Fluorescence Lifetime Imaging of NAD(P)H T Cells Autofluorescence in the Lymphatic Nodes to Assess the Effectiveness of Anti-CTLA-4 Immunotherapy.

The main problem in the field of tumor immunotherapy is the lack of reliable biomarkers that allow pre-determining the susceptibility of individual patients to treatment, as well as insufficient knowledge about the resistance mechanisms. Biomarkers based on the autofluorescence of metabolic coenzymes in immune cells can become a powerful new predictor of early tumor response to treatment, whereas the optical FLIM method can be a tool to predict the effectiveness of immunotherapy, which allows preserving the spatial structure of the sample and obtaining results on the metabolic status of immune cells in real time. The aim of the study is to conduct a metabolic autofluorescence imaging study of the NAD(P)H metabolic coenzyme in immune cells of freshly isolated lymph nodes as a potential marker for assessing the effectiveness of an early response to immunotherapy. Materials and Methods: The study was carried out on C57Bl/6 FoxP3-EGFP mice with B16F0 melanoma implanted near the inguinal lymph node. The mice were injected with antibodies to CTLA-4 (Bio X Cell, USA) (250 µg per mouse, intraperitoneally on days 7, 8, 11, and 12 of the tumor growth). FLIM images in the nicotinamide adenine dinucleotide (phosphate) coenzyme (NAD(P)H) channel (excitation - 375 nm, reception - 435-485 nm) were received using an LSM 880 fluorescent confocal laser scanning microscope (Carl Zeiss, Germany) equipped with a FLIM Simple-Tau module 152 TCSPC (Becker & Hickl GmbH, Germany). Flow cytometry was conducted using a BD FACSAria III cell sorter (BD Biosciences, USA). Results: Immunotherapy with checkpoint inhibitors resulted in marked metabolic rearrangements in T cells of freshly isolated lymph nodes in responder mice, with inhibition of the tumor growth. Fluorescence lifetime imaging data on NAD(P)H indicated an increase in the free fraction of NADH α1, a form associated with glycolysis to meet high demands of the activated T cells and pro-inflammatory cytokine synthesis. In contrast, non-responder mice with advanced tumors showed low values of the ratio of free fraction to bound α1/α2, which may be related to mechanisms of resistance to therapy.The response to immunotherapy was verified by data on the expression of activation and proliferation markers by means of flow cytometry. The authors observed an increase in the production of the pro-inflammatory cytokine IFN-γ in effector T cells in responder mice compared to untreated controls and non-responders. In addition, an increase in the expression of the surface activation markers CD25 and CD69 was registered compared to untreated controls. Conclusion: Use of the FLIM method allowed to demonstrate that autofluorescence of the NAD(P)H coenzyme is sensitive to the response to checkpoint immunotherapy and can be used as a reliable marker of the effectiveness of response to treatment.

Multiphoton Microscopy and Mass Spectrometry for Revealing Metabolic Heterogeneity of Hepatocytes in vivo.

The aim of the investigation was to study the possibility of revealing the heterogeneity of normal liver hepatocytes in terms of metabolic status using the modern methods of multiphoton microscopy and mass spectrometry. Materials and Methods: Heterogeneity of hepatocytes in terms of total metabolic activity was assessed using multiphoton microscopy based on the autofluorescence intensity of intracellular cofactors NAD(P)H and FAD. Hepatocyte heterogeneity in terms of intensity of intracellular metabolic processes was determined using the fluorescence lifetime imaging (FLIM) method based on the data about fluorescence lifetime contributions of various forms of NAD(P)H. The method of time-of-flight secondary ion mass spectrometry (TоF-SIMS) was used to study the lipid and amino acid composition of hepatocytes. Results: It has been revealed using multiphoton microscopy that hepatocytes are heterogeneous in terms of general metabolic activity. Using FLIM, it was established that the heterogeneity degree was high in terms of intensity of oxidative phosphorylation, glycolysis, and synthetic processes (lipogenesis, nucleic acid synthesis, and the pentose phosphate pathway). The TоF-SIMS method revealed the presence of hepatocyte heterogeneity in terms of amino acid and lipid composition, which points to various intensities of synthetic processes in individual hepatocytes. Moreover, differences in the content of PO3 ions were revealed. The results of ToF-SIMS study correlate with the data obtained by multiphoton microscopy and FLIM, confirming the revealed heterogeneity of hepatocytes in terms of general metabolic activity and intensity of intercellular metabolic processes. Conclusion: The latest methods of fluorescence bioimaging and mass spectrometry proved to be effective in revealing hepatocyte heterogeneity in terms of metabolic status. The presence of heterogeneity should be taken into account in studying the liver tissue under various conditions with the application of fluorescence bioimaging methods.

Distinct organization of adaptive immunity in the long-lived rodent Spalax galili.

A balanced immune response is a cornerstone of healthy aging. Here, we uncover distinctive features of the long-lived blind mole-rat (Spalax spp.) adaptive immune system, relative to humans and mice. The T-cell repertoire remains diverse throughout the Spalax lifespan, suggesting a paucity of large long-lived clones of effector-memory T cells. Expression of master transcription factors of T-cell differentiation, as well as checkpoint and cytotoxicity genes, remains low as Spalax ages. The thymus shrinks as in mice and humans, while interleukin-7 and interleukin-7 receptor expression remains high, potentially reflecting the sustained homeostasis of naive T cells. With aging, immunoglobulin hypermutation level does not increase and the immunoglobulin-M repertoire remains diverse, suggesting shorter B-cell memory and sustained homeostasis of innate-like B cells. The Spalax adaptive immune system thus appears biased towards sustained functional and receptor diversity over specialized, long-lived effector-memory clones-a unique organizational strategy that potentially underlies this animal's extraordinary longevity and healthy aging.

Optical coherence angiography for pre-treatment assessment and treatment monitoring following photodynamic therapy: a basal cell carcinoma patient study.

Microvascular networks of human basal cell carcinomas (BCC) and surrounding skin were assessed with optical coherence angiography (OCA) in conjunction with photodynamic therapy (PDT). OCA images were collected and analyzed in 31 lesions pre-treatment, and immediately/24 hours/3-12 months post-treatment. Pre-treatment OCA enabled differentiation between prevalent subtypes of BCC (nodular and superficial) and nodular-with-necrotic-core BCC subtypes with a diagnostic accuracy of 78%; this can facilitate more accurate biopsy reducing sampling error and better therapy regimen selection. Post-treatment OCA images at 24 hours were 98% predictive of eventual outcome. Additional findings highlight the importance of pre-treatment necrotic core, vascular metrics associated with hypertrophic scar formation, and early microvascular changes necessary in both tumorous and peri-tumorous regions to ensure treatment success.

Accurate early prediction of tumour response to PDT using optical coherence angiography.

Prediction of tumour treatment response may play a crucial role in therapy selection and optimization of its delivery parameters. Here we use optical coherence angiography (OCA) as a minimally-invasive, label-free, real-time bioimaging method to visualize normal and pathological perfused vessels and monitor treatment response following vascular-targeted photodynamic therapy (PDT). Preclinical results are reported in a convenient experimental model (CT-26 colon tumour inoculated in murine ear), enabling controlled PDT and post-treatment OCA monitoring. To accurately predict long-term treatment outcome, a robust and simple microvascular metric is proposed. It is based on perfused vessels density (PVD) at t = 24 hours post PDT, calculated for both tumour and peri-tumour regions. Histological validation in the examined experimental cohort (n = 31 animals) enabled further insight into the excellent predictive power of the derived early-response OCA microvascular metric. The results underscore the key role of peri-tumour microvasculature in determining the long-term PDT response.

In-vivo longitudinal imaging of microvascular changes in irradiated oral mucosa of radiotherapy cancer patients using optical coherence tomography.

Mucositis is the limiting toxicity of radio(chemo)therapy of head and neck cancer. Diagnostics, prophylaxis and correction of this condition demand new accurate and objective approaches. Here we report on an in vivo longitudinal monitoring of the oral mucosa dynamics in 25 patients during the course of radiotherapy of oropharyngeal and nasopharyngeal cancer using multifunctional optical coherence tomography (OCT). A spectral domain OCT system with a specially-designed oral imaging probe was used. Microvasculature visualization was based on temporal speckle variations of the full complex signal evaluated by high-pass filtering of 3D data along the slow scan axis. Angiographic image quantification demonstrated an increase of the vascular density and total length of capillary-like-vessels before visual signs or clinical symptoms of mucositis occur. Especially significant microvascular changes compared to their initial levels occurred when grade two and three mucositis developed. Further, microvascular reaction was seen to be dose-level dependent. OCT monitoring in radiotherapy offers a non-invasive, convenient, label-free quantifiable structural and functional volumetric imaging method suitable for longitudinal human patient studies, furnishing fundamental radiobiological insights and potentially providing useful feedback data to enable adaptive radiotherapy (ART).

Photodynamic therapy monitoring with optical coherence angiography.

Photodynamic therapy (PDT) is a promising modern approach for cancer therapy with low normal tissue toxicity. This study was focused on a vascular-targeting Chlorine E6 mediated PDT. A new angiographic imaging approach known as M-mode-like optical coherence angiography (MML-OCA) was able to sensitively detect PDT-induced microvascular alterations in the mouse ear tumour model CT26. Histological analysis showed that the main mechanisms of vascular PDT was thrombosis of blood vessels and hemorrhage, which agrees with angiographic imaging by MML-OCA. Relationship between MML-OCA-detected early microvascular damage post PDT (within 24 hours) and tumour regression/regrowth was confirmed by histology. The advantages of MML-OCA such as direct image acquisition, fast processing, robust and affordable system opto-electronics, and label-free high contrast 3D visualization of the microvasculature suggest attractive possibilities of this method in practical clinical monitoring of cancer therapies with microvascular involvement.

Microbiota Induces Expression of Tumor Necrosis Factor in Postnatal Mouse Skin.

Tumor necrosis factor (TNF) is a pleiotropic cytokine that regulates many important processes in the body. TNF production in a physiological state supports the structure of lymphoid organs and determines the development of lymphoid cells in hematopoiesis. However, chronic TNF overexpression leads to the development of various autoimmune disorders. Sites of TNF production in the naïve state remain unclear due to the lack of in vivo models. In the present study, we used TNF-2A-Kat reporter mice to monitor the expression of TNF in different tissues. Comparative analysis of tissue fluorescence in TNF-2A-Kat reporter mice and wild type mice revealed constitutive expression of TNF in the skin of naïve adult mice. In the skin of TNF-2A-Kat reporter mouse embryos, no statistically significant differences in the expression of TNF compared to wild type animals were observed. Furthermore, we established that local depletion of microflora with topical antibiotics leads to a reduction in the fluorescence signal. Thus, we assume that the skin microflora is responsible for the expression of TNF in the skin of mice.

Green-to-red primed conversion of Dendra2 using blue and red lasers.

Recently, an unusual phenomenon of primed conversion of fluorescent protein Dendra2 by combined action of blue (488 nm) and near-infrared (700-780 nm) lasers was discovered. Here we demonstrate that primed conversion can be induced by red lasers (630-650 nm) common for most confocal and single molecule detection microscopes.

Comparative Analysis of Proliferation and Viability of Multipotent Mesenchymal Stromal Cells in 3D Scaffolds with Different Architectonics.

3D biodegradable materials (scaffolds) containing bioactive hydroxyapatite molecules fabricated by foaming in supercritical carbon dioxide and by selective laser sintering were used for culturing of mesenchymal stromal cells from the human adipose tissue. Experiments showed that stromal cells from the human adipose tissue adhered and proliferated on all studied types of structures. Addition of hyproxyapatite to the scaffold stimulated proliferation of stromal adipose tissue cells.

In Vitro Effect of Laser-Induced Hydrodynamics on Cancer Cells.

We studied the effect of laser-induced hydrodynamic on viability of Colo-26 murine colon carcinoma cells in vitro. Laser-induced hydrodynamics was generated by a laser (λ=1.56 µ, power 3 W, 5 min exposure); to this end, the fiber end was submersed into a buffer above the cell monolayer. It was found that laser-induced hydrodynamics destructed the monolayer at standoff distances of between the working end of the laser fiber to cell monolayer of 1 and 5 mm and triggers apoptotic and necrotic death in remaining cells at a distance of 4 mm from the emitter.

Contrasting properties of gold nanoparticles for optical coherence tomography: phantom, in vivo studies and Monte Carlo simulation.

The possibility of using silica-gold nanoshells with 150 nm silica core size and 25 nm thick gold shell as contrasting agents for optical coherence tomography (OCT) is analyzed. Experiments on agar biotissue phantoms showed that the penetration of nanoshells into the phantoms increases the intensity of the optical coherence tomography (OCT) signal and the brightness of the corresponding areas of the OCT image. In vivo experiments on rabbit skin demonstrated that the application of nanoshells onto the skin provides significant contrasting of the borders between the areas containing nanoshells and those without. This effect of nanoshells on skin in vivo is manifested by the increase in intensity of the OCT signal in superficial parts of the skin, boundary contrast between superficial and deep dermis and contrast of hair follicles and glands. The presence of nanoshells in the skin was confirmed by electron microscopy. Monte Carlo simulations of OCT images confirmed the possibility of contrasting skin-layer borders and structures by the application of gold nanoshells. The Monte Carlo simulations were performed for two skin models and exhibit effects of nanoparticles similar to those obtained in the experimental part of the study, thus proving that the effects originate exactly from the presence of nanoparticles.