The ability of stem cells to differentiate into specialized cell types presents a number of opportunities for regenerative medicine, stem cell therapy and developmental biology. Because traditional assessments of stem cells are destructive, time consuming, and logistically intensive, the use of a non-invasive, label-free approach to study of cell differentiation provides a powerful tool for rapid, high-content characterization of cell and tissue cultures. Here, we elucidate the metabolic changes in MSCs during adipogenic differentiation, based on the fluorescence of the metabolic co-factors NADH, NADPH, and FAD using the methods of two-photon fluorescence microscopy combined with FLIM. To estimate the contribution of energy metabolism and lipogenesis in the observed changes of the metabolic profile, a separate analysis of NADH and NADPH is required. In our study we demonstrated, for the first time, an increased contribution of protein-bound NADPH in adipocytes that is associated with lipogenesis. The optical redox ratio FAD/NAD(P)H decreased during adipogenic differentiation, and that this was likely to be explained by the intensive biosynthesis of lipids and the enhanced NADPH production associated with this. Based on the data on the fluorescence lifetime contribution of protein-bound NAD(P)H, we registered a metabolic switch from glycolysis to oxidative phosphorylation in adipocytes.

IntroductionIn recent years, mesenchymal stem cells (MSCs) have been demonstrated to play an important role in carcinogenesis. However, the effect of MSCs on tumor and metastasis development and the mechanisms underlying the interaction of cancer and stem cells are not completely understood. This study investigated the effect of MSCs on breast cancer metastasis formation by using the methods of in vivo fluorescence and luminescence imaging.MethodsMSCs were isolated from bone marrow of normal donors, characterized, and genetically labeled with luciferase (luc2). The effects of MSCs on MDA-MB-231 cancer cell proliferation were evaluated in conditioned medium from MSCs. To generate lung metastases, MDA-MB-231 cells stably expressing red fluorescent protein Turbo FP650 were injected intravenously into nude mice. On day 10 after the cancer cell injection, mice were injected via the tail vein with MSCs-luc2 cells (the MET + MSCs group). Animals that received the injection of MDA-MB-231-Turbo FP650 alone (the MET group) and no injections (the intact control group) served as controls. Fluorescence and bioluminescence imaging was performed for monitoring of the metastasis formation and MSC distribution in the recipient’s body.ResultsWe found that the proliferative activity of the cancer cells in the presence of MSC conditioned medium was lower than that of the cells grown in conventional culture medium. The metastasis formation in the MET + MSCs group was delayed in time as compared with the MET group. Macroscopic and histological examination of isolated lungs 8 weeks after cancer cell injection showed that the total number of metastases in animals of the MET + MSCs group was significantly lower. Using bioluminescence imaging in vivo, we found that MSCs-luc2 cells survived in the host animal for at least 7 weeks and re-migrated to the lung 6 to 7 weeks after injection. Immunohistochemical analysis revealed the presence of MSCs-luc2 in metastases and lung tissue.ConclusionsLong-term in vivo bioluminescence imaging of intravenously injected MSCs-luc2 cells showed distribution of MSCs to the lungs and abdominal organs within the first 2 to 3 weeks and re-migration to the lungs in weeks 6 to 7. It was found that MSCs reduced the proliferative activity of cancer cells in vitro and lung metastasis formation in mice.

Tumor resistance to therapy is associated with the 3D organization and peculiarities of the tumor microenvironment, of which intercellular adhesion is a key participant. In this work, the abundance of contact proteins was compared in SKOV-3 and SKOV-3.ip human ovarian adenocarcinoma cell lines, cultivated in monolayers, tumor spheroids and collagen hydrogels. Three-dimensional models were characterized by extremely low expression of basic molecules of adherens junctions E-cadherin and demonstrated a simultaneous decrease in desmosomal protein desmoglein-2, gap junction protein connexin-43 and tight junction proteins occludin and ZO-1. The reduction in the level of contact proteins was most pronounced in collagen hydrogel, accompanied by significantly increased resistance to treatment with doxorubicin and targeted anticancer toxin DARPin-LoPE. Thus, we suggest that 3D models of ovarian cancer, especially matrix-based models, tend to recapitulate tumor microenvironment and treatment responsiveness to a greater extent than monolayer culture, so they can be used as a highly relevant platform for drug efficiency evaluation.