Mimicking the cellular environment does not cause monocyte-derived macrophages to become phenotypically similar to Kupffer cells.

Resident macrophages of various mammalian organs are characterized by several distinctive features in their gene expression profile and phenotype, including involvement in the regulation of organ functions, as well as reduced sensitivity to proinflammatory activation factors. The reasons for the formation of such a specific phenotype remain the subject of intensive research. Some papers emphasize the role of the origin of organ macrophages. Other studies indicate that monocytes that develop in the red bone marrow are also able to form resident macrophages with a phenotype characteristic of a particular organ, but this requires appropriate microenvironmental conditions. In this article, we studied the possibility of differentiation of monocyte-derived macrophages into cells with a Kupffer-like phenotype under the influence of the main stromal components of Kupffer cells macrophage niche: Ito cells, liver sinusoid endotheliocytes and hepatocyte growth factor (HGF). It was found that Kupffer cells are characterized by several features, including increased expression of transcription factors Spic and Id3, as well as MUP family genes, Clusterin and Ngp genes. In addition, Kupffer cells were characterized by a higher proliferative activity. The expression of marker genes of Kupffer cells (i.e. Id3, Spic, Marco and Timd4) increased in monocyte-derived macrophages during coculture with Ito cells, liver sinusoid endothelial cells, macrophage colony-stimulating factor and HGF cells only by 3 days. However, the expression level of these genes was always higher in Kupffer cells. In addition, a complete coincidence of the expressed gene profile in monocyte-derived macrophages and Kupffer cells did not occur even after 3 days of culturing.

Altered Glycolysis, Mitochondrial Biogenesis, Autophagy and Apoptosis in Peritoneal Endometriosis in Adolescents.

Energy metabolism plays a pivotal role in the pathogenesis of endometriosis. For the initial stages of the disease in adolescents, this aspect remains unexplored. The objective of this paper was to analyze the association of cellular and endosomal profiles of markers of glycolysis, mitochondrial biogenesis, apoptosis, autophagy and estrogen signaling in peritoneal endometriosis (PE) in adolescents. We included 60 girls aged 13-17 years in a case-control study: 45 with laparoscopically confirmed PE (main group) and 15 with paramesonephric cysts (comparison group). Samples of plasma and peritoneal fluid exosomes, endometrioid foci and non-affected peritoneum were tested for estrogen receptor (Erα/ß), hexokinase (Hex2), pyruvate dehydrogenase kinase (PDK1), glucose transporter (Glut1), monocarboxylate transporters (MCT1 and MCT2), optic atrophy 1 (OPA1, mitochondrial fusion protein), dynamin-related protein 1 (DRP1, mitochondrial fission protein), Bax, Bcl2, Beclin1, Bnip3, P38 mitogen-activated protein kinase (MAPK), hypoxia-inducible factor 1 (Hif-1α), mitochondrial voltage-dependent anion channel (VDAC) and transforming growth factor (TGFß) proteins as markers of estrogen signaling, glycolysis rates, mitochondrial biogenesis and damage, apoptosis and autophagy (Western-Blot and PCR). The analysis identified higher levels of molecules associated with proliferation (ERß), glycolysis (MCT2, PDK1, Glut1, Hex2, TGFß and Hif-1α), mitochondrial biogenesis (OPA1, DRP1) and autophagy (P38, Beclin1 and Bnip3) and decreased levels of apoptosis markers (Bcl2/Bax) in endometrioid foci compared to non-affected peritoneum and that in the comparison group (p < 0.05). Patients with PE had altered profiles of ERß in plasma and peritoneal fluid exosomes and higher levels of Glut1, MCT2 and Bnip3 in plasma exosomes (p < 0.05). The results of the differential expression profiles indicate microenvironment modification, mitochondrial biogenesis, estrogen reception activation and glycolytic switch along with apoptosis suppression in peritoneal endometrioid foci already in adolescents.

Fluorescence lifetime imaging microscopy as an instrument for human sperm assessment.

Mammalian spermatozoa are highly energized cells in which most of the proteins and activated signaling cascades are involved in the metabolic pathways. Flavin adenine dinucleotide (FAD) has one of the most important roles in the correct functional activity of spermatozoa since it acts as a cofactor for flavoenzymes, critical for proper metabolism and predominantly located in mitochondria. Non-invasive, vital and non-traumatic examination of sperm FAD level and microenvironment could be performed by fluorescence lifetime imaging microscopy (FLIM). In this study, we assessed the metabolic status of spermatozoa from healthy donors and found that FLIM could be used to segregate and separate the male germ cells according to the type of metabolic activity which corresponds with spermatozoa motility measured in standard spermogram tests.

Spleen regeneration after subcutaneous heterotopic autotransplantation in a mouse model.

BACKGROUND: Splenectomy may lead to severe postoperative complications, including sepsis and cancers. A possible solution to this problem is heterotopic autotransplantation of the spleen. Splenic autografts rapidly restore the regular splenic microanatomy in model animals. However, the functional competence of such regenerated autografts in terms of lympho- and hematopoietic capacity remains uncertain. Therefore, this study aimed to monitor the dynamics of B and T lymphocyte populations, the monocyte-macrophage system, and megakaryocytopoiesis in murine splenic autografts. METHODS: The model of subcutaneous splenic engraftment was implemented in C57Bl male mice. Cell sources of functional recovery were studied using heterotopic transplantations from B10-GFP donors to C57Bl recipients. The cellular composition dynamics were studied by immunohistochemistry and flow cytometry. Expression of regulatory genes at mRNA and protein levels was assessed by real-time PCR and Western blot, respectively. RESULTS: Characteristic splenic architecture is restored within 30 days post-transplantation, consistent with other studies. The monocyte-macrophage system, megakaryocytes, and B lymphocytes show the highest rates, whereas the functional recovery of T cells takes longer. Cross-strain splenic engraftments using B10-GFP donors indicate the recipient-derived cell sources of the recovery. Transplantations of scaffolds populated with splenic stromal cells or without them afforded no restoration of the characteristic splenic architecture. CONCLUSIONS: Allogeneic subcutaneous transplantation of splenic fragments in a mouse model leads to their structural recovery within 30 days, with full reconstitution of the monocyte-macrophage, megakaryocyte and B lymphocyte populations. The circulating hematopoietic cells provide the likely source for the cell composition recovery.

The prospects of cell therapy for endometriosis.

Endometriosis is a chronic inflammatory estrogen-dependent disease characterized by the growth of endometrial-like tissue outside the physiological region. Despite the fact that this disease is common, laparoscopic surgery is currently the gold standard in the treatment of endometriosis. In this regard, it is necessary to develop new effective methods of minimally invasive therapy for endometriosis. One of the promising areas in the treatment of endometriosis is cell therapy. Cellular therapy is a vast branch of therapeutic methods with various agents. Potential cell therapies for endometriosis may be based on the principle of targeting aspects of the pathogenesis of the disease: suppression of estrogen receptor activity, angiogenesis, fibrosis, and a decrease in the content of stem cells in endometriosis foci. In addition, immune cells such as NK cells and macrophages may be promising agents for cell therapy of endometriosis. Standing apart in the methods of cell therapy is the replacement therapy of endometriosis. Thus, many studies in the field of the pathogenesis of endometriosis can shed light not only on the causes of the disease and may contribute to the development of new methods for personalized cell therapy of endometriosis.

The Therapeutic Potential of Multipotent Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Endometrial Regeneration.

Disruption of endometrial regeneration, fibrosis formation, and intrauterine adhesions underlie the development of "thin" endometrium and/or Asherman's syndrome (AS) and are a common cause of infertility and a high risk for adverse obstetric outcomes. The methods used (surgical adhesiolysis, anti-adhesive agents, and hormonal therapy) do not allow restoration of the regenerative properties of the endometrium. The experience gained today with cell therapy using multipotent mesenchymal stromal cells (MMSCs) proves their high regenerative and proliferative properties in tissue damage. Their contribution to regenerative processes is still poorly understood. One of these mechanisms is based on the paracrine effects of MMSCs associated with the stimulation of cells of the microenvironment by secreting extracellular vesicles (EVs) into the extracellular space. EVs, whose source is MMSCs, are able to stimulate progenitor cells and stem cells in damaged tissues and exert cytoprotective, antiapoptotic, and angiogenic effects. This review described the regulatory mechanisms of endometrial regeneration, pathological conditions associated with a decrease in endometrial regeneration, and it presented the available data from studies on the effect of MMSCs and their EVs on endometrial repair processes, and the involvement of EVs in human reproductive processes at the level of implantation and embryogenesis.

Therapeutic Efficiency of Proteins Secreted by Glial Progenitor Cells in a Rat Model of Traumatic Brain Injury.

Traumatic brain injuries account for 30-50% of all physical traumas and are the most common pathological diseases of the brain. Mechanical damage of brain tissue leads to the disruption of the blood-brain barrier and the massive death of neuronal, glial, and endothelial cells. These events trigger a neuroinflammatory response and neurodegenerative processes locally and in distant parts of the brain and promote cognitive impairment. Effective instruments to restore neural tissue in traumatic brain injury are lacking. Glial cells are the main auxiliary cells of the nervous system, supporting homeostasis and ensuring the protection of neurons through contact and paracrine mechanisms. The glial cells' secretome may be considered as a means to support the regeneration of nervous tissue. Consequently, this study focused on the therapeutic efficiency of composite proteins with a molecular weight of 5-100 kDa secreted by glial progenitor cells in a rat model of traumatic brain injury. The characterization of proteins below 100 kDa secreted by glial progenitor cells was evaluated by proteomic analysis. Therapeutic effects were assessed by neurological outcomes, measurement of the damage volume by MRI, and an evaluation of the neurodegenerative, apoptotic, and inflammation markers in different areas of the brain. Intranasal infusions of the composite protein product facilitated the functional recovery of the experimental animals by decreasing the inflammation and apoptotic processes, preventing neurodegenerative processes by reducing the amounts of phosphorylated Tau isoforms Ser396 and Thr205. Consistently, our findings support the further consideration of glial secretomes for clinical use in TBI, notably in such aspects as dose-dependent effects and standardization.

An Eye on Kupffer Cells: Development, Phenotype and the Macrophage Niche.

Macrophages are key participants in the maintenance of tissue homeostasis under normal and pathological conditions, and implement a rich diversity of functions. The largest population of resident tissue macrophages is found in the liver. Hepatic macrophages, termed Kupffer cells, are involved in the regulation of multiple liver functionalities. Specific differentiation profiles and functional activities of tissue macrophages have been attributed to the shaping role of the so-called tissue niche microenvironments. The fundamental macrophage niche concept was lately shaken by a flood of new data, leading to a revision and substantial update of the concept, which constitutes the main focus of this review. The macrophage community discusses contemporary evidence on the developmental origins of resident macrophages, notably Kupffer cells and the issues of heterogeneity of the hepatic macrophage populations, as well as the roles of proliferation, cell death and migration processes in the maintenance of macrophage populations of the liver. Special consideration is given to interactions of Kupffer cells with other local cell lineages, including Ito cells, sinusoidal endothelium and hepatocytes, which participate in the maintenance of their phenotypical and functional identity.

Endometriosis and Cancer: Exploring the Role of Macrophages.

Endometriosis and cancer have much in common, notably their burgeoning of cells in hypoxic milieus, their invasiveness, and their capacity to trigger remodeling, vascularization, and innervation of other tissues. An important role in these processes is played by permissive microenvironments inhabited by a variety of stromal and immune cells, including macrophages. Remarkable phenotypical plasticity of macrophages makes them a promising therapeutic target; some key issues are the range of macrophage phenotypes characteristic of a particular pathology and the possible manners of its modulation. In both endometriosis and cancer, macrophages guard the lesions from immune surveillance while promoting pathological cell growth, invasion, and metastasis. This review article focuses on a comparative analysis of macrophage behaviors in endometriosis and cancer. We also highlight recent reports on the experimental modulation of macrophage phenotypes in preclinical models of endometriosis and cancer.

Therapeutic Effects of hiPSC-Derived Glial and Neuronal Progenitor Cells-Conditioned Medium in Experimental Ischemic Stroke in Rats.

Transplantation of various types of stem cells as a possible therapy for stroke has been tested for years, and the results are promising. Recent investigations have shown that the administration of the conditioned media obtained after stem cell cultivation can also be effective in the therapy of the central nervous system pathology (hypothesis of their paracrine action). The aim of this study was to evaluate the therapeutic effects of the conditioned medium of hiPSC-derived glial and neuronal progenitor cells in the rat middle cerebral artery occlusion model of the ischemic stroke. Secretory activity of the cultured neuronal and glial progenitor cells was evaluated by proteomic and immunosorbent-based approaches. Therapeutic effects were assessed by overall survival, neurologic deficit and infarct volume dynamics, as well as by the end-point values of the apoptosis- and inflammation-related gene expression levels, the extent of microglia/macrophage infiltration and the numbers of formed blood vessels in the affected area of the brain. As a result, 31% of the protein species discovered in glial progenitor cells-conditioned medium and 45% in neuronal progenitor cells-conditioned medium were cell type specific. The glial progenitor cell-conditioned media showed a higher content of neurotrophins (BDNF, GDNF, CNTF and NGF). We showed that intra-arterial administration of glial progenitor cells-conditioned medium promoted a faster decrease in neurological deficit compared to the control group, reduced microglia/macrophage infiltration, reduced expression of pro-apoptotic gene Bax and pro-inflammatory cytokine gene Tnf, increased expression of anti-inflammatory cytokine genes (Il4, Il10, Il13) and promoted the formation of blood vessels within the damaged area. None of these effects were exerted by the neuronal progenitor cell-conditioned media. The results indicate pronounced cytoprotective, anti-inflammatory and angiogenic properties of soluble factors secreted by glial progenitor cells.

Inherent control of hepatocyte proliferation after subtotal liver resection.

At the normal physiological conditions, hepatocytes predominantly reside in G0 phase of cell cycle; they actively proceed to G1 phase upon damage to the organ. As it was shown in experiments with restoration of liver mass in rats after subtotal hepatectomy (resection of 80% of the organ mass may be considered as a model of the 'small for size' liver syndrome), the growth inhibition is due to prolonged arrest of hepatocyte proliferation, molecular mechanisms of which remain understudied. In a rat model of liver regeneration after surgical removal of 80% of its mass, we observe a delayed onset of hepatocyte proliferation: Ki67+ hepatocytes begin to appear as late as at 30 h after liver subtotal resection. Their appearance coincides with the beginning of transcription of genes for cyclins A2, B1, D 1 , and E 1 at 24-30 h after surgery. The corresponding increase in concentrations of cyclin D 1 and E proteins is further delayed till 48 h after liver resection. We have also observed a prolonged decrease in the expression of proto-oncogene c-met (the hepatocyte growth factor receptor-encoding gene Met), an increase in expression of the transforming growth factor ß1 (TGFß 1 ) receptor-encoding gene Tgfbr2. At the same time, irreversible block of hepatocyte proliferation is prevented by expression of certain factors, notably of the TWEAK/Fn14 signaling pathway: concentrations of the corresponding proteins in remnant livers have peaked from 24 to 48 h after liver subtotal resection.

Role of the Monocyte-Macrophage System in Normal Pregnancy and Preeclampsia.

The proper functioning of the monocyte-macrophage system, an important unit of innate immunity, ensures the normal course of pregnancy. In this review, we present the current data on the origin of the monocyte-macrophage system and its functioning in the female reproductive system during the ovarian cycle, and over the course of both normal and complicated pregnancy. Preeclampsia is a crucial gestation disorder characterized by pronounced inflammation in the maternal body that affects the work of the monocyte-macrophage system. The effects of inflammation at preeclampsia manifest in changes in monocyte counts and their subset composition, and changes in placental macrophage counts and their polarization. Here we summarize the recent data on this issue for both the maternal organism and the fetus. The influence of estrogen on macrophages and their altered levels in preeclampsia are also discussed.

Dynamics of macrophage populations of the liver after subtotal hepatectomy in rats.

BACKGROUND: In many clinical cases of extensive liver resection (e.g. due to malignancy), the residual portion is too small to maintain the body homeostasis. The resulting acute liver failure is associated with the compensatory growth inhibition, which is a typical manifestation of the 'small for size' liver syndrome. The study investigates possible causes of the delayed onset of hepatocyte proliferation after subtotal hepatectomy (80% liver resection) in rats. RESULTS: The data indicate that the growth inhibition correlates with delayed upregulation of the Tnf gene expression and low content of the corresponding Tnfα protein within the residual hepatic tissue. Considering the involvement of Tnf/Tnfα, the observed growth inhibition may be related to particular properties of liver macrophages - the resident Kupffer cells with CD68+CX1CR3-CD11b- phenotype. CONCLUSIONS: The delayed onset of hepatocyte proliferation correlates with low levels of Tnfα in the residual hepatic tissue. The observed growth inhibition possibly reflects specific composition of macrophage population of the liver. It is entirely composed of embryonically-derived Kupffer cells, which express the 'proregeneratory' M2 macrophage-specific marker CD206 in the course of regeneration.

Mechanisms and physiological relevance of acid-base exchange in functional units of the kidney.

This review discusses the importance of homeostasis with a particular emphasis on the acid-base (AB) balance, a crucial aspect of pH regulation in living systems. Two primary organ systems correct deviations from the standard pH balance: the respiratory system via gas exchange and the kidneys via proton/bicarbonate secretion and reabsorption. Focusing on kidney functions, we describe the complexity of renal architecture and its challenges for experimental research. We address specific roles of different nephron segments (the proximal convoluted tubule, the loop of Henle and the distal convoluted tubule) in pH homeostasis, while explaining the physiological significance of ion exchange processes maintained by the kidneys, particularly the role of bicarbonate ions (HCO3-) as an essential buffer system of the body. The review will be of interest to researchers in the fields of physiology, biochemistry and molecular biology, which builds a strong foundation and critically evaluates existing studies. Our review helps identify the gaps of knowledge by thoroughly understanding the existing literature related to kidney acid-base homeostasis.

M1 macrophages as promising agents for cell therapy of endometriosis.

Endometriosis is a chronic estrogen-dependent disease characterized by the presence of endometrial glands and stroma outside their normal anatomical location. While laparoscopic removal of foci remains the gold standard therapy, it has limited efficacy and certain risks. However, cell therapy using pro-inflammatory M1 macrophages presents a promising and minimally invasive alternative for treating endometriosis. This approach showcases the potential for innovative and effective treatments for this condition. This study aims to explore the anti-endometriosis properties of M1 macrophages. A reproducible syngeneic mouse model of endometriosis was utilized, revealing that formed foci are primarily composed of macrophages with an anti-inflammatory M2 phenotype rather than M1 macrophages. To investigate further, chemically reprogrammed M1 macrophages were labeled with the membrane fluorescent tag PKH26 and administered to animals with endometriosis. Therapy resulted in a decrease in the number and size of foci, accompanied by a shift in the phenotypic composition of peritoneal macrophages. Specifically, the content of M2 macrophages decreased while that of M1 macrophages increased, resembling the composition of healthy animals. Our study conclusively demonstrates the anti-endometriosis properties of M1 macrophages, providing a strong foundation for future research in the cell therapy of endometriosis.

Gene-Expression Patterns of Tumor and Peritumor Tissues of Smoking and Non-Smoking HPV-Negative Patients with Head and Neck Squamous Cell Carcinoma.

We studied the gene-expression patterns in specimens of tumor and peritumor tissue biopsies of 26 patients with head and neck carcinomas depending on smoking status. Histological and immunohistochemical examinations verified that all tumors belonged to the "classical" subgroup of head and neck carcinomas, and the HPV-negative tumor status was confirmed. The expression of 28 tumor-associated genes determined by RT-PCR was independent of patients' sex or age, TNM status, degree of differentiation, or tissue localization. Moreover, in peritumor tissue, none of the 28 genes were differentially expressed between the groups of smoking and nonsmoking patients. During oncotransformation in both studied groups, there were similar processes typical for HNSCC progression: the expression levels of paired keratins 4 and 13 were reduced, while the expression levels of keratin 17 and CD44 were significantly increased. However, further investigation revealed some distinctive features: the expression of the genes EGFR and TP63 increased significantly only in the nonsmoking group, and the expression of IL6, CDKN2A, EGF, and PITX1 genes changed only in the smoking group. In addition, correlation analysis identified several clusters within which genes displayed correlations in their expression levels. The largest group included 10 genes: TIMP1, TIMP2, WEE1, YAP, HIF1A, PI3KCA, UTP14A, APIP, PTEN, and SLC26A6. The genetic signatures associated with smoking habits that we have found may serve as a prerequisite for the development of diagnostic panels/tests predicting responses to different therapeutic strategies for HNSCC.

Hyaluronic Acid Prevents Fusion of Brain Tumor-Derived Spheroids and Selectively Alters Their Gene Expression Profile.

Hyaluronic acid (HA), a major glycosaminoglycan of the brain extracellular matrix, modulates cell behaviors through binding its receptor, Cd44. In this study, we assessed the influence of HA on high-grade brain tumors in vitro. The model comprised cell cultures derived from six rodent carcinogen-induced brain tumors, forming 3D spheroids prone to spontaneous fusion. Supplementation of the standard culture medium with 0.25% HA significantly inhibited the fusion rates, preserving the shape and size uniformity of spheroids. The 3D cultures were assigned to two groups; a Cd44lo group had a tenfold decreased relative expression of Cd44 than another (Cd44hi) group. In addition, these two groups differed by expression levels of Sox2 transcription factor; the correlation analysis revealed a tight negative association for Cd44 and Sox2. Transcriptomic responses of spheroids to HA exposure also depended on Cd44 expression levels, from subtle in Cd44lo to more pronounced and specific in Cd44hi, involving cell cycle progression, PI3K/AKT/mTOR pathway activation, and multidrug resistance genes. The potential HA-induced increase in brain tumor 3D models' resistance to anticancer drug therapy should be taken into account when designing preclinical studies using HA scaffold-based models. The property of HA to prevent the fusion of brain-derived spheroids can be employed in CNS regenerative medicine and experimental oncology to ensure the production of uniform, controllably fusing neurospheres when creating more accurate in vitro brain models.

SP1 Gene Methylation in Head and Neck Squamous Cell Cancer in HPV-Negative Patients.

There is still much to learn about the epigenetic mechanisms controlling gene expression during carcinogenesis. When researching aberrant DNA methylation, active proliferative tumor cells from head and neck squamous cell cancer (HNSCC) can be used as a model. The aim of the study was to investigate the methylation status of CDKN1, CDKN2A, MYC, Smad3, SP1, and UBC genes in tumor tissue (control-normal tissue) in 50 patients (37 men and 13 women) with HPV-negative HNSCC. Methods: Bisulfite conversion methods and methyl-sensitive analysis of high-resolution melting curves were used to quantify the methylation of genes. In all patients and across various subgroups (tongue carcinoma, laryngeal and other types of carcinomas T2, T3, T4 status; age before and after 50 years; smoking and non-smoking), there are consistent differences in the methylation levels in the SP1 gene in tumor DNA compared to normal. Results: The methylation of the SP1 gene in tumor DNA suppresses its expression, hinders HNSCC cell proliferation regulation, and could be a molecular indicator of malignant cell growth. The study of DNA methylation of various genes involved in carcinogenesis is promising because hypermethylated promoters can serve as potential biomarkers of disease.

Oral Microbiome Dysbiosis as a Risk Factor for Stroke: A Comprehensive Review.

Stroke represents a significant global health burden, with a substantial impact on mortality, morbidity, and long-term disability. The examination of stroke biomarkers, particularly the oral microbiome, offers a promising avenue for advancing our understanding of the factors that contribute to stroke risk and for developing strategies to mitigate that risk. This review highlights the significant correlations between oral diseases, such as periodontitis and caries, and the onset of stroke. Periodontal pathogens within the oral microbiome have been identified as a contributing factor in the exacerbation of risk factors for stroke, including obesity, dyslipidemia, atherosclerosis, hypertension, and endothelial dysfunction. The alteration of the oral microbiome may contribute to these conditions, emphasizing the vital role of oral health in the prevention of cardiovascular disease. The integration of dental and medical health practices represents a promising avenue for enhancing stroke prevention efforts and improving patient outcomes.

A single-center experience of the upright proton therapy for skull-base chordomas and chondrosarcomas: Updated results.

Aim: To access efficacy and safety of the upright proton therapy for the skull-base chordomas and chondrosarcomas. Materials and methods: The study encompasses single-center experience of proton therapy in chordomas (CA) and chondrosarcomas (CSA) of skull-base localization. We evaluate overall survival, local control and toxicity. Tumor response was assessed in accordance with RANO criteria. Treatment-related toxicity was evaluated with the help of CTCAE v 5.0 scale. Results: Proton therapy in the upright position was utilized for 51pts (patients) with CA-CSA (40 pts with chordoma and 11pts with chondrosarcoma) at the A. Tsyb Medical Radiological Research Center in 2016-2023. Median tumor volume constituted 30 cm3 (IQR (interquartile range) 15-41 cm3). Median total dose was 70 GyRBE. Median number of fractions was 35. Overall survival (OS) at 1-, 2- and 3-year rates reached 98.0 %, 88.6 % and 82.7 %, respectively. Median follow-up time was 36 months. The 1-, 2- and 3-year local control (LC) rates constituted, respectively, 98 %, 78.6 % and 66.3 %. Prior surgery showed statistically significant association with better prognosis (p = 0.023). Brainstem-to-tumor dose coverage compromise became the major pattern of LC failure (p = 0.03). The late radiation toxicity reactions included temporal lobe necrosis grade 2 in 2 pts, xerostomia grade 1 in 1pt, radiation cataract grade 2 in 1pt and persistent headache grade 2 in 4 pts. Severe late toxicity reactions were observed in 2 cases (4 %): 1 myelitis grade 3 and brainstem damage grade 5 in 1pt. Conclusion: Local control was achieved in the majority of patients receiving the scanning-beam upright proton therapy for skull-base CA-CSA. The LC rates after a surgery-radiotherapy combination treatment were higher compared with irradiation alone. Pattern of failure is mostly brainstem-tumor dose compromise. The high OS and LC rates were accompanied by low toxicity incidence. Even in complex case of the skull base CA-CSA upright proton therapy shows promising clinical outcomes.