Abstract
The tumor microenvironment is composed of heterogeneous populations of cells, including cancer, immune, and stromal cells. Progression of tumor growth and initiation of metastasis is critically dependent on the reciprocal interactions between cancer cells and stroma. Through RNA-Seq and protein analyses, we found that cancer-associated fibroblasts derived from human breast cancer brain metastasis express significantly higher levels of chemokines CXCL12 and CXCL16 than fibroblasts from primary breast tumors or normal breast. To further understand the interplay between cancer cells and cancer-associated fibroblasts from each site, we developed three-dimensional organoids composed of patient-derived primary or brain metastasis cancer cells with matching cancer-associated fibroblasts. Three-dimensional CAF aggregates generated from brain metastasis promote migration of cancer cells more effectively than cancer-associated fibroblast aggregates derived from primary tumor or normal breast stromal cells. Treatment with a CXCR4 antagonist and/or CXCL16 neutralizing antibody, alone or in combination, significantly inhibited migration of cancer cells to brain metastatic cancer-associated fibroblast aggregates. These results demonstrate that human brain metastasis cancer-associated fibroblasts potently attract breast cancer cells via chemokines CXCL12 and CXCL16, and blocking CXCR6-CXCL16/CXCR4-CXCL12 receptor–ligand interactions may be an effective therapy for preventing breast cancer brain metastasis.
Highlights
Brain metastasis is the most lethal outcome of breast cancer, leading to death within 4–6 months in 10–15% of patients once detected.[1, 2] For brain metastasis to occur, cancer cells from the primary tumor must migrate to the brain, traverse the blood–brain barrier, and proliferate within the brain parenchyma.[3]
We obtained six normal breast tissue samples from either the contralateral breast of breast cancer patients, or patients who underwent prophylactic mastectomy. Histological analysis of both human primary breast and brain metastatic tumor samples showed the presence of vimentin-positive stromal cells surrounding cytokeratin-positive breast cancer cells (Fig. 1a). To study these cells and develop an ex-vivo culture system that allows expansion of both patient-specific breast cancer cells and Cancer-associated fibroblasts (CAFs), human breast tumor tissue was mechanically dissociated into small fragments, and plated onto tissue culture plate in medium supplemented with epidermal and keratinocyte growth factor
Based on live cell imaging, immunofluorescent microscopy, and FACS analysis, we found that significantly higher numbers of MCF-HER2 cells or patient-derived cancer cells migrated to brain metastatic CAF aggregates than primary CAF or normal breast fibroblasts aggregates (Fig. 4b and Supplemental Fig. 6)
Summary
Brain metastasis is the most lethal outcome of breast cancer, leading to death within 4–6 months in 10–15% of patients once detected.[1, 2] For brain metastasis to occur, cancer cells from the primary tumor must migrate to the brain, traverse the blood–brain barrier, and proliferate within the brain parenchyma.[3]. Cell–cell and cell–extracellular matrix (ECM) interactions in 3D spatial environment are critical for understanding the complex cross-talk mechanisms between cancer and stromal cells Both gene and protein expressions in an ex vivo 3D culture system appear to conserve various paracrine-dependent cellular interactions that occur in vivo microenvironment.[5,6,7] studies have shown that testing of chemotherapy treatments or immunotherapies based on 2D monolayer systems does not correspond with results in an in vivo setting, further demonstrating the limitations of 2D monolayer systems.[8] developing and testing the effectiveness of novel therapies for breast cancer in vitro require recreation of the 3D breast cancer microenvironment composed of stroma and cancer cells, ideally derived from the same patient, as one functional unit
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