Preliminary Assessment of the Comparison of the Response of Mitochondrial-based Biomarker in Endometriosis Cells and Normal Cells Using Single-cell Analysis

Background: A lot of research is focusing on endometriosis in order to understand its mechanisms which are still poorly understood nowadays. There is no treatment and the only way to diagnose endometriosis is invasively by laparoscopy. To date, only the biomarker CA 125 is recognized as an indicator of endometriosis, despite the fact that its specificity is low. Furthermore, endometriosis has been identified as a precursor lesion for a number of epithelial ovarian cancer.
Research purpose: This research, utilizing the microscopic platform for single-cell analysis, focuses on studying mitochondria and their different responses to
Rhodamine 123, a green fluorescent dye. This study aims to compare the
mitochondrial fragmentation count, the mitochondrial membrane potential intensity (ΔΨm) and its relative ATP production, and the mitochondrial morphology to identify potential variations between cancer cells (A549), endometriosis cells (VK2/E6E7) and normal cells (MeT-5A).
Material and methods: A549, VK2 E6/E7, and MeT-5A which are all epithelial
cells were seeded into a microfluidic flow chamber to allow the single cell analysis and incubated for 24 hours at a temperature of 37°C and 5% of CO2. VK2/E6E7 and A549 were then treated for 40 minutes with 7.5 M of the biomarker
Rhodamine 123 compared with 10 M for MeT-5A cells to obtain respectively the
ΔΨm and the mitochondrial fragmentation count (MFC) within the single living
cells. The cells were then observed under the microscope using the brightfield mode and the fluorescent mode.
Results: The preliminary data indicate that the A549 cell line has a significantly higher number of mitochondrial fragmentations (MFC=2.459) than the VK2 (MFC=1.373) and MeT-5A lines (MFC=1.422). No significant differences were
identified among the three lines regarding the ΔΨm.
Conclusion: It was demonstrated during this research that the A549 cell line had a higher number of mitochondrial fragmentations than the MeT-5A and VK2/E6E7
cell lines. Mitochondrial fragmentation results from an imbalance between the fusion and fission mechanisms. An increase in fission favours fragmentation of the mitochondria. It is therefore normal for A549 to have a high MFC. With regard to the VK2/E6E7 cell line, its MFC is close to the MeT-5A cell line. Numerous studies qualify endometriosis as an intermediate stage between benign and malignant cells. However, the data indicate a lower MFC than that of healthy cells. We therefore think that this may be due to the concentration of probe used in this experimentwhich was 10M for MeT-5A cell line and 7.5M for the VK2/E6E7 and A549 cell lines. Further experiments will be carried out to investigate potential factors of variation in cell response to the Rhodamine123 biomarker. The investigations will initially be carried out using the same Rhd123 concentration for all three cell lines. In a second phase, it would also be relevant to study two endometriosis cell lines from different regions of the
body.