(466b) An in Vitro Model Utilizing Cell Spheroids and Hyaluronic Acid Hydrogels to Study the Role of Extracellular Matrix Stiffness on Tumor Dormancy in Brain Metastatic Breast Cancer

The ability of breast cancer cells (BCCs) to metastasize to distant organs contributes to approximately 90% of breast cancer related mortalities. These BCCs might metastasize even before the patient is diagnosed with a primary tumor. The common metastatic sites for BCCs are lungs, liver, brain, bone and lymph nodes. Upon arrival to these metastatic sites, these BCCs cells could exhibit either a dormant or proliferative phenotype by sensing the mechanical/biochemical/cellular cues from the microenvironment of that particular site. Among these breast cancer metastases, breast cancer brain metastasis (BCBM) is most aggressive, with a survival rate of approximately 4-9 months. In recent years, tumor cell clusters, specifically, circulating tumor cell clusters, have been shown to possess higher metastatic potential compared to single cells. However, the interactions between BCBM cell clusters and the brain microenvironment are not well understood due to the lack of relevant biomimetic in vitro models to study such interactions.

To address this need, herein, we employed hyaluronic acid (HA) hydrogels to mimic the native brain microenvironment, as it is one of the most abundant extracellular matrix component in the brain. Tumor cell spheroids were employed to mimic tumor cell clusters, and in our studies, we utilized MDA-MB-231Br BCBM cells. To investigate the impact of mechanical cues (extracellular matrix stiffness) on cell spheroids, HA hydrogels of varying stiffnesses (i.e., soft - 0.4 kPa, stiff - 4.5 kPa) were prepared. To evaluate the effect of tumor size on dormancy of BCBM clusters, different sizes of spheroids were prepared using 100 – 10,000 tumor cells. In this model, over a period of 7 days, we observed that areas of spheroids composed of 100 and 500 cells remained largely constant and the area of spheroids containing ≥ 1000 cells increased on stiff HA hydrogel. On the soft HA hydrogel, spheroid areas remained constant irrespective of spheroid size indicating a dormant phenotype. This was further supported by EdU and Ki-67 staining wherein we observed that the percentage of EdU+ and Ki-67+ cells was significantly higher on the stiff vs. soft HA hydrogel (in the case of spheroids made using 10,000 cells). We also demonstrated the reversibility of observed dormant phenotype on the soft HA hydrogel, wherein spheroids composed of 10,000 cells cultured on soft HA hydrogel for 7 days were transferred to a stiff and soft HA hydrogel respectively and cultured for another 7 days. Ki-67 staining results demonstrated an increase in the percentage of Ki-67+ cells for stiff HA hydrogel compared to spheroids cultured on soft HA hydrogel. Overall, such systems provide useful tools to probe cell-matrix interactions in BCBM and also for testing of drugs.