Competitive bone marrow transplantation assay measures multi-lineage reconstitution of hematopoiesis in irradiated transplant recipient mice. Thus this assay is routinely used to determine haematopoietic stem and progenitor cells (HSPCs) functionality in vivo. The principle of the method is to transplant bone marrow donor cells (derived from transgenic mice of choice) on C57BL6 background together with normal competitor bone marrow. In order to distinguish donor from competitor cells upon transplantation, usually competitor mice are congenic and carry the differential B cell antigen originally designated Ly5.1 and CD45.1.A typical competitive bone marrow transplantation experiment will contain two transplantation groups, donor (transgenic mice of choice and their controls) are transplanted in competition with normal competitors and engraftment efficiency is evaluated in both blood and bone marrow.
Haematopoietic stem cells (HSCs) are characterized by their self-renewal potential associated to dormancy. Here we identify the cell surface receptor neogenin-1 as specifically expressed in dormant HSCs. Loss of neogenin-1 initially leads to increased HSC expansion but subsequently to loss of self-renewal and premature exhaustion in vivo. Its ligand netrin-1 induces Egr1 expression and maintains quiescence and function of cultured HSCs in a Neo1 dependent manner. Produced by arteriolar endothelial and periarteriolar stromal cells, conditional netrin-1 deletion in the bone marrow niche reduces HSC numbers, quiescence and self-renewal, while overexpression increases quiescence in vivo. Ageing associated bone marrow remodelling leads to the decline of netrin-1 expression in niches and a compensatory but reversible upregulation of neogenin-1 on HSCs. Our study suggests that niche produced netrin-1 preserves HSC quiescence and self-renewal via neogenin-1 function. Decline of netrin-1 production during ageing leads to the gradual decrease of Neo1 mediated HSC self-renewal.
11524 Background: The sympathetic nervous system (SNS) has been implicated in the pathogenesis of epithelial malignancies. Clinical studies have shown that elevated SNS activity is associated with increased prostate cancer risk and mortality, whereas use of sympathetic β-adrenergic receptor (bAR) blockers reduces the risk. The prostate stroma is highly innervated by the SNS, and depletion of bARs (Adrb2 + Adrb3) in this compartment inhibits tumor growth. We aim to identify the stromal populations that mediate SNS signaling in the tumor microenvironment, and elucidate the mechanism by which loss of bAR signaling inhibits prostate cancer progression. Methods: In vivo xenograft tumor growth was measured in Adrb2-/-; Adrb3-/- mice using an orthotopic PC3-luciferase model. Cancer progression in the transgenic HI-myc model was assessed after conditional Adrb2 deletion in stromal populations by intercrossing Adrb2fl/fl line to stromal Cre lines: endothelial Cdh5CreERT2, myeloid Csf1riCre, and pericyte NG2CreERTM. A novel prostate-specific collagen matrix transplantation assay was employed to assess angiogenesis. Results: Deletion of the SNS bAR neural receptors Adrb2 and Adrb3 in the microenvironment synergized to arrest tumor growth. Imaging the tumor vasculature revealed a defect in vessel branching, suggesting altered angiogenesis in the absence of bAR stimulation. Histology revealed that vascular innervation was greatest during the pre-neoplastic PIN stage, and that conditional Adrb2 deletion in endothelial cells, but not myeloid cells or pericytes, reduced progression to high-grade PIN, and maintained inhibition of disease progression throughout all histopathological cancer stages. In the orthotopic matrix assay, endothelial deletion of Adrb2 was confirmed to inhibit angiogenesis as did sympathetic denervation. Conclusions: The SNS mediates a pro-angiogenic switch during the early stages of prostate carcinogenesis. We identified endothelial cells as the stromal targets of the SNS, and that abrogation of bAR signaling in the vasculature inhibits angiogenesis and delays prostate cancer progression, suggesting a novel therapeutic avenue for prostate cancer treatment.
