Co-Isolation of Cytokines and Exosomes: Implications for Immunomodulation Studies.

Exosomes play a vital role in intercellular communication and their immunomodulatory potential have become an important focus in cancer research. Various methods have been developed for the isolation although each method differs in the number and purity of exosomes they yield. In melanoma, tumor-derived exosomes drive immunosuppression within the tumor microenvironment. The co-elution of exosomes and soluble factors such as cytokines during isolation, however, make it difficult to ascertain the contribution of exosome cargo, as soluble cytokines are equally capable of immune suppression. In this review we will expound upon the biological relevance that exosome-associated cytokines possess. Furthermore, we discuss the technical challenges that arise during exosome isolation and what this means for further studies into the TME and in vivo work.

A Rapid Exosome Isolation Using Ultrafiltration and Size Exclusion Chromatography (REIUS) Method for Exosome Isolation from Melanoma Cell Lines.

Cells release extracellular vesicles (EVs) that can be detected both in vivo and in cell culture medium. Among EVs, exosomes are 50-150 nm vesicles that are systematically packaged into multivesicular bodies for release into the external environment. In cancer, these intentionally packaged exosomes carry a payload of proteins such as RNAs and surface receptors that facilitate the reprogramming of proximal cells to assemble a protumor microenvironment. Exosomes have been implicated as an important intermediary extracellular communication pathway between cells, including in melanoma. Human melanoma-derived exosomes (HMEX) have been demonstrated to modulate the extracellular environment and inhibit immune cell activation. There are many methods to isolate and enrich for exosomes and the method applied can impact yield and purity of the isolates. In this chapter we describe the REIUS (rapid exosome isolation using ultrafiltration and size exclusion chromatography) method to isolate HMEX from melanoma cell cultures and then demonstrate their enrichment using molecular and microscopic approaches.

Purity and yield of melanoma exosomes are dependent on isolation method.

Both exosomes and soluble factors have been implicated in the generation of an immunosuppressive tumour microenvironment. Determining the contribution of each requires stringent control of purity of the isolated analytes. The present study compares several conventional exosome isolation methods for the presence of co-enriched soluble factors while isolating exosomes from human melanoma-derived cell lines. The resultant preparations were analysed by multiplex bead array analysis for cytokine profiles, and by electron microscopy and nanotracking analysis for exosome size distribution and concentration. It is demonstrated that the amount and repertoire of soluble factors in exosome preparations is dependent upon the isolation method used. A combination of ultrafiltration and size exclusion chromatography yielded up to 58-fold more exosomes than ultracentrifugation, up to 836-fold lower concentrations of co-purified soluble factors when adjusted for exosome yield, and a greater than two-fold increase in PD-L1 expressing exosomes. Mechanistically, in context of the immunomodulatory effects of exosomes, the exosome isolation method should be carefully considered in order to limit any effects due instead to co-eluted soluble factors.

Ap4A Regulates Directional Mobility and Antigen Presentation in Dendritic Cells.

The significance of intracellular Ap4A levels over immune activity of dendritic cells (DCs) has been studied in Nudt2fl/fl/CD11c-cre mice. The transgenic mice have been generated by crossing floxed NUDT2 gene mice with DC marker CD11c recombinase (cre) mice. The DCs derived from these mice have higher levels of Ap4A (≈30-fold) compared with those derived from Nudt2+/+ mice. Interestingly, the elevated Ap4A in DCs has led them to possess higher motility and lower directional variability. In addition, the DCs are able to enhance immune protection indicated by the higher cross-presentation of antigen and priming of CD8+ OT-I T cells. Overall, the study denotes prominent impact of Ap4A over the functionality of DCs. The Nudt2fl/fl/CD11c-cre mice could serve as a useful tool to study the influence of Ap4A in the critical immune mechanisms of DCs.

A Review of Exosomes and their Role in The Tumor Microenvironment and Host-Tumor "Macroenvironment".

Tumor-derived exosomes (TEX) are important intercellular messengers that contribute to tumorigenesis and metastasis through a variety of mechanisms such as immunosuppression and metabolic reprogramming that generate a pre-metastatic niche favorable to tumor progression. Our lab has contributed further to the understanding of the miRNA payloads in TEX by demonstrating that human melanoma-derived exosome (HMEX) associated miRNAs contribute to the metabolic reprogramming of normal stroma. This mini-review highlights the role of TEX in the tumor microenvironment (TME) and the hypothesis that exosomes may also generate a host-tumor "macroenvironment" beyond the TME through their miRNA and protein payloads, so to speak "fertilizing the soil for cancer seeding."

Publisher Correction: Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Metabolic reprogramming of stromal fibroblasts by melanoma exosome microRNA favours a pre-metastatic microenvironment.

Local acidification of stroma is proposed to favour pre-metastatic niche formation but the mechanism of initiation is unclear. We investigated whether Human Melanoma-derived exosomes (HMEX) could reprogram human adult dermal fibroblasts (HADF) and cause extracellular acidification. HMEX were isolated from supernatants of six melanoma cell lines (3 BRAF V600E mutant cell lines and 3 BRAF wild-type cell lines) using ultracentrifugation or Size Exclusion Chromatography (SEC). Rapid uptake of exosomes by HADF was demonstrated following 18 hours co-incubation. Exposure of HDAF to HMEX leads to an increase in aerobic glycolysis and decrease in oxidative phosphorylation (OXPHOS) in HADF, consequently increasing extracellular acidification. Using a novel immuno-biochip, exosomal miR-155 and miR-210 were detected in HMEX. These miRNAs were present in HMEX from all six melanoma cell lines and were instrumental in promoting glycolysis and inhibiting OXPHOS in tumour cells. Inhibition of miR-155 and miR-210 activity by transfection of miRNA inhibitors into HMEX reversed the exosome-induced metabolic reprogramming of HADF. The data indicate that melanoma-derived exosomes modulate stromal cell metabolism and may contribute to the creation of a pre-metastatic niche that promotes the development of metastasis.