(294b) Stiffness in a Bone Marrow Mimetic Microenvironment Dictates Breast Cancer Cell Invasiveness and Proliferation

Breast cancer (BC) poses a major health risk domestically and globally. In the United States, over 280,000 new cases of invasive BC are expected in 2021. Metastasis, the spreading of cancer cells to other organs, is the primary reason for complications and deaths related to BC. Bone metastasis is highly prevalent in breast cancer patients with metastatic disease, which is the 3^rd most common location of metastasis with BC patients. Metastasized cancer cells in the bone marrow can remain undetected for long periods of time, typically from months to years. These dormant cells will eventually form osteolytic or osteoblastic lesions, causing pathological fracture and inhibiting patient quality of life. However, the physical cues of the bone metastatic environment and how these dormant BC cells interact is highly understudied. Previous research has implicated increased stiffness in the growth and invasion of BC cells. The bone marrow microenvironment is molecularly and cellularly complex, and the mechanical properties within the bone marrow range from 0.5 kPa in the sinusoidal region to 35 kPa in the endosteal region. It has been shown that cells in stiffer environments present an epithelial-to-mesenchymal transition (EMT) and a higher invasiveness, which is seen in malignant cancer cell progression. Here, we hypothesize that breast cancer cells experiencing higher stiffnesses in a bone marrow-like microenvironment will exhibit greater invasive potential when compared to lower stiffness regions.

We fabricated alginate-Matrigel hydrogels as a bone marrow model and varied crosslinking with calcium sulfate (CaSO₄) from 10 mM to 50 mM to modulate stiffness. We conducted rheology to determine the physical properties of the hydrogels. We encapsulated luciferase-labeled triple negative breast cancer (mouse 4T1, human MDA-MB-231) and estrogen receptor positive (human MCF7) cells into these hydrogels and cultured for them for 2 or 7 days. Cells were fixed and stained with phalloidin and Hoechst to visualize the cytoskeleton and nuclei, respectively, via fluorescence microscopy. Additionally, proliferation was assessed via bioluminescence imaging and Ki67 staining. Cells were also stained with cortactin, vimentin, N-Cadherin and E-Cadherin to evaluate EMT.

Through rheology, we found that the alginate-Matrigel hydrogels recapitulated the stiffness range observed in the bone marrow microenvironment (0.5 kPa to 25 kPa). Cells encapsulated in hydrogels with higher stiffness displayed increased elongation and proliferation (p<0.05). Stiffer environments also enhanced cellular invasive capacity and EMT.

This work establishes a system that can replicate bone marrow mechanical properties and demonstrates that stiffnesses within the range of the bone marrow alter the cellular response of BC cells. Future studies include developing co-cultures of tumor cells with macrophages to examine how physical properties influence tumor-immune cell interactions. Overall, this work will elucidate the physical factors that cause cancer cells to exit from dormancy.