Adenocarcinomas and their derived lymphovascular emboli contain dual compartmental populations of tumor microvesicles which differ in size and possibly content and function.

Adenocarcinomas and their associated lymphovascular emboli metastasize as tight aggregates of tumor cells. Using a xenograft model of human inflammatory breast cancer (Mary-X), which exhibited florid lymphovascular emboli in mice and high density tumor aggregates (spheroids) in vitro, we previously demonstrated that both emboli and spheroids are mediated by an intact overexpressed E-cadherin axis which regulates homotypic tumor cell adhesion. We now report that Mary-X spheroids secrete 5-10 fold higher levels of microvesicles (MVs) than most other carcinoma cell lines. Despite the high density of tumor cells and the strong juxtaposition of tumor cells to each other in the spheroids, intercellular spaces exist which contain numerous MVs. These intraspheroidal (IS) MVs entrapped between the E-cadherin adherens junctions can be isolated by dissociating the spheroids in Ca+2-free media with EDTA. These IS MVs compared to the extraspheroidal (ES) MVs found in the conditioned media (CM) comprise a significant (approximately 25%) subpopulation of MVs which persisted for several days in culture (day 3: 0.48 v 2.0; day 7: 1.1 v 3.9; day 14: 0.64 v 3.2 (# x 10E10)). The IS MVs differed in size compared to the ES MVs. By nanoparticle tracking analysis (Nanosight), IS MV mean volume was 79 nm compared with the ES MV mean volume of 152 nm. These size differences were also confirmed by transmission electron microscopy (TEM), though both were smaller (50 nm v 110 nm). The proteome profile of the IS MVs can be readily compared with that of the ES MVs found in CM. Cellular target differences between the IS MVs and the ES MVs can also be studied. It would be attractive to hypothesize that IS MVs preferentially function in autocrine signaling whereas ES MVs signal in a paracrine manner.