Biomechanical Profiling of Murine Fibroblasts in Triple-Negative Breast Cancer Microenvironments

Introduction: : Triple-negative breast cancer (TNBC) is a highly metastatic subtype of breast cancer representing 15-20% of all cases. TNBC differs from other types of breast cancer as it lacks all 3 receptors used to define other subtypes: estrogen receptor (ER), progesterone receptor (PR), and HER2. These differences contribute to the high mortality rate of TNBC as traditional breast cancer therapies lack efficacy in clinical practice. There is limited knowledge of the specific mechanisms working to drive the increased risk of metastasis. Our previous studies suggest that biomechanical strain can alter TNBC migration. The mechanical forces present in the tumor microenvironment (TME) have been indicated to play a role in the invasiveness of TNBC. Cancer-associated fibroblasts (CAFs) are the most abundant stromal cells in the TME, stromal cells that play a crucial role in inducing strain on cancer cells. CAFs contain high levels of α-smooth muscle actin (aSMA), causing them to exhibit higher contractility levels compared to normal fibroblasts. The objective of this project was to develop and characterize murine CAFs from TNBC tumors for use in advanced 3D TME models.

Materials and

Methods: : To generate mCAFs for TNBC models, 4T1 tumor cells were implanted in the 4th mammary fat pad of Balb/C mice; tumors were harvested after reaching 2cm and digested with collagenase. Resulting cell suspensions were allowed to adhere to tissue culture plastic for 30min before aspiration of non-adhered cells. Cells that stably grew after multiple passages were characterized for markers of CAFs, including aSMA and Snail1 via immunofluorescence. To investigate if the cells could generate matrix deformations, similar to human CAFs, we

embedded these cells into a 3D microtissue containing 1 μm blue fluorophore beads within a 10 mg/mL fibrin gel. Matrix deformations driven by the contractile activity of human CAFs, mouse cancer-associated fibroblasts (mCAFs), and HUVEC-TERT2 (HT2s) were monitored using a bead displacement assay. The HT2 were used as a control with known small matrix deformations. For bead displacements, microtissues were imaged on Day 2 after loading with an inverted epifluorescence microscope equipped with an environmental chamber. Imaging was performed by capturing 100μm z-stacks at 1μm step size. Matrix deformation was measured over every 30 minutes.

Results, Conclusions, and Discussions:: The objective of this study was to characterize the biomechanical strain generated by murine CAFs to later investigate how their contractile behavior may contribute to TNBC progression in vivo. The ability of CAFs to deform the extracellular matrix likely promotes mechanotransduction signaling pathways and enhances the expression of intracellular markers such as αSMA and Snail1. Upregulation of aSMA and Snail1 indicated an activated CAF phenotype that is associated with cancer progression. Immunofluorescence staining validated that our mCAF populations expressed high levels of both α-SMA and SNAIL1 (Fig. 1). Our bead displacement analysis revealed that on average human CAF containing microtissues exhibited significantly greater matrix deformation compared to the mCAFs and the HT2 controls. Both mCAFs lines (denoted as -1 and -2) generated similar magnitudes of matrix deformations, indicating both may be robust models for future studies (Fig,2). Future work will involve looking at the effects of mCAF induced strains on TNBC migration and proliferation in a multi-chamber microfluidic model of the TME. Additionally, long term plans include implanting these mCAF and TNBC cells into mice to investigate the effect on tumor burden. By developing good mCAF models for TNBC, we are able to accurately replicate the TME in vivo. This allows us to further understand the effects of CAFs induced strain on TNBC progression, accelerating the development of more effective targeted therapies.

Acknowledgements and/or References (Optional)::