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.

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.

BACKGROUND: The spatial organization of immune cells within tumors is an important area of investigation in studies of interactions between tumor cells and the tumor microenvironment. This is particularly relevant because some immune cells function through direct contact with their target cells, whereas others communicate over a distance via paracrine signaling involving cytokines. Thus, the topography of tumor cells and microenvironmental cells may determine the possibility and nature of intercellular interactions and thereby influence the functional effects of immune cells. AIM: This study aimed to compare the spatial transcriptomic profiles of tumor and stromal regions enriched in macrophages in triple-negative breast cancer. METHODS: Eight patients with triple-negative breast cancer were included. Spatial transcriptomic analysis was performed on formalin-fixed, paraffin-embedded tissue sections using high-throughput RNA sequencing with the 10X Visium platform. The annotated spots enriched in intraepithelial and stromal macrophages were used for downstream bioinformatic analysis. RESULTS: A total of 437 differentially expressed genes were identified between the two groups of spots containing macrophages with distinct spatial localization. Spots with intraepithelial macrophages were characterized by activation of processes related to cytokine and chemokine signaling, regulation of regulatory T-cell differentiation, organization of cell–cell contacts, wound healing, and inhibition of viral activity. Spots enriched in stromal macrophages demonstrated activation of biological processes associated with the regulation of angiogenesis, cell migration and recruitment, cell adhesion, and stromal remodeling. CONCLUSION: Macrophage topography within primary tumors of triple-negative breast cancer is associated with their functional characteristics. These fundamental findings may be useful for developing prognostic criteria and therapeutic approaches aimed at modulating the tumor microenvironment to improve long-term outcomes in patients with triple-negative breast cancer.

Objective. To study the microenvironment features of MMR-proficient (pMMR) and MMR-deficient (dMMR) endometrial cancer. Material and methods. The study included 34 patients with pMMR endometrial cancer and 10 patients with dMMR endometrial cancer. Using the method of multiplex TSA-associated (tyramide signal amplification) immunofluorescence, phenotyping of the tumor microenvironment was performed with an assessment of stromal and intratumor cell localization and PD-1 expression. Results. In pMMR endometrial tumors, the predominant cell population was CD163+ macrophages, and in tumors with dMMR, CD163+ macrophages were found equally with CD8+ T lymphocytes and CD20+ B lymphocytes. In dMMR tumors, the number of CD8+ T lymphocytes and CD20+ B lymphocytes (as well as CD20+ B lymphocytes expressing PD-1) is higher compared to pMMR tumors, while the number of FoxP3+ T lymphocytes was significantly lower. The number of CD163+ macrophages did not differ depending on the MMR status, while the number of CD163+ macrophages was higher in the stroma compared to the number of these cells located intraepithelially. In patients with dMMR tumors, the number of CD8+ T lymphocytes was higher in the stroma, and did not differ in pMMR tumors. Conclusion. In this study, it was found that dMMR endometrial tumors are characterized by an increase in the proportion of CD8+ T lymphocytes and CD20+ B lymphocytes, which may be associated with a better response to immunotherapy. The differences in spatial distribution of CD8+ T lymphocytes and CD163+ macrophages confirmed the importance of immune cell localization for prognosis and treatment efficacy. These results highlight the need for further study of the endometrial cancer microenvironment in order to personalize therapy.

Objective. To evaluate the impact of combined anti-PD-1 immunotherapy on the cellular composition of the tumor microenvironment in patients with gastric cancer. Material and methods. The study included 9 patients with morphologically confirmed gastric adenocarcinoma (stages T2-4N0-1M0) and positive PD-L1 status (CPS >1). All patients received 8 courses of preoperative chemotherapy according to the FLOT regimen, combined with additional immunotherapy using pembrolizumab (400 mg every 6 weeks). Changes in the tumor microenvironment were assessed using multicolor immunofluorescence followed by quantitative analysis of the proportions of CD8+ cytotoxic T-lymphocytes, CD20+ B-lymphocytes, CD163+ macrophages, and FoxP3+ regulatory T-cells. Results. A statistically significant increase in the number of CD8+ T-lymphocytes (from 3.18% to 6.23%, p=0.0391) and, in particular, their PD-1-expressing subpopulation (from 0.00% to 0.22%, p=0.0156) was observed, while the numbers of CD20+, CD163+, and FoxP3+ cells remained unchanged. As a result, the CD8+/FoxP3+ cell ratio significantly increased during combined anti-PD-1 immunotherapy (from 0.69 to 3.22, p=0.0039). Additionally, associations were identified between immune parameters and clinicopathological characteristics, suggesting that gender and tumor stage may influence the immune response. Conclusion. The obtained data indicate a remodeling of the tumor microenvironment under the influence of anti-PD-1 therapy, which potentially enhances the antitumor immune response and opens prospects for further optimization of gastric cancer treatment.