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.

The molecular mechanisms underlying constitutive nuclear factor-jB (NF-jB) activation in solid tumors has not been elucidated. We show that Annexin-1 (ANXA1) is involved in this process, and suppression of ANXA1 in highly metastatic breast cancer cells impedes migration and metastasis capabilities in vitro and in vivo. ANXA1 expression correlates with NF-jB activity, suggesting that ANXA1 may be required for the constitutive activity of IjB kinase (IKK) and NF-jB in highly metatstatic breast cancer. Gel-filtration analysis demonstrated that ANXA1 co-elutes with the members of the IKK complex and NF-jB signaling pathway, and immunoprecipitation confirmed that ANXA1 can bind to and interact with IKKc or NEMO, but not IKKa or IKKb. Importantly, silencing of ANXA1 prevents the interaction of NEMO and RIP1, which indicates that ANXA1 is required for the recruitment of RIP1 to the IKK complex, which may be important for the activation of NF-jB. Downstream targets of NF-jB include uPA and CXCR4, which can be modulated by ANXA1 silencing. CXCR4-mediated migration of breast cancer cell lines in response to CXCL12 was significantly modulated by ANXA1, indicating its importance in the tissue-specific migration of breast cancer cells. Chromatin immunoprecipitation experiments confirmed that in ANXA1 overexpressed cells, NF-jB was recruited to CXCR4 promoter without external stimulation, indicating that ANXA1 is critical for the constitutive activation of NF-jB in breast cancer to promote metastasis. Finally, we show that ANXA1 overexpression enhances metastasis and reduces survival in an intracardiac metastasis model, while ANXA1-deficient mice crossed with MMTV-PyMT mice display significantly less metastasis than their heterozygous littermates, indicating that ANXA1 is an important gene in breast cancer metastasis. Our data reveal that ANXA1 can constitutively activate NF-jB in breast cancer cells through the interaction with the IKK complex, and suggests that modulating ANXA1 levels has therapeutic potential to suppress breast cancer metastasis.

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 copurified soluble factors when adjusted for exosome yield, and a greater than twofold 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.

TLRs play a pivotal role in the recognition of bacteria and viruses. Members of the family recognize specific pathogen sequences to trigger both MyD88 and TRIF-dependent pathways to stimulate a plethora of cells. Aberrant activation of these pathways is known to play a critical role in the development of autoimmunity and cancer. However, how these pathways are entirely regulated is not fully understood. In these studies, we have identified Annexin-A1 (ANXA1) as a novel regulator of TLR-induced IFN-β and CXCL10 production. We demonstrate that in the absence of ANXA1, mice produce significantly less IFN-β and CXCL10, and macrophages and plasmacytoid dendritic cells have a deficiency in activation following polyinosinic:polycytidylic acid administration in vivo. Furthermore, a deficiency in activation is observed in macrophages after LPS and polyinosinic:polycytidylic acid in vitro. In keeping with these findings, overexpression of ANXA1 resulted in enhanced IFN-β and IFN-stimulated responsive element promoter activity, whereas silencing of ANXA1 impaired TLR3- and TLR4-induced IFN-β and IFN-stimulated responsive element activation. In addition, we show that the C terminus of ANXA1 directly associates with TANK-binding kinase 1 to regulate IFN regulatory factor 3 translocation and phosphorylation. Our findings demonstrate that ANXA1 plays an important role in TLR activation, leading to an augmentation in the type 1 IFN antiviral cytokine response.

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.”