A multilayered cancer-on-a-chip model to analyze the effectiveness of new-generation photosensitizers

Three-dimensional (3D) cellular models of cancer tissue are necessary tools to analyze new anticancer drugs under in vitro conditions. Diagnostics and treatment of ovarian cancer are major challenges for current medicine. In our report we propose a new three-dimensional (3D) cellular model of ovarian cancer which can mimic a fragment of heterogeneous cancer tissue. We used Lab-on-a-chip technology to create a microfluidic system that allows cellular multilayer to be cultured. Cellular multilayer mimics the structure of two important elements of cancer tissue: flesh and stroma. For this reason, it has an advantage over other in vitro cellular models. We used human ovarian fibroblasts (HOF) and human ovarian cancer cells in our research (A2780). In the first stage of the study, we proved that the presence of non-malignant fibroblasts in co-culture with ovarian cancer cells stimulates the proliferation of cancer cells, which is important in the progression of ovarian cancer. In the next stage of the research, we tested the usefulness of the newly-developed cellular model in the analysis of anticancer drugs and therapies under in vitro conditions. We tested two photosensitizers (PS): free and nanoencapsulated meso-tetrafenylporphyrin, and we evaluated the potential of these drugs in anticancer photodynamic therapy (PDT) of ovarian cancer. We also studied the mechanism of PDT based on the analysis of the level of reactive oxygen species (ROS) in cell cultures. Our research confirmed that the use of new-generation PS can significantly increase the efficacy of PDT in the treatment of ovarian cancer. We also proved that the newly-developed 3D cellular model is suitable for rapid screening of anticancer drugs and has the potential to be used clinically in the future, e.g. in the selection of treatment methods for anticancer personalized medicine.

two independent monolayer cultures or A2780/HOF co-culture. The cell cultures were daily monitored using flow cytometry. Fig. S1 shows changes in populations of cultured cells during five-day culture.

Fig. S1
The results of analysis of the population of A2780 and HOF cells cultured as monoculture and coculture obtained by flow cytometry. In the last row the ratio of the population of cancer cells Electronic Supplementary Material (ESI) for Analyst. This journal is © The Royal Society of Chemistry 2020 2 (A2780) and fibroblasts (HOF) in co-culture on the following days of culture was presented. 1st day ratio of A2780/HOF was 50% ÷ 50%.
It was found that the presence of fibroblasts stimulated growth of cancer cells. On 5th day of culture, the percent of cancer cells increased from 50 % to 96 % in co-culture. In addition, quantitative changes in the number of cells in the following populations: A2780 monoculture, HOF monoculture and A2780 / HOF co-culture were showed in Table 1.

Table 1
Changes in the number of cells in populations: A2780, HOF and A2780/HOF in the following days of culture.
The results obtained using flow cytometry confirmed that after 96 hours of culture the number of nonmalignant cells did not change significantly (Table 1). However, the number of cells cultured in the A2780/HOF culture on the last day of culture (96 h) increased about 8 times compared to the first day of culture (24 h). The observations confirmed previous conclusions that the presence of stromal cells affects the proliferation of cancer cells.

Efficiency of photodynamic therapy using nano-TPP -macroscale tests
Two types of ovarian cells: A2780 cancer cells and HOF non-malignant cells were seeded on standard 96-well plate at a density of 10 4 cells/well. The cells were cultured as monocultures (A2780 and HOF) and co-culture (A2780+HOF). To obtain the co-culture, non-malignant cells were mixed with cancer cells in a 1:1 ratio. Then the PDT procedure was performed. For this purpose, the solutions of nano-TPP (0 ÷ 10 μM) were added. After 24 h incubation of the cells with PS (37 °C, 5 % 3 CO 2 ), the cells were exposed to light (640 nm, 40 mW cm -2 ). After 24 hours, cell viability was assessed by MTT test. Fig S2 shows the obtained results. cells. In the case of co-culture of A2780 and HOF cells, the total cell viability also decreased with increasing of PS concentration. For high drug concentrations: 5 µM, 10 µM and 15 µM, the cell viability in co-culture was: 61.37% ± 9.24%, 50.23% ± 3.62% and 38.17% ± 4.19%, respectively (Fig.  S2 A). In the case of microscale studies on a 3D cellular model, the total cell viability in co-culture already for concentration of 5 µM was lower than 40 % (Fig. 5 A).
The results obtained in the macroscale are different from the results obtained on 3D culture obtained by microfluidic system. In the standard 2D cell culture stromal cells and ovarian cancer cells grow side by side. However, in cell monolayer, the number of intercellular connections is lower than in 3D structure. Limitation of direct contact stroma-cancer may be the reason for weakened interactions between fibroblasts and cancer cells. Finally, in the macroscale conditions we can get a different response of cancer cells to the drug than in the microscale. Based on microscopic images, we conclude that after PDT procedure performed on cell co-culture, mainly cancer cells were dead, and the signal, which indicated the cell viability greater than in the microscale, came from live fibroblasts ( Fig. S2 B). Probably in the macroscale, the non-malignant cells do not induce the activation of resistance mechanisms in cancer cells or stroma affect metabolism of PS in the other way, than under microfluidic conditions.

Comparison of cell viability in irradiated (LIGHT) and non-irradiated (DARK) controls
Two types of controls were used during the experiments in the microfluidic system. In the case of the photosensitizer cytotoxicity testing (Fig. 4A, Fig. 5A) control sample was the cells untreated with compound and untreated with a light. In the case of the photosensitizer photocytotoxicity studying (Fig. 4A, Fig. 5A) a control sample was the cells without photosensitizer (C PS = 0 µM), but irradiated with LED. Differential cell staining in the irradiated and non-irradiated controls with Propidium Iodide and Calcein-AM was performed. The results are shown in Fig. S3. The light used in PDT was not cytotoxic. No differences in cell viability were observed in the irradiated and non-irradiated controls. In both cases, the cell viability was about 100 %.