Elena Shmakova

Trophoblast cell surface antigen 2 (TROP2) has emerged as a promising therapeutic target in non-small cell lung cancer (NSCLC) in the context of the development of antibody-drug conjugates. In this setting, reproducible and biologically meaningful immunohistochemical assessment of TROP2 expression, taking into account its intracellular localization, is of particular importance. Objective. To characterize TROP2 expression in lung adenocarcinomas and squamous cell carcinomas and to evaluate its association with clinicopathological parameters using different immunohistochemical assessment methods. Material and methods. This retrospective single-center study included 60 cases of NSCLC (30 adenocarcinomas and 30 squamous cell carcinomas) diagnosed between 2019 and 2023. TROP2 expression was evaluated by immunohistochemistry using the semi-quantitative H-score and the normalized membrane ratio (NMR), reflecting the relative contribution of membranous and cytoplasmic expression. Associations between TROP2 expression and clinicopathological characteristics were analyzed. Results. TROP2 expression was detected in all examined NSCLC samples. TROP2 expression assessed by H-score was significantly higher in squamous cell carcinoma compared with lung adenocarcinoma (p=0.0001), whereas NMR values did not differ between histological subtypes. No associations were observed between H-score and clinicopathological parameters. In contrast, NMR demonstrated histotype-dependent associations: with tumor stage in lung adenocarcinoma (p=0.0483) and with lymphovascular invasion in squamous cell carcinoma (p=0.0139). The proportion of cases meeting the NMR-based criteria for TROP2 positivity was low. Conclusion. The results indicate that the choice of TROP2 assessment method substantially influences data interpretation. Consideration of intracellular protein localization using the NMR appears to be more informative than conventional semi-quantitative approaches and may reflect biological features of tumor progression in NSCLC.

Background: Fibroblast activation protein (FAP) is a promising target for cancer diagnosis and therapy, but the specificity and sensitivity of anti-FAP antibodies used for immunohistochemistry (IHC) vary considerably. A head-to-head comparison of commercially available clones is needed to guide antibody selection, thereby standardizing FAP IHC protocols and ensuring reliable patient stratification for FAP-directed therapies. Methods: We evaluated three anti-FAP clones—FAP/4851 (mouse monoclonal), JA56-11 (recombinant rabbit), and EPR20021 (rabbit monoclonal)—in formalin-fixed, paraffin-embedded tissues from 137 patients with breast (n = 29), colorectal (n = 27), lung (n = 42), and head and neck squamous cell carcinoma (n = 39). FAP expression was assessed by manual semiquantitative scoring (H-score and percentage of positive cells) and, for a validation subset, by QuPath-based digital image analysis. The study included negative (normal liver/skeletal muscle) and positive (WI-38 cell block) controls. Results: Clones JA56-11 and EPR20021 showed specific stromal staining, whereas FAP/4851 exhibited intense non-specific reactivity and was excluded from quantitative analysis. EPR20021 demonstrated significantly higher H-scores than JA56-11 in all four cancer types (Wilcoxon test, p ≤ 0.0179), resulting in a higher overall detection rate under the tested conditions. Aberrant tumor cell expression was detected almost exclusively by EPR20021 in head and neck (59% of cases) and colorectal (59%) cancers. QuPath validation showed good correlation with manual scoring for EPR20021 in the tumor compartment of lung cancer (Spearman’s ρ = 0.701, p = 0.024) with small bias (Bland–Altman). Conclusions: Under the conditions tested, EPR20021 (Abcam) demonstrated higher staining intensity and detection rates than JA56-11 across several tumor types. Clone FAP/4851 showed nonspecific staining under the conditions used in this study and was therefore not suitable for quantitative analysis in the present experimental setting. Consequently, careful selection of well-validated antibody clones is essential for ensuring reproducible and reliable FAP immunohistochemical analysis.

Immunotherapy is a promising treatment strategy for treating colorectal cancer (CRC). Despite significant advances in this field, resistance and low efficacy of immunotherapy remain a principal problem. One of the most important factors affecting the response to immunotherapy is the tumor microenvironment (TME). Among the components of the TME, tumor-associated macrophages (TAMs) are key immune cells involved in cancer progression by stimulating tumor cell proliferation, angiogenesis, epithelial-mesenchymal transition, metastasis, and tumor immune evasion. This review presents currently investigated combination therapy based on the immune checkpoint inhibitors and inhibitors of diverse components of the TME, including TAMs, that can potentially increase the effectiveness of CRC treatment. Therapeutic efficacy, together with the functional activity of TAMs, is estimated in multiple preclinical data obtained with diverse in vitro and in vivo models. Ongoing clinical trials demonstrated the association of treatment effectiveness with TAM phenotypes and functions.

Colorectal cancer is the third most common cancer worldwide, making lymph node recovery critical for treatment decisions and prognosis. Within the colorectal tumor microenvironment, the metabolic programming of tumor-associated macrophages (TAMs) can drive both pro- and anti-tumor responses, yet the specific glycolytic pathways governing their pro-metastatic conversion present promising therapeutic targets. This study investigated the role of glycolysis activating enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in mediating TAMs metabolic polarization, and its potential as a therapeutic target. PFKFB3 expression was found to be predominant in TAMs in CRC tumor samples. Lipidomic analysis performed by HPLC-MS/MS revealed that PFKFB3 inhibition altered glycerophospholipid metabolism (p = 6.13 × 10⁻¹⁰), and shifted TAMs toward sphingolipid-mediated immunosuppressive metabolism. PFKFB3 activity was associated with a specific reduction in asparagine availability, potentially pointing to a targeted reprogramming of amino acid metabolism supporting distinct TAM functions under conditions of intra-tumoral metabolic stress. These findings highlight PFKFB3 as an essential regulator of TAMs pro-tumoral metabolism in CRC, particularly in colon cancer.

Type 2 diabetes mellitus (T2DM) is characterized by an uncontrolled increase in blood glucose levels and insulin resistance in cells of various tissues. Vascular complications in T2DM have an inflammatory nature. Drugs with different mechanisms of action have been developed and used to treat T2DM, initially aimed at controlling blood glucose levels. Among them, sodium-glucose cotransporter 2 inhibitors (SGLT2-i) were developed as specific inhibitors of glucose reabsorption in the kidneys, but along with lowering blood glucose levels, they demonstrated multiple (including non-glycemic) positive effects in the treatment of T2DM related to their beneficial effects on the immune system. SGLT2 inhibitors can reduce the risk of diabetic cardiomyopathy (DCM) and chronic kidney disease (CKD) development in patients with and without diabetes. SGLT2-is improve cardio-renal complications through a number of signaling pathways, including those dependent on the involvement of non-coding RNAs (ncRNAs) and their targets. The best-studied classes of ncRNAs are microRNAs, which are short (less than 200 bases) RNAs (miRNAs), long non-coding RNAs (lncRNAs) (more than 200 bases), and circular RNAs (circRNAs). The regulatory effect of ncRNAs has broad physiological significance, and changes in the ncRNAs’ expression are associated with the pathogenesis of different diseases, including T2DM. RNA-seq allows the construction of networks of interactions of lncRNA/circRNA-miRNA-mRNA called competitive endogenous RNA (ceRNA) networks, to identify clinically significant molecular markers, to improve the mechanistic understanding of pathogenesis, and to contribute to the development of new diagnostics and therapies. Our review summarizes the role of non-coding RNA in the action of SGLT2 inhibitors in cardio-renal complications in T2DM. We focus on methods of detection, genetics, and the effects of non-coding RNA. Specific attention is given to the role of non-coding RNAs in the inflammatory reactions of innate immune cells in relation to the SGLT2 inhibitors.

Colorectal cancer is the third most common cancer worldwide, making lymph node re-covery critical for treatment decisions and prognosis. Within the colorectal tumor mi-croenvironment, the metabolic programming of tumor-associated macrophages (TAMs) can drive both pro- and anti-tumor responses, yet the specific glycolytic pathways gov-erning their pro-metastatic conversion present promising therapeutic targets. This study investigated the role of glycolysis activating enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) in mediating TAM metabolic polarization, and its potential as a therapeutic target. PFKFB3 expression was found to be predominant in tumor-associated macrophages in CRC human tumor sam-ples. Lipidomic analysis performed by HPLC-MS/MS revealed that PFKFB3 inhibition altered glycerophospholipid metabolism (p=6.13E-10), and shifted TAMs toward sphingolipid-mediated immunosuppressive metabolism. PFKFB3 activity was associated with a specific reduction in asparagine availability, potentially pointing to a targeted reprogramming of amino acid metabolism supporting distinct macrophage functions under conditions of intra-tumoral metabolic stress. These findings highlight PFKFB3 as an essential metabolic regulator of TAMs pro-tumoral activity in CRC, particularly in colon cancer.

Type 2 diabetes mellitus (T2DM) is characterized by uncontrolled increase in blood glucose levels, insulin resistance and metabolic inflammation. Vascular complication in T2DM have the inflammatory nature. Drugs with different mechanisms of action have been developed and used to treat T2DM, initially aimed to control blood glucose levels. Among them sodium-glucose cotransporter 2 inhibitors (SGLT2-i) were developed as specific inhibitors of glucose reabsorption in the kidneys, but along with lowering blood glucose levels, they demonstrated multiple (including non-glycemic) positive effects in the treatment of T2DM related to their beneficial effects on the immune system. SGLT2 inhibitors can reduce the risk of diabetic cardiomyopathy (DCM) and chronic kidney disease (CKD) development in patients with and without diabetes. SGLT2-is improve cardio-renal complications through a number of signalling pathways, including those dependent on the involvement of non-coding RNAs (ncRNAs) and their targets. The best-studied classes of ncRNAs are microRNAs, which are short (are less than 200 bases) RNAs (miRNAs), long non-coding RNAs (lncRNAs) (are more than 200 bases), and circular RNAs (circRNAs). The regulatory effect of ncRNAs has broad physiological significance, and changes in the ncRNAs expression are associated with the pathogenesis of different diseases including T2DM. RNA-seq allowed to construct networks of interactions of lncRNA/circRNA-miRNA-mRNA called competitive endogenous RNA (ceRNA) networks, to identify clinically significant molecular markers, to improve mechanistic understanding of pathogenesis, and to contribute to the development of new diagnostics and therapies. Our review summarizes the role of non-coding RNA in the action of SGLT2 inhibitors in cardio-renal complications in T2DM. We focus on methods of detection, genetics and mechanism of action of non-coding RNA. Specific attention is given to the role of non-coding RNAs in the inflammatory reactions of innate immune cells in relation to the SGLT2 inhibitors.

Background/Objectives: In gastric cancer, only a subset of patients benefit clinically from neoadjuvant chemoimmunotherapy, underscoring the need for robust biomarkers that can predict treatment responses and guide personalized immunotherapy. This study aimed to characterize the immune microenvironment of gastric tumors and identify predictive markers associated with therapeutic efficacy. Methods: We prospectively enrolled 16 patients with histologically confirmed, PD-L1–positive (CPS ≥ 1) gastric adenocarcinoma (T_2–4N_0–1M₀). All patients received eight cycles of FLOT chemotherapy combined with pembrolizumab. Treatment response was assessed by Mandard tumor regression grading. Spatial transcriptomic profiling (10x Genomics Visium) and multiplex immunofluorescence were used to evaluate tumor-infiltrating immune cell subsets and PD-1 expression at baseline and after treatment. Results: Transcriptomic analysis differentiated the immune landscapes of responders from non-responders. Responders exhibited elevated expression of IL1B, CXCL5, HMGB1, and IFNGR2, indicative of an inflamed tumor microenvironment and type I/II interferon signaling. In contrast, non-responders demonstrated upregulation of immunosuppressive genes such as LGALS3, IDO1, and CD55, along with enrichment in oxidative phosphorylation and antigen presentation pathways. Multiplex immunofluorescence confirmed a higher density of FoxP3⁺ regulatory T cells in non-responders (median 5.36% vs. 2.41%; p = 0.0032). Notably, PD-1⁺ CD8⁺ T cell and PD-1⁺ FoxP3⁺ Treg frequencies were significantly elevated in non-responders, suggesting that PD-1 expression within cytotoxic and regulatory compartments may contribute to immune evasion. No substantial differences were observed in PD-L1 CPS or PD-1⁺ B cells and PD-1⁺ macrophages. Conclusions: Our findings identify PD-1⁺ CD8⁺ T cells and PD-1⁺ FoxP3⁺ Tregs as potential biomarkers of resistance to neoadjuvant chemoimmunotherapy in gastric cancer. Transcriptional programs centered on IL1B/CXCL5 and LGALS3/IDO1 define distinct immune phenotypes that may guide future combination strategies targeting both effector and suppressive arms of the tumor immune response.

A growing body of evidence indicates that nonglycemic effects of sodium–glucose cotransporter 2 (SGLT2) inhibitors play an important role in the protective effects of these drugs in diabetes, chronic kidney disease, and heart failure. In recent years, the anti-inflammatory potential of SGLT2 inhibitors has been actively studied. This review summarizes results of clinical and experimental studies on the anti-inflammatory activity of SGLT2 inhibitors, with a special focus on their effects on macrophages, key drivers of metabolic inflammation. In patients with type 2 diabetes, therapy with SGLT2 inhibitors reduces levels of inflammatory mediators. In diabetic and non-diabetic animal models, SGLT2 inhibitors control low-grade inflammation by suppressing inflammatory activation of tissue macrophages, recruitment of monocytes from the bloodstream, and macrophage polarization towards the M1 phenotype. The molecular mechanisms of the effects of SGLT2 inhibitors on macrophages include an attenuation of inflammasome activity and inhibition of the TLR4/NF-κB pathway, as well as modulation of other signaling pathways (AMPK, PI3K/Akt, ERK 1/2-MAPK, and JAKs/STAT). The review discusses the state-of-the-art concepts and prospects of further investigations that are needed to obtain a deeper insight into the mechanisms underlying the effects of SGLT2 inhibitors on the molecular, cellular, and physiological levels.