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.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is a common clinical problem, leading to significant morbidity and mortality, and no effective pharmacotherapy exists. The ...problem of ARDS causing mortality became more apparent during the COVID-19 pandemic. Biotherapeutic products containing multipotent mesenchymal stromal cell (MMSC) secretome may provide a new therapeutic paradigm for human healthcare due to their immunomodulating and regenerative abilities. The content and regenerative capacity of the secretome depends on cell origin and type of cultivation (two- or three-dimensional (2D/3D)). In this study, we investigated the proteomic profile of the secretome from 2D- and 3D-cultured placental MMSC and lung fibroblasts (LFBs) and the effect of inhalation of freeze-dried secretome on survival, lung inflammation, lung tissue regeneration, fibrin deposition in a lethal ALI model in mice. We found that three inhaled administrations of freeze-dried secretome from 2D- and 3D-cultured placental MMSC and LFB protected mice from death, restored the histological structure of damaged lungs, and decreased fibrin deposition. At the same time, 3D MMSC secretome exhibited a more pronounced trend in lung recovery than 2D MMSC and LFB-derived secretome in some measures. Taking together, these studies show that inhalation of cell secretome may also be considered as a potential therapy for the management of ARDS in patients suffering from severe pneumonia, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, their effectiveness requires further investigation.
Cellular redox status and the level of reactive oxygen species (ROS) are important regulators of apoptotic potential, playing a crucial role in the growth of cancer cell and their resistance to ...apoptosis. However, the relationships between the redox status and ROS production during apoptosis remain poorly explored. In this study, we present an investigation on the correlations between the production of ROS, the redox ratio FAD/NAD(P)H, the proportions of the reduced nicotinamide cofactors NADH and NADPH, and caspase-3 activity in cancer cells at the level of individual cells. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) was applied to monitor simultaneously apoptosis using the genetically encoded sensor of caspase-3, mKate2-DEVD-iRFP, and the autofluorescence of redox cofactors in colorectal cancer cells upon stimulation of apoptosis with staurosporine, cisplatin or hydrogen peroxide. We found that, irrespective of the apoptotic stimulus used, ROS accumulation correlated well with both the elevated pool of mitochondrial, enzyme-bound NADH and caspase-3 activation. Meanwhile, a shift in the contribution of bound NADH could develop independently of the apoptosis, and this was observed in the case of cisplatin. An increase in the proportion of bound NADPH was detected only in staurosporine-treated cells, this likely being associated with a high level of ROS production and their resulting detoxification. The results of the study favor the discovery of new therapeutic strategies based on manipulation of the cellular redox balance, which could help improve the anti-tumor activity of drugs and overcome apoptotic resistance.
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.
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.
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.
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.
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.
•We developed a method for pHi mapping in living cancer cells in vitro and in tumors in vivo.•The novel genetically encoded indicator, SypHer2, was used.•Intracellular pH was measured in HeLa cells in monolayer and tumor spheroids.•We obtained fluorescence ratio maps, representing the pHi distribution, for HeLa tumors in vivo and ex vivo.•A correspondence of the zones with higher pHi to the necrotic and hypoxic areas was demonstrated.
There is a need for better strategies to promote burn wound healing and prevent infection. The aim of our study was to develop an easy-to-use placental multipotent mesenchymal stromal cell (MMSC) ...secretome-based chitosan hydrogel (MSC-Ch-gel) and estimate its antimicrobial and regenerative activity in
-infected burn wounds in rats.
Proteomic studies of the MMSC secretome revealed proteins involved in regeneration, angiogenesis, and defence responses. The MMSC secretome was collected from cultured cells and mixed with water-soluble chitosan to prepare the placental MSC-Ch-gel, which was stored in liquid phase at 4 °C. The wounds of rats with established II-IIIa-degree burns were then infected with
and externally covered with the MSC-Ch-gel. Three additional rat groups were treated with medical Vaseline oil, the antiseptic drug Miramistin
, or the drug Bepanthen
Plus. Skin wound samples were collected 4 and 8 days after burning for further microbiological and histological analysis. Blood samples were also collected for biochemical analysis.
Application of the MSC-Ch-gel cleared the wound of microorganisms (
wasn't detected in the washings from the burned areas), decreased inflammation, enhanced re-epithelialisation, and promoted the formation of well-vascularised granulation tissue.
MSC-Ch-gel effectively promotes infected wound healing in rats with third-degree burns. Gel preparation can be easily implemented into clinical practice.
The phase of the cell cycle determines numerous aspects of cancer cell behaviour including invasiveness, ability to migrate and responsiveness to cytotoxic drugs. To non-invasively monitor ...progression of cell cycle in vivo, a family of genetically encoded fluorescent indicators, FUCCI (fluorescent ubiquitination-based cell cycle indicator), has been developed. Existing versions of FUCCI are based on fluorescent proteins of two or more different colors fused to cell-cycle-dependent degradation motifs. Thus, FUCCI-expressing cells emit light of different colors in different phases providing a robust way to monitor cell cycle progression by fluorescence microscopy and flow cytometry but limiting the possibility to simultaneously visualize other markers. To overcome this limitation, we developed a single-color variant of FUCCI, called FUCCI-Red, which utilizes two red fluorescent proteins with distinct fluorescence lifetimes, mCherry and mKate2. Similarly to FUCCI, these proteins carry cell cycle-dependent degradation motifs to resolve G1 and S/G2/M phases. We showed utility of FUCCI-Red by visualizing cell cycle progression of cancer cells in 2D and 3D cultures and monitoring development of tumors in vivo by confocal and fluorescence lifetime imaging microscopy (FLIM). Single-channel registration and red-shifted spectra make FUCCI-Red sensor a promising instrument for multiparameter in vivo imaging applications, which was demonstrated by simultaneous detection of cellular metabolic state using endogenous fluorescence in the blue range.
The effects of chemotherapy on the tumor microenvironment remain poorly investigated, although these are crucial for the drug delivery and anti-tumor activity and can modify physiological behavior of ...cancer cells. Optical coherence angiography and phosphorescence lifetime spectroscopy are promising optical methods to characterize both tumor vasculature and its oxygenation, respectively. In this study, we used these methods to explore the influence of irinotecan, a widely used chemotherapeutic agent, on tumor angiogenesis and oxygen content in a mouse model of colorectal cancer in vivo. For the first time, it was shown that irinotecan at conventional dosing reduces tumor oxygenation and blood perfusion via antiangiogenic mechanism of action. Immunohistochemistry for hypoxia and for the endothelial cell marker CD31 verified the in vivo findings. These data show the prospects of the optical techniques to monitor tumor microenvironment in the course of chemotherapy, which can help to optimize and individualize the treatment.
Genetically encoded photosensitizers are a promising optogenetic instrument for light-induced production of reactive oxygen species in desired locations within cells in vitro or whole body in vivo. ...Only two such photosensitizers are currently known, GFP-like protein KillerRed and FMN-binding protein miniSOG. In this work we studied phototoxic effects of miniSOG in cancer cells.
HeLa Kyoto cell lines stably expressing miniSOG in different localizations, namely, plasma membrane, mitochondria or chromatin (fused with histone H2B) were created. Phototoxicity of miniSOG was tested on the cells in vitro and tumor xenografts in vivo.
Blue light induced pronounced cell death in all three cell lines in a dose-dependent manner. Caspase 3 activation was characteristic of illuminated cells with mitochondria- and chromatin-localized miniSOG, but not with miniSOG in the plasma membrane. In addition, H2B-miniSOG-expressing cells demonstrated light-induced activation of DNA repair machinery, which indicates massive damage of genomic DNA. In contrast to these in vitro data, no detectable phototoxicity was observed on tumor xenografts with HeLa Kyoto cell lines expressing mitochondria- or chromatin-localized miniSOG.
miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor.
This is the first study to assess phototoxicity of miniSOG in cancer cells. The results suggest an effective ontogenetic tool and may be of interest for molecular and cell biology and biomedical applications.
•Phototoxic effects of miniSOG in cancer cells in vitro and in vivo were studied.•HeLa cells expressed miniSOG in plasma membrane, mitochondria or chromatin.•We showed that miniSOG is an effective genetically encoded photosensitizer for cells in vitro.
The use of polymeric carriers to deliver hydrophobic photosensitizers has been widely discussed as a way to improve both fluorescence diagnostic and photodynamic therapy (PDT) of cancers; however, ...the photophysical and pharmacokinetic parameters, as well as the PDT activity, of such modifications have, until now, only been poorly investigated. The purpose of the present study was to explore the efficacy of PDT with the formulation of the photosensitizer chlorin e6 (Ce6) in combination with polyvinyl alcohol (PVA) in comparison with Ce6 alone and with the clinical drug, Photodithazine in a mouse tumor model. We also investigated the photoactivity of the Ce6-PVA in a model reaction of tryptophan oxidation, analyzed the polymer–Ce6 interaction using fluorescence spectroscopy and atomic-force microscopy, and tested the phototoxicity in vitro. Using fluorescence imaging in vivo we found that injection to mice of Ce6 in a formulation with PVA resulted in a higher tumor-to-normal ratio and greater photobleaching when compared with either the use of Ce6 alone, or with the effects of Photodithazine. Tumor growth study and histological examination of CT26 tumors revealed fast, reproducible tumor regression and more advanced necrosis after PDT with Ce6-PVA. The higher photoactivity of the Ce6-PVA complex was confirmed in a model reaction of tryptophan oxidation and in cultured cells. Therefore, encapsulation of Ce6 in PVA represents a promising strategy for further increasing the selectivity and efficacy of PDT.
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•A comparison of Ce6-PVA complex with Ce6 alone and with Photodithazine was performed in vitro and in vivo.•A higher photoactivity of Ce6-PVA was detected in a reaction of tryptophan oxidation and in cultured cancer cells.•Ce6-PVA showed a higher selectivity for the mouse tumor and a greater photobleaching rate.•PDT with Ce6-PVA resulted in fast, reproducible tumor regression and advanced necrosis.•Encapsulation of Ce6 in PVA represents a promising strategy for increasing the efficacy of PDT.
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