用于表征炎性乳腺癌细胞系淋巴血管间隙浸润的血管化3D微流控乳腺肿瘤平台

Highly aggressive cancers such as inflammatory breast cancer (IBC) are characterized by the presence of tumor cell emboli within lymphatic and blood vessels, a phenomenon known as lymphovascular space invasion (LVSI). In this study, we developed a vascularized 3D in vitro tumor microfluidic platform to evaluate LVSI mechanisms including tumor invasion, cancer cells intravasation, vascular and Extracellular Matrix (ECM) remodeling, and emboli formation in IBC cell lines (MDA-IBC3, A3250, and SUM149).Initially, various collagen concentrations (2,3,4,5and 6 mg/mL) were evaluated using optical fiber-based interferometry nanoindentation to measure collagen hydrogel stiffness and identify a concentration representative of normal breast tissue (0.5-1 kPa). A 4 mg/mL collagen concentration was selected, yielding an average stiffness of approximately 0.7 kPa, and was used to assess how the incorporation of cancer cells alters ECM stiffness. Platforms were fabricated by polymerizing a 4 mg/mL collagen type I solution containing cancer cells (2 million cells/mL) around two 22G needles to form vascular channels. These channels were seeded with mKate-tagged telomerase-immortalized endothelial (TIME) cells at 10 million cells/mL, and a shear stress of 4 dyn/cm² was applied to establish aligned, functional endothelium. Confocal microscopy was used to monitor vessel sprouting, permeability, emboli formation, and tumor cell intravasation for one week. Effluent media was collected on Days 3 and 7 for cytokine analysis. By day 7, vessel coverage was significantly reduced in TIME+A3250 and TIME+SUM149 platforms compared to TIME-only and TIME+IBC3 platforms (p < 0.001). Although the number of endothelial sprouts was significantly higher in TIME+A3250 platforms compared to TIME+IBC3, the average sprout length did not differ significantly. A3250 cells also exhibited a higher frequency of invadopodia-positive tumor cells compared to MDA-IBC3, indicating a greater invasive phenotype. Consistent with this, the number of intravasated A3250 cells was approximately 15-fold higher than that of MDA-IBC3 cells. Spatial analyses of the platforms' midplanes images further revealed that A3250 cells accumulated around the vessel to a much greater extent than the area farther from the vessel over time. Notably, platforms containing A3250 cells showed an extreme hydrogel deformation, including central separation, suggesting profound collagen remodeling driven by this cell line. This trend was not observed with the other IBC lines (MDA-IBC3 and SUM149).In conclusion, our results indicate that the A3250 IBC cell line shows a more aggressive phenotype compared to the other IBC cell lines. We observed significantly higher intravasation relative to MDA-IBC3, greater disruption of endothelial vessel coverage, and a higher frequency of invadopodia-positive cells.