Mycoplasma infection modifies attachment, proliferation, and the expression of immune checkpoint antigen CD276 (B7‐H3) on the Cal29 bladder cancer cell line

Abstract

Introduction and aimBladder cancer is among the most prevalent malignancies in Western societies. Despite intensive research, progress towards better diagnosis or therapy was sparse in the last decades. However, many studies documented elevated expression of immune checkpoint antigens including PD‐1, PD‐L1, CTLA‐4, or CD276 (alias B7‐H3) on bladder tumor cells. Immune checkpoint antigens are capable to suppress immune responses against the tumor. But novel therapies targeting immune checkpoint antigens are promising. Some of these agents are already part of clinical studies. At the same time mycoplasma infections were reported in about 18% of sexually experienced adults. We therefore investigated if mycoplasma contaminated urothelial carcinoma cells Cal29 differed in attachment, appearance, proliferation, and expression of immune checkpoint antigen CD276 from mycoplasma‐free Cal29.Materials and MethodsMycoplasma infected Cal29 cells (mcCal29) and clean Cal29 cells (Cal29, from ATCC) were expanded and infection vs. sterility was monitored by PCR throughout the experiment. Cells were seeded and attachment, appearance and size of the cells were observed by microscopy. Proliferation was enumerated in consecutive passages using a hematocytometer and trypan blue dye exclusion. Expression of CD276 was studies on the mRNA level by quantitative PCR of cDNA (RT‐qPCR). Analyses of GAPDH and PPIAγ served as controls. CD276 protein levels were explored by immunoblot, and ß‐actin served a control. CD276 expression on cell surfaces was explored by flow cytometry and is documented as mean fluorescence intensity (MFI). Unstained cells and beads served as controls.ResultsMycoplasma‐contaminated Cal29 proliferated significantly faster than clean Cal29 (n=5, 132%, p<7,8E‐09). Upon seeding in flasks, Cal29 adhered faster then mcCal29, attached more firm to the flasks as microscopy showed up to double diameters in comparison to mcCal29. Expression of CD276 transcripts and total proteins did not differ, but on cell surfaces mcCal29 (MFI 4334) expressed less than half of CD276 in comparison to Cal29 (MFI 9729).ConclusionsUrinary infection by mycoplasma may indirectly contribute to tumor spready by accelerating proliferation of bladder cancer cells and by reducing cell‐cell and/or cell‐matrix interactions as documented by attachment and cell growth studies. However, mcCal29 presented less CD276 on tumor cell surfaces. Thus, mycoplasma infection may facilitate immune responses to bladder cancer cells by reducing the expression of CD276. But these preliminary results must be conformed or challenged by additional studies with other urothelial cells.