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BackgroundBreast cancer (BC) progression to metastatic disease is the leading cause of death in women worldwide. Metastasis is driven by cancer stem cells (CSCs) and signals from their microenvironment. Interleukin (IL) 30 promotes BC progression, and its expression correlates with disease recurrence and mortality. Whether it acts by regulating BCSCs is unknown and could have significant therapeutic implications.MethodsHuman (h) and murine (m) BCSCs were tested for their production of and response to IL30 by using flow cytometry, confocal microscopy, proliferation and sphere-formation assays, and PCR array. Immunocompetent mice were used to investigate the role of BCSC-derived IL30 on tumor development and host outcome. TCGA PanCancer and Oncomine databases provided gene expression data from 1084 and 75 hBC samples, respectively, and immunostaining unveiled the BCSC microenvironment.ResultshBCSCs constitutively expressed IL30 as a membrane-anchored glycoprotein. Blocking IL30 hindered their proliferation and self-renewal efficiency, which were boosted by IL30 overexpression. IL30 regulation of immunity gene expression in human and murine BCSCs shared a significant induction ofIL23andCXCL10. Both immunoregulatory mediators stimulated BCSC proliferation and self-renewal, while their selective blockade dramatically hindered IL30-dependent BCSC proliferation and mammosphere formation. Orthotopic implantation of IL30-overexpressing mBCSCs, in syngeneic mice, gave rise to poorly differentiated and highly proliferating MYC+KLF4+LAG3+tumors, which expressed CXCL10 and IL23, and were infiltrated by myeloid-derived cells, Foxp3+T regulatory cells and NKp46+RORγt+type 3 innate lymphoid cells, resulting in increased metastasis and reduced survival. In tumor tissues from patients with BC, expression ofIL30overlapped with that ofCXCL10andIL23,and ranked beyond the 95th percentile in a Triple-Negative enriched BC collection from the Oncomine Platform. CIBERSORTx highlighted a defective dendritic cell, CD4+T and γδ T lymphocyte content and a prominent LAG3 expression in IL30highversusIL30lowhuman BC samples from the TCGA PanCancer collection.ConclusionsConstitutive expression of membrane-bound IL30 regulates BCSC viability by juxtacrine signals andviasecond-level mediators, mainly CXCL10 and IL23. Their autocrine loops mediate much of the CSC growth factor activity of IL30, while their paracrine effect contributes to IL30 shaping of immune contexture. IL30-related immune subversion, which also emerged from computational analyses, strongly suggests that targeting IL30 can restrain the BCSC compartment and counteract BC progression.