Denys Leonidovych Kolesnyk

Cell mechanical properties are fundamental to numerous cellular functions, including growth, division, motility, adhesion, and responsiveness to pharmacological treatments. The mechanics of cellular components collectively contribute to overall cell mechanical behavior. Structures such as the membrane, cytoskeleton, nucleus, and other organelles constitute an interconnected network that is central to the mechanobiology of individual cells. During the progression of tumor cells to a metastatic phenotype, significant alterations in mechanical properties occur, playing a pivotal role in this transformation. To model metastasizing tumor cells, cultivation under deadhesive conditions has been employed. In this study, two cell lines with distinct metastatic potentials — Lewis lung carcinoma (LLC) and LLC/R9 — were cultured under either adhesive (representing typical tumor cells) or deadhesive (representing metastatic tumor cells) conditions. The quantitative assessment of cell mechanical properties was found to depend upon the cell cycle phase, measurement technique, and theoretical modeling approach. This article examines alterations in mechanical properties, specifically membrane tension and elastic shear modulus, in circulating metastatic tumor cells as determined via micropipette aspiration. Results indicate that cells grown under deadhesive conditions exhibit greater population homogeneity and a narrower distribution of elastic shear modulus values compared to those grown under adhesive conditions. Furthermore, LLC cells demonstrated lower elastic shear moduli than LLC/R9 cells, correlating with their respective metastatic activities. These mechanical parameters may serve as valuable markers for determining the metastatic potential of tumor cells.

Lactate dehydrogenase (LDH) is a key glycolytic enzyme that catalyzes the interconversion of pyruvate and lactate, with LDHA gaining particular attention for its overexpression in various malignancies and pivotal role in the Warburg effect-driven metabolic reprogramming. Elevated LDHA activity supports rapid ATP production under hypoxic conditions, maintains NAD⁺ regeneration, and promotes lactate accumulation, creating an acidic tumor microenvironment (TME) that favors invasion and immune evasion. Accumulating evidence demonstrates that LDHA is essential for primary tumor growth and critically involved in circulating tumor cell (CTC) survival, anoikis resistance, and metastatic spread. These functions are mediated by its regulation of adhesion molecules, cytoskeletal remodeling, and energy adaptation that enable CTCs to withstand mechanical shear stress and immune surveillance in the bloodstream. Pharmacological inhibition of LDHA, particularly via sodium oxamate (oxamate), has shown substantial potential in reducing metastasis and enhancing chemotherapy sensitivity in preclinical models. Oxamate has emerged as a promising candidate for metabolic cancer therapy due to its unique double effects on tumor metabolism and anti-tumor immunity, which are an advantage rarely highlighted in broader LDHA-focused reviews. This review synthesizes the molecular mechanisms through which LDHA drives tumor progression, dissects its context-specific functions in CTC biology, and evaluates the translational potential of LDHA-targeted strategies, with a focused emphasis on oxamate, as a transformative anti-metastatic therapeutic paradigm. By filling a critical gap in synthesizing oxamate’s distinct metabolic–immune regulatory actions, this work addresses an unmet need in the management of advanced, treatment-refractory cancers.

Journal of Medicinal Plants for Economic Development

Background: Microscopy is a key tool in biophysical research for visualizing living cells' morphology, structure, and dynamic processes. Depending on the specific research goals, various optical and electron microscopy techniques can be applied, each offering unique benefits. Determining the structural features of the cytoskeleton, nucleus, and membrane of circulating tumor cells in the model Lewis lung carcinoma (LLC) is an important biophysical and biological task, as it will allow identifying targets for antitumor and antimetastatic therapy, as well as investigating the mechanisms of action of antitumor drugs. However, comprehensive multimodal imaging of such cells — particularly under non-adherent conditions — remains limited in the literature due to the difficulty of working with such models. Objectives: This study aimed to visualize Lewis lung carcinoma (LLC) cells cultured under de-adhesive conditions using various microscopy techniques for their characterization and to examine the cancer cells' structure and functionality. The goal was to evaluate each method's individual capabilities and combined strengths in revealing cellular morphology, internal structure, and nanoparticle interactions. Materials and Methods: LLC cells were obtained from the National Bank of Cell Lines and Tumor Strains of the IEPOR (NAS of Ukraine) and cultured in RPMI-1640 medium under conditions of de-adhesive growth. Imaging was performed using inverted optical microscopy (Euromex Oxion), fluorescence and confocal microscopy (Carl Zeiss LSM 510) with F-actin (Alexa Fluor 488-phalloidin) and nuclear (Hoechst 33342) staining, and label-free Coherent Anti-Stokes Raman Scattering (CARS) microscopy (Leica TCS SP8). Scanning electron microscopy (TESCAN MIRA3 LMU) was used for high-resolution surface imaging. 2D-MoS₂ nanoparticles, as well as 2D-MoS₂ and doxorubicin simultaneously, were applied to investigate nanoparticle-mediated labeling and cellular uptake. Results: A comparative analysis of multiple imaging modalities — including optical, fluorescence, confocal, CARS, and SEM — was applied to Lewis lung carcinoma (LLC) cells. Fluorescence microscopy with specific fluorophores made it possible to analyze the size and properties of actin fibers and showed that nuclei occupy most of the deadhesive cells. Electron microscopy revealed numerous filopodia on the cell surface. CARS showed the presence of lipid droplets in the cells. Conclusions: Each microscopy method provided complementary insights into cell morphology, cytoskeletal organization, lipid content, and surface ultrastructure. Nanoparticles demonstrated high utility as dual imaging and therapeutic agents. This work represents the first detailed SEM study of non-adherent LLC cells and highlights the potential of integrated multimodal imaging for studying circulating tumor cell models.

Background: Osteosarcoma (OS) is the most common primary malignant bone tumor. Doxorubicin (DOX) is extensively used in OS chemotherapy, yet improving patient outcomes remains challenging. This study investigated the effect of free DOX combined with inductive moderate hyperthermia (IMH) on Saos-2 human OS cells. Methods: Cell viability was assessed by trypan blue exclusion. Flow cytometry analyzed apoptosis, necrosis, and reactive oxygen species (ROS) in cells exposed to control (no treatment), IMH (42 MHz frequency, 500 μT magnetic field induction, 564 V/m electric field strength, 15 W output power, and 30 min duration) alone, DOX (0.06 μg/mL) alone, or DOX combined with IMH. The expression of p14^ARF tumor suppressor and epidermal growth factor receptor (EGFR) was evaluated by immunocytochemistry. Spatial autocorrelation analysis quantified the heterogeneity of p14^ARF and EGFR distributions in acquired images. Results: The half maximal inhibitory concentration (IC₅₀) of DOX in Saos-2 cells had minimal variation between 48 h (0.060 ± 0.01 μg/mL) and 72 h (0.055 ± 0.003 μg/mL). DOX + IMH resulted in a 15% increase in early apoptosis and a 20% elevation in ROS levels compared with DOX alone. Immunocytochemical analysis revealed a 37% increase in p14^ARF and a 32% reduction in EGFR expression following combined treatment in comparison to DOX alone. Image analysis showed that DOX + IMH treatment caused the highest Moran’s index values for p14^ARF and EGFR, reflecting less heterogeneous spatial distributions (p < 0.05). Conclusions: IMH enhanced DOX-induced cytotoxicity in Saos-2 cells by initiating ROS-mediated apoptosis and reducing heterogeneity of cellular responses.

Background. The predominance of aerobic glycolysis over oxidative phosphorylation (the Warburg effect) is characteristic of almost all invasive forms of cancer. The specificity of cancer (especially metastatic) cell energy metabolism represents multiple targets for therapeutic intervention using glycolysis inhibitors. Although the transition of cells to the bloodstream (i.e., anchorage-independent growth) is an important step of the metastatic cascade, the question of the cytotoxic efficacy of glycolysis inhibitors against these cells remains largely open. Methods. The cytotoxicity of 2-deoxy-D-glucose, sodium oxamate, metformin, cisplatin, and paclitaxel against high and low metastatic Lewis lung carcinoma cells during anchorage-independent growth (using polyHEMA coated plates) compared to anchorage-dependent growth was studied. The survival, proliferative activity, metabolic plasticity, and FTIR-ATR spectra of the sodium oxamate-resistant cells under different growth conditions were investigated Results. Unlike cisplatin and paclitaxel (which are the basic drugs for the treatment of lung cancer) glycolysis inhibitors such as 2-deoxy-D-glucose and sodium oxamate exhibit high cytotoxicity against carcinoma cells during anchorage-independent growth (compared to anchorage-dependent growth), which correlates with an enhanced glucose consumption rate of these cells. The cytotoxicity of glycolysis inhibitors was shown to be limited by the emergence of resistant cells whose survival was maintained at low glucose consumption rates especially in cells under anchorage-independent growth, most likely due to their ability to activate glutaminolysis. Conclusions. The enhanced cytotoxicity of glycolysis inhibitors against cells during anchorage-independent growth and the emergence of resistant cells, whose survival is presumably related to the activation of mitochondrial metabolism, indicates the potential antimetastatic efficacy of a combination of glycolysis and OXPHOS inhibitors

Metastasis in oncological diseases remains one of the main reasons for negative prognosis regarding treatment. Any new data on the biophysical and biochemical characteristics of circulating metastatic cells will help to develop a concept for antimetastatic therapy. In this study, we found a number of differences in the spectroscopic and morphological features of circulating metastatic cells. FT-IR and Raman spectra cultivated by adhesive and de-adhesive methods (with the latter used as a model for metastatic cells) have shown spectroscopic features, namely in FT-IR spectra in the region of CH stretching vibrations, which are associated with structural rearrangements in the cell membrane, as well as changes in the intensity and position of the PO₂⁻ group vibration bands correlated with proliferative activity. The spectral features in the regions of OH stretching and Amide I vibrations as well as other spectral markers of the metastatic cells grown under different cultivation conditions were derived. Raman spectra showed a redistribution of the amino acid Tyr/Trp (tryptophan to tyrosine) ratio and in Tyr doublet intensity in the region of 500–900 cm⁻¹, as well as varying glycogen levels in different cells. The spectroscopic markers are in accordance with biochemical data. CARS and confocal optical microscopy were applied to determine the state of the cells and the F-actin expression level, which turned out to be higher for adhesive cells in comparison with de-adhesive cells. The shape and the morphological properties of the cells differ drastically. The correlation of vibrational markers with biochemical data and the cytofluorometric method was discussed.

Metastasis in oncological diseases remains one of the main reasons for negative prognosis regarding treatment. Any new data on the biophysical and biochemical characteristics of circulating metastatic cells will help to develop the concept of antimetastatic therapy. In this study we found a number of differences in spectroscopic and morphological features of circulating metastatic cells. FTIR and Raman spectra of cultivated by adhesive and de-adhesive methods (last as a model for metastatic cells) have showed spectroscopic features, namely in FTIR in the region of CH - stretching vibrations associated with structural rearrangements in the cell membrane, as well as changes in the intensity and position of the PO2- group vibrations bands correlated with a proliferative activity. The spectral features in the regions of OH stretching and Amid I vibrations and other spectral markers of the metastatic cells under different cultivation conditions were derived. Raman spectra showed redistribution of amino acid Tyr/Trp ratio and in Tyr duplet intensity in the region of 500-900 cm-1 , as well different glycogen level in different cells. The spectroscopic markers are in the accordance with biochemical data. CARS and confocal optical microscopy were applied for determination a state of the cells and F-actin expression level that turned out to be higher for adhesive cells in comparison with de-adhesive cells. Shape and morphological property of the cells drastically differs. The correlation of vibrational markers with biochemical data and cytofluorometric method were discussed.

Despite efforts in osteosarcoma (OS) research, the role of inductive moderate hyperthermia (IMH) in delivering and enhancing the antitumor effect of liposomal doxorubicin formulations (LDOX) remains unresolved. This study investigated the effect of a combination treatment with LDOX and IMH on Saos-2 human OS cells. We compared cell viability using a trypan blue assay, apoptosis and reactive oxygen species (ROS) measured by flow cytometry and pro-apoptotic Bax protein expression examined by immunocytochemistry in response to IMH (42 MHz frequency, 15 W power for 30 min), LDOX (0.4 μg/mL), and LDOX plus IMH. The lower IC₅₀ value of LDOX at 72 h indicated increased accumulation of the drug in the OS cells. LDOX plus IMH resulted in a 61% lower cell viability compared to no treatment. Moreover, IMH potentiated the LDOX action on the Saos-2 cells by promoting ROS production at temperatures of p < 0.05). Therefore, we determined that IMH could enhance LDOX delivery and its antitumor effect via altered membrane permeabilization, ROS generation, and a lower level of visualized Bax heterogeneity in the Saos-2 cells, suggesting the potential translation of these findings into in vivo studies.

The majority of tumor-associated macrophages exhibit the M2 phenotype, which is an important determinant of tumor development and metastasis. Reducing the number of intratumoral M2 macrophages is an urgent task. It has been revealed that blockade of glycolysis by sodium oxamate or the glutamine-free medium both suppresses the ability of high-metastatic (LLC) and low-metastatic (LLCR9) Lewis lung carcinoma cells to macrophage reprogramming into the M2 phenotype.