The canonical view of the origin of tumor lymphovascular emboli is that they usually originate from lymphovascular invasion as part of a multistep metastatic process. Recent experimental evidence has suggested that metastasis can occur earlier than previously thought and we found evidence that tumor emboli formation can result from the short-circuiting step of encircling lymphovasculogenesis. Experimentally, we used a xenograft of human inflammatory breast cancer (MARY-X), a model that exhibited florid tumor emboli, to generate tumoral spheroids in vitro. In observational studies, we chose human breast carcinoma cases where there appeared to be a possible transition of in situ carcinoma to lymphovascular emboli without intervening stromal invasion. These cases were studied by morphometry as well as IHC with tumor proliferation (Ki-67) and adhesion (E-cadherin) markers, myoepithelial (p63), as well as endothelial (podoplanin [D2-40], CD31, VEGFR-3, Prox-1) markers. Unlabelled spheroids coinjected with either GFP or RFP-human myoepithelial cells or murine embryonal fibroblasts (MEFs) gave rise to tumors which exhibited GFP/RFP immunoreactivity within the cells lining the emboli-containing lymphovascular channels. In vitro studies demonstrated that the tumoral spheroids induced endothelial differentiation of cocultured myoepithelial cells and MEFs, measured by real time PCR and immunofluorescence. In humans, the in situ clusters exhibited similar proliferation, E-cadherin immunoreactivity and size as the tumor emboli (p =.5), suggesting the possibility that the latter originated from the former. The in situclusters exhibited a loss (50%-100%) of p63 myoepithelial immunoreactivity but not E-cadherin epithelial immunoreactivity. The tumor emboli were mainly present within lymphatic channels whose dual p63/CD31, p63/D2-40 and p63/VEGFR-3 and overall weak patterns of D2-40/CD31/VEGFR-3 immunoreactivities suggested that they represented immature and newly created vasculature derived from originally myoepithelial-lined ducts. Collectively both experimental as well as observational studies suggested the possibility that these breast cancer emboli resulted from encircling lymphovasculogenesis rather than conventional lymphovascular invasion.
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