Comparison of primary and passaged tumor cell cultures and their application in personalized medicine.

Passaged cell lines represent currently an integral component in various studies of malignant neoplasms. These cell lines are utilized for drug screening both in monolayer cultures or as part of three-dimensional (3D) tumor models. They can also be used to model the tumor microenvironment in vitro and in vivo through xenotransplantation into immunocompromised animals. However, immortalized cell lines have some limitations of their own. The homogeneity of cell line populations and the extensive passaging in monolayer systems make these models distant from the original disease. Recently, there has been a growing interest among scientists in the use of primary cell lines, as these are passaged directly from human tumor tissues. In this case, cells retain the morphological and functional characteristics of the tissue from which they were derived, an advantage often not observed in passaged cultures. This review highlights the advantages and limitations of passaged and primary cell cultures, their similarities and differences, as well as existing test systems that are based on primary and passaged cell cultures for drug screening purposes.

Mitochondrial Protein Density, Biomass, and Bioenergetics as Predictors for the Efficacy of Glioma Treatments.

The metabolism of glioma cells exhibits significant heterogeneity and is partially responsible for treatment outcomes. Given this variability, we hypothesized that the effectiveness of treatments targeting various metabolic pathways depends on the bioenergetic profiles and mitochondrial status of glioma cells. To this end, we analyzed mitochondrial biomass, mitochondrial protein density, oxidative phosphorylation (OXPHOS), and glycolysis in a panel of eight glioma cell lines. Our findings revealed considerable variability: mitochondrial biomass varied by up to 3.2-fold, the density of mitochondrial proteins by up to 2.1-fold, and OXPHOS levels by up to 7.3-fold across the cell lines. Subsequently, we stratified glioma cell lines based on their mitochondrial status, OXPHOS, and bioenergetic fitness. Following this stratification, we utilized 16 compounds targeting key bioenergetic, mitochondrial, and related pathways to analyze the associations between induced changes in cell numbers, proliferation, and apoptosis with respect to their steady-state mitochondrial and bioenergetic metrics. Remarkably, a significant fraction of the treatments showed strong correlations with mitochondrial biomass and the density of mitochondrial proteins, suggesting that mitochondrial status may reflect glioma cell sensitivity to specific treatments. Overall, our results indicate that mitochondrial status and bioenergetics are linked to the efficacy of treatments targeting metabolic pathways in glioma.

Electrospinning Approach for Preparing Nanostructured Scaffolds for Stem Cell Seeding and/or Implantation in Neurotrauma.

Preparation of highly porous biocompatible and bioresorbable nerve conduit or scaffold by electrospinning based on synthetic polycaprolactone with a molecular weight of 80 kDa (PCL 80 kDa) has significance in the context of regenerative medicine with special emphasis on their application in neurotrauma. PCL conduits/scaffolds serving as a support structure for seeded stem cells show promising regenerative potential to promote functional recovery and tissue regeneration in models of neurotrauma. Here we describe a standard protocol for the production of conduits by electrospinning at high field-forming voltages (24kB) using a 6% solution of PCL 80 kDa in a chloroform/methanol mixture.

Cross-reactivity of hantavirus antibodies after immunization with PUUV antigens.

Nephropathia epidemica (NE), caused by Puumala (PUUV) orthohantavirus, is endemic in the Republic of Tatarstan (RT). There are limited options for NE prevention in RT. Currently, available vaccines are made using Haantan (HNTV) orthohantavirus antigens. In this study, the efficacy of microvesicles (MVs) loaded with PUUV antigens to induce the humoral immune response in small mammals was analyzed. Additionally, the cross-reactivity of serum from immunized small mammals and NE patients with HNTV, Dobrava, and Andes orthohantaviruses was investigated using nucleocapsid (N) protein peptide libraries. Finally, the selected peptides were analyzed for allergenicity, their ability to induce an autoimmune response, and their interaction with Class II HLA. Several N protein peptides were found to be cross-reactive with serum from MVs immunized small mammals. These cross-reactive epitopes were located in oligomerization perinuclear targeting and Daxx-interacting domains. Most cross-reactive peptides lack allergenic and autoimmune reactivity. Molecular docking revealed two cross-reacting peptides, N6 and N19, to have good binding with three Class II HLA alleles. These peptides could be candidates for developing vaccines and therapeutics for NE.

Development of a Three-Dimensional Multicellular Model of Human Neuroblastoma Using Matrigel as an Extracellular Matrix Analogue.

Neuroblastoma, the most prevalent extracranial solid tumor in children, poses therapeutic challenges due to its variable clinical course and propensity for metastasis. Despite advances in treatment strategies like chemotherapy, drug resistance remains a significant concern, highlighting the need for improved models to study tumor behavior and drug responses. This chapter proposes the development of a three-dimensional multicellular model of human neuroblastoma using Matrigel as an ECM analogue. Such models aim to replicate the complexity of the tumor microenvironment, providing valuable insights into tumor progression and drug resistance mechanisms. By recapitulating key features of neuroblastoma within a physiologically relevant context, these models offer a platform for preclinical drug screening and therapeutic development.

The Impact of Treadmill Training on Tissue Integrity, Axon Growth, and Astrocyte Modulation.

Spinal cord injury (SCI) presents a complex challenge in neurorehabilitation, demanding innovative therapeutic strategies to facilitate functional recovery. This study investigates the effects of treadmill training on SCI recovery, emphasizing motor function enhancement, neural tissue preservation, and axonal growth. Our research, conducted on a rat model, demonstrates that controlled treadmill exercises significantly improve motor functions post-SCI, as evidenced by improved scores on the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale and enhanced electromyography readings. Notably, the training facilitates the preservation of spinal cord tissue, effectively reducing secondary damage and promoting the maintenance of neural fibers in the injured area. A key finding is the significant stimulation of axonal growth around the injury epicenter in trained rats, marked by increased growth-associated protein 43 (GAP43) expression. Despite these advancements, the study notes a limited impact of treadmill training on motoneuron adaptation and highlights minimal changes in the astrocyte and neuron-glial antigen 2 (NG2) response. This suggests that, while treadmill training is instrumental in functional improvements post-SCI, its influence on certain neural cell types and glial populations is constrained.

NF-κB and JAK/STAT Signaling Pathways as Crucial Regulators of Neuroinflammation and Astrocyte Modulation in Spinal Cord Injury.

Spinal cord injury (SCI) leads to significant functional impairments below the level of the injury, and astrocytes play a crucial role in the pathophysiology of SCI. Astrocytes undergo changes and form a glial scar after SCI, which has traditionally been viewed as a barrier to axonal regeneration and functional recovery. Astrocytes activate intracellular signaling pathways, including nuclear factor κB (NF-κB) and Janus kinase-signal transducers and activators of transcription (JAK/STAT), in response to external stimuli. NF-κB and STAT3 are transcription factors that play a pivotal role in initiating gene expression related to astrogliosis. The JAK/STAT signaling pathway is essential for managing secondary damage and facilitating recovery processes post-SCI: inflammation, glial scar formation, and astrocyte survival. NF-κB activation in astrocytes leads to the production of pro-inflammatory factors by astrocytes. NF-κB and STAT3 signaling pathways are interconnected: NF-κB activation in astrocytes leads to the release of interleukin-6 (IL-6), which interacts with the IL-6 receptor and initiates STAT3 activation. By modulating astrocyte responses, these pathways offer promising avenues for enhancing recovery outcomes, illustrating the crucial need for further investigation into their mechanisms and therapeutic applications in SCI treatment.

Astrocytes in Spinal Cord Injury: Current Opportunities and Prospects for Directional Polarization.

Activation of astrocytes during spinal cord injury (SCI) is accompanied by changes in their morphology and functional activity, possibly having severity-, localization-, and time-dependent features. The understanding of the role of reactive astrocytes has undergone significant changes over the last decades, and new data are still emerging to assess the diversity of functional manifestations of reactive cells. This review discusses the current understanding of astrocyte behavior, possible manifestations of their negative and positive roles in SCI, and the prospects for using various methods of directed polarization of astrocytes to improve post-traumatic outcomes. Despite the existing difficulties regarding the disclosure of the complex cascade of molecular changes of reactive astrocytes in different posttraumatic periods, researchers do not give up hope for the development of astrocyte-targeted methods that could reduce the severity of secondary injury by regulating the negative effects of these cells.

Immunology of SARS-CoV-2 Infection.

According to the data from the World Health Organization, about 800 million of the world population had contracted coronavirus infection caused by SARS-CoV-2 by mid-2023. Properties of this virus have allowed it to circulate in the human population for a long time, evolving defense mechanisms against the host immune system. Severity of the disease depends largely on the degree of activation of the systemic immune response, including overstimulation of macrophages and monocytes, cytokine production, and triggering of adaptive T- and B-cell responses, while SARS-CoV-2 evades the immune system actions. In this review, we discuss immune responses triggered in response to the SARS-CoV-2 virus entry into the cell and malfunctions of the immune system that lead to the development of severe disease.

Development of a novel prostate Cancer-Stroma Sphere (CSS) model for In Vitro tumor microenvironment studies.

Tumor employs non-cancerous cells to gain beneficial features that promote growth and survival of cancer cells. Despite intensive research in the area of tumor microenvironment, there is still a lack of reliable and reproducible in vitro model for tumor and tumor-microenvironment cell interaction studies. Herein we report the successful development of a heterogeneous cancer-stroma sphere (CSS) model composed of prostate adenocarcinoma PC3 cells and immortalized mesenchymal stem cells (MSC). The CSS model demonstrated a structured spatial layout of the cells, with stromal cells concentrated at the center of the spheres and tumor cells located on the periphery. Significant increase in the levels of VEGFA, IL-10, and IL1a has been detected in the conditioned media of CSS as compared to PC3 spheres. Single cell RNA sequencing data revealed that VEGFA was secreted by MSC cells within heterogeneous spheroids. Enhanced expression of extracellular membrane (ECM) proteins was also shown for CSS-derived MSCs. Furthermore, we demonstrated that the multicellular architecture altered cancer cell response to chemotherapeutic agents: the inhibition of sphere formation by topotecan was 74.92 ± 4.56 % for PC3 spheres and 45.95 ± 7.84 % for CSS spheres (p < 0.01), docetaxel showed 37,51± 20,88 % and 15,67± 14,08 % inhibition, respectively (p < 0.05). Thus, CSS present an effective in vitro model for examining the extracellular matrix composition and cell-to-cell interactions within the tumor, as well as for evaluating the antitumor activity of drugs.

Cancer of unknown primary and the «seed and soil¼ hypothesis.

The worldwide incidence rate of cancer of unknown primary (CUP) reaches 5% (Kang et al, 2021; Lee, Sanoff, 2020; Yang et al, 2022). CUP has an alarmingly high mortality rate, with 84% of patients succumbing within the first year following diagnosis (Registration and Service, 2018). Under normal circumstances, tumor cell metastasis follows the «seed and soil¼ hypothesis, displaying a tissue-specific pattern of cancer cell homing behavior based on the microenvironment composition of secondary organs. In this study, we questioned whether seed and soil concept applies to CUP, and whether the pattern of tumor and metastasis manifestations for cancer of known primary (CKP) can be used to inform diagnostic strategies for CUP. We compared data from metastatic and primary CUP foci to the metastasis patterns observed in CKP. Furthermore, we evaluated several techniques for identifying the tissue-of-origin (TOO) in CUP profiling, including DNA, RNA, and epigenetic TOO techniques.

Rosuvastatin as a Supplemental Treatment for the Clinical Symptoms of Nephropathia Epidemica: A Pilot Clinical Study.

Nephropathis epidemica (NE), a mild form of hemorrhagic fever with renal syndrome (HFRS), is an acute zoonotic disease endemic in the Republic of Tatarstan. This study aimed to assess the impact of rosuvastatin on the clinical and laboratory results of NE. A total of 61 NE patients and 30 controls were included in this study; 22 NE patients and 7 controls received a daily dose of rosuvastatin (10 mg) for ten consecutive days. Serum samples were collected on days 1, 5, and 10 after admission to the hospital. These samples were analyzed to determine the levels of lipids, cytokines, and kidney toxicity markers. Our findings indicate that rosuvastatin reduced the duration of the second wave of fever and alleviated back pain and headache symptoms. Additionally, low-density lipoprotein cholesterol (LDL-C) serum levels were significantly decreased on days 5 and 10 upon rosuvastatin treatment. Furthermore, rosuvastatin decreased the levels of cytokines in the serum, particularly proinflammatory cytokines IL-1ß and IL-8. NE patients had significantly altered levels of the kidney toxicity markers albumin and osteopontin. The data from our study provide evidence supporting the therapeutic potential of rosuvastatin in NE cases.

Immunotherapy Strategy for Systemic Autoimmune Diseases: Betting on CAR-T Cells and Antibodies.

Systemic autoimmune diseases (SAIDs), such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and rheumatoid arthritis (RA), are fully related to the unregulated innate and adaptive immune systems involved in their pathogenesis. They have similar pathogenic characteristics, including the interferon signature, loss of tolerance to self-nuclear antigens, and enhanced tissue damage like necrosis and fibrosis. Glucocorticoids and immunosuppressants, which have limited specificity and are prone to tolerance, are used as the first-line therapy. A plethora of novel immunotherapies have been developed, including monoclonal and bispecific antibodies, and other biological agents to target cellular and soluble factors involved in disease pathogenesis, such as B cells, co-stimulatory molecules, cytokines or their receptors, and signaling molecules. Many of these have shown encouraging results in clinical trials. CAR-T cell therapy is considered the most promising technique for curing autoimmune diseases, with recent successes in the treatment of SLE and SSc. Here, we overview novel therapeutic approaches based on CAR-T cells and antibodies for targeting systemic autoimmune diseases.

Eternal Youth: A Comprehensive Exploration of Gene, Cellular, and Pharmacological Anti-Aging Strategies.

The improvement of human living conditions has led to an increase in average life expectancy, creating a new social and medical problem-aging, which diminishes the overall quality of human life. The aging process of the body begins with the activation of effector signaling pathways of aging in cells, resulting in the loss of their normal functions and deleterious effects on the microenvironment. This, in turn, leads to chronic inflammation and similar transformations in neighboring cells. The cumulative retention of these senescent cells over a prolonged period results in the deterioration of tissues and organs, ultimately leading to a reduced quality of life and an elevated risk of mortality. Among the most promising methods for addressing aging and age-related illnesses are pharmacological, genetic, and cellular therapies. Elevating the activity of aging-suppressing genes, employing specific groups of native and genetically modified cells, and utilizing senolytic medications may offer the potential to delay aging and age-related ailments over the long term. This review explores strategies and advancements in the field of anti-aging therapies currently under investigation, with a particular emphasis on gene therapy involving adeno-associated vectors and cell-based therapeutic approaches.

Adipose-Derived Mesenchymal Stem Cell (MSC) Immortalization by Modulation of hTERT and TP53 Expression Levels.

Mesenchymal stem cells (MSCs) are pivotal players in tissue repair and hold great promise as cell therapeutic agents for regenerative medicine. Additionally, they play a significant role in the development of various human diseases. Studies on MSC biology have encountered a limiting property of these cells, which includes a low number of passages and a decrease in differentiation potential during in vitro culture. Although common methods of immortalization through gene manipulations of cells are well established, the resulting MSCs vary in differentiation potential compared to primary cells and eventually undergo senescence. This study aimed to immortalize primary adipose-derived MSCs by overexpressing human telomerase reverse transcriptase (hTERT) gene combined with a knockdown of TP53. The research demonstrated that immortalized MSCs maintained a stable level of differentiation into osteogenic and chondrogenic lineages during 30 passages, while also exhibiting an increase in cell proliferation rate and differentiation potential towards the adipogenic lineage. Long-term culture of immortalized cells did not alter cell morphology and self-renewal potential. Consequently, a genetically stable line of immortalized adipose-derived MSCs (iMSCs) was established.

T-Lymphocytes Activated by Dendritic Cells Loaded by Tumor-Derived Vesicles Decrease Viability of Melanoma Cells In Vitro.

Immunotherapy represents an innovative approach to cancer treatment, based on activating the body's own immune system to combat tumor cells. Among various immunotherapy strategies, dendritic cell vaccines hold a special place due to their ability to activate T-lymphocytes, key players in cellular immunity, and direct them to tumor cells. In this study, the influence of dendritic cells processed with tumor-derived vesicles on the viability of melanoma cells in vitro was investigated. Dendritic cells were loaded with tumor-derived vesicles, after which they were used to activate T-cells. The study demonstrated that such modified T-cells exhibit high activity against melanoma cells, leading to a decrease in their viability. Our analysis highlights the potential efficacy of this approach in developing immunotherapy against melanoma. These results provide new prospects for further research and the development of antitumor strategies based on the mechanisms of T-lymphocyte activation using tumor-derived vesicles.

The Potential of Dendritic Cell Subsets in the Development of Personalized Immunotherapy for Cancer Treatment.

Since the discovery of dendritic cells (DCs) in 1973 by Ralph Steinman, a tremendous amount of knowledge regarding these innate immunity cells has been accumulating. Their role in regulating both innate and adaptive immune processes is gradually being uncovered. DCs are proficient antigen-presenting cells capable of activating naive T-lymphocytes to initiate and generate effective anti-tumor responses. Although DC-based immunotherapy has not yielded significant results, the substantial number of ongoing clinical trials underscores the relevance of DC vaccines, particularly as adjunctive therapy or in combination with other treatment options. This review presents an overview of current knowledge regarding human DCs, their classification, and the functions of distinct DC populations. The stepwise process of developing therapeutic DC vaccines to treat oncological diseases is discussed, along with speculation on the potential of combined therapy approaches and the role of DC vaccines in modern immunotherapy.

Сytotoxic effect of CAR-T cells against modified MCF-7 breast cancer cell line.

The most often diagnosed and fatal malignancy in women is breast cancer. The International Agency for Research on Cancer (IARC) estimates that there are 2.26 million new cases of cancer in 2020. Adoptive cell therapy using T cells with chimeric antigen receptor shows potential for the treatment of solid tumors, such as breast cancer. In this work the effectiveness of CAR-T cells against monolayer and three-dimensional bioprinted tumor-like structures made of modified MCF-7 breast cancer cells was assessed. The cytokine profile of supernatants after co-cultivation of MCF-7 tumor cell models with CAR-T cells was also measured to reveal the inflammatory background associated with this interaction.

Comparative Analysis of Cytokine Profiles in Cerebrospinal Fluid and Blood Serum in Patients with Acute and Subacute Spinal Cord Injury.

BACKGROUND: Cytokines are actively involved in the regulation of the inflammatory and immune responses and have crucial importance in the outcome of spinal cord injuries (SCIs). Examining more objective and representative indicators of the patient's condition is still required to reveal the fundamental patterns of the abovementioned posttraumatic processes, including the identification of changes in the expression of cytokines. METHODS: We performed a dynamic (3, 7, and 14 days post-injury (dpi)) extended multiplex analysis of cytokine profiles in both CSF and blood serum of SCI patients with baseline American Spinal Injury Association Impairment Scale grades of A. RESULTS: The data obtained showed a large elevation of IL6 (>58 fold) in CSF and IFN-γ (>14 fold) in blood serum at 3 dpi with a downward trend as the post-traumatic period increases. The level of cytokine CCL26 was significantly elevated in both CSF and blood serum at 3 days post-SCI, while other cytokines did not show the same trend in the different biosamples. CONCLUSIONS: The dynamic changes in cytokine levels observed in our study can explore the relationships with the SCI region and injury severity, paving the way for a better understanding of the pathophysiology of SCI and potentially more targeted and personalized therapeutic interventions.

Emerging Gene Therapy Approaches in the Management of Spinal Muscular Atrophy (SMA): An Overview of Clinical Trials and Patent Landscape.

Spinal muscular atrophy (SMA) is a rare autosomal recessive neuromuscular disease that is characterized by progressive muscle atrophy (degeneration), including skeletal muscles in charge of the ability to move. SMA is caused by defects in the SMN1 gene (Survival of Motor Neuron 1) which encodes a protein crucial for the survival and functionality of neuron cells called motor neurons. Decreased level of functioning SMN protein leads to progressive degeneration of alpha-motor neurons performing muscular motility. Over the past decade, many strategies directed for SMN-level-restoration emerged, such as gene replacement therapy (GRT), CRISPR/Cas9-based gene editing, usage of antisense oligonucleotides and small-molecule modulators, and all have been showing their perspectives in SMA therapy. In this review, modern SMA therapy strategies are described, making it a valuable resource for researchers, clinicians and everyone interested in the progress of therapy of this serious disorder.