Perifocal Tissues Research Articles

Optical coherence tomography-angiography for anterior uveal tract evaluation in normal subjects and in pathology

Purpose: to assess the potentials of optical coherence tomography-angiography (OCTA) in the evaluation of anterior segment (AS) uveal vessels in normal and pathological conditions.Material and methods. 20 healthy volunteers (40 eyes) with no ophthalmic pathology (the control group) and 85 previously untreated patients (85 eyes) with suspected tumors of the irido-ciliary area (the iris, the ciliary body and the peripheral sections of the choroid) were examined. All participants had optical coherence tomography (OCT) and OCTA of AS, with the qualitative assessment of scans (vessels pattern, lumen, tortuosity) and the quantitative assessment (vessel density index, VD%, for the affected zone). In ciliary body or choroid pathologies, VD index was measured for the conjunctiva in the focus projection. VD index included mean and local VD, as well as VD of perifocal tissues. In addition, B-mode ultrasound scanning and ultrasound biomicroscopy were performed.Results. OCTA scans in 4 sectors of the normal iris showed a predominantly radial pattern of conjunctival vessels positioning, with their lumen remaining the same along their entire visible length. The lowest VD value (38.5%) was registered in the temporal iris segment, while the highest (43.9%) was revealed in the inferior quadrant. In the iris tumors area, intrinsic vascularity molded in various vascular patterns was observed. Melanomas could be suspected because of the vessels oriented along the axis of the tumor and by non-uniform lumen of the vessels.Conclusion. AS-OCTA is an informative method for the visualization of iris vessels in normal conditions and in iris pathology and may be considered a valuable addition to the standard visualization techniques.

Anti-PD-1 treatment protects against seizure by suppressing sodium channel function.

Although programmed cell death protein 1 (PD-1) typically serves as a target for immunotherapies, a few recent studies have found that PD-1 is expressed in the nervous system and that neuronal PD-1 might play a crucial role in regulating neuronal excitability. However, whether brain-localized PD-1 is involved in seizures and epileptogenesis is still unknown and worthy of in-depth exploration. The existence of PD-1 in human neurons was confirmed by immunohistochemistry, and PD-1 expression levels were measured by real-time quantitative PCR (RT-qPCR) and western blotting. Chemoconvulsants, pentylenetetrazol (PTZ) and cyclothiazide (CTZ), were applied for the establishment of invivo (rodents) and invitro (primary hippocampal neurons) models of seizure, respectively. SHR-1210 (a PD-1 monoclonal antibody) and sodium stibogluconate (SSG, a validated inhibitor of SH2-containing protein tyrosine phosphatase-1 [SHP-1]) were administrated to investigate the impact of PD-1 pathway blockade on epileptic behaviors of rodents and epileptiform discharges of neurons. A miRNA strategy was applied to determine the impact of PD-1 knockdown on neuronal excitability. The electrical activities and sodium channel function of neurons were determined by whole-cell patch-clamp recordings. The interaction between PD-1 and α-6 subunit of human voltage-gated sodium channel (Nav1.6) was validated by performing co-immunostaining and co-immunoprecipitation (co-IP) experiments. Our results reveal that PD-1 protein and mRNA levels were upregulated in lesion cores compared with perifocal tissues of surgically resected specimens from patients with intractable epilepsy. Furthermore, we show that anti-PD-1 treatment has anti-seizure effects both invivo and invitro. Then, we reveal that PD-1 blockade can alter the electrophysiological properties of sodium channels. Moreover, we reveal that PD-1 acts together with downstream SHP-1 to regulate sodium channel function and hence neuronal excitability. Further investigation suggests that there is a direct interaction between neuronal PD-1 and Nav1.6. Our study reveals that neuronal PD-1 plays an important role in epilepsy and that anti-PD-1 treatment protects against seizures by suppressing sodium channel function, identifying anti-PD-1 treatment as a novel therapeutic strategy for epilepsy.

Indices of insulin-like growth factors family in the lung tissue of patients with non-small cell lung cancer after COVID-19 of various severity

Purpose of the study. An analysis of levels of IGF and their carrying proteins in lung tissues of cancer patients depending on the severity of the previous COVID-19 infection.Patients and methods. The study included 60 patients with histologically verified non-small cell lung cancer (NSCLC) T1–3NхM0 receiving treatment at the Thoracic Department, National Medical Research Centre for Oncology, in 2020–2021. The control group included 30 NSCLC patients after asymptomatic or mild COVID-19 disease (15 males and 15 females); the main group included 30 (15 men and 15 women) patients after severe or moderate to severe COVID-19 infection. The mean age of patients was 59.11 ± 2.89 years; no significant differences were noted between the control and main groups. All participants gave their informed consent prior to the study approved by the Ethics Committee of National Medical Research Centre for Oncology. Qualitative assessment of IGF-I, IGF-II and IGFBP-1,2,3 levels in the tissues of the tumor, peritumoral area and resection line were measured by ELISA (Mediagnost, Germany). The statistical analysis was performed in the Statistica 10 program, the differences were considered statistically significant at p < 0.05.Results. Regardless of the gender, levels of IGF-I and IGF-II in tumor and resection line samples in patients of the main group were higher than in the control group on average by 1.5–2.2 times, and IGFBP-1 in the tumor was lower by 1.3 times in men and by 5 times in women. The ratio of IGF and IGFBP-1-3 in patients of the control group in perifocal tissues changed towards the parameters in the tumor tissue. IGF/IGFBP-1-3 in men of the main group were lower or did not differ from the indices in the intact tissue, while in women they increased, similarly to the tumor tissue.Conclusion. An increase in the ratio of IGF and carrier proteins in the tumor tissue of patients in the main group indicated an excessive accumulation of IGF in it, which may contribute to more aggressive growth of malignant tumors. The most pronounced disorders in the system of insulin-like growth factors were found in the tissues of the tumor and intact lung of patients with previous severe and moderate to severe COVID-19.

Local immunity features in patients with non-invasive muscular bladder cancer of various degrees of malignance

Purpose of the study. To study the features of the local distribution of populations of immune system cells in patients with non-invasive muscular bladder cancer of various degrees of malignancy.Materials and methods. The study included 51 patients with newly diagnosed non-muscle-invasive bladder cancer (papillary urothelial carcinoma) who received complex treatment and follow-up after 9 months at the oncourological department of the National Medical Research Center of Oncology. Patients were divided into two groups: group 1 – with a tumor of low malignant potential (Low grade – LG), n = 31; group 2 – with a tumor of high malignant potential (High grade – HG), n = 20. After 6–9 months, 24 patients were diagnosed with a relapse of the disease – in 48,4 % in patients of group 1 (n = 15) and in 45 % – in group 2 (n = 9). In cell suspensions obtained from the primary and recurrent tumors, as well as the perifocal zone, the relative number of populations of immunocompetent cells was estimated using flow cytometry. A comparison was made of the content of individual populations of lymphocytes in the tumor tissue, the perifocal zone of primary and recurrent lesions of various degrees of malignancy. Statistical processing was performed using Statistica 13.0.Results. The development of a recurrent tumor of low malignant potential is accompanied by the involvement of cells of innate immunity (NK- and NKT-lymphocytes) into its microenvironment, which is associated with an imbalance in the number of main cells of adaptive immunity – a fairly pronounced decrease in the tumor of T-lymphocytes of the helper-inductor type was noted with a constant content cytotoxic T-lymphocytes, as well as the multidirectional nature of changes in DP- (decrease) and DN-lymphocytes (increase). A feature of the development of a recurrent tumor of high malignant potential is that it is accompanied by the involvement of innate immunity cells (NK- and NKT-lymphocytes) into its microenvironment, as well as multidirectional changes in DP- (decrease) and DN-lymphocytes (increase).Conclusion. Studies of the population composition of tumors and their perifocal tissues of NMIBC revealed a number of features that are reflected in the redistribution of cytolytic cells, the formation of immunosuppressive conditions, which are reflected both in the manifestation of the biological properties of tumor cells and in changes in the cellular composition of bladder tissues involved in the process. development and progression of cancer.

ВЛИЯНИЕ MMP-9 НА РОСТ И РАЗВИТИЕ ПЕРВИЧНО- МНОЖЕСТВЕННЫХ ЭКСПЕРИМЕНТАЛЬНЫХ ОПУХОЛЕЙ В УСЛОВИЯХ ПЕРВИЧНОГО ИММУНОДЕФИЦИТА

MMP9 is deeply involved in invasion, metastasis and angiogenesis of various tumors, and can also affect the tumor microenvironment. The aim of this study was to analyze the MMP9 levels in tissues with independent and com- bined growth (MPT): B16/F10 melanoma and Lewis lung carcinoma. Primary immunodeficiency presented in a model of Balb/c Nude mice was chosen as a comorbid pathology. The study included 24 females divided into groups (n=6 each): 1 – intact animals, 2 – animals with B16/F10 melanoma, 3 – animals with Lewis lung carcinoma (LLC), 4 - ani- mals with a combination of B16/F10 and LLC - multiple primary malignancies (MPMs). Levels of MMP9 were measured by ELISA in homogenates of tumor and perifocal tissues in all animals. In animals with MPT, levels of MMP9 in B16/ F10 were 3.9 times higher than in LLC, and the levels in B16/F10 perifocal tissues were 1.9 times higher than in LLC perifocal tissues. MMP9 in non-cancer tissues in animals with MPT 2.8 times exceeded the levels in intact animals. In animals with MPT, MMP9 in tumor samples positively correlated with the primary node volumes. Whatever the level of MMP9 in the tumor and perifocal tissues, it is clearly directly involved in cancer development.

Local cellular immunity in colorectal cancer depending on tumor site.

e15613 Background: Colorectal cancer (CRC) is the third most commonly diagnosed cancer. More than 60% of all cases of CRC are colon cancer (CC). The role of individual units of the immune system in the development of this tumor is ambiguous. The purpose of this study was to characterize local populations and subpopulations of immunocompetent cells in colorectal cancer with different tumor locations. Methods: The study included 50 CC (adenocarcinoma) patients aged 35-86 years, women n = 26 (52%). Stage I tumors were registered in 4 patients (8%), stage II 25 (50%), stage III 21 (42%). 20 patients (40%) had right-sided CC (group 1), 9 (18%) left-sided CC (group 2), and 21 (42%) sigmoid CC (group 3). All patients received surgical treatment. Cell suspensions were obtained from tumor (TT) and peritumoral tissues (PT) (1-3 cm from the tumor), and then processed with an antibody panel in accordance with the manufacturer instructions (Becton Dickinson, USA) to identify the main populations and subpopulations of leukocytes and lymphocytes. The relative number of major populations and subpopulations of lymphocytes was determined on the BD FACSCanto flow cytometer. Results: A decrease in lymphocytic infiltration was noted left-sided tumors and sigmoid tumors compared to right-sided CC, by 46% and 51%, respectively. The percentage of CD3+ cells was almost the same regardless of the colon tumor location, and the number of main populations of T lymphocytes differed: in group 3, the content of CD4+ cells was 21% higher, and CD8+ cells were 22% lower compared with group 1, while group 2 had no differences. Group 2 differed in the tumor infiltration with both NK and NKT lymphocytes, which were higher by 25% and 22%, respectively, than in group 1. An increase in NK cells was noted in the sigmoid colon (group 3), and the relative number of NKT lymphocytes decreased. A common feature of TT in groups 2 and 3 was an increase in the content of B lymphocytes by 98% and 133%, respectively. PT of group 2 showed a decrease by 72%, 33%, 66%, and 46% in the relative number of lymphocytes, CD4+, NKT and B lymphocytes compared with group 1, together with an increased content of NK and CD8+ lymphocytes. PT in patients of group 3 had a decrease in the number of total and NK lymphocytes by 46% and 26%, respectively, and a significant increase by 85% in the content of CD8+ cells, with no changes in CD3+, CD4+ cells, B and NKT lymphocytes. Conclusions: The local immune status of CRC patients demonstrated a number of differences: right-sided tumors were characterized by a higher T-lymphocytic infiltration with the same tendency in the perifocal tissues, while left-sided and sigmoid tumors showed higher B-lymphocytic infiltration, which can help in the disease prognosis and choice of treatment strategy.

Gender features of the chronic pain effect on some vascular endothelial growth factors and their skin receptors on the B16/F10 melanoma model.

e24061 Background: Patients with oncological pathology, especially with the progression of the disease, often have chronic pain[1]. Pain effects singly can cause dysfunction of the vascular system, which could be an additional risk of the tumor process progression. The aim of the study was to identify gender-specific chronic pain effects on some vascular endothelial growth factors and their skin receptors in the B16/F10 melanoma model. Methods: The study was conducted on mice of the C57BL/6 line (females, males; age 8 weeks, weight 21-22 g, n = 48, 12 animals in each group). Two weeks after the ligation of the sciatic nerve melanoma B16/F10 was transplanted under the skin in the back of the main groups of animals (I- females, II-males); control groups were held the same procedure without ligation (III-females, IV-males). The levels of VEGFA, sVEGFR1, VEGFC, sVEGFR3 were determined in intact skin by ELISA. All manipulations were carried out in accordance with the European Convention for the Protection of Vertebrates Used for Experimental and Other Scientific Purposes (ETS 123). Results: Chronic pain contributed to the accumulation of VEGF-A, sVEGF-R1, VEGF-C in the skin and the reduction of sVEGF-R3 in all animals: in females - 2.7 times, 2.9 times, 6.1 times and 6.1 times, accordingly, in males - 3.1 times, 3.7 times, 3.5 times and 7.2 times, accordingly. Consequently, in males the amplitude of fluctuations of VEGF and their receptors in the skin against the background of pain was higher than in females, except for VEGF-C, the amount of which increased in females more than in males, despite its identical initial level in the skin of animals of both sexes. As a result, the skin of males with pain contained more VEGF-A, sVEGF-R1 and sVEGF-R3, accordingly, by 1.4 times (p < 0.05), 2.0 times and 1.9 times (p < 0.05) and less VEGF-C - by 1.8 times (p < 0.05). Conclusions: Chronic neuropathic pain had different effects on changes in the level of vascular endothelial growth factors and their receptors in intact skin in females and males of melanoma B16/F10 tumor carriers. The gender characteristics of the effect of chronic pain on neoangiogenesis may be associated with different rates of growth and development of the malignant process in male and female mice in the experiment.

The number of cancer stem cells in the tumor tissue and perifocal tissue of non-muscle invasive bladder cancer

Purpose of the study. Determine the content of cancer stem cells (CSCs) in the tumor tissue (TT) and perifocal tissues (PT) in muscle-non-invasive bladder cancer.Materials and methods. We’ve examined fragments of TT and PT of 7 muscle-non-invasive bladder cancer (NMIBC) after surgical intervention – transurethral resection of the urinary bladder (TUR). In tissue samples that were used to obtain cell suspension of TT and PT using the BD Medimachine apparatus (BD, USA) was treated with monoclonal antibodies CD45-APCCy7, CD44-FITC, CD133-РЕ, CD24-PE (BD, USA) and were assessed on flow cytometer FacsCantoII (BD, USA). The percentage of cells with CSC phenotypic markers was determined in the analysis sample: CD45-CD44+CD24+, CD45-CD44+, CD45-CD24+, CD45-CD133+, CD45-CD44+CD133+. The presence of significant differences in the groups was evaluated using the STATISTICA 13 software package and the differences between the samples were considered significant at p < 0.05. The percentage of cells of the corresponding phenotype was calculated relative to the total number of cells. The percentage of cells with the corresponding phenotype was calculated relative to the total number of cells.Results. The relative numbers of cells with CSC phenotypic markers, such as CD24, CD44, were 77 % and 58 % higher in TT than in PT: 18.3 ± 3.5 vs. 4.3 ± 2.1, p ≤ 0.044 and 15.5 ± 5.3 vs. 6.5 ± 0.8, p ≤ 0.043, respectively. The number of CD133+ cells was 83 % higher in PT compared to TT – 41.6 ± 12.1 vs. 22.7 ± 7.6, p ≤ 0.047.Conclusion. The study of CSCs is a promising direction for the study of oncogenesis and can be used to assess the nature of the further development of relapse and / or progression of the disease, as well as various therapeutic approaches that are aimed at eliminating with CSC phenotypic markers and blocking the pathways leading to the emergence and maintenance of this cell population in patients with NMIBC.

The impact of diabetes mellitus on levels of sex hormones and their receptors in tumor tissues in female rats with Guerin’s carcinoma

Purpose of the study. Analysis of the effect of DM on levels of sex hormones and their receptors in tumor and perifocal tissues in outbred white female rats.Materials and methods. Outbred white female rats were divided into 2 groups, 18 rats each: control group – with transplantable Guerin’s carcinoma, main group – with transplantable Guerin’s carcinoma growing in presence of DM. DM was reproduces in animals by the single intraperitoneal alloxan injection (150 mg/kg body weight). Animals were killed after 10 days of the tumor growth. Levels of hormones (estradiol, testosterone, progesterone and prolactin) and their receptors (RЕα, RЕβ, RA and RP4) were measured by ELISA in tumor and perifocal tissues in animals of the control and main groups.Results. Female rats with Guerin’s carcinoma growing in presence of DM showed an increase in glucose in tumor and perifocal tissues by 1.8 times (р < 0.05) and 7.6 times, respectively, compared to the levels in the control group. Levels of E2 were increased, respectively, by 2.4 and 9.5 times; P4 – by 2.1 and 3.0 times; PRL – by 2.7 and 4.4 times. T was increased in perifocal tissues by 2.2 times and was unchanged in tumor tissues. RЕα and RP4 were elevated by 3.9 and 3.0 times, respectively, only in tumor tissues, and RA by 3.9 times only in perifocal tissues. The REα/RЕβ ratio was increased only in the tumor by 3.2 times. The Е2/RЕβ ratio was increased in tumor and perifocal tissues by 2.0 and 9.6 times, respectively. The T/RA ratio was decreased in the tumor and its perifocal area by 1.4 (р < 0.05) and 2.0 times, respectively. The opposite changes were observed in the Е2/RЕα and P4/RP4 ratios: a decrease in the tumor by 1.6 and 1.4 times, respectively (р < 0.05), and an increase in the perifocal area by 9.8 and 2.5 times, respectively.Conclusion. Female rats with Guerin’s carcinoma growing in presence of DM demonstrated local hyperglycemia, changes in the levels of sex steroids and a misbalance in their receptors in tumor and perifocal tissues causing active metastasis and reduced average survival of animals.

Use of optical coherence tomography angiography in assessment in conjunctival vascular architecture in health and pathology

Investigation of the capabilities of anterior segment (AS) optical coherence tomography angiography (OCTA) in evaluation of conjunctival vascular architecture in healthy individuals and in various pathological conditions. The study included 17 healthy volunteers (34 eyes; the control group) and 62 patients (68 eyes) with conjunctival lesions of various nature. All participants underwent AS-OCT and AS-OCTA with assessment of qualitative (vessels pattern, lumen, pathologic tortuosity) and quantitative parameters (vessel density (VD, %) in the lesion area). Mean VD (MVD) and local VD (LVD) were determined, as well as VD in perifocal tissues (PVD). OCTA scans in 8 conjunctival sectors showed mostly radial pattern of the vascular architecture, with vessel lumen remaining the same over their entire visible length. Larger-sized vessels in deeper conjunctival layers were discovered in most cases. The lowest VD value (33.3%) was registered in the superotemporal quadrant, and the highest (38.9%) - in the nasal. Tortuosity of the vessels with course disruption, uneven lumen over the length of the vessels and increase in VD were observed in the area of conjunctival lesions in all cases excluding congenital abnormalities, pingueculae and conjunctival melanocytic intraepithelial neoplasia. The malignant nature of the tumors was indicated by dense vessel distribution and difficulties for visualization of intravascular space, and confirmed by pathohistological analysis. An increase in the number of areas with a lace-like pattern was characteristic for melanomas, with mean VD of more than 50% in the most vascularized areas. AS-OCTA is an informative method for the visualization of vessels in healthy conjunctiva and in conjunctival pathology. Local VD in the lesion area should be measured when the vessels are unevenly distributed.

Effect of diabetes mellitus on levels of insulin-like growth factors and their carrier proteins in Guerin’s carcinoma and it’s perifocal tissue in rats

Purpose of the study. Diabetes mellitus (DM) is considered an independent risk factor for higher cancer incidence and death rates. The system of insulin-like growth factors and their carrier proteins (IGF and IGFBP) and hyperglycemia create favorable conditions for the proliferation and metastasis of cancer cells.Materials and methods. Outbred male and female rats were divided into groups (n = 8 each): controls - with Guerin's carcinoma; main group - Guerin's carcinoma growing in presence of DM. Experimental DM was reproduces in animals by the single intraperitoneal alloxan injection (150 mg/kg body weight). After 10 days of the carcinoma growth, levels of IGF and IGFBP in the tumor and in it's perifocal area were measured using ELISA.Results. DM in females upregulated levels of glucose both in the tumor and in perifocal tissues by 1.8 (p < 0.05) and 8.1 times, respectively, but caused opposite changes in IGF-I - it's increase by 6.3 times in the tumor and decrease by 3.2 times in the perifocal area; as a result, such tumors with small primary nodes were more "aggressive" and actively metastasized. In males, induced DM downregulated levels of glucose, IGF-II and IGFBP2 in the carcinoma by 8.4, 3.1 and 1.7 (p < 0.05) times, respectively, and increased levels of IGF-I and IGFBP2 by 1.4 and 1.3 times (p < 0.05) in the perifocal area without changing glucose levels; as a result, tumor volumes exceeded the values in the standard growth, without metastasizing into visceral organs.Conclusion. We revealed gender differences in changing levels of glucose and IGF both in the tumor and in it's perifocal tissue in rats with Guerin's carcinoma growing in presence of DM; these differences could determine different tumor growth dynamics in male and female rats.

Effect of modified neoadjuvant chemotherapy on levels of E6 oncoprotein in HPV-infected cervical tumor tissue

. Background. Human papillomavirus (HPV) has been established to be the etiological factor of cervical cancer (CC). HPV infection and CC progression involve the direct participation of the E6 oncoprotein. Aim. An analysis of the E6 oncoprotein levels in tissues of the tumor and its perifocal area in HPV-associated cervical squamous cell cancer as an objective indicator of the effect of treatment depending on preoperative chemotherapy. Material and methods. The study included clinical and laboratory data of 237 patients with high-risk HPV infection of the cervix. The patients were divided into 4 groups: two main groups (CC T2а2–2bN0–1M0) and two control groups. Patients in the main group 1 (n=84) received standard neoadjuvant chemotherapy (NACT), in the main group 2 (n=93) — modified NACT with prior plasmapheresis session and a parallel course of nonspecific immunotherapy with Allokin-alpha. Control group 1 (n=40) included patients with CC T1b2–2a1N0–1M0, surgical treatment; control group 2 (n=20) — HPV-positive patients without CC. Levels of E6 were measured in samples of the cervical tumor and perifocal tissues. Results. The lowest levels of the E6 oncoprotein were registered in the group of HPV-positive patients without CC. After modified NACT, E6 levels in tumor tissues remained 4.6 times higher than in intact tissues, and even so, these patients demonstrated minimal E6 levels compared to other CC patients. E6 in tumor tissues was significantly lower than in main group 1 (by 3.3 times) and 8 times lower than in control group 1. E6 levels in the perifocal tissues of patients in main group 2 were 1.9 times lower than in the corresponding tissues of patients in main group 1 and 2.2 times lower than in control group 1. Conclusions. Inclusion of plasmapheresis and inducers of endogenous interferonogenesis into neoadjuvant treatment for cervical cancer can be considered pathogenetically justified, since it affects the key unit of cervical carcinogenesis.

Parameters of local cellular immunity in patients with bladder cancer.

e16508 Background: Bladder cancer (BC), which accounts for 4.5% of the total cancer incidence, is the cause of frequent severe disability and a significant deterioration in the quality of life of patients. BC development is determined by a number of factors, involving the immune system; however, its role is poorly understood. The purpose of the study was to determine characteristics of parameters of the cellular immunity in patients with non-muscle invasive BC. Methods: The study included 14 patients with newly diagnosed stage I BC. Patients were divided into two groups: with low-grade tumors (LG), n = 7, mean age 57 years; with high-grade tumors (HG), n = 6, mean age 60 years. All patients underwent transurethral resection of the bladder, and tumor tissues (TT, size up to 1.5 cm) and perifocal tissues (PT, at least 0.5 cm from the tumor) were obtained. The specimens were disintegrated to obtain the cell suspension. The relative numbers of the main populations of leukocytes and T- and B-lymphocytes, as well as some subpopulations of T-lymphocytes, were determined using the BD FACSCanto II flow cytometer and a panel of antibodies according to the manufacturer's instructions (Becton Dickinson, USA). Results: Reduction of lymphoid tissue infiltration by 78% was observed in TT in HG patients, compared to the LG group; levels of CD8+ lymphocytes, CD3+CD4-CD8- and CD3+CD4+CD8+ cells, NK- and B-lymphocytes were lower by 27%, 33%, 51%, 45% and 62%, respectively. Levels of Treg in HG TT were 32% lower than in LG. The numbers of activated CD4+, CD8+ cells were unchanged, but TT infiltration by naive lymphocytes of both populations was increased by 28% and 31%, respectively. In HG PT, a decrease in the total number of lymphocytes by 27% was observed, together with higher levels of CD3+CD4+CD8+ cells and NK- and B-lymphocytes by 200%, 213% and 31%, respectively. Conclusions: BC patients with various tumor grades demonstrate different distributions of lymphocyte populations in tumor and perifocal tissues; the functional activity of these cells can both contribute to the disease development and determine the risk of recurrence.

Cancer stem cells as markers of bladder cancer recurrence.

e16511 Background: Bladder cancer (BC) is an urgent problem of oncology in the world, and without appropriate and timely treatment it can lead to severe disability and a significant deterioration in the quality of life of patients. BC accounts for 4.5% of the total cancer incidence. Cancer stem cells (CSCs) are actively involved in the development of recurrent malignant tumors, and also play an important role in the development of chemotherapy and radioresistance of tumor cells. CSCs in BC are poorly studied. Our purpose was to determine the CSC numbers in patients with non-muscle invasive BC. Methods: The study included 7 patients with newly diagnosed stage I BC, G2, intermediate prognosis according to the EORTC scoring system. All patients underwent transurethral resection of the bladder, and tumor tissues (TT, size up to 1.5 cm) and perifocal tissues (PT, at least 1.0 cm from the tumor) were obtained. The specimens were disintegrated to obtain the cell suspension, and the CSC percentages were determined using the FACS Canto II flow cytometer with monoclonal antibodies to CD45-APC-Cy7, CD24-FITC, and CD133–PE according to the manufacturer's instructions (BD, USA). Numbers of cells with CSC markers (CD24+, CD44+, CD133+, CD24+CD44+, CD44+CD133+) were calculated as the percentage from the total number of CD45--cells. The results of statistical processing were presented as the arithmetic mean and the standard error of the arithmetic mean. The significance of differences between the samples was assessed using the Mann-Whitney’s nonparametric test. Results: The numbers of CD45--cells were similar in TT and PT (61.3±5.8 and 71.8±12.6). The relative numbers of cells with CSC phenotypic markers, such as CD24, CD44, were 77% and 58% higher in TT than in PT: 18.3±3.5 vs. 4.3±2.1 and 15.5±5.3 vs. 6.5±0.8, respectively; p≤0.05. The number of CD133+ cells was 83% higher in PT compared to TT - 41.6±12.1 vs. 22.7±7.6. The numbers of CD44+CD24+ and CD44+CD133+ cells in TT were higher than in PT by 80% and 63%, respectively: 10.3±4.9 vs. 2.1±0.4, 9.0±4.5 vs. 3.3±0.9, p≤0.05. CD44-CD24+ CD133+ cells were not detected in PT. Conclusions: The results indicate the peculiarities of the distribution of CSCs in TT and PT, which can be used to predict the risk of the disease recurrence and/or progression, and also help to evaluate results of the treatment.

Inhibitory effect of PAI-I in perifocal and resection line tissues on tumor growth in women with melanoma.

e22083 Background: The ability of malignant tumors to invasion and metastasis is associated with increased activity of the plasmin/plasminogen family proteases. The purpose of the study was to analyze changes in the plasmin/plasminogen system in tumors and adjacent tissues in patients with cutaneous melanoma in dependence on their gender. Methods: The study included 13 men and 22 women with cutaneous melanoma pT1-2N0M0. Levels of plasmin (PAP), content and activity of uPA, content and activity of tPA, content and activity of PAI were determined by ELISA in homogenates of melanoma (M), perifocal (P) and resection line (RL) tissues after surgical treatment. All patients gave a voluntary informed consent for the study. Skin tissues obtained from 20 non-cancer patients after plastic surgery served as the control. Results: The maximal PAP content in women was found in M – 16.5 times higher than in control, in RL – twice higher; PAP in all tissues of men was on average 7 times higher than in control. The content and activity of uPA and tPA in M were elevated in patients of both genders: in women by more than 3 times, in men by 4.9 and 3.7 times for uPA, respectively, and on average by 1.4 times for tPA. Increased content and activity of PAI-I were observed only in M of women by 74.6 and 2.3 times respectively, while PAI-I levels in men were similar to control values, and its activity was 2.9 times higher. Content and activity of PAI in P and RL tissues of men did not exceed the norm despite an increased activity of uPA and tPA, while in women they were elevated on average by 4 and 1.8 times, respectively. Conclusions: A local increase in the content and activity of the PAI-I plasminogen activator inhibitor in P and RL tissues only in women is a distinctive gender characteristics that can inhibit the spread of melanoma.

Local hormonal status of tumor and adjacent tissues in patients with melanoma depending on their gender.

e22084 Background: Gender is an important independent prognostic factor for cutaneous melanoma incidence, and survival in women is better. The purpose of the study was to determine the content of sex steroids and prolactin in tumor, perifocal and resection line tissues in male and female patients with melanoma. Methods: The study included 13 men and 22 women with cutaneous melanoma (M) pT1-2N0M0. Levels of free testosterone (fT), estradiol (E2), estrone (E1), progesterone and prolactin were measured by ELISA in M, perifocal (P) and resection line (RL) tissues after surgical treatment. The average age of patients was in men 54±3.56 years (median age 53, 32-81), in women 65±2.67 years (median age 63, 39-82). Skin tissues obtained from 20 non-cancer patients after plastic surgery served as the control. Results: In M of women, levels of fT, E2 and E1 were elevated on average by 1.3 times, while in men fT was decreased by 1.7 times, and estrogens were increased – E2 by 1.7 and E1 by 3.7 times, compared to the corresponding control levels. In women, fT in P tissues was increased by 1.6 times, while parameters in RL were similar to control values. In men, fT in P and RL tissues was decreased by 3.8 and 2.3 times respectively, and E2 was increased by 1.6 and 1.3 times respectively. E1 in P tissues was decreased by 1.7 times, in RL – similar to control levels. Conclusions: M in patients of both genders has an altered hormonal profile characterized by hyperestrogenia, with the prevalence of E1 and androgen deficiency in men. The expansion of the “tumor field” due to changes in the hormonal profile of P and even RL tissues was established only in male patients with the same morphological prevalence of the process, which may be one of the reasons for the more aggressive tumor course.

Influence of urokinase gene-knockout in C57BL/6-PlautmI. IBugThisPlau6FDhu/GFDhu mice on growth factors in malignant melanoma

Purpose of the study. Studying characteristics of the growth factor dynamics in the intact skin, tumors and perifocal tissues of melanoma in urokinase (uPA) gene-knockout mice.Materials and methods. The study included male and female С57 ВL/6 mice (n=47) and C57BL/6‑Plautm1.1BugThisPlauGFDhu/GFDhu mice with uPA gene-knockout (n=31). В16/F10 melanoma was transplanted subcutaneously at a dose of 0.5 mL (1:10 in normal saline). Intact mice of the same strain served as controls. Levels of VEGFA, VEGFC, sVEGFR1, sVEGFR3, IGF1, IGF2, TGFβ1 and FGF21 were determined by ELISA in the skin, tumor and perifocal tissues isolated on the 21st day of the tumor growth.Results. uPA gene-knockout inhibited the growth (mostly in females) and metastasis (predominantly in males) of melanoma in mice. Inhibition of the migration of malignant cells in males could be due to low levels of TGF-β1 compared to С57 ВL/6 mice: in the skin – by 5.0 times, in tumors – by 1.8 times and in perifocal tissues – by 6.1 times. In uPA gene-knockout females, lower levels of TGF-β1 were observed in tumors – by 1.4 times inhibited metastasis, but not completely, and solitary metastatic foci were registered in the lungs. Нigh levels of IGF1 in tissues of all uPA gene-knockout mice (males: in tumors by 1.4 times, in perifocal tissues by 2.6 times, in the skin by 3.6 times; females: in tumors by 2.6 times, in perifocal tissues by 25.0 times, in the skin by 13.9 times, compared to С57 ВL/6 mice) could maintain the metastatic phenotype of cancer cells (in females) or hiher proliferative activity of melanoma cells (in males). Lower levels of FGF‑21 in tumors (males – by 5.3 times, females – by 18.4 times), perifocal tissues (males – by 9.6 times, females – by 8,5 times) and skin (males – by 6.7 times, females – by 3.3 times) in uPA gene-knockout animals could be due to the IGF‑1 growth, as their reciprocal interaction is known. Interestingly, a significant, although lesser than in mice with a normal genotype, accumulation of VEGFA in melanoma tissues was observed: in males – in tumors by 44.9 times, in perifocal tissues by 6.8 times, in the skin by 2.4 times; in females – in tumors by 5.6 times, in perifocal tissues by 2.6 times, in the skin by 3.3 times, compared to the corresponding intact controls, due to the probable involvement of the uPA receptor (uPAR) in the implementation of VEGF-induced processes.Conclusion. Changing the activity of a system of some growth factors, uPA gene-knockout modifies melanoma metabolism by inhibiting its growth and eliminating or reducing its metastatic activity.

INFLUENCE OF CHRONIC NEUROGENIC PAIN ON LOCAL LEVELS OF SEX HORMONES AND RECEPTORS IN SKIN, TUMOR AND PERIFOCAL AREA IN MALE MICE WITH TRANSPLANTABLE В16/F10 MELANOMA

Local synthesis and metabolism of sex hormones allow the skin to regulate autonomously both nociceptive reactions and proliferative processes in various pathologies. T he purpose of the study was to reveal the influence of chronic neurogenic pain on the balance of sex hormones in the skin, tumor and perifocal area in male mice in the dynamics of В16/F10 melanoma growth. Material and methods. The study included С57ВL/6 male mice. Animals were divided into the main group – animals with В16/F10 melanoma transplanted after the development of chronic neurogenic pain (n=25), comparison group (n=27) – animals with standard tumor transplantation, controls – males with chronic neurogenic pain (n=8), and intact mice (n=8). Levels of estradiol (E2), estrone (E1), testosterone (T), progesterone (P4) (CUSABIO, China), estrogen receptors – ERα, ERβ (Сloud-Clone Corp. USA, China), progesterone (RP4) and androgens (RA) (CUSABIO, China) were determined in 10 % homogenates of skin, tumor and perifocal tissues 1, 2 and 3 weeks after В16/F10 melanoma transplantation using standard ELISA test systems adapted for experimental animals. R esults. Chronic neurogenic pain causes an imbalance of sex steroids with marked hyperestrogenism. The imbalance is achieved by increased content of estradiol and receptors of estrogen, increased concentration of progesterone with inhibition of its receptor apparatus, as well as reduced levels of testosterone with increased concentration of androgen receptors. Melanoma in animals with chronic neurogenic pain grows and develops in a target organ with an altered steroid balance, which leads to changes in the tumor characteristics. Chronic neurogenic pain and B16/F10 melanoma has a unidirectional effect on the local hormonal status of the skin.

Possibilities of ultrasound methods in diagnostics of eyelid tumors

To determine the informative value of ultrasound biomicroscopy (UBM) with A-scan for assessment of qualitative and quantitative characteristics of eyelid tumors less than 5 mm in size. The study included 25 patients (25 eyes) with eyelid tumors less than 5 mm in size. In addition to standard ophthalmic examination, complex ultrasound diagnostics including B-scan, Color Doppler imaging and UBM with A-scan were performed. The localization, size, structure of eyelid tumors and the state of perifocal tissues were evaluated. All patients underwent surgical treatment with following histological examinations of dissected tissues. Due to qualitative analysis of the studied formations and small number of included patients, there was no need in statistical analysis of the data. Complex application of UBM and A-scan allowed specifying the localization, size, structure of the small-sized tumors and detecting typical echographic signs of benign or malignant properties of the pathological process. Ultrasound data (UBM and A-scan) of eyelid tumors was highly correlated to histological features. UBM with A-scan can be recommended for differential diagnostics of small-sized tumors and optimizing their management.

The urokinase gene knockout effects on growth factor dynamics in mice with melanoma, developing on the background of chronic neurogenic pain

Purpose of the study . Studying the dynamics of growth factors (GF) in urokinase (uPA)-deficient mice with chronic neurogenic pain (CNP) and B16/F10 melanoma. Materials and methods . Levels of VEGFA, VEGFC, sVEGFRl, sVEGFR3, IGF1, IGF2, TGFp1 and FGF21 were determined by ELISA in tumors, perifocal tissues (PT) and the skin of male and female С 57 BL/6 mice (with a normal genome, n = 75) and C57BL/6-Plautm1.1BugThisPlauGFDhu/GFDhu mice (uPA-deficient animals, n = 46) at 3nd week of the carcinogenesis and CNP. Results . The skin of intact uPA-deficient mice demonstrated higher GF levels than in C57BL/6 mice, but VEGF- А and TGF-p1 in males (unlike females) were 4.4 and 5 times lower than in C57BL/6 males. This changes were generally similar in the skin of C57BL/6 mice with CNP. uPA-deficient females showed elevated GF in PT, especially VEGF- А and IGF1 — by 21.5 and 8.1 times, respectively in simultaneously CNP and growth of the melanoma. uPA gene-knockout males had similar changes in GF, although less marked. The levels of all studied GF in tumor tissue were lower than levels in PT in both males and females, except for VEGFA in males — 5.6 times higher in tumor tissue. Changes in PT of C57BL/6 mice were similar: maximally increased levels of all GF, especially VEGF, IGF and TGF-p1 — in females on average by 6.2, 15.9 and 5.5 times, respectively, in males by 9.4, 5.9 and 6.7 times, respectively, compared to the skin levels. While the absolute values of GF concentrations and the intensity of changes were higher than in uPA-deficient mice. Conclusion . In general, skin levels of GF in intact uPA-deficient mice were similar to the levels in mice without uPA-deficient with CNP. The GF dynamics was analogous in mice of both lines at simultaneously CNP and growth of the melanoma, but the intensity of changes in mice without uPA-deficient was significantly higher implying a synergic effect of CNP and paracrine influence of melanoma on the GF levels.