Iliya P. Romanov

Progesterone receptors are involved in the regulation of tumor cell proliferation and therefore can be considered as a target of antitumor drugs. However,

Background Progesterone receptors (PRs) play a role in the regulation of cell proliferation and are expressed in non-small cell lung cancer (NSCLC) tissue. Therefore, they represent a potential target for novel antitumor therapies. A survival analysis of NSCLC patients based on PR expression in tumor tissue may help assess the feasibility of using PR modulators in the treatment of this disease. Aim This study aims to evaluate the prognostic significance of PR expression in NSCLC to determine the potential utility of PR modulators as a therapeutic strategy. Methods PR expression was assessed in 130 surgically resected NSCLC samples using immunofluorescence analysis combined with flow cytometry. Primary antibodies against PR (NBP2-4638, Novus Biologicals, USA) and secondary antibodies conjugated with DyLight650 (ab98729, Abcam, UK) were used. The percentage of PR-expressing cells was quantified using FlowJo software. Statistical analyses were conducted in GraphPad Prism and RStudio using the “survival” package. The prognostic impact of PR expression in NSCLC tissue was evaluated in the overall patient cohort and after excluding censored events (n = 56) to minimize the influence of confounding factors on survival analysis. Results After excluding censored events and stratifying patients based on the median PR expression level (57%), survival analysis revealed that high PR expression in NSCLC tissue is associated with a poorer prognosis (p = 0.05). Patients with high PR expression (≥ 57%) had a median survival of 12.8 months, whereas those with low PR expression (< 57%) had a median survival of 25.8 months (HR = 1.7). Conclusions Elevated PR expression in NSCLC tumors is associated with reduced patient survival. These findings suggest that PR modulators may have potential therapeutic value for NSCLC patients with PR-positive tumors.

Background. The cytoskeletal protein β-tubulin class III (Tubb3) is associated with tumor resistance to taxanes and vinca alkaloids, as well as with the metastatic potential of neoplasm, however, data from immunohistochemical analysis of Tubb3 expression in non-small cell lung cancer (NSCLC) tissue are few and contradictory. Purpose. Characterization of the level and intensity of Tubb3 expression in NSCLC tissue and analysis of the identified parameters correlation with clinically significant characteristics of the disease. Methods. Quantitative assessment of the level and intensity of Tubb3 expression in 120 surgical samples of NSCLC was carried out by immunofluorescence method associated with flow cytometry. Primary rabbit monoclonal antibodies specific to Tubb3 and secondary anti-rabbit antibodies conjugated with fluorescent dye DyLight650 (ab98510, UK) were used. The expression of the marker was assessed by two parameters: the level of expression measured as the percentage of the cells expressing Tubb3 and the intensity of expression in conventional units (CU) represented as the ratio of the geometric mean fluorescence intensity in the experimental and control samples (cells incubated with secondary antibodies only). Results. 1. Tubb3 expression was detected in all NSCLC samples studied. The median level and intensity of Tubb3 expression was 30.5% and 2.0 CU with significant differences (up to 10 times) in the quantitative values of both parameters in different patients. 2. The distribution of the studied tumors in terms of the level and intensity of Tubb3 expression differs from normal (P<0.001), the associative relationship between the assessed parameters is very strong (Spearman's rank correlation coefficient was 0.91; P<0.0001). 3. Statistical analysis did not reveal correlations between the level of Tubb3 expression and the gender and smoking status of the patients, with the degree of tumor differentiation, as well as with the stage of NSCLC. 4. In the group of lung adenocarcinomas, the median level of Tubb3 expression is higher compared with squamous cell lung cancer in male and female patients (P=0.01). Conclusion. High heterogeneity of Tubb3 expression level in NSCLC tissue in the patients and differences in the parameters between the tumors of various histotypes indicate the importance of further correlation analysis of Tubb3 expression level with the patients' life span in order to identify the prognostic value of the marker.

Quantitative assessment of estrogen receptors alpha (ERα) expression was carried out in 115 samples of non-small cell lung cancer (NSCLC) by an immuno uorescent assay and ow cytometry. It has been shown that high level of ERα ≥20% predicts higher aggressiveness of NSCLC than at low level <20%: median survival at 78 mos of follow-up is increased by 1.5 times; the risk of death is reduced by almost 2.0 times (p = 0.04). Time to death was increased by an average of 18 mos in about 20% of patients with low ERα expression. The results validate informative value of immuno uorescence analysis and owcytometry for quantifying the ERα expression and substantiate the prospects of antiestrogen therapy as a new option for NSCLC treatment, in particular, by analogy with breast cancer - in a long-term adjuvant therapy in ERα+ NSCLC patients.

Background. Progesterone receptors (PR) are regulators of cell proliferation and therefore can be considered as an aim for targeted medications in the treatment of oncological diseases. At the same time, a quantitative assessment of PR expression in the tissue of non-small cell lung cancer (NSCLC), which has not yet been carried out in other studies, will determine the possibility of using PR modulators for the treatment of this disease and identify the potential category of patients most susceptible to these drugs.Purpose. To characterize NSCLC by quantitative indicators of PR expression and to determine the correlation of clinically significant characteristics of patients and clinical and morphological parameters of a NSCLC tumor with the PR expression to assess the possibility of using PR modulators in the treatment of this disease.Methods. The PR expression in 130 surgical samples of NSCLC was quantified using an immunofluorescence method associated with flow cytometry. Primary antibodies to PR (NBP2-46388) and secondary antibodies conjugated with DyLight650 (ab98729) were used.Results. The expression of progesterone receptors was detected in all the studied tumors; an abnormal distribution of the marker expression level was noted (P=0.01). The mean expression level was 55.3±16.2%, and the median was 57% with a range of 70%, which indicates heterogeneity of PR expression in tumors of different patients. There were no statistically significant differences in the level of PR expression depending on the histotype and stage of NSCLC, as well as on the sex of patients. At the same time, the level of expression and the frequency of overexpression of PR (>67%) in tumors in non-smoking patients are higher than in smokers (P⩽0.02).Conclusion. The high frequency of occurrence and level of PR expression in NSCLC indicate the possible effectiveness of the use of their modulators in the treatment of this disease, especially in non-smoking patients.

Pulmonary fibrosis is a common and threatening post-COVID-19 complication with poorly resolved molecular mechanisms and no established treatment. The plasminogen activator system, including urokinase (uPA) and urokinase receptor (uPAR), is involved in the pathogenesis of COVID-19 and contributes to the development of lung injury and post-COVID-19 pulmonary fibrosis, although their cellular and molecular underpinnings still remain obscure. The aim of the current study was to assess the role of uPA and uPAR in the pathogenesis of pulmonary fibrosis. We analyzed uPA and uPAR expression in human lung tissues from COVID-19 patients with pulmonary fibrosis using single-cell RNA-seq and immunohistochemistry. We modeled lung fibrosis in Plau-/- and Plaur-/- mice upon bleomycin instillation and explored the effect of uPAR downregulation in A549 and BEAS-2B lung epithelial cells. We found that uPAR expression drastically decreased in the epithelial airway basal cells and monocyte/macrophage cells, whereas uPA accumulation significantly increased in tissue samples of COVID-19 patients. Lung injury and fibrosis in Plaur-/- vs. WT mice upon bleomycin instillation revealed that uPAR deficiency resulted in pro-fibrogenic uPA accumulation, IL-6 and ACE2 upregulation in lung tissues and was associated with severe fibrosis, weight loss and poor survival. uPAR downregulation in A549 and BEAS-2B was linked to an increased N-cadherin expression, indicating the onset of epithelial–mesenchymal transition and potentially contributing to pulmonary fibrosis. Here for the first time, we demonstrate that plasminogen treatment reversed lung fibrosis in Plaur-/- mice: the intravenous injection of 1 mg of plasminogen on the 21st day of bleomycin-induced fibrosis resulted in a more than a two-fold decrease in the area of lung fibrosis as compared to non-treated mice as evaluated by the 42nd day. The expression and function of the plasminogen activator system are dysregulated upon COVID-19 infection, leading to excessive pulmonary fibrosis and worsening the prognosis. The potential of plasminogen as a life-saving treatment for non-resolving post-COVID-19 pulmonary fibrosis warrants further investigation.