Most studies on CTCs have focused on isolating cells that express EpCAM. In this study, we emphasize the presence of EpCAM-negative and EpCAM^low CTCs, in addition to EpCAM^high CTCs, in early BC. We evaluated stem cell markers (CD44/CD24 and CD133) and EMT markers (N-cadherin) in each subpopulation. Our findings indicate that all stemness variants were present in both EpCAM^high and EpCAM-negative CTCs, whereas only one variant of stemness (nonCD44+CD24−/CD133+) was observed among EpCAM^low CTCs. Nearly all EpCAM^high CTCs were represented by CD133+ stem cells. Notably, the hybrid EMT phenotype was more prevalent among EpCAM-negative CTCs. scRNA-seq of isolated CTCs and primary tumor partially confirmed this pattern. Therefore, further investigation is imperative to elucidate the prognostic significance of EpCAM-negative and EpCAM^low CTCs.

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: 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.

Understanding subtype-specific variability of functional programs in FAP+ tumor-associated fibroblasts (TAFs) is fundamental for developing effective therapeutic strategies targeting stromal components. The aim of this study was to identify subtype-specific signaling pathways, markers, and molecular features of FAP+ TAFs. Using spatial transcriptomic analysis, we demonstrated that FAP+ TAFs in luminal breast cancer exhibit a phenotype characterized by extracellular matrix organization (GO:0030198, FDR q-value = 0.0307) and expression of genes associated with metastasis (COL10A1, MMP13, CXCL14, TSPAN8). In contrast, FAP+ TAFs in triple-negative breast cancer display a pronounced immunomodulatory phenotype with overexpression of immunosuppressive genes (CD36, PLA2G2A, CHI3L1) and enrichment of immune response-related pathways (immune response (GO:0006955, FDR q-value = 7.85e-17), inflammatory response (GO:0006954, FDR q-value = 2.79e-11), regulation of cytokine production (GO:0001817, FDR q-value = 3.39e-10)). We also identified subtype-specific gene signatures related to radioresistance: luminal A and B subtypes showed activation of DNA repair pathways (IGF1R, ERBB3, CRIP1), while triple-negative tumors demonstrated enrichment of epithelial-mesenchymal transition and stemness markers (ZEB2, NOTCH4, FOXM1). These findings emphasize that FAP+ fibroblasts are not a homogeneous population but functionally specialize depending on tumor subtype — acting as stromal architects in luminal breast cancer and as regulators of immune response in triple-negative breast cancer.

Objective : to characterize the transcriptomic features of CD68 ⁺ and CD163 ⁺ macrophage clusters within the tumor microenvironment of patients with triple-negative breast cancer (TNBC), depending on the PD-L1 status of the tumor. Material and Methods . Eleven patients with TNBC were included in the study. PD-L1 status was assessed by immunohistochemistry. Spatial transcriptomic profiling was performed using the Visium 10x Genomics platform. Cluster annotation for CD68 ⁺ and CD163 ⁺ cells was carried out in Loupe Browser 8.0.0. Differential gene expression and pathway enrichment analysis were performed for CD68 ⁺ and CD163 ⁺ clusters in PD-L1-positive and PD-L1-negative groups. Results. PD-L1-positive tumors showed compensatory recruitment of immune cells with features of functional exhaustion, in which CD68 ⁺ macrophages were involved. In contrast, PD-L1-negative tumors were characterized by an active adaptive immune response, with CD68 ⁺ macrophages functioning as antigen-presenting cells, together with evidence of negative regulation of inflammation associated with CD163 ⁺ macrophages. Conclusion. The functional state of macrophages and other cellular components of the TNBC microenvironment varies significantly with PD-L1 status. These results may contribute to the identification of biomarkers for predicting immunotherapy response in this patient group.

Aim: The aim was to determine the transcriptomic features of tumor and stromal cells in direct contact within tumor nodules in luminal and triple-negative breast cancer. Methods: Spatial transcriptomic profiling was performed using the Visium 10x platform on FFPE tumor tissue sections from 10 patients with luminal breast cancer and 9 patients with triple-negative breast cancer. Manual morphological annotation of spots and evaluation of differentially expressed genes (DEGs) in identified spot clusters were performed using Loupe Browser v8.0.0 (10X Genomics, USA). Activated biological processes were assessed using the Enrichr online resource and the GO Biological Process 2025 database. Ligand-receptor pairs were identified using the CellChat package (v2.0) in R (v4.4.2). Results: In luminal breast cancer, mixed cluster (tumor cells colocalized with stromal cells) was characterized by overexpression of genes encoding S100A family Ca2+-binding proteins (S100A4, S100A8, S100A9), matrix metalloproteinases (MMP2, MMP7, MMP14), cytokeratins (KRT5, KRT7, KRT15, KRT23, KRT81), the mesenchymal marker VIM, and epithelial-mesenchymal transition (EMT)-associated genes (ICAM1, PRRX1) compared to tumor-only cluster. In triple-negative breast cancer, mixed cluster showed overexpression of S100A2, S100A8, S100A9, the epithelial gene KRT6B, the cancer stem cell marker CD44, and NOTCH2, which is associated with negative regulation of EMT. In both breast cancer subtypes, mixed cluster showed transcriptomic enrichment of gene sets associated with regulation of the ERK/MAPK cascade, apoptosis, and cell adhesion and migration. Ligand-receptor pairs associated with cell-cell contact, EMT, and immune response were also detected in colocalized cells, with a broader spectrum of these pairs observed in luminal breast cancer. Conclusions: This study assessed the transcriptomic characteristics of directly contacting tumor and stromal cells and identified the spectrum of ligand-receptor pairs mediating their interactions. Characterizing the properties of cells at the tumor-stroma interface helps unravel mechanisms of breast cancer progression and identify novel diagnostic markers and therapeutic targets.