Macrophage-like phenotypes of tumor-immune hybrid cells in primary breast cancer

Aim: Tumor-immune hybrid cells (THCs) co-expressing leukocyte and epithelial markers arise from cell fusion and may contribute to cancer progression. However, the phenotypic heterogeneity of THCs, their association with macrophage lineage markers, and their clinical significance in breast cancer (BC) remain poorly characterized. Methods: Using multicolor flow cytometry, we analyzed primary breast tumors from 42 patients to characterize THCs expressing pan-leukocyte antigen cluster of differentiation 45 (CD45) together with epithelial markers epithelial cell adhesion molecule (EpCAM) and/or pan-cytokeratin. Expression of macrophage/monocyte-associated markers (CD68, CD163, CD16, CD206) was assessed across THC subpopulations. Results: THCs were heterogeneous, with CD45+EpCAM+panCK– and CD45+EpCAM+panCK+ subpopulations being most frequent, whereas CD45+EpCAM–panCK+ hybrids were rare. Within EpCAM+ THCs, a substantial proportion expressed M2‑like markers (CD163+, CD206+), indicating an immunosuppressive, pro‑tumor phenotype. The proportion of THCs carrying macrophage markers was increased in larger tumors (T2) and in node‑positive cases (N1–3). Patients with the luminal B [human epidermal growth factor receptor 2 (HER2)–] subtype—characterized by higher proliferative activity—showed a significant enrichment of hybrids expressing CD163 and CD206. Notably, specific rare THC subpopulations were associated with distant metastasis during follow-up: the CD68+CD163+CD16–CD206– phenotype within the CD45+EpCAM–panCK+ fraction and the CD68–CD163–CD16–CD206+ phenotype within the CD45+EpCAM+panCK+ fraction. The former, despite its very low frequency (cutoff 0.65%), showed the highest diagnostic accuracy (95.65%) for predicting poorer survival (p = 0.012). Conclusions: The predominance of M2-like THCs in more aggressive breast tumors, along with the identification of rare hybrid phenotypes associated with distant metastasis, underscores the clinical significance of these cells. Validation in larger cohorts is warranted to establish their utility as prognostic biomarkers and to elucidate their functional role in BC progression.