Abstract B01: The metabolic adaptations during cancer-stroma co-evolution: The intracellular pH, NAD(P)H changes and hydrogen peroxide production in cancer cells

Abstract

Abstract Intoduction: It is generally accepted that cancer cells and cancer-associated fibroblasts (CAFs) can change their metabolism to maintain tumor progression and adapt cancer cells to unfavorable conditions. Recently it was found that in some tumors cancer cells have oxidative metabolism and CAFs switch their metabolism to aerobic glycolysis when interplay with cancer cells (“reverse Warburg effect”) (1). It is supposed that cancer cells produce hydrogen peroxide to initiate oxidative stress in CAFs and drive aerobic glycolysis (2). Also it is known that intracellular pH (pHi) level is an important regulator of many cells functions. And the pHi in cancer cells differ from pHi in normal cells. The pHi alterations in cancer cell may be a result of metabolic changes during neoplastic processes (3). The goal of our study was to investigate metabolic interaction between cancer cells and fibroblasts in the co-culture model. The following parameters were studied In cancer cells: pHi hydrogen peroxide level and NAD(P)H. Materials and Methods: The experiments were performed on Hela Kyoto cancer cells and normal skin fibroblasts. HeLa Kyoto, stably expressing genetically encoded fluorescent pHi-sensor SypHer2 (HeLa-SypHer2) or H2O2-sensor HyPer2 (HeLa-HyPer2) were used for detection of intracellular and hydrogen peroxide levels, correspondently (4). Fluorescence of NAD(P)H was detected using two-photon excitation, and fluorescence lifetime and relative contributions of free and protein-bound forms were measured. Cancer cells and fibroblasts were co-plated with a 1:5 fibroblast-to-cancer cell ratio or plated in parallel as a monotypic culture (5). Cells were analyzed daily for 3 days. Fluorescence confocal microscopy and fluorescence lifetime imaging (FLIM) were performed using the fluorescence laser-scanning microscope LSM 710. For FLIM investigation FLIM module based on time-correlated single photon counting Simple Tau 152 TCSPC was used. The obtained images were processed using ImageJ 1.39p and SPCImage software. Results: In our work we demonstrate that during tumor-stroma co-evolution in model system based on Hela Kyoto cells and fibroblasts cancer cells switch their metabolism from oxidative phosphorylation (OXPHOS) to glycolysis while fibroblast switch their metabolism from glycolysis to OXPHOS. We found that these metabolic changes happened after hydrogen peroxide production in cancer cells and spreading of H2O2 to adjusted fibroblasts. The peak of hydrogen peroxide production took place at the second day of co-cultivation. In the corresponded mono-culture the flash of H2O2 was not observed. It was shown that the pHi level in cancer cells became slightly more acidic in co-culture conditions in comparison with corresponded mono-culture. Conclusions: To our knowledge, these three parameters reflecting cancer cells metabolism were analyzed for the first time in a model of tumor-stroma co-evolution. The understanding of metabolic features of cancer and normal cells during neoplastic transformation have fundamental and practical importance as it can be used for tumor diagnostics, development of new anticancer drugs targeting metabolic pathways.