A Multi-Layer Breast Cancer Model to Study the Synergistic Effect of Photochemotherapy

Abstract

Breast cancer is one of the most common cancers among women. The development of new and effective therapeutic approaches in the treatment of breast cancer is an important challenge in modern oncology. Two-dimensional (2D) cell cultures are most often used in the study of compounds with potential anti-tumor nature. However, it is necessary to develop advanced three-dimensional (3D) cell models that can, to some extent, reflect the physiological conditions. The use of miniature cancer-on-a-chip microfluidic systems can help to mimic the complex cancer microenvironment. In this report, we developed a 3D breast cancer model in the form of a cell multilayer, composed of stromal cells (HMF) and breast cancer parenchyma (MCF-7). The developed cell model was successfully used to analyze the effectiveness of combined sequential photochemotherapy, based on doxorubicin and meso-tetraphenylporphyrin. We proved that the key factor that allows achieving the synergistic effect of combination therapy are the order of drug administration to the cells and the sequence of therapeutic procedures. To the best of our knowledge, studies on the effectiveness of combination photochemotherapy depending on the sequence of the component drugs were performed for the first time under microfluidic conditions on a 3D multilayered model of breast cancer tissue.

Keywords: breast cancer; cancer-on-a-chip; meso-tetraphenylporphyrin; multilayered cell model; sequential photochemotherapy; three-dimensional (3D) cell culture.

Figure 1

The geometry of the PDMS/PDMS/glass microfluidic system for a spatial multilayer cell culture and a scheme of the cell’s arrangement in the culture chamber.

Figure 2

The scheme of cell introduction into the microsystem.

Figure 3

The imaging of three-dimensional cell multilayer in the microsystem. The breast fibroblasts (HMF) were stained with the red fluorescent dye (CMTPX), while the cancer cells (MCF-7) were stained with the green fluorescent compound (CMFDA). The image was acquired on the fourth day of culture (96 h).

Figure 4

Analysis of the proliferation of non-malignant (HMF) and cancer (MCF-7) breast cells in the microsystem. The intensity of proliferation of (A) HMF and (B) MCF-7 cell monocultures. (C) The intensity of proliferation of co-cultures of HMF and MCF-7 cells (cell multilayer). The images show changes in cell morphology on the first (24 h or 48 h) and last (96 h) day of culture. The blue line marks the doubling of the number of cells in the population (2 times stronger signal). Asterisks indicate data that differ statistically from the control (24 h). The red frames in the microscopic images are close-ups of the cell morphology in the central area of the chamber.

Figure 5

The ratio of the population of cancer cells (MCF-7) and fibroblasts (HMF) in co-culture on the following days of culture. The results were obtained via flow cytometry. First-day ratio of MCF-7/HMF was 50%:50%.

Figure 6

(A) Viability of non-malignant (HMF) and cancer (MCF-7) breast cells before and after the photochemotherapy procedure (PDT→DOX). Asterisks indicate statistically significant differences (ANOVA, α = 0.05). (B) Microscopic images of HMF/MCF-7 co-culture performed in the microsystem after the photochemotherapy procedure (PDT→DOX) (green cells—alive; red cells—dead). Scale bar: 500 µm.

Figure 7

(A). Viability of non-malignant (HMF) and cancer (MCF-7) breast cells before and after the photochemotherapy procedure (DOX→PDT). (B) Microscopic images of HMF/MCF-7 co-culture performed in the microsystem after the photochemotherapy procedure (DOX→PDT) (green cells—alive; red cells—dead). Scale bar: 500 μm.