Finally, 25 ng of cDNA was used for qPCR

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Finally, 25 ng of cDNA was used for qPCR. signalling triggered migration of cancer cells from early lesions shortly after HER2 activation, but promoted proliferation in advanced primary tumour cells. The switch from migration to proliferation was regulated by elevated HER2 expression and increased tumour cell Hyal2 density involving miRNA-mediated progesterone receptor (PGR) down-regulation and was reversible. Cells from early, low-density lesions displayed more stemness features than cells from dense, advanced tumours, migrated more and founded more metastases. Strikingly, we found that at least 80% of metastases were derived from early disseminated cancer cells (DCC). Karyotypic and phenotypic analysis of human disseminated cancer cells and primary tumours corroborated the relevance of these findings for Deracoxib human metastatic dissemination. Introduction Systemic cancer (the dissemination and subsequent distant outgrowth of cells from a solid tumour) occurs in two phases: a clinically latent stage of hidden cancer spread and then manifest metastasis. Manifest metastasis remains mostly incurable. Clinically undetectable minimal residual disease (MRD), defined by disseminated Deracoxib cancer cells (DCCs) that are left behind after primary tumour (PT) surgery, offers a time-window to prevent metastasis1,2. However, only circumstantial knowledge is available about MRD and systemic (adjuvant) therapies consequently improve outcome in only about 20% of patients3,4. This situation indicates that our current understanding of early systemic cancer is insufficient to prevent metastasis. The first direct evidence for a characteristic biology of early-disseminated cancer and MRD came from analyses of disseminated cancer cells (DCCs) isolated from bone marrow of breast cancer patients before (M0 stage) and after (M1 stage) manifestation of metastasis5,6, indicating that M0-DCCs might have disseminated early and evolved in parallel with the primary tumour7. Studies in transgenic mouse models8C10 and in patients with pre-malignant lesions or carcinomas8,11,12 corroborated this concept but the relevance of DCCs remains hotly contested13. We therefore addressed the issue of breast cancer cell dissemination soon after cancer initiation and asked whether mechanisms exist that reduce metastatic seeding from advanced cancer. Finally, we addressed whether early DCCs are able to form metastases. We report a mechanism involving cell density, HER2 and progesterone signalling that reconciles early and late dissemination models. Results Progesterone and HER2 signalling regulate gene expression in early mammary lesions In Balb-NeuT mice, dissemination starts shortly after expression of the Her2-transgene at puberty (around week 4), when first hyperplastic lesions become apparent8. From weeks 4C9 we observed micro-invasion8, and a sharp decline in the ratio of DCCs to total tumour area (a measure of cell numbers at risk to disseminate) during primary tumour growth (Extended Data Fig 1a). The genetic program governing dissemination from early lesions (ELs) in microdissected tissue samples (Extended Data Fig 1b and Supplementary Table 1) revealed signature gene expression profiles compared to healthy mammary glands, primary tumours (PT) and lung metastases (Figure 1a). We defined 1278 gene transcripts unique to ELs of which 300 were highly conserved between mouse and human (Supplementary File 1). Open in a separate window Figure 1 Identification of a gene expression signature linked to early dissemination(a) Heatmaps of Deracoxib genes differentially expressed between different sample types: normal mammary glands from BALB/c, early lesions (EL), primary tumours (PT) and metastases (MET) from Balb-NeuT mice; yellow, upregulation; blue, downregulation. (b) Five-gene surrogate signature (qPCR) for EL profile. (c) Progesterone (P) activates EL-signature (up-regulated HER2 in 4T1 and MM3MG cells, respectively (Extended Data Fig 1hCi). Collectively, these results suggested that the genetic program Deracoxib of ELs depends on the combined activation of progesterone and HER2 pathways. Progesterone induces migration and stemness of EL- but not of PT cells Since progesterone mediates branching14 in mammary gland development we explored the role of the progesterone-induced EL signature for cancer cell migration. We found the mRNA of the progesterone-induced paracrine signals (PIPS) and up-regulated in EL samples (Extended Data Fig 2a). Treatment of EL-derived cells with PIPS mimicked the effect of progesterone.