Abstract Understanding breast tumor progression requires insights both at the molecular and cellular levels. In particular, the transition of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) is a key, yet poorly understood event. Membrane-Type 1 matrix metalloproteinase (MT1-MMP) is critical for pericellular remodelling of the basement membrane and interstitial collagen during invasion by carcinoma cells. The role of MT1-MMP during the transition from DCIS to IDC was investigated using an intraductal human-in-mouse xenograft model. Breast tumor-derived MCF10.DCIS.com cells, which express MT1-MMP were injected into the primary mammary ducts of female SCID mice. Histological analysis revealed a progression from DCIS, to microinvasive and invasive lesions by 5, 7-8 and 10-12 weeks post-injection, respectively. Immunohistochemistry (IHC) staining showed homogeneous expression of MT1-MMP over the tumor section at DCIS stage, and up-regulation at the edge of the tumours at microinvasive and invasive states. In microinvasives lesion, up-regulation of MT1-MMP in tumour cells coincided with disruption of the basement membrane. At later stages, one characteristic of invasive lesions is the increase in collagen fibers deposition. Interestingly, MT1-MMP expression was also increased at the front of infiltrating tumours in contact with the stroma. After 10 weeks, intraductally injected cells knocked down for MT1-MMP developed invasive tumours only in 20% of injected mammary glands, while 100% of glands injected with control cells developed infiltrating lesions. All together, these results demonstrated that MT1-MMP is instrumental for the transition from in situ to invasive breast tumours. IHC staining revealed a strong correlation of the expression of the basal marker p63 and MT1-MMP in tumour cells both at the microinvasive and invasive stages. One hypothesis was that contact of tumor cells with the stroma triggered induction of p63 that in turn up-regulated MT1-MMP. Along this line, we found that MCF10.DCIS.com cells plated on type I collagen up-regulated p63 and MT1-MMP, both at the mRNA and protein levels, while silencing of p63 abolished collagen-dependent MT1-MMP increase. At the functional level, silencing of MT1-MMP or p63 inhibited invasion of multicellular spheroids into 3D type collagen as well as the cells capacity to degrade collagen I fibers. Finally, MT1-MMP expression was analysed by IHC staining on human tissue microarray comprising 432 IDCs, 40 microinfiltrated lesions and 68 DCIS. MT1-MMP was upregulated in IDC as compared to DCIS. Invasive triple-negative breast (TNBC) and grade III tumours expressed highest MT1-MMP levels. To our knowledge, this is the first demonstration that up-regulation of MT1-MMP is associated with invasiveness, histopathologic grade and molecular subtypes in human breast cancer. We also observed 10% of TNBC expressing p63 and found a positive correlation with MT1-MMP expression suggesting an interplay between p63 and MT1-MMP during progression. Citation Format: Catalina Lodillinsky, Elvira Infante, Laetitia Fuhrmann, Alan Guichard, Joanna Cyrta, Emilie Lagoutte, Marie Irondelle, Sophie Vacher, Ivan Bieche, Marina Glukhova, Anne Vincent-Salomon, Philippe Chavrier. Membrane-anchored MT1-MMP downstream of p63 is essential for the transition of in situ to invasive breast carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-30. doi:10.1158/1538-7445.AM2014-LB-30