(4cs) Development of Translational Microscale Systems to Interrogate How Biophysical and Biochemical Cues Alter the Phenotype of Metastatic Hormone Positive (HR+) Breast Cancer

The tumor microenvironment (TME) is a complex, heterogeneous system containing both healthy and cancerous cells whose interactions with each other, along with the extracellular matrix (ECM), drives cancer progression and drug resistance. Recent studies have suggested that metastasizing cancer cells are exposed to both biochemical and biophysical stressors which enhances their proliferative behavior and resistance to therapeutics resulting in a poorer prognosis. Specifically, the hormone receptor positive (HR⁺) breast cancer subtype, which accounts for approximately ~70% of breast cancer patients and relies on estrogen signaling for proliferation and tumor growth, has been shown to become resistant to standard of care endocrine therapy at the metastatic site. The underlying mechanisms driving this pro-survival, drug resistant phenotype are currently unknown due, in part, to limited bioanalytical technologies capable of recreating the in vivo conditions of the TME and metastasis. The goal of this work was to develop a suite of microfluidic technologies that better reproduce in situ conditions to study how (1) external biophysical stressors like fluid shear stress (FSS) or (2) three-dimensional (3D) co-culture with stromal cells alters the phenotype metastatic HR⁺ breast cancer. First, we developed a modular microfluidic platform to study how exposure to FSS, a fluid-induced force cells experience while in transit through the circulatory system, enhanced proliferation and protein phosphorylation in single HR⁺ breast cancer cells. Results show that, after shearing, cells enhanced their Ki67 expression (a proliferation marker) when compared to non-sheared cells. Moreover, increased activation of pro-growth and survival pathways such as AKT, mTOR, and STAT3 were also detected in the sheared populations. Next, we utilized a droplet microfluidic platform coupled with a thiol-acrylate (TA) hydrogel to study intracellular communication between cancer and stromal cells grown as 3D spheroids. Studies have shown that adipose derived stem cells (ASCs) have the ability to repair, maintain, and enhance surrounding tissue via paracrine signaling while fibroblasts play a pivotal role in cancer progression through ECM remodeling and production of different ECM proteins such as collagen-I. 3D co-culture of HR⁺ breast cancer cells and primary fibroblasts showed enhanced production of collagen-I; however, the distribution of collagen-I was found to vary resulting in two distinct subpopulations of spheroids independent of size. One group with a single ‘hotspot’ (a localized region of collagen-I) with diminished expression while a second group displayed multiple hotspots with enhanced collagen-I expression. 3D co-culture of HR⁺ breast cancer cells and ASCs resulted in enhanced proliferation in the absence of estrogen or the presence of the endocrine therapy Fulvestrant. These findings provide new insight into how HR⁺ breast cancer interacts with different biochemical and biophysical stressors in the TME and how it leverages these external factors to drive a more aggressive, pro-survival phenotype at the metastatic site.