A microfluidic 3D in vitro model for specificity of breast cancer metastasis to bone

Abstract

Cancer metastases arise following extravasation of circulating tumor cells with certain tumors exhibiting high organ specificity. Here, we developed a 3D microfluidic model to analyze the specificity of human breast cancer metastases to bone, recreating a vascularized osteo-cell conditioned microenvironment with human osteo-differentiated bone marrow-derived mesenchymal stem cells and endothelial cells. The tri-culture system allowed us to study the transendothelial migration of highly metastatic breast cancer cells and to monitor their behavior within the bone-like matrix. Extravasation, quantified 24 h after cancer cell injection, was significantly higher in the osteo-cell conditioned microenvironment compared to collagen gel-only matrices (77.5 ± 3.7% vs. 37.6 ± 7.3%), and the migration distance was also significantly greater (50.8 ± 6.2 μm vs. 31.8 ± 5.0 μm). Extravasated cells proliferated to form micrometastases of various sizes containing 4 to more than 60 cells by day 5. We demonstrated that the breast cancer cell receptor CXCR2 and the bone-secreted chemokine CXCL5 play a major role in the extravasation process, influencing extravasation rate and traveled distance. Our study provides novel 3D in vitro quantitative data on extravasation and micrometastasis generation of breast cancer cells within a bone-like microenvironment and demonstrates the potential value of microfluidic systems to better understand cancer biology and screen for new therapeutics.

Keywords: Bone; Breast cancer; Extravasation; Hydrogel; Metastasis; Microfluidics.

Fig. 1

Generation of the osteo-cell conditioned microenvironment. (A) hBM-MSCs (brown) were cultured for 2–3 weeks within osteogenic medium and seeded within microfluidic devices where they started depositing extracellular matrix (yellow filaments). After 3 days endothelial cells (ECs) (red) were seeded and a monolayer covering the media channel was generated. Finally, cancer cells (green) were introduced after 3 additional days and their extravasation ability and micrometastasis generation were monitored for 1 to 5 days. (B and C) Three-dimensional reconstruction of a confocal stack represents a top (B) and a front view (C) of a single ROI. HUVECs (RFP) completely covered the channel walls while osteo-differentiated hBM-MSCs were homogeneously distributed within the collagen gel. Cells were stained with DAPI (nuclei, blue) and phalloidin (F-actin, green). (D) Alizarin Red staining of calcium deposits (dark region) within a gel channel (optical microscopy, bright field). (E) Two-dimensional projection of a confocal stack highlights the ability of osteo-differentiated hBM-MSCs to secrete osteocalcin (green) within a 3D microenvironment. Cells were stained with DAPI (nuclei, blue) and phalloidin (F-actin, yellow). Osteo-differentiated hBM-MSCs are labeled as osteo-cells. Scale bars: 50 µm.

Fig. 2

Extravasation of cancer cells into the collagen gel matrix with and without osteo-differentiated hBM-MSCs (labeled as osteo-cells). (A) Three-dimensional confocal reconstruction shows MDA-MB-231 cancer cells (GFP) transmigrated across the endothelial monolayer into the collagen gel containing osteo-differentiated hBM-MSCs. VE-cadherin (red) and DAPI (nuclei, blue) staining. (B) Average percentage of extravasated cancer cells was significantly higher (n=27(min)-39(max) regions, p<0.005) in the collagen gel with osteo-differentiated hBM-MSCs. (C) Projected images show extravasated cancer cells (GFP) travelled farther into the osteo-cell conditioned microenvironment (ii) compared to the collagen gel-only matrix (i). Cells were stained with (i) DAPI (nuclei, blue) and (ii) VE-cadherin (red) + DAPI (nuclei, blue). HUVECs were RFP labeled. (D) Average distance travelled by extravasated cells into the gel matrix increased significantly in osteo-cell conditioned microenvironment (n=11(min)-17(max) regions, p<0.05). Scale bars: 50 µm.

Fig. 3

Effect of CXCL5 and CXCR2 in cancer cell extravasation. (A) Addition of CXCR2 blocking antibody significantly reduced cancer cell average extravasation within collagen gel containing osteo-differentiated hBM-MSCs compared to non-treated cancer cells (ctrl) or IgG incubated cancer cells (n=9(min)-24(max) regions, p<0.005). (B) Two-dimensional projection of a confocal stack shows that cancer cells (GFP, white arrows) generally remained within the endothelial cell (RFP)-coated channel and did not extravasate into the collagen gel containing osteo-differentiated hBM-MSCs when anti-CXCR2 antibody was added. Cells were stained with DAPI (nuclei, blue) and phalloidin (F-actin, yellow). (C) Addition of CXCL5 within collagen gel-only devices significantly enhanced the average percentage of cancer cell extravasation compared to unconditioned devices (ctrl) and addition of IgG (n=9(min)-24(max) regions, p<0.05). (D) Two-dimensional projection of a confocal stack showing two extravasated cancer cells (GFP, white arrows) inside CXCL5-conditioned collagen gel-only devices. Cells were stained with DAPI (nuclei, blue) and phalloidin (F-actin, yellow). HUVECs were RFP labeled. (E) The average extravasation distance was significantly higher with the addition of CXCL5 (condition) within collagen gel-only devices compared to unconditioned devices (p<0.05) whereas there was no significant difference within a osteo-cell conditioned matrix with (condition) or without (control) cancer cell incubation in CXCR2 blocking antibody (n=11(min)-17(max) regions). Scale bars: 50 µm.

Fig. 4

Generation of micrometastases within the osteo-cell conditioned microenvironment. Three-dimensional confocal images of a single gel region show a representative large (A) and small (B) cancer cell (GFP) micrometastasis. Ki-67 staining (red) demonstrates all cancer cells were proliferating within the colonized microenvironment. Cells were stained with DAPI (nuclei, blue). (C) Histogram showing micrometastasis distribution. Although 4 large clusters (>35 cells) were detected, micrometastases generally contained fewer than 10 cells. (D) Histogram represents the number of micrometastases per gel region (n=15 regions). Scale bars: 50 µm.