Elucidating the mechanisms underlying hematopoietic stem cell (HSC) specification and expansion in the embryo has been hampered by the lack of analytical cell culture systems that recapitulate in vivo development. Here, we describe an ex vivo model that facilitates a rapid and robust emergence of multipotent long-term repopulating HSCs in the embryonic AGM region. Because this method includes a cell dissociation step prior to reconstruction of a three-dimensional functional tissue and preserves both stromal and hematopoietic elements, it allowed us to identify the direct ancestry of the rapidly expanding HSC pool. We demonstrate that extensive generation of definitive HSCs in the AGM occurs predominantly through the acquisition of stem characteristics by the VE-cadherin+CD45+ population.

BackgroundIntegrin αvβ6 promotes migration, invasion, and survival of cancer cells; however, the relevance and role of αvβ6 has yet to be elucidated in breast cancer.MethodsProtein expression of integrin subunit beta6 (β6) was measured in breast cancers by immunohistochemistry (n > 2000) and ITGB6 mRNA expression measured in the Molecular Taxonomy of Breast Cancer International Consortium dataset. Overall survival was assessed using Kaplan Meier curves, and bioinformatics statistical analyses were performed (Cox proportional hazards model, Wald test, and Chi-square test of association). Using antibody (264RAD) blockade and siRNA knockdown of β6 in breast cell lines, the role of αvβ6 in Human Epidermal Growth Factor Receptor 2 (HER2) biology (expression, proliferation, invasion, growth in vivo) was assessed by flow cytometry, MTT, Transwell invasion, proximity ligation assay, and xenografts (n ≥ 3), respectively. A student’s t-test was used for two variables; three-plus variables used one-way analysis of variance with Bonferroni’s Multiple Comparison Test. Xenograft growth was analyzed using linear mixed model analysis, followed by Wald testing and survival, analyzed using the Log-Rank test. All statistical tests were two sided.ResultsHigh expression of either the mRNA or protein for the integrin subunit β6 was associated with very poor survival (HR = 1.60, 95% CI = 1.19 to 2.15, P = .002) and increased metastases to distant sites. Co-expression of β6 and HER2 was associated with worse prognosis (HR = 1.97, 95% CI = 1.16 to 3.35, P = .01). Monotherapy with 264RAD or trastuzumab slowed growth of MCF-7/HER2-18 and BT-474 xenografts similarly (P < .001), but combining 264RAD with trastuzumab effectively stopped tumor growth, even in trastuzumab-resistant MCF-7/HER2-18 xenografts.ConclusionsTargeting αvβ6 with 264RAD alone or in combination with trastuzumab may provide a novel therapy for treating high-risk and trastuzumab-resistant breast cancer patients.