Exosomes are naturally occurring biological nanomembranous vesicles (～40 to 100 nm) of endocytic origin that are released from Ptprc diverse cell types into the extracellular space. identification in EpCAM-Exos of the classical apical trafficking molecules CD63 (LAMP3) mucin 13 and the apical intestinal enzyme sucrase isomaltase and increased expression of dipeptidyl peptidase IV and the apically restricted pentaspan membrane glycoprotein prominin 1. In contrast the A33-Exos preparation was enriched with basolateral trafficking molecules such as early endosome antigen 1 the Golgi membrane protein ADP-ribosylation factor and clathrin. Our observations are consistent with EpCAM- and A33-Exos being released from the apical and basolateral surfaces respectively and the EpCAM-Exos proteome profile with widely published stereotypical exosomes. A proteome analysis of LIM1863-derived shed microvesicles (sMVs) was also performed in order to BRL-15572 clearly distinguish A33- and EpCAM-Exos from sMVs. Intriguingly several members of the MHC class I family of antigen presentation molecules were exclusively observed in A33-Exos whereas neither MHC class I nor MHC class II molecules were observed via MS in EpCAM-Exos. Additionally we report for the first time in any extracellular vesicle study the colocalization of EpCAM claudin-7 and CD44 in EpCAM-Exos. Given that these molecules are known to complex together to promote tumor progression further characterization of exosome subpopulations will enable a deeper understanding of their possible role in regulation of the tumor microenvironment. The microenvironment in which a tumor originates plays a critical role in its initiation progression and metastasis (1-3). Recent advances have indicated that although the microenvironment provides crucial signaling to maintain tissue architecture inhibit cell growth and constrain the malignant phenotype it can also promote and induce cancer (4). In addition to cancer cells the tumor microenvironment comprises normal cells blood cells secreted proteins and peptides and constituents of the extracellular matrix that actively influence cell behavior. Secreted proteins peptides and physiological small molecules such as soluble cytokines and chemokines are currently recognized as the main exocrine and juxtacrine factors underlying cell-to-cell communication within the tumor microenvironment and providing the metastatic niche in distant organs (5). In addition to soluble secreted proteins and peptides most cell types also release extracellular membrane vesicles (eMVs)1 that transfer information between cells in the microenvironment; it is now recognized that eMVs can also influence cell-to-cell communication during tumor progression (6 7 BRL-15572 Another emerging means by which cells relay information to other cells is long thin interconnecting membranous bridges that connect neighboring cells through adhesion mechanisms BRL-15572 (actin-based cytonemes or filopodial bridges) or tunneling nanotubes which can establish direct tubular conduits between the cytoplasms of adjacent cells (for a review see Ref. 8). Over the past decade eMVs have been shown to exhibit important pleiotropic roles in many biological processes. For example eMVs are enriched in various bioactive molecules such as growth factors lipids membrane receptors (adhesion molecules oncogenic receptors) mRNA microRNA transcriptional BRL-15572 factors splicing factors and infectious particles (HIV prions) (9; see reviews in Refs. 6 and 10-12). These bioactive molecules have been reported to (i) directly stimulate target cells via BRL-15572 bioactive lipids or by acting like soluble cell-surface signaling complexes; (ii) transfer oncogenic cargo and cancer cell properties to nearby indolent or normal cells; (iii) epigenetically reprogram recipient cells via the transfer of mRNA microRNA and transcription factors; and (iv) serve as a delivery vehicle in a “Trojan horse” manner to transfer pathological cargo such as plant toxins prions or HIV particles. Although many of these properties have been ascribed to exosomes it should be noted that functional studies were commonly performed on eMV preparations which in many cases are heterogeneous mixtures of shed microvesicles (sMVs) exosomes exosome-like particles and apoptotic blebs (13). Exosomes along with sMVs that bud off from the plasma membrane (PM) and apotopic bodies represent specific subtypes of eMVs (reviewed in Ref. 13). Of these exosomes have been the most widely.