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Genetically encoded pH-sensors are the promising instrument for intracellular pH (pHi) registration. In tumor tissue the reversed pH gradient is known to be the important hallmark of cancer and regulator of tumor response on chemotherapy. However the effect of chemotherapeutic drugs on the pHi of tumor cells is largely unknown. Here we using genetically encoded ratiometric pH-sensor SypHer2 were able to monitor pHi in vitro in cell monolayer and tumor spheroids and in vivo in tumor xenografts. In tumor cell monolayer different pHi dynamic was revealed in the dying cell and division-arrested surviving cells. The treatment effect of taxol varied in monolayer and tumor spheroids and pHi changes were able to reflect these difference. The tend to pHi decrease in respect to taxol in vivo matched with results obtained for the cell monolayer. Also in both cases the cell cycle-arrest was the main treatment effect in contrast to tumor spheroid, where the cell death was the primary result. These findings elucidate the significance of pHi in the mechanisms of taxol action on cervical cancer cells and will be valuable for development of new approaches for cancer treatment.

Significance: Despite the importance of the cell membrane in regulation of drug activity, the influence of drug treatments on its physical properties is still poorly understood. The combination of fluorescence lifetime imaging microscopy (FLIM) with specific viscosity-sensitive fluorescent molecular rotors allows the quantification of membrane viscosity with high spatiotemporal resolution, down to the individual cell organelles. Aim: The aim of our work was to analyze microviscosity of the plasma membrane of living cancer cells during chemotherapy with cisplatin using FLIM and correlate the observed changes with lipid composition and cell’s response to treatment. Approach: FLIM together with viscosity-sensitive boron dipyrromethene-based fluorescent molecular rotor was used to map the fluidity of the cell’s membrane. Chemical analysis of membrane lipid composition was performed with time-of-flight secondary ion mass spectrometry (ToF-SIMS). Results: We detected a significant steady increase in membrane viscosity in viable cancer cells, both in cell monolayers and tumor spheroids, upon prolonged treatment with cisplatin, as well as in cisplatin-adapted cell line. ToF-SIMS revealed correlative changes in lipid profile of cisplatin-treated cells. Conclusions: These results suggest an involvement of membrane viscosity in the cell adaptation to the drug and in the acquisition of drug resistance.

Colorectal cancer is one of the most commonly diagnosed and poorly responding to chemotherapy types of cancer, which emphasizes the importance of personalized approach to treatment selection. Short-term primary cell cultures established from patients’ tumors represent a valuable model for testing drug response. In this study, we developed protocols for generation of the short-term primary cell cultures from colorectal cancer tissue and assessment their chemosensitivity using MTT test. Additionally, we showed the possibility of metabolic analysis of patient-derived cancer cells using fluorescence lifetime imaging (FLIM) of autofluorescent cofactor NAD(P)H. Since FLIM of NAD(P)H demonstrates the potential to detect early responses to cancer treatment, we assume that this method, alone or in combination with MTT assay, can be used for choosing the optimal chemotherapy for patients.

Fluorescent proteins in combination with fluorescence imaging technologies offer unique opportunities to explore cancer. Using new fluorescent proteins, we developed methodologies for in vivo monitoring of different functional processes in mouse tumor models and for tumor treatment.

OX40 receptor-expressing regulatory T cells (Tregs) populate tumors and suppress a variety of immune cells, posing a major obstacle for cancer immunotherapy. Different ways to functionally inactivate Tregs by triggering OX40 receptor have been suggested, including anti-OX40 antibodies and Fc:OX40L fusion proteins. To investigate whether the soluble extracellular domain of OX40L (OX40Lexo) is sufficient to enhance antitumor immune response, we generated an OX40Lexo-expressing CT26 colon carcinoma cell line and studied its tumorigenicity in immunocompetent BALB/c and T cell deficient nu/nu mice. We found that soluble OX40L expressed in CT26 colon carcinoma favors the induction of an antitumor response which is not limited just to cells co-expressing EGFP as an antigenic determinant, but also eliminates CT26 cells expressing another fluorescent protein, KillerRed. Tumor rejection required the presence of T lymphocytes, as indicated by the unhampered tumor growth in nu/nu mice. Subsequent re-challenge of tumor-free BALB/c mice with CT26 EGFP cells resulted in no tumor growth, which is indicative of the formation of immunological memory. Adoptive transfer of splenocytes from mice that successfully rejected CT26 OX40Lexo EGFP tumors to naïve mice conferred 100% resistance to subsequent challenge with the CT26 EGFP tumor.

Collagen, the main non-cellular component of the extracellular matrix (ECM), is profoundly reorganized during tumorigenesis and has a strong impact on tumor behavior. The main source of collagen in tumors is cancer-associated fibroblasts. Cancer cells can also participate in the synthesis of ECM, however, the contribution of both types of cells to collagen rearrangements during the tumor progression is far from being clear. Here, we investigated the processes of collagen biosynthesis and remodeling in parallel with the transcriptome changes during cancer cells and fibroblasts interactions. Combining immunofluorescence, RNA sequencing, and second harmonic generation microscopy, we have explored the relationships between the ratio of epithelial (E) and mesenchymal (M) components of hybrid E/M cancer cells, their ability to activate fibroblasts, and the contributions of both cell types to collagen remodeling. To this end, we studied (i) co-cultures of colorectal cancer cells and normal fibroblasts in a collagen matrix, (ii) patient-derived cancer-associated fibroblasts, and (iii) mouse xenograft models. We found that the activation of normal fibroblasts that form dense collagen networks consisting of large, highly oriented fibers depends on the difference in E/M ratio in the cancer cells. The more-epithelial cells activate the fibroblasts more strongly, which correlates with a dense and highly ordered collagen structure in tumors in vivo. The more-mesenchymal cells activate the fibroblasts to a lesser degree, on the other hand, this cell line has a higher innate collagen remodeling capacity. Normal fibroblasts activated by cancer cells contribute to the organization of the extracellular matrix in a way that is favorable for migratory potency. At the same time, in co-culture with epithelial cancer cells, the contribution of fibroblasts to the reorganization of ECM is more pronounced. Therefore, one can expect that targeting the ability of epithelial cancer cells to activate normal fibroblasts may provide a new anticancer therapeutic strategy.

Background Measuring intracellular pH (pHi) in tumors is essential for the monitoring of cancer progression and the response of cancer cells to various treatments. The purpose of the study was to develop a method for pHi mapping in living cancer cells in vitro and in tumors in vivo, using the novel genetically encoded indicator, SypHer2. Methods A HeLa Kyoto cell line stably expressing SypHer2 in the cytoplasm was used, to perform ratiometric (dual excitation) imaging of the probe in cell culture, in 3D tumor spheroids and in tumor xenografts in living mice. Results Using SypHer2, pHi was demonstrated to be 7.34 ± 0.11 in monolayer HeLa cells in vitro under standard cultivation conditions. An increasing pHi gradient from the center to the periphery of the spheroids was displayed. We obtained fluorescence ratio maps for HeLa tumors in vivo and ex vivo. Comparison of the map with the pathomorphology and with hypoxia staining of the tumors revealed a correspondence of the zones with higher pHi to the necrotic and hypoxic areas. Conclusions Our results demonstrate that pHi imaging with the genetically encoded pHi indicator, SypHer2, can be a valuable tool for evaluating tumor progression in xenograft models. General significance We have demonstrated, for the first time, the possibility of using the genetically encoded sensor SypHer2 for ratiometric pH imaging in cancer cells in vitro and in tumors in vivo. SypHer2 shows great promise as an instrument for pHi monitoring able to provide high accuracy and spatiotemporal resolution.

Intracellular pH (pHi) is one of the most important parameters that regulate the physiological state of cells and tissues. pHi homeostasis is crucial for normal cell functioning. Cancer cells are characterized by having a higher (neutral to slightly alkaline) pHi and lower (acidic) extracellular pH (pHe) compared to normal cells. This is referred to as a "reversed" pH gradient, and is essential in supporting their accelerated growth rate, invasion and migration, and in suppressing anti-tumor immunity, the promotion of metabolic coupling with fibroblasts and in preventing apoptosis. Moreover, abnormal pH, both pHi and pHe, contribute to drug resistance in cancers. Therefore, the development of methods for measuring pH in living tumor cells is likely to lead to better understanding of tumor biology and to open new ways for cancer treatment. Genetically encoded, fluorescent, pH-sensitive probes represent promising instruments enabling the subcellular measurement of pHi with unrivaled specificity and high accuracy. Here, we describe a protocol for pHi imaging at a microscopic level in HeLa tumor spheroids, using the genetically encoded ratiometric (dual-excitation) pHi indicator, SypHer2.

Although cisplatin plays a central role in cancer chemotherapy, the mechanisms of cell response to this drug have been unexplored. The present study demonstrates the relationships between the intracellular pH (pHi), cell bioenergetics and the response of cervical cancer to cisplatin. pHi was measured using genetically encoded sensor SypHer2 and metabolic state was accessed by fluorescence intensities and lifetimes of endogenous cofactors NAD(P)H and FAD. Our data support the notion that cisplatin induces acidification of the cytoplasm early after the treatment. We revealed in vitro that a capacity of cells to recover and maintain alkaline pHi after the initial acidification is the crucial factor in mediating the cellular decision to survive and proliferate at a vastly reduced rate or to undergo cell death. Additionally, we showed for the first time that pHi acidification occurs after prolonged therapy in vitro and in vivo, and this, likely, favors metabolic reorganization of cells. A metabolic shift from glycolysis towards oxidative metabolism accompanied the cisplatin-induced inhibition of cancer cell growth in vitro and in vivo. Overall, these findings contribute to an understanding of the mechanisms underlying the responsiveness of an individual cell and tumor to therapy and are valuable for developing new therapeutic strategies.

OCT-based local strain relaxation/creep evaluation is an emerging tool for tissue viscoelasticity characterization. We present a tool for 2D visualization of local strain relaxation and creep time/rate inside the tissue.

Intracellular pH (pHi) is one of the most important parameters that regulate the physiological state of cells and tissues. pHi homeostasis is crucial for normal cell functioning. Cancer cells are characterized by having a higher (neutral to slightly alkaline) pHi and lower (acidic) extracellular pH (pHe) compared to normal cells. This is referred to as a “reversed” pH gradient, and is essential in supporting their accelerated growth rate, invasion and migration, and in suppressing anti-tumor immunity, the promotion of metabolic coupling with fibroblasts and in preventing apoptosis. Moreover, abnormal pH, both pHi and pHe, contribute to drug resistance in cancers. Therefore, the development of methods for measuring pH in living tumor cells is likely to lead to better understanding of tumor biology and to open new ways for cancer treatment. Genetically encoded, fluorescent, pH-sensitive probes represent promising instruments enabling the subcellular measurement of pHi with unrivaled specificity and high accuracy. Here, we describe a protocol for pHi imaging at a microscopic level in HeLa tumor spheroids, using the genetically encoded ratiometric (dual-excitation) pHi indicator, SypHer2.

The aim of the present work was to study energy metabolism in human cervical carcinoma (HeLa) cells in vitro and in vivo using two-photon FLIM. Cellular metabolism was examined by monitoring of the fluorescence lifetimes of free and protein-bound forms of NAD(P)H and FAD and their relative contributions. Two-photon fluorescence and second harmonic generation microscopy as well as standard histopathology with hematoxylin and eosin were used to characterize tissue structure. Cellular metabolism was analyzed in cancer cells co-cultured with human fibroblasts and in tumor xenografts transplanted to nude mice. In the HeLa-huFB co-culture we observed a metabolic shift from OXPHOS toward glycolysis in cancer cells, and from glycolysis to OXPHOS in fibroblasts, starting from Day 2 of co-culturing. In the tumor tissue we detected metabolic heterogeneity with more glycolytic metabolism of cancer cells in the stroma-rich zones. The results of the study are of a great importance for understanding metabolic behavior of tumors and for development of anticancer drugs targeted to metabolic pathways.

Alteration in the cellular energy metabolism is a principal feature of tumors. An important role in modifying cancer cell metabolism belongs to the cancer-associated fibroblasts. However, the regulation of their interaction has been poorly studied to date. In this study we monitored the metabolic status of both cell types by using the optical redox ratio and the fluorescence lifetimes of the metabolic co-factors NAD(P)H and FAD, in addition to the intracellular pH and the hydrogen peroxide levels in the cancer cells, using genetically encoded sensors. In the co-culture of human cervical carcinoma cells HeLa and human fibroblasts we observed a metabolic shift from oxidative phosphorylation toward glycolysis in cancer cells, and from glycolysis toward OXPHOS in fibroblasts, starting from Day 2 of co-culturing. The metabolic switch was accompanied by hydrogen peroxide production and slight acidification of the cytosol in the cancer cells in comparison with that of the corresponding monoculture. Therefore, our HeLa-huFb system demonstrated metabolic behavior similar to Warburg type tumors. To our knowledge, this is the first time that these 3 parameters have been investigated together in a model of tumor-stroma co-evolution. We propose that determination of the start-point of the metabolic alterations and understanding of the mechanisms of their realization can open a new ways for cancer treatment.

Introduction An increasing number of recent studies suggest that tumour response to chemotherapy cannot be fully described in terms of only interaction of the drug with the main cellular target (e.g. DNA or microtubules) but can include multiple physiological and physicochemical changes. Insight into drug-induced functional alterations in cancer cells and tissues is crucially important for understanding of mechanisms of a drug action and for development new approaches to enable monitoring of the early-treatment response. Material and methods In our studies, we focus on multiparameteric analysis of tumour responses to chemotherapy using advanced fluorescence imaging techniques. Previously, we developed methodologies for in vivo probing several parameters, including cytosolic pH, metabolic status, and viscosity of plasma membrane, in mouse tumour models. Mapping of cytosolic pH in tumours is performed using ratiometric genetically encoded sensor SypHer2 and fluorescence whole-body imaging [Shirmanova et al. BBA-GS 2015]. Imaging of cellular metabolism is based on the visualisation of fluorescence intensities and lifetimes of intrinsic metabolic cofactors NAD(P)H and FAD [Shirmanova et al. Sci.Rep. 2017]. Viscosity is measured using molecular BODIPY-based rotor and fluorescence lifetime imaging microscopy (FLIM) [Shimolina et al. Sci.Rep. 2017]. Results and discussions We showed that acidification of cytosolic pH occurs after therapy with cisplatin in vivo , and this, likely, favours metabolic reorganisation of cells. Treated tumours exhibited a decreased relative contribution from free (cytosolic, protein-unbound) NAD(P)H, indicating a metabolic shift from glycolysis towards oxidative metabolism. It is interesting that optical metabolic imaging could allow early detection of tumour response to chemotherapy, before there are changes in tumour size, and metabolic changes were the same for therapeutic agents with different mechanisms of action (cisplatin, paclitaxel). Moreover, tumours treated with cisplatin displayed decreased plasma membrane viscosity. Presumably, this alteration of viscosity participates in cisplatin-induced apoptosis. Similar changes of pH, energy metabolism and viscosity were previously reported for cisplatin-treated cultured cancer cells. Conclusion These results, therefore, suggest that all investigated parameters play a role in the cytotoxicity of the drugs and may provide a useful approach for monitoring tumour responses to chemotherapy. The study was supported by the Russian Science Foundation (#14-25-00129).

This work is aimed at the development of new approach to register intracellular pH with genetically encoded ratiometric sensor. Intracellular pH of cancer cells was studied in vitro and in vivo. Changes of intracellular pH under conditions of co-culturing with fibroblast were investigated.

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. References: 1. S. Pavlides et.al. Cell Cycle, 2009, 8:23, 3984-4001 2. M. P. Lisanti, et.al. Cell Cycle 10:15, 2011, 2440-2449 3. R. A. Cardone, et.al. Nature Reviews. Cancer V.5. 2005. 786-795 4. K.N. Markvicheva et al., Bioorganic& Medicinal Chemistry, 2011, 19, 1079-1084 5. U. E. Martinez-Outschoorn, et.al Cell Cycle, 2011, 10:15, 2504-2520 Acknowledgments: This work was supported by the Russian Foundation for Basic Research (project # 15-32-20324). Citation Format: Irina N. Druzhkova, Marina V. Shirmanova, Maria M. Lukina, Varvara V. Dudenkova, Vsevolod V. Belousov, Sergey A. Lukiyanov, Elena V. Zagaynova. The metabolic adaptations during cancer-stroma co-evolution: The intracellular pH, NAD(P)H changes and hydrogen peroxide production in cancer cells. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B01.