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The present study constitutes an investigation into the interconnection between tumor, endothelial and stromal cells in heterotypic breast cancer spheroids. The generation of models was achieved through the utilization of MCF7, MDA-MB-231, and SK-BR-3 tumor cell lines, in conjunction with endothelial TIME-RFP cells and either cancer-associated (BrC4f) or normal (BN120f) fibroblasts, within ultra-low attachment plates. It was established that stromal cells, most notably fibroblasts, were conducive to the aggregation of tumor cells into spheroids and the formation of pseudovessels in close proximity to fibroblast bands. In contrast to the more aggressive tumor models MDA-MB-231 and SK-BR-3, microenvironment cells do not influence the migration ability of MCF7 tumor cells. Heterotypic spheroids incorporating CAFs demonstrated a more aggressive and immunosuppressive phenotype. Multiplex immunoassay analysis of cytokines, followed by STRING cluster analysis, was used to identify key processes including angiogenesis, invasion, stem cell maintenance, and immunosuppression. Furthermore, a cluster of cytokines (LIF, SDF-1, HGF, SCGFb) was identified as potentially involved in the regulation of PD-L1 expression by tumor cells. This finding reveals a potential mechanism of immune evasion and suggests new avenues for therapeutic investigation.<\/jats:p>","DOI":"10.3390\/cells15020145","type":"journal-article","created":{"date-parts":[[2026,1,14]],"date-time":"2026-01-14T11:01:14Z","timestamp":1768388474000},"page":"145","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Heterotypic 3D Model of Breast Cancer Based on Tumor, Stromal and Endothelial Cells: Cytokines Interaction in the Tumor Microenvironment"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0005-1477-1118","authenticated-orcid":false,"given":"Anastasia","family":"Leonteva","sequence":"first","affiliation":[{"name":"Scientific Center of Genetics and Life Sciences, Sirius University of Science and Technology, 1 Olimpiysky Avenue, Krasnodar Region, Sirius 354340, Russia"},{"name":"Institute of Chemical Biology and Fundamental Medicine Siberian Branch of the Russian Academy of Sciences, Akad. Lavrentiev Ave. 8, Novosibirsk 630090, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alina","family":"Kazakova","sequence":"additional","affiliation":[{"name":"Scientific Center of Genetics and Life Sciences, Sirius University of Science and Technology, 1 Olimpiysky Avenue, Krasnodar Region, Sirius 354340, Russia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ekaterina","family":"Berezutskaya","sequence":"additional","affiliation":[{"name":"Institute of Chemical Biology and Fundamental Medicine Siberian Branch of the Russian Academy of Sciences, Akad. 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