In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes.

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Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other Extracellular Vesicles

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Biological properties of extracellular vesicles and their physiological functions

Biogenesis, Secretion, and Intercellular Interactions of Exosomes and Other

Communication by Extracellular Vesicles: Where We Are and Where We Need to Go.

The ability of exosomes to transfer cargo from donor to acceptor cells, thereby triggering phenotypic changes in the latter, has generated substantial interest in the scientific community. However, the extent to which exosomes differ from other extracellular vesicles in terms of their biogenesis and functions remains ill-defined. Here, we discuss the current knowledge on the specificities of exosomes and other types of extracellular vesicles, and their roles as important agents of cell-to-cell communication.

Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication.

Activation of myofibroblast rich stroma is a rate-limiting step essential for cancer progression. The responsible factors are not fully understood, but TGFβ1 is probably critical. A proportion of TGFβ1 is associated with extracellular nano-vesicles termed exosomes, secreted by carcinoma cells, and the relative importance of soluble and vesicular TGFβ in stromal activation is presented. Prostate cancer exosomes triggered TGFβ1-dependent fibroblast differentiation, to a distinctive myofibroblast phenotype resembling stromal cells isolated from cancerous prostate tissue; supporting angiogenesis in vitro and accelerating tumour growth in vivo. Myofibroblasts generated using soluble TGFβ1 were not pro-angiogenic or tumour-promoting. Cleaving heparan sulphate side chains from the exosome surface had no impact on TGFβ levels yet attenuated SMAD-dependent signalling and myofibroblastic differentiation. Eliminating exosomes from the cancer cell secretome, targeting Rab27a, abolished differentiation and lead to failure in stroma-assisted tumour growth in vivo. Exosomal TGFβ1 is therefore required for the formation of tumour-promoting stroma.

Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes.

BACKGROUND The post-translational modification of proteins, including glycosylation, is known to differ between normal and tumour cells. In this study, the expression profile of two glycosyltranferases, UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 (ppGalNAc-T6) and alpha6-sialyl-transferase-I (ST6GalNAc-I) was assessed, in a cohort of women with breast cancer. PATIENTS AND METHODS Breast cancer tissues (n=127) and normal background tissues (n=33) were collected immediately after excision during surgery. Following RNA extraction, reverse transcription was carried out and transcript levels were determined using real-time quantitative PCR and normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Transcript levels within the breast cancer specimens were compared to the normal background tissues and analyzed against conventional pathological parameters and clinical outcome over a 10 year follow-up period. RESULTS Significantly higher levels of ppGalNAc-T6 were found in the breast cancer specimens compared to the background tissue (p=0.015). There was a non-significant trend for levels to increase with the Nottingham Prognostic Index (NPI) and TNM stage and those who died from breast cancer. ST6GalNAc-I expression was associated with better prognosis, reaching significance when comparing patients who remained disease free to those with distant recurrence (p=0.0096). The relationship approached significance when comparing NPI 2 to NPI 3 (p=0.058) and disease free patients to non-disease free patients (p=0.052) or those who died of breast cancer (p=0.060). For both enzymes a significant association with ductal type was found. CONCLUSION Expression of ppGalNAc-T6 is significantly higher in breast cancer compared to 'normal'/benign breast tissue samples. ST6GalNAc-I expression in breast cancer is associated with better prognosis.

Prognostic Utility of Glycosyltransferase Expression in Breast Cancer

Our recent study showed that a novel member of bone morphogenetic protein (BMP) family, BMP-10, was decreased in prostate cancer. In the present study, we investigated the implication of BMP-10 in breast cancer, particularly the relation of its expression with clinical aspects. The expression of BMP-10 was examined in a cohort of human breast cancer specimens (normal, n = 23; cancer, n = 97), using both quantitative real-time PCR and immunohistochemical staining. The full-length human BMP-10 was cloned into a mammalian expression plasmid vector and then transfected into breast cancer cells. The effect on growth, cell matrix adhesion, motility, and invasion of MDA-MB-231 cells by BMP-10 was then investigated using in vitro growth assays. Immunohistochemical staining and quantitative real-time PCR revealed a decreased expression of BMP-10 in breast cancer. Further analysis of BMP-10 transcript level against the clinical aspect demonstrated that the decreased BMP-10 expression correlated with disease progression, bone metastasis, and poor prognosis. The disease-free survival of the patients with a higher level of BMP-10 was 132.8 (95% CI, 122.0–143.5) months, significantly longer compared to 93.7 (95% CI, 60.3–127.2) months for patients with a lower level of BMP-10 expression (P = 0.043). The overexpression of BMP-10 has broad inhibitory effects on the in vitro growth, invasion, and motility of breast cancer cells. Taken together, BMP-10 can inhibit the cell growth of breast cancer cells, and decreased BMP-10 expression correlates to poor prognosis and disease progression, particularly the lymphatic and bone metastasis. Bone morphogenetic protein-10 (BMP-10) may function as a tumor suppressor in breast cancer. (Cancer Sci 2010)

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1349-7006.2010.01648.x

Bone morphogenetic protein-10 (BMP-10) inhibits aggressiveness of breast cancer cells and correlates with poor prognosis in breast cancer

Background: Ubiquitin modification of proteins influences cellular processes relevant to carcinogenesis. CHIP (carboxyl-terminus of Hsc70-interacting protein) is a chaperone-dependent E3 ubiquitin ligase, regulating the stability of heat shock protein 90 (HSP90) interacting proteins. CHIP is implicated in the modulation of estrogen receptor (ESR1) and Her-2/neu (ERBB2) stability. LOX (lysyl-oxidase) serves intracellular roles and catalyses the cross-linking of extracellular matrix (ECM) collagens and elastin. LOX expression is altered in human malignancies and their peri-tumoral stroma. However, paradoxical roles are reported. In this study, the level of mRNA expression of CHIP and LOX were assessed in normal and malignant breast tissue and correlated with clinico-pathological parameters. Materials and Methods: Breast cancer (BC) tissues (n = 127) and normal tissues (n = 33) underwent RNA extraction and reverse transcription; transcript levels were determined using real-time quantitative PCR and normalized against CK-19. Transcript levels were analyzed against TNM stage, nodal involvement, tumor grade and clinical outcome over a ten-year follow-up period. Results: CHIP expression decreased with increasing Nottingham Prognostic Index (NPI): NPI-1 vs. NPI-3 (12.2 vs. 0.2, P = 0.0264), NPI-2 vs. NPI-3 (3 vs. 0.2, P = 0.0275). CHIP expression decreased with increasing TNM stage: TNM-1 vs. TNM-2 (12 vs. 0, P = 0.0639), TNM-1 vs. TNM-2-4 (12 vs. 0, P = 0.0434). Lower transcript levels were associated with increasing tumor grade: grade 1 vs. grade 3 (17.7 vs. 0.3, P = 0.0266), grade 2 vs. grade 3 (5 vs. 0.3, P = 0.0454). The overall survival (OS) for tumors classified as 'low-level expression', was poorer than those with 'high-level expression' (118.1 vs. 152.3 months, P = 0.039). LOX expression decreased with increasing NPI: NPI-1 vs. NPI-2 (3 vs. 0, P = 0.0301) and TNM stage: TNM-1 = 3854639, TNM-2 = 908900, TNM-3 = 329, TNM-4 = 1.232 (P = NS). Conclusion: CHIP expression is associated with favorable prognostic parameters, including tumor grade, TNM stage and NPI. CHIP expression predicts OS. LOX expression is associated with improved NPI. In addition to their prognostic utility, mechanistic insights into tumor suppressor function may offer potential therapeutic strategies.

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Wen Jiang

Papers

Differentiation of tumour-promoting stromal myofibroblasts by cancer exosomes.

Prognostic Utility of Glycosyltransferase Expression in Breast Cancer

Bone morphogenetic protein-10 (BMP-10) inhibits aggressiveness of breast cancer cells and correlates with poor prognosis in breast cancer

Prognostic implications of carboxyl-terminus of Hsc70 interacting protein and lysyl-oxidase expression in human breast cancer.

Expressed in high metastatic cells (Ehm2) is a positive regulator of keratinocyte adhesion and motility: The implication for wound healing