(162au) Stiffness in a Bone Marrow Mimetic Microenvironment Alters Breast Cancer Cell Invasiveness and Proliferation

Breast cancer poses a major health risk domestically and globally: in the United States, over 230,000 new cases of invasive breast cancer were diagnosed, and over 40,000 women died of the disease in 2019 alone. One major cause of cancer death is metastasis or cancer cells spreading to other organs. Bone metastasis is highly prevalent in breast cancer patients with metastatic disease, which is the 3^rd most common location of metastasis. Cancer cells in the bone marrow can remain undetected for long periods of time. These dormant cells will eventually form osteolytic lesions, causing pathological fracture and inhibiting patient quality of life. However, the cues that cause these dormant cells to awaken and grow uncontrollably are unknown. Previous research has implicated stiffness in increased breast cancer growth and invasion. 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 5 mM to 50 mM to modulate stiffness. We conducted rheology to understand the physical properties of the hydrogels. We also encapsulated 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 nuclei counts.

Through rheology, it was determined that the alginate-Matrigel hydrogels recapitulated the range observed in the bone marrow microenvironment (0.5 kPa to 22 kPa). Cells encapsulated in hydrogels with higher stiffness displayed increased elongation and proliferation (p<0.05). This suggests that stiffer environments enhance cellular invasive capacity and potentially 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 breast cancer 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.