(73c) Multicellular Cancer Cell-Monocyte Co-Culture Spheroids Recapitulate Immunosuppressive and Pro-Tumorigenic Microenvironments in Brain Metastatic Breast Cancer

Brain metastatic breast cancer (BMBC) is one of the major causes of breast cancer-related morbidity. Brain metastasis occurs in 15 to 30% of breast cancer patients. Human Epidermal Growth Factor Receptor 2 (HER2) positive and triple negative breast cancer (TNBC) bears the highest propensity to metastasize to the brain, with highly aggressive TNBC accounting for early fatality. The extremely poor prognosis of BMBC patients is attributed to the lack of any effective treatment measures against BMBC. A major hurdle in the development of treatment strategies against BMBC is the highly specialized tumor microenvironment (TME) of BMBC. BMBC microenvironment is an extremely complex and heterogeneous niche of cellular and acellular components of which the innate immune cells, resident microglia, and blood derived macrophages comprise the major fraction. So far, the role of these BMBC TME prevalent innate immune cells in mediating BMBC progression and response to therapy remains largely unexplored and poorly understood, in part, due to limited access to samples of brain metastases and lack of model systems to study these crucial interactions.

To address this need, herein, we developed a three-dimensional (3D) co-culture spheroid platform using brain metastatic TNBC cell line MDA-MB-231Br and patient derived brain metastatic TNBC cell line F2-7 with blood derived innate immune cells, monocytes. Using our co-culture spheroids, we found that the close contact interaction of BMBC cells and monocytes imparts proliferative advantage to BMBC cells. BMBC and monocyte co-culture spheroids exhibited significantly elevated levels of markers associated with pro tumor M2 macrophages (CD163, CD206 and c-Myc) along with immunosuppressive and evasive markers PD1, PDL1, CD47, and CD24 suggesting that BMBC cells in its milieu modulates immune response to pro tumor outcomes. In addition, F2-7 and monocyte co-culture spheroids showed significant upregulation of M1 type macrophage markers (CD80 and HLA-DRA) along with both pro and anti-inflammatory cytokines (IL6 and IL10). F2-7 and monocyte co-culture spheroids also exhibited variation in the magnitude of the fold change in the levels of various immune related markers (i.e., CD163, c-Myc and PDL1) when compared to MDA-MB-231Br and monocyte co-culture spheroids. These findings indicate that our 3D multicellular co-culture spheroid platform captures the heterogeneity in the BMBC cell induced modulation of the tumor immune microenvironment. Co-culture spheroids were treated with Paclitaxel to assess the impact of innate immune cells on the response of BMBC cells to therapeutic challenge. We found that the viability of Paclitaxel treated co-culture spheroids of BMBC and M0 macrophages was significantly higher than monoculture spheroids of respective BMBC cells treated with Paclitaxel. Similar outcomes were observed for Paclitaxel treated BMBC and M2 macrophage co-culture spheroids. Furthermore, we observed no significant difference in viability of co-culture spheroids of BMBC cells and M2 macrophages without or with Paclitaxel treatment. These findings suggest that the presence of innate immune cells in the BMBC TME alters the response of BMBC cells to Paclitaxel, rendering them more resistant to this clinically relevant chemotherapeutic agent.

In sum, our in vitro co-culture spheroid platform comprising of BMBC cells and monocytes/macrophages successfully captured the pro-tumorigenic, immunosuppressive and therapy resistant microenvironments observed in vivo. In addition, our system is also reflective of the pro and anti-inflammatory heterogeneity observed in the BMBC niche. Thus, this system could serve as a platform for studying tumor-immune cell interactions, drug screening applications, and discovering novel drug combination strategies.