Human Tumor-Lymphatic Microfluidic Model Reveals Differential Conditioning of Lymphatic Vessels by Breast Cancer Cells

Abstract

Breast tumor progression is a complex process involving intricate crosstalk between the primary tumor and its microenvironment. In the context of breast tumor-lymphatic interactions, it is unclear how breast cancer cells alter the gene expression of lymphatic endothelial cells and how these transcriptional changes potentiate lymphatic dysfunction. Thus, there is a need for in vitro lymphatic vessel models to study these interactions. In this work, a tumor-lymphatic microfluidic model is developed to study the differential conditioning of lymphatic vessels by estrogen receptor-positive (i.e., MCF7) and triple-negative (i.e., MDA-MB-231) breast cancer cells. The model consists of a lymphatic endothelial vessel cultured adjacently to either MCF7 or MDA-MB-231 cells. Quantitative transcriptional analysis reveals expression changes in genes related to vessel growth, permeability, metabolism, hypoxia, and apoptosis in lymphatic endothelial cells cocultured with breast cancer cells. Interestingly, these changes are different in the MCF7-lymphatic cocultures as compared to the 231-lymphatic cocultures. Importantly, these changes in gene expression correlate to functional responses, such as endothelial barrier dysfunction. These results collectively demonstrate the utility of this model for studying breast tumor-lymphatic crosstalk for multiple breast cancer subtypes.

Keywords: breast cancer; estrogen receptor-positive; lymphatic; microfluidic; triple-negative.

Figure 1.

Concept and operation of the breast tumor-lymphatic microfluidic device. A) The device consists of two polydimethylsiloxane (PDMS) layers. The bottom layer comprises a chamber and microchannels for suspending two adjacent lumen rods. The top layer contains the ports for fluid exchange. Cell culture in the device allows the formation of a lumen lined with primary human lymphatic endothelial cells (HLECs) and a second lumen filled with breast cancer cells. B) Illustration of key steps in the operation of the co-culture device. C) Representative confocal image of a 3D lymphatic vessel. D-F) Representative images of vessel monoculture, co-culture with MCF7 cells (in yellow), and co-culture with MDA-MB-231 cells (in green) in the device.

Figure 2.

Influence of MCF7 and MDA-MB-231 cells on lymphatic lumen gene expression. A-H) Lymphatic endothelial lumens were co-cultured alone and in the presence of MCF7 or MDA-MB-231 cells for 24 hours. Then, lymphatic endothelial cells were isolated and gene expression was analyzed. The graphs show the genes that were differentially expressed when lymphatic endothelial cells were co-cultured with MCF7 (blue columns) or MDA-MB-231 (red columns). I) Clustergram showing the changes in all the genes analyzed. The clustering algorithm revealed the co-culture with MCF7 led to a more different gene expression signature. J) Radar plot showing the number of genes affected in HLECs by the presence of MCF7 (in blue) and MAD-MB-231 (in red).

Figure 3.

Endothelial cell coverage and lymphangiogenic sprouting. A) Confocal images showing the middle plane of lymphatic vessels in monoculture and co-culture with MCD7 and MDA-MB-231 cells. B) Z-projected images of vessels in monoculture and co-culture with MCF7 and MDA-MB-231 cells. C) Quantification of cell coverage for each culture condition showing an average percentage of cell coverage >99% for all conditions. D) Crosstalk with the breast cancer cells induced lymphangiogenic sprouting in the vessels. There were no observable sprouts in the monoculture control. Three individual vessels (n = 3) were measured for each culture condition to determine the average permeability value (mean ± s.d.).

Figure 4.

Vessel permeability in monoculture and co-culture. Representative images at t = 0 min. and t = 10 min. for diffusion of A) a glucose analog and B) 70 kDa dextran. Dashed boxes indicate the region of interest used for measuring diffusion profiles. Dashed lines indicate the walls of the vessel. C) Diffusion profiles at t = 10 min. for monoculture and co-culture conditions. D) Glucose and dextran permeability values calculated for monoculture and co-culture conditions. MCF7-conditioned vessels were the leakiest in comparison to MDA-MB-231-conditioned vessels and monoculture controls. Three individual vessels (n = 3) were measured for each culture condition to determine the average permeability value (mean ± s.d.).