Mycoplasma Infection Modifies the Proliferation and Attachment of Cal29 Bladder Cancer Cells, as Well as the Expression of Immune Checkpoint Antigen CD276

Abstract

Purpose

Bladder cancer is among the most prevalent malignancies. Despite intensive research, progress toward better diagnosis or therapy has been sparse in recent decades. However, many studies documented elevated expression of immune checkpoint antigens including CD276 on urothelial tumor cells, and novel therapies targeting immune checkpoint antigens are promising. At the same time, mycoplasma infections were reported in about 18% of sexually active adults. We, therefore, investigated if mycoplasma-contaminated urothelial carcinoma cells Cal29 cells differed in proliferation, attachment, appearance, and expression of immune checkpoint antigen CD276 from mycoplasma-free Cal29.

Methods

Mycoplasma-infected Cal29 cells (myCal29) and clean Cal29 cells (cCal29) were expanded separately and infection vs. sterility was monitored by PCR throughout the experiment. Mycoplasma-infected normal urothelial cells (myNUCs) and clean normal urothelial cells (cNUCs) served as controls. Cells were seeded and attachment, appearance, and size of the cells were observed by microscopy. Proliferation was enumerated in consecutive passages. Expression of CD276 was studied on the mRNA level by quantitative PCR of cDNA (RT-qPCR) and on protein levels by Western blot. CD276 amounts on cell surfaces were explored by flow cytometry (FC).

Results

Mycoplasma-infected Cal29 proliferated significantly faster than cCal29 (n=5, 132%, p<0.001). Upon seeding in flasks, cCal29 adhered faster than myCal29, and attached more firmly to the flasks as microscopy showed up to double diameters in comparison to myCal29. Expression of CD276 transcripts and total proteins in cCal29 (p<0.001) and cNUCs (p<0.05) was significantly higher when compared to the corresponding infected cells. By flow cytometry, myCal29 (MFI 4334) presented less than half of CD276 on the cell surface when compared to cCal29 (MFI 9729).

Conclusions

On one hand, urinary infection by mycoplasma may indirectly contribute to tumor spreading by accelerating the proliferation of bladder cancer cells and by reducing cell-cell and/or cell-matrix interactions as documented by our cell growth and attachment studies. On the other hand, on myCal29 and myNUCs cell surfaces less CD276 was recorded. Thus, mycoplasma infection of bladder cancer cells may facilitate T-cell-mediated immune responses by reducing the amounts of immune-checkpoint antigen CD276 on cell surfaces.