Microvesicle-mediated Wnt/β-Catenin Signaling Promotes Interspecies Mammary Stem/Progenitor Cell Growth

Abstract

Signaling mechanisms that regulate mammary stem/progenitor cell (MaSC) self-renewal are essential for developmental changes that occur in the mammary gland during pregnancy, lactation, and involution. We observed that equine MaSCs (eMaSCs) maintain their growth potential in culture for an indefinite period, whereas canine MaSCs (cMaSCs) lose their growth potential in long term cultures. We then used this system to investigate the role of microvesicles (MVs) in promoting self-renewal properties. We found that Wnt3a and Wnt1 were expressed at higher levels in MVs isolated from eMaSCs compared with those from cMaSCs. Furthermore, eMaSC-MVs were able to induce Wnt/β-catenin signaling in different target cells, including cMaSCs. Interestingly, the induction of Wnt/β-catenin signaling in cMaSCs was prolonged when using eMaSC-MVs compared with recombinant Wnt proteins, indicating that MVs are not only important for transport of Wnt proteins, but they also enhance their signaling activity. Finally, we demonstrate that the eMaSC-MVs-mediated activation of the Wnt/β-catenin signaling pathway in cMaSCs significantly improves the ability of cMaSCs to grow as mammospheres and, importantly, that this effect is abolished when eMaSC-MVs are treated with Wnt ligand inhibitors. This suggests that this novel form of intercellular communication plays an important role in self-renewal.

Conserved and variable: Understanding mammary stem cells across species

“…Two other studies reported that primary canine mammary epithelial cells are able to form colonies in suspension (mammospheres), representing the stem cell property of anchorage‐independent growth . The in vitro self‐renewal potential of the mammospheres, however, varied between these two reports: in one study, primary canine mammospheres could be passaged up to 8–10 times , while in the other study mammosphere regeneration was limited to 2–3 passages . Our unpublished data show that primary canine mammary epithelial cells with mammosphere‐forming ability express CD49f, CD29 and CD44 (Table ).…”

“…The function of this subpopulation has not been confirmed in vitro or in vivo yet, as these markers are intracellular and do not enable isolation and characterization of live cells . Two other studies reported that primary canine mammary epithelial cells are able to form colonies in suspension (mammospheres), representing the stem cell property of anchorage‐independent growth . The in vitro self‐renewal potential of the mammospheres, however, varied between these two reports: in one study, primary canine mammospheres could be passaged up to 8–10 times , while in the other study mammosphere regeneration was limited to 2–3 passages .…”

Pathogenic roles of microvesicles in diabetic retinopathy

“…MVs are shed directly from the cell membrane by a "budding" process and typically range in size from approximately 100 to 1000 nm, although these values are somewhat arbitrary and subclass overlap may exist [29] . MVs are identified by the expression of phosphatidylserine (PS) on their surface, which is indicative of their release from apoptotic or activated cells [30] . In these cells, PS is externalized, whereas the membrane PS has a cytosolic orientation in quiescent cells [31] .…”

Differential signaling pathway activation in 7,12-dimethylbenz[a] anthracene (DMBA)-treated mammary stem/progenitor cells from species with varying mammary cancer incidence

“…We recently found differences in the intrinsic behavior of mammary stem/progenitor cells (MaSC) from horse and dog [ 14 ]. Specifically, we found that MaSC from these species showed a striking difference in growth potential in long-term cultures, which led to the identification of a novel form of intercellular communication involving microvesicle-mediated Wnt/β-catenin signaling [ 14 ].…”

“…We recently found differences in the intrinsic behavior of mammary stem/progenitor cells (MaSC) from horse and dog [ 14 ]. Specifically, we found that MaSC from these species showed a striking difference in growth potential in long-term cultures, which led to the identification of a novel form of intercellular communication involving microvesicle-mediated Wnt/β-catenin signaling [ 14 ]. Since MaSC have a unique capacity for self-renewal and persist for the lifetime of the animal [ 15 , 16 ], they are thought to represent a primary target cell for the origin and development of mammary cancer [ 17 – 19 ].…”