Inhaled Placental Mesenchymal Stromal Cell Secretome from Two- and Three-Dimensional Cell Cultures Promotes Survival and Regeneration in Acute Lung Injury Model in Mice.

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.

Insight into redox regulation of apoptosis in cancer cells with multiparametric live-cell microscopy.

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.

Antimicrobial and Regenerative Effects of Placental Multipotent Mesenchymal Stromal Cell Secretome-Based Chitosan Gel on Infected Burns in Rats.

BACKGROUND: 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 Staphylococcus aureus-infected burn wounds in rats. METHODS: 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 S. aureus 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. RESULTS: Application of the MSC-Ch-gel cleared the wound of microorganisms (S. aureus wasn't detected in the washings from the burned areas), decreased inflammation, enhanced re-epithelialisation, and promoted the formation of well-vascularised granulation tissue. CONCLUSIONS: MSC-Ch-gel effectively promotes infected wound healing in rats with third-degree burns. Gel preparation can be easily implemented into clinical practice.

FUCCI-Red: a single-color cell cycle indicator for fluorescence lifetime imaging.

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.

In vivo metabolic and SHG imaging for monitoring of tumor response to chemotherapy.

Although chemotherapy remains one of the main types of treatment for cancer, treatment failure is a frequent occurrence, emphasizing the need for new approaches to the early assessment of tumor response. The aim of this study was to search for indicators based on optical imaging of cellular metabolism and of collagen in tumors in vivo that enable evaluation of their response to chemotherapy. The study was performed on a mouse colorectal cancer model with the use of cisplatin, paclitaxel, and irinotecan. The metabolic activity of the tumor cells was assessed using fluorescence lifetime imaging of the metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H. Second harmonic generation (SHG) imaging was used to analyze the extent and properties of collagen within the tumors. We detected an early decrease in the free/bound NAD(P)H ratio in all treated tumors, indicating a shift toward a more oxidative metabolism. Monitoring of collagen showed an early increase in the amount of collagen followed by an increase in the extent of its orientation in tumors treated with cisplatin and paclitaxel, and decrease in collagen content in the case of irinotecan. Our study suggests that changes in cellular metabolism and fibrotic stroma organization precede morphological alterations and tumor size reduction, and that this indicates that NAD(P)H and collagen can be considered as intrinsic indicators of the response to treatment. This is the first time that these parameters have been investigated in tumors in vivo in the course of chemotherapy with drugs having different mechanisms of action. © 2018 International Society for Advancement of Cytometry.

Can "Indirect" Contact Laser Surgery be Used for Fluorescence-Image Guided Tumor Resections? Preliminary Results.

Ensuring the complete removal of tumor tissue is the main challenge during resection operations. Recently, a technique of "indirect" contact laser surgery has been developed. In this study we assess the possibility of using such surgery for fluorescence image-guided tumor resection. Mouse colon adenocarcinoma CT-26 cells stably expressing the fluorescent protein mKate-2 was used as the tumor model. Resections of the tumor nodes were performed with either a scalpel blade, a laser scalpel with a bare tip, or a laser scalpel with a strongly absorbing coating on the fiber tip. Tumor-positive resection margins were detected using an IVIS Spectrum fluorescence imaging system. After tumor resection with the scalpel blade over half of the animals needed one additional resection to remove residual tumor cells. Animals in this group showed tumor recurrence within 7 days. Fluorescence imaging of the tumor bed, performed after resection to assess the presence of tumor cell clusters, was sufficiently effective only with a bloodless resection. The laser scalpels both with the bare tip and with the strongly absorbing coating on the tip provided such bloodless tumor resection in contact mode. Fewer animals required additional resections when the bare tipped scalpel was used and this also resulted in a reduction in tumor recurrence. After resections were carried out with the laser scalpel with the strongly absorbing coating on the tip, fluorescence was detected in the operative field and this led to undertaking additional resections, although subsequent investigation suggested that this was "false" fluorescence, resulting from the effects of the scalpel rather than the presence of residual tumor cells. The method of laser resection with a strongly absorbing coating on the tip therefore did not appear to demonstrate definite advantages over laser resection with a bare tip when removing tumors.

Photodynamic therapy of mouse tumor model using chlorin e6- polyvinyl alcohol complex.

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.

Chemotherapy with cisplatin: insights into intracellular pH and metabolic landscape of cancer cells in vitro and in vivo.

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.

Fluorescence Imaging of Actin Fine Structure in Tumor Tissues Using SiR-Actin Staining.

BACKGROUND: The rearrangement of actin cytoskeleton is being increasingly considered a marker of cancer cell activity, but the fine structure and remodeling of microfilaments within tumor tissue still remains unclear. MATERIALS AND METHODS: We used the recently introduced silicon-rhodamine (SiR)-actin dye to visualize endogenous actin within tissues by confocal or total internal reflection fluorescence microscopy. We established imaging conditions for robust blinking of SiR-actin, which makes this dye applicable for super-resolution localization microscopy, as well as for an efficient background elimination. RESULTS: We studied tumor tissue samples in two mouse models at high resolution and revealed a complex network of thick curved bundles of actin in cancer cells in tumors. This actin pattern differed strongly from that in cancer cells in vitro and in normal tissues. CONCLUSION: Localization microscopy with SiR-actin provides an efficient way to visualize fine actin structure in tumor tissues. It is potentially applicable to a variety of biological and clinical samples.

Intracellular pH imaging in cancer cells in vitro and tumors in vivo using the new genetically encoded sensor SypHer2.

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.

Flavoprotein miniSOG as a genetically encoded photosensitizer for cancer cells.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: miniSOG is an excellent genetically encoded photosensitizer for mammalian cells in vitro, but it is inferior to KillerRed in the HeLa tumor. GENERAL SIGNIFICANCE: 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 fluorescent protein KillerRed on tumor cells in mice.

KillerRed is known to be a unique red fluorescent protein displaying strong phototoxic properties. Its effectiveness has been shown previously for killing bacterial and cancer cells in vitro. Here, we investigated the photototoxicity of the protein on tumor xenografts in mice. HeLa Kyoto cell line stably expressing KillerRed in mitochondria and in fusion with histone H2B was used. Irradiation of the tumors with 593 nm laser led to photobleaching of KillerRed indicating photosensitization reaction and caused significant destruction of the cells and activation of apoptosis. The portion of the dystrophically changed cells increased from 9.9% to 63.7%, and the cells with apoptosis hallmarks from 6.3% to 14%. The results of this study suggest KillerRed as a potential genetically encoded photosensitizer for photodynamic therapy of cancer.

Comparative study of tumor hypoxia by diffuse optical spectroscopy and immunohistochemistry in two tumor models.

The capabilities of diffuse optical spectroscopy for noninvasive assessing of oxygen status in experimental tumors have been demonstrated. Specific features of the distribution of total hemoglobin, oxygenated hemoglobin, deoxygenated hemoglobin, and blood-oxygen saturation were shown on two tumor models having different histological structure and functional characteristics. The results obtained by the optical technique were verified by immunohistochemical study of tissue samples marked with exogenous marker of hypoxia--pimonidazole.

OCT-guided laser hyperthermia with passively tumor-targeted gold nanoparticles.

The goal of this study is the development of a method of local laser hyperthermia with gold nanoparticles under noninvasive optical monitoring of nanoparticle accumulation in tumor tissue in vivo. Bifunctional plasmon resonant nanoparticles that are optimal for OCT diagnostics and laser heating at the wavelength of 810 nm were used in the study. The OCT examination showed that the accumulation of gold nanoparticles in the tumor invading into skin was maximal 4-5 h after intravenous injection. It was demonstrated that nanoparticle accumulation in tumor allowed more local heating and enhanced thermal sensitivity of tumor tissue. Laser hyperthermia that heated tumor up to 44-45 °C at maximum nanoparticle accumulation induced apoptotic death of tumor cells and inhibited tumor growth by 104% on the 5th day after treatment.

In vivo visualization of Tradescantia leaf tissue and monitoring the physiological and morphological states under different water supply conditions using optical coherence tomography.

The optical coherence tomography (OCT) capabilities of plants were evaluated using leaves of Tradescantia pallida (Rose) D. Hunt. The internal structure of the leaf tissues was visualized in vivo and the physiological and morphological states of the tissues under different water supply conditions were monitored using OCT. The OCT technique provides non-invasive two-dimensional images directly on intact plants. The acquisition time of a two-dimensional image with a size of 200x200 pixels and a spatial resolution of 15 microm is 1-3 s. It was shown that OCT is a useful tool for monitoring the physiological and morphological states of plant tissues supplied with varying amounts of water and under the influence of different chemical factors.

In vivo optical coherence tomography feasibility for bladder disease.

PURPOSE: Optical coherence tomography is a new imaging modality capable of imaging luminal surface of biological tissue in the near infrared range with a spatial resolution close to the cellular level. We identified characteristic optical coherence tomography patterns for nonproliferative and proliferative inflammation, and malignant alterations of the bladder. MATERIALS AND METHODS: Optical coherence tomography was performed to image the bladder of 66 patients. The probe passed through the operating channel of a cystoscope and was pressed onto the mucosal site of interest. A mucosal biopsy of the image site was obtained. Optical coherence tomography was used to construct 680 images of the bladder and the images were compared with histology slides. RESULTS: Optical coherence tomography images of normal bladder showed 3 layers, namely the mucosa or transitional epithelium, submucosa and smooth muscle. In exudative processes there were poor light scattering areas in the connective tissue layer. Images of bladders with proliferative cystitis revealed nonuniform thickening of the epithelium or hyperplasia. Squamous metaplasia appeared as thicker and less transparent epithelium with a jagged boundary. Images of transitional cell carcinoma were characterized by the complete loss of a regular layered structure of the bladder wall and the penetration depth of optical imaging was slight. CONCLUSIONS: This study provides the characteristic optical coherence tomography pattern of nonproliferative and proliferative inflammation, and the characteristic appearance of severe dysplasia and transitional cell carcinoma. This technique may be useful as a guide for biopsy and for assisting in establishing resection margins.