invasive breast cancer (IBC), a disease with a significantly worse prognosis. While genetic alterations fail to explain this transition, structural changes in the tumor microenvironment (TME) may play a critical role in enabling disease progression. TME alterations, including activation of cancer-associated fibroblasts (CAFs) and increased extracellular matrix (ECM) stiffness have been correlated to disease progression, yet the precise mechanisms by which these factors initiate the transition from DCIS to IBC remain unclear. In this proposal, we utilize ex vivo analyses to identify transcriptomic, proteomic, and mechanical features associated with CAF populations and determine how these features correlate with invasive progression. With these findings, we will develop an in vitro stromal model and, by employing advanced perturbation techniques, we will determine if these features of the stromal matrix are causal drivers of disease progression. The overall hypothesis of this proposal is that CAF-driven matrix remodeling, through fibronectin secretion or contractility-driven collagen bundling, generates a mechanical niche that drives carcinoma invasion. Preliminary single-cell sequencing work of patient samples reveals collagen and fibronectin signaling as the dominant cell-cell signaling pathways between CAF populations and invasive carcinoma cells within invasive breast cancer. Additionally, preliminary work from our 3D in vitro stromal model reveals CAFs to be mechanoresponsive to matrix properties and are necessary to facilitate carcinoma invasion. In Aim 1, we will determine the correlative relationships between CAF phenotype, transcriptomic profiles, and matrix mechanics within ex vivo DCIS and IBC patient samples. In Aim 2, we will identify how matrix mechanics shape CAF phenotype and whether CAF-driven ECM deposition and remodeling drive mechanical changes in ECM within a 3D stromal model. In aim 3, we will establish whether CAF-mediated matrix alterations are sufficient to enable carcinoma invasion within a 3D co-culture stromal model. Using cutting edge analysis techniques, the proposed work will establish a comprehensive understanding of the biophysical, molecular, and cellular landscape distinguishing the location, phenotype, and context of CAFs in DCIS versus IBC. Additionally, this proposal features an innovative in vitro experimental design that seeks to uncover how CAFs respond to and remodel 3D viscoelastic matrices, and to establish the mechanical interactions between CAFs and carcinoma cells that drive invasion. Completion of this proposed work will improve our understanding of the mechanisms underlying breast cancer invasion and inform therapeutic strategies.
