Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer.

Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3' UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.

Exosomes: Dynamic Mediators of Extracellular Communication in the Tumor Microenvironment.

It is becoming increasingly recognized that the tumor microenvironment significantly contributes to the development, progression, and metastasis of cancer and also plays a role in response to treatment. The tumor microenvironment is a complex and heterogeneous niche comprised of stromal cells, cancer cells, blood vessels, areas of hypoxia and necrotic tissue, fibrosis, and extracellular matrix. Cellular communication takes place within the tumor microenvironment, both via cell to cell contact, and through extracellular mechanisms such as exosomal signaling. Exosomes are very small membrane-bound vesicles that have been shown to play key roles in the progression of cancer including modulation of the tumor microenvironment through the induction of angiogenesis, the transfer of genetic information that confers drug resistance, and increased cell migration, invasion, proliferation, and survival, as well as the modulation of immune cell interactions. The role of exosomes in several different cancers has been investigated. In the context of osteosarcoma, understanding how exosomes may modulate the tumor microenvironment to support metastatic growth particularly in the lung, the most common site of metastases, may identify novel therapeutic targets for relapsed patients.

Exosomal communication by metastatic osteosarcoma cells modulates alveolar macrophages to an M2 tumor-promoting phenotype and inhibits tumoricidal functions.

Osteosarcoma metastasizes to the lung, and there is a link between the predominance of tumor-promoting immunosuppressive M2 macrophages in the metastases and poor patient survival. By contrast, M1 macrophage predominance correlates with longer survival. M2 macrophages can be induced by various stimuli in the tumor microenvironment, including exosomes, which are 40- to 150-nm vesicles that are involved in intercellular communication and contribute to tumor progression and immune evasion. Recognizing that tumor cells can influence the tumor microenvironment to make it more permissive and because of the link between M2 dominance and curtailed patient survival, we evaluated the effect of exosomes from non-metastatic K7 and Dunn osteosarcoma cells and the metastatic sublines K7M3 and DLM8 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophages with K7M3 and DLM8 exosomes induced expression of IL10, TGFB2, and CCL22 mRNA (markers of M2 macrophages) and decreased phagocytosis, efferocytosis, and macrophage-mediated tumor cell killing. In contrast, exosomes from non-metastatic K7 or Dunn cells did not inhibit phagocytosis, efferocytosis, and macrophage-mediated cytotoxicity or induce increased expression of IL10, TGFB2 or CCL22 mRNA. In addition, metastatic osteosarcoma cell exosomes significantly increased the secretion of TGFB2, a key signaling pathway associated with tumor- mediated immune suppression. Finally, the inhibition of TGFB2 reversed the suppressive activity of alveolar macrophages exposed to metastatic osteosarcoma cell exosomes. Our data suggest that the exosomes from metastatic osteosarcoma cells can modulate cellular signaling of tumor-associated macrophages, thereby promoting the M2 phenotype and creating an immunosuppressive, tumor-promoting microenvironment through the production of TGFB2.