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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., Competing Interests: MD Anderson Cancer Center and R.K. hold patents in the area of exosome biology and are licensed to Codiak Biosciences Inc. MD Anderson Cancer Center and R.K. are stock equity holders in Codiak Biosciences Inc. R.K. receives research support from Codiak Biosciences Inc. and serves as a member of the board of directors. V.S.L. served as a paid consultant for Codiak Biosciences Inc., (© 2021 The Authors.)

The Stimulator of Interferon Genes (STING) pathway is implicated in the innate immune response and is important in both oncogenesis and cancer treatment. Specifically, activation of the cytosolic DNA sensor STING in antigen-presenting cells (APCs) induces a type I interferon response and cytokine production that facilitates antitumor immune therapy. However, use of STING agonists (STINGa) as a cancer therapeutic has been limited by unfavorable pharmacological properties and targeting inefficiency due to rapid clearance and limited uptake into the cytosol. Exosomes, a class of extracellular vesicles shed by all cells are under consideration for their use as effective carriers of drugs owing to their innate ability to be taken up by cells and their biocompatibility for optimal drug biodistribution. Therefore, we engineered exosomes to deliver the STING agonist cyclic GMP-AMP (iExo STINGa ), to exploit their favorable pharmacokinetics and pharmacodynamics. Selective targeting of the STING pathway in APCs with iExo STINGa was associated with superior potency compared with STINGa alone in suppressing B16F10 tumor growth. Moreover, iExo STINGa showed superior uptake of STINGa into dendritic cells compared with STINGa alone, which led to increased accumulation of activated CD8 + T-cells and an antitumor immune response. Our study highlights the potential of exosomes in general, and iExo STINGa specifically, in enhancing cancer therapy outcomes., Competing Interests: Conflict of interest V. S. L. is a paid consultant for Codiak Biosciences. MD Anderson Cancer Center and R. K. hold patents in the area of exosome biology (unrelated to the topic of this publication) and are licensed to Codiak Biosciences Inc. MD Anderson Cancer Center and R. K. are stock equity holders in Codiak Biosciences Inc. R. K. is a consultant and a scientific advisor of Codiak Biosciences Inc., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tissue factor (TF)-expressing tumor-derived extracellular vesicles (EVs) can promote metastasis and pre-metastatic niche formation, but the mechanisms by which this occurs remain largely unknown. We hypothesized that generation of activated factor X (FXa) by TF expressed on tumor-derived EV could activate protease-activated receptors (PARs) on non-activated endothelial cells to induce a pro-adhesive and pro-inflammatory phenotype. We obtained EV from TF-expressing breast (MDA-MB-231) and pancreatic (BxPC3 and Capan-1) tumor cell lines. We measured expression of E-selectin and secretion of interleukin-8 (IL-8) in human umbilical vein endothelial cells after exposure to EV and various immunologic and chemical inhibitors of TF, FXa, PAR-1, and PAR-2. After 6 h of exposure to tumor-derived EV (pretreated with factor VIIa and FX) in vitro , endothelial cells upregulated E-selectin expression and secreted IL-8. These changes were decreased with an anti-TF antibody, FXa inhibitors (FPRCK and EGRCK), and PAR-1 antagonist (E5555), demonstrating that FXa generated by TF-expressing tumor-derived EV was signaling through endothelial PAR-1. Due to weak constitutive PAR-2 expression, these endothelial responses were not induced by a PAR-2 agonist peptide (SLIGKV) and were not inhibited by a PAR-2 antagonist (FSLLRY) after exposure to tumor-derived EV. In conclusion, we found that TF-expressing cancer-derived EVs activate quiescent endothelial cells, upregulating E-selectin and inducing IL-8 secretion through generation of FXa and cleavage of PAR-1. Conversion of resting endothelial cells to an activated phenotype by TF-expressing cancer-derived EV could promote cancer metastases.