Bladder cancer (BC) patients face high rates of disease recurrence, partially driven by the cancer field effect. This effect is mediated in part by the release of pro-tumorigenic cargos in membrane-enclosed extracellular vesicles (EVs), but the specific underlying mechanisms remain poorly understood. Protein disulfide isomerase (PDIA1) catalyze disulfide bond formation and can help mitigate endoplasmic reticulum (ER) stress, potentially supporting tumor survival. Here, BC cells were found to exhibit better survival under ER stress when PDIA1 was downregulated. These cells maintained homeostatic PDIA1 levels through the EV-mediated release of PDIA1. Chronic exposure of urothelial cells to these PDIA1-enriched BCEVs induced oxidative stress and DNA damage, ultimately leading to the malignant transformation of recipient cells. The EV-transformed cells exhibited DNA damage patterns potentially attributable to oxidative damage, and PDIA1 was found to be a key tumorigenic cargo within EVs. Tissue microarray analyses of BC recurrence confirmed a significant correlation between tumor recurrence and the levels of both PDIA1 and ER stress. Together, these data suggest that cancer cells selectively sort oxidized PDIA1 into EVs for removal, and these EVs can, in turn, induce oxidative stress in recipient urothelial cells, predisposing them to malignant transformation and thereby increasing the risk of recurrence.

You have accessJournal of UrologyCME1 May 2022MP54-19 PROTEIN DISULFIDE ISOMERASE AS A PREDICTIVE BIOMARKER FOR NON-MUSCLE INVASIVE BLADDER CANCER RECURRENCE Kit L. Yuen, Chia-Hao Wu, Christopher R. Silvers, Alexis Steinmetz, Hiroshi Miyamoto, Edward M. Messing, and Yi-Fen Lee Kit L. YuenKit L. Yuen More articles by this author , Chia-Hao WuChia-Hao Wu More articles by this author , Christopher R. SilversChristopher R. Silvers More articles by this author , Alexis SteinmetzAlexis Steinmetz More articles by this author , Hiroshi MiyamotoHiroshi Miyamoto More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002633.19AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: High recurrence rates found in non-muscle invasive bladder cancer (NMIBC) pose a financial and psychological burden for patients. We demonstrated that bladder cancer (BC) cells release protein disulfide isomerase (PDI), a redox-regulated ER-resident protein, via extracellular vesicles (EVs) when experiencing high levels of ER stress for their own survival. Chronic exposure to PDI-enriched BC EVs induced malignant transformation in recipient non-transformed cells. We hypothesized that the dynamic redox state in BC cells determines PDI cellular localization and tumors with elevated PDI preferentially incorporate PDI into EVs for removal. Furthermore, PDI expression levels and subcellular localization in tumors from NMIBC patients may predict NMIBC recurrence. METHODS: Retrospective chart review identified 122 patients with newly diagnosed low-grade NMIBC classified into 2 groups: recurrent (N=55) and non-recurrent (N=67) during follow-up period. Formalin fixed paraffin embedded tumor specimens from index transurethral resection of bladder tumor (TURBT) were obtained and a tissue microarray (TMA) was constructed. Immunofluorescent staining of TMA sections was conducted using antibodies against PDI and BiP, an ER stress marker. Stained tissues were photographed and tumor regions in each field were manually masked and confirmed by a genitourinary pathologist. Within each tumor region, total antibody labeling was determined by measuring the mean pixel intensity values with NIH ImageJ/Fiji. Manders coefficient was used to assess the colocalization of epitopes using JACoP plugin in ImageJ. RESULTS: Tumor tissues from patients that recurred expressed significantly higher levels of PDI and BiP. Moreover, high PDI and BiP expression predicted worse recurrence-free survival (Fig 1A and B). These data suggest that tissues under higher levels of ER stress—as indicated by higher levels of PDI and BiP expression—have a higher risk of recurrence. Significantly higher levels of PDI were detected in non-ER cytosol in recurrent cohort tumors (Fig 1D). CONCLUSIONS: This study provides a novel mechanism for BC recurrence and valuable clinical predictive biomarkers for NMIBC recurrence and progression. Future clinical applications targeting this pathway may be an avenue to prevent NMIBC recurrence. Source of Funding: NCI R01 CA173 © 2022 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 207Issue Supplement 5May 2022Page: e934 Advertisement Copyright & Permissions© 2022 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kit L. Yuen More articles by this author Chia-Hao Wu More articles by this author Christopher R. Silvers More articles by this author Alexis Steinmetz More articles by this author Hiroshi Miyamoto More articles by this author Edward M. Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement PDF downloadLoading ...

INTRODUCTION AND OBJECTIVE: High recurrence rates found in non-muscle invasive bladder cancer (NMIBC) pose a financial and psychological burden for patients. We demonstrated that bladder cancer (BC) cells release protein disulfide isomerase (PDI), a redox-regulated ER-resident protein, via extracellular vesicles (EVs) when experiencing high levels of ER stress for their own survival. Chronic exposure to PDI-enriched BC EVs induced malignant transformation in recipient non-transformed cells. We hypothesized that the dynamic redox state in BC cells determines PDI cellular localization and tumors with elevated PDI preferentially incorporate PDI into EVs for removal. Furthermore, PDI expression levels and subcellular localization in tumors from NMIBC patients may predict NMIBC recurrence. METHODS: Retrospective chart review identified 122 patients with newly diagnosed low-grade NMIBC classified into 2 groups: recurrent (N=55) and non-recurrent (N=67) during follow-up period. Formalin fixed paraffin embedded tumor specimens from index transurethral resection of bladder tumor (TURBT) were obtained and a tissue microarray (TMA) was constructed. Immunofluorescent staining of TMA sections was conducted using antibodies against PDI and BiP, an ER stress marker. Stained tissues were photographed and tumor regions in each field were manually masked and confirmed by a genitourinary pathologist. Within each tumor region, total antibody labeling was determined by measuring the mean pixel intensity values with NIH ImageJ/Fiji. Manders coefficient was used to assess the colocalization of epitopes using JACoP plugin in ImageJ. RESULTS: Tumor tissues from patients that recurred expressed significantly higher levels of PDI and BiP. Moreover, high PDI and BiP expression predicted worse recurrence-free survival. These data suggest that tissues under higher levels of ER stress—as indicated by higher levels of PDI and BiP expression—have a higher risk of recurrence. Significantly higher levels of PDI were detected in non-ER cytosol in recurrent cohort tumors. CONCLUSIONS: This study provides a novel mechanism for BC recurrence and valuable clinical predictive biomarkers for NMIBC recurrence and progression. Future clinical applications targeting this pathway may be an avenue to prevent NMIBC recurrence. Citation Format: Kit L. Yuen, Chia-Hao Wu, Christopher R. Silvers, Alexis R. Steinmetz, Hiroshi Miyamoto, Edward M. Messing, Yi-Fen Lee. Protein disulfide isomerase as a predictive biomarker for non-muscle invasive bladder cancer recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 517.

Markers of stromal activation at future metastatic sites may have prognostic value and may allow clinicians to identify and abolish the pre-metastatic niche to prevent metastasis. In this study, we evaluate tenascin-C as a marker of pre-metastatic niche formation in bladder cancer patient lymph nodes.Tenascin-C expression in benign lymph nodes was compared between metastatic (n = 20) and non-metastatic (n = 27) patients with muscle-invasive bladder cancer. Urinary extracellular vesicle (EV) cytokine levels were measured with an antibody array to examine potential correlation with lymph node inflammation. The ability of bladder cancer EVs to activate primary bladder fibroblasts was assessed in vitro.Lymph node tenascin-C expression was elevated in metastatic patients vs. non-metastatic patients, and high expression was associated with worse survival. Urinary EVs contained four cytokines that were positively correlated with lymph node tenascin-C expression. Bladder cancer EVs induced tenascin-C expression in fibroblasts in an NF-κB-dependent manner.Tenascin-C expression in regional lymph nodes may be a good predictor of bladder cancer metastasis and an appropriate imaging target. It may be possible to interrupt pre-metastatic niche formation by targeting EV-borne tumour cytokines or by targeting tenascin-C directly.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-04 PROTEIN DISULFIDE ISOMERASE IN EXTRACELLULAR VESICLES DRIVE MALIGNANT TRANSFORMATION IN BLADDER CANCER TUMOR MICROENVIRONMENT Chia-Hao Wu, Kit L. Yuen, Christopher R. Silvers, Edward M. Messing, and Yi-Fen Lee Chia-Hao WuChia-Hao Wu More articles by this author , Kit L. YuenKit L. Yuen More articles by this author , Christopher R. SilversChristopher R. Silvers More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Non-muscle invasive bladder cancer (NMIBC) exhibits a high recurrence rate and tumor multifocality, placing significant financial and psychological burden on patients. Extracellular vesicles (EVs) are membrane-bound nano-particles containing bioactive molecules critical for intercellular communication. Recent studies provide evidence that BCEVs promote the malignant transformation of predisposed cells by activating the oncogenic ER stress response. We sought to identify responsible cargo proteins and underlying mechanisms that drive tumorigenesis. METHODS: Mass spectrometry was used to identify the candidate EV protein: protein disulfide isomerase (PDI). Loss/gain-of-function approaches were applied to define the functional roles of EV-borne PDI. Various assays including Western blot, DCFDA, immunofluorescence staining, and colony-forming assay were performed to characterize cellular molecular alterations during transformation. Whole-genome sequencing (WGS) analysis was performed to assess the mutations in the transformed cells. Urinary EVs isolated from NMIBC patients were analyzed for the presence of PDI. RESULTS: EV-borne PDI, a redox chaperone of ER, was identified for its exclusive and abundant presence in BCEVs. Treating normal urothelial SV-HUC cells with PDI-enriched BCEVs increased cellular oxidative stress and DNA damage. Knock down of PDI in BCEVs reduced cellular ROS and DNA damage, suppressing tumorigenesis in SV-HUC recipient cells. Restoring PDI by extrusion rescues tumorigenic phenotypes. In transformed cells, WGS identified 114,978 unique variants and 875 genes harboring mutations in coding regions. Importantly, several of these genes are tumor suppressors, negative regulators of cell proliferation, and regulators of apoptosis- essential alterations described as seminal hallmarks of cancer. PDI was detectable in urinary EVs derived from NMIBC patients, making PDI a viable candidate marker for liquid biopsy. CONCLUSIONS: This supports a model wherein PDI-enriched EVs induce cellular stress responses in recipient normal urothelial cells and promote mutational events. Accumulation of somatic mutations facilitates the selection for cells that gain a survival advantage and eventually evolve into fully transformed malignant cells. This study offers a potential therapeutic target in BCEVs to prevent BC recurrence. PDI detection in urinary EVs may provide a novel noninvasive biomarker for NMIBC recurrence. Source of Funding: NCI R01 CA173986 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e656-e656 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Chia-Hao Wu More articles by this author Kit L. Yuen More articles by this author Christopher R. Silvers More articles by this author Edward M. Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Loading ...

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV (PD47)1 Apr 2020PD47-08 URINARY EXOSOME CYTOKINE LEVELS CORRELATE WITH PELVIC LYMPH NODE INFLAMMATION Christopher Silvers*, Hiroshi Miyamoto, Edward M. Messing, and Yi-Fen Lee Christopher Silvers*Christopher Silvers* More articles by this author , Hiroshi MiyamotoHiroshi Miyamoto More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000934.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Pre-clinical evidence suggests that inflammatory cytokines carried by cancer exosomes can cause inflammation at pre-metastatic sites, enabling metastatic colonization and outgrowth. A role for exosomes in pre-metastatic niche formation has not yet been demonstrated in humans. Pelvic lymph nodes are frequent sites of bladder cancer metastasis, and we hypothesized that tumor exosomes may drive pre-metastatic niche formation in tumor-draining lymph nodes by delivering encapsulated inflammatory cytokines that activate the lymph node stroma. In this study, we examined whether the cytokine levels found in bladder cancer patient urinary exosomes (which include tumor-derived exosomes) correlate with the expression of stromal activation marker tenascin C in the corresponding patient pelvic lymph nodes. METHODS: The study group included 13 bladder cancer patients with muscle-invasive disease (pT2-pT4) and no history of treatment with bacillus Calmette-Guérin. Exosomes were isolated from the patients’ urine by differential ultracentrifugation, and the exosomal levels of 13 inflammatory cytokines and growth factors were measured using an antibody array. One benign pelvic lymph node was randomly selected from each patient, sectioned, and stained for tenascin C, a prospectively chosen marker of stromal activation that is up-regulated during inflammation. Tenascin C expression was calculated as the positive-staining area fraction. Correlations between individual cytokine expression and tenascin C staining were determined using Spearman’s coefficient. P-values were corrected for multiple comparisons using the Benjamini-Hochberg procedure. RESULTS: Four of the 13 cytokines examined (HGF, bFGF, TGFβ, KGF) were positively correlated with lymph node tenascin C expression (P < 0.05). The Spearman’s correlation coefficients (rs) of the four cytokines ranged from 0.70-0.81. CONCLUSIONS: The correlation between bladder exosome cytokine levels and pelvic lymph node inflammation suggests that exosomes may be a mechanism by which primary tumors generate inflammation at distant sites. Our data suggest that tumor exosomes containing inflammatory cytokines may reprogram lymph node stromal cells in ways that support metastatic colonization. Understanding the roles that tumor exosomes play in metastatic processes may aid in their early detection and provide targets for therapeutic intervention. Source of Funding: NCI, National Institutes of Health grant RO1 CA173986. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e928-e928 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christopher Silvers* More articles by this author Hiroshi Miyamoto More articles by this author Edward M. Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement PDF downloadLoading ...

INTRODUCTION AND OBJECTIVE: Non-muscle invasive bladder cancer is characterized by a high recurrence rate and multifocal tumors that may have different driver mutations. This feature contributes greatly to the challenges in clinical management and financial burden. Increasing evidence has indicated that cancer extracellular vesicles (EV) carry oncogenic cargos and play roles in tumorigenesis. Our recent study reported that normal urothelial cells are malignantly transformed by long-term exposure to cancer EVs through inducing endoplasmic reticulum (ER) stress. This study aims to identify responsible EV-borne molecules that drive tumorigenesis. METHODS: Proteomic analysis of EVs was performed by TMT mass spectrometry and Online database (PANTHER and Human Protein Atlas) analysis. Cell lines, including grade 4 bladder cancer TCCSUP cells, and non-malignant urothelial SV-HUC cells were used. We applied a loss-of-function strategy to study the functional roles of the candidate protein in both cell lines. The impacts on SV-HUC cells after receiving EVs were assessed by Western blot, DCFDA assay, and gH2AX staining to respectively detect ER stress signaling, reactive oxygen species (ROS) level, and DNA damage. TCCSUP was analyzed by Western blot to determine apoptosis and PDI signals. Redox state of the candidate protein was determined by immunoprecipitation followed by reduced thiol quantification. RESULTS: Protein disulfide isomerase (PDI) was found enriched and exclusively present in cancer EVs and was selected for further investigation. Its expression level is inversely correlated with bladder cancer patient survival. Under ER stress, TCCSUP bladder cancer cells maintain a PDI homeostasis through releasing PDI by EVs while retaining non-active reduced-PDI within the cells. Knocking down PDI in cancer cells increases their resistance to ER stress-induced apoptosis. Surprisingly, TCCSUP cells export EVs contain mostly oxidized-PDI, which can induce ER stress and oxidative stress. Correspondingly, treating normal SV-HUC cells with TCCSUP EVs results in increased cellular ROS, PERK/ER stress signaling, and DNA damage levels that could eventually lead to malignant transformation. CONCLUSIONS: Bladder cancer cells export oxidized PDI via EVs into extracellular space to reduce cellular stress. Exposing to PDIcontaining EVs in the surrounding tumor microenvironment induces oncogenic insults in normal cells. This study provides a novel mechanism for the field cancerization effect and its contribution to bladder cancer recurrence.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP17)1 Apr 2020MP17-02 TENASCIN C EXPRESSION IN BENIGN PELVIC LYMPH NODES CORRELATES WITH METASTATIC STATUS AND SURVIVAL Christopher Silvers*, Hiroshi Miyamoto, Edward M. Messing, and Yi-Fen Lee Christopher Silvers*Christopher Silvers* More articles by this author , Hiroshi MiyamotoHiroshi Miyamoto More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000842.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Increasing evidence suggests that inflammation and stromal activation at pre-metastatic sites constitute a niche that supports the colonization and outgrowth of disseminated tumor cells. The objective of this study was to use clinical samples to determine whether bladder cancer metastasis is preceded by the formation of a pre-metastatic niche in pelvic lymph nodes. We hypothesized that stromal activation would be detected in benign lymph nodes and that uninvolved nodes from metastatic patients would contain more evidence of stromal activation than would nodes from non-metastatic patients. METHODS: The study group included 47 muscle-invasive bladder cancer patients with metastatic (n=20) or non-metastatic (n=27) disease. One benign pelvic lymph node was randomly selected from each patient, sectioned, and immunohistochemically stained. Benign node status was confirmed using a cytokeratin stain which revealed the presence of occult micrometastases in three of the initial 50 candidate nodes. As a marker of activation in fibroblasts and endothelial cells, we prospectively chose tenascin C, a glycoprotein that is otherwise down-regulated in healthy adult tissues. Tenascin C expression in each node was calculated as the positive-staining area fraction, and expression was compared between the metastatic and non-metastatic patient groups using the Wilcoxon rank-sum test. Disease-specific survival curves for high and low tenascin C expressing patients were compared using the log-rank test. RESULTS: Tenascin C staining in the uninvolved nodes of both groups was variable, with some showing minimal expression and others containing regions of strong, diffuse stromal staining. Patients with metastatic bladder cancer had a 1.64-fold higher mean tenascin C expression in the benign lymph nodes in comparison to non-metastatic patients (P = 0.0049). Patients with high tenascin C expression in benign lymph nodes had worse disease-specific survival (P = 0.05). CONCLUSIONS: These results suggest that bladder cancer metastasis is preceded by stromal activation in tumor-draining lymph nodes. Furthermore, lymph node stromal activation is associated with poor patient survival. Pelvic lymph node dissection following cystectomy may be a valuable source of clinical specimens allowing the investigation of pre-metastatic niche formation in bladder cancer. Source of Funding: NCI, National Institutes of Health grant RO1 CA173986. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e225-e226 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christopher Silvers* More articles by this author Hiroshi Miyamoto More articles by this author Edward M. Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement PDF downloadLoading ...

Recent data suggest that patients with a basal/stem-like bladder cancer (BC) subtype tend to have metastatic disease, but this is unconfirmed. Here we report the identification of murine MB49 cell line sub-clones with stem-like characteristics in culture. Subcutaneous implantation of S2 and S4 MB49 sub-clones into immunocompetent mice resulted in lung metastases in 50% and 80% of mice respectively, whereas none of the mice implanted with the parental cells developed metastasis. Gene profiling of cells cultured from S2 and S4 primary and metastatic tumors revealed that a panel of genes with basal/stem-like/EMT properties is amplified during metastatic progression. Among them, ITGB1, TWIST1 and KRT6B are consistently up-regulated in metastatic tumors of both MB49 sub-clones. To evaluate clinical relevance, we examined these genes in a human public dataset and found that ITGB1 and KRT6B expression in BC patient tumor samples are positively correlated with tumor grade. Likewise, the expression levels of these three genes are correlated with worse clinical outcomes. This MB49 BC metastatic pre-clinical model provides a unique opportunity to validate and recapitulate results discovered in patient studies and to pursue future mechanistic therapeutic interventions for BC metastasis.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP57)1 Apr 2019MP57-16 TUMOR DERIVED EXTRACELLULAR VESICLES PROMOTE BLADDER TUMORIGENESIS BY INDUCING ENDOPLASMIC RETICULUM STRESS. ChiaHao Wu*, Christopher Silvers, Edward Messing, and Yi-Fen Lee ChiaHao Wu*ChiaHao Wu* More articles by this author , Christopher SilversChristopher Silvers More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/01.JU.0000556620.32600.efAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVES: Many types of cancer undergo endoplasmic reticulum (ER) stress. It is a stress response when unfolded protein accumulates, that leads to the activation of a series of signaling cascade, including apoptosis and inflammation. Tumors secrete a higher number of extracellular vesicles (EVs) containing oncogenic cargo that can be taken up and disrupt homeostasis in normal cells. We hypothesize that EVs derived from bladder cancer (BC) cells, by causing ER stress, impose a selection force in normal recipient cells that lead to a higher malignant transformation frequency. METHODS: EVs from TCC-SUP, a BC line, and SV-HUC cells, a non-malignant immortalized urothelial line, were collected by serial centrifugation, counted by nanoparticle tracking analysis, and examined by mass spectrometry. SV-HUC cells were chronically treated with TCC-SUP EVs and were determined by an in vitro colony formation assay and an in vivo xenograft mouse model. Some cancer cell properties: loss of contact inhibition, genome instability, and invasion were investigated. EVs provoked ER stress responses were assayed by Western blot. RESULTS: BC cells secreted a higher number of EVs than normal urothelial cells. These BCEVs carried distinct proteins; some were oncogenic and could induce normal recipient cells′ pro-apoptotic ER stress response. Prolonged activation of ER stress led cells to switch to a pro-survival branch of ER stress response, and eventually, those cells were transformed and gained several cancerous properties. More importantly, inhibition of ER stress response by docosahexaenoic acid reduced BCEV-induced tumorigenesis. CONCLUSIONS: Our data provide a novel mechanism whereby prolonged ER stress induced by persistent exposure to tumor-derived EVs drives the malignant transformation of clinically indolent recipient cells. This study provides insight into bladder cancer′s multifocal and recurrent nature and suggests EVs as potential markers of disease recurrence and progression. Source of Funding: RO1CA173986 Rochester, NY© 2019 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 201Issue Supplement 4April 2019Page: e818-e819 Advertisement Copyright & Permissions© 2019 by American Urological Association Education and Research, Inc.MetricsAuthor Information ChiaHao Wu* More articles by this author Christopher Silvers More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement PDF downloadLoading ...

The field cancerization effect has been proposed to explain bladder cancer's multifocal and recurrent nature, yet its mechanisms remain unknown. In this work we show that chronic exposure to tumor-derived extracellular vesicles (TEVs) resulted in the neoplastic transformation of non-malignant human SV-HUC urothelial cells. Inhibition of EV uptake prevented transformation. Transformed cells not only possessed several oncogenic properties, such as genome instability, loss of cell-cell contact inhibition, and invasiveness, but also displayed altered morphology where cells show enlarged cytoplasm with disrupted ER alignment and the accumulation of smaller mitochondria. Treatment of SV-HUC cells with TEVs provoked the unfolded protein response of endoplasmic reticulum (UPRER). Prolonged induction of UPRER signaling led to the activation of the survival branch of the UPRER pathway, where cells had elevated expression of the IRE1, NFB and the inflammatory cytokine leptin, and loss of CHOP, a pro-apoptotic protein. More importantly, inhibition of ER stress by docosahexaenoic acid prevented TEV-induced transformation. We propose that TEVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress-induced unfolded protein response and inflammation. This study provides insight into the mechanisms of the bladder cancer field effect and tumor recurrence.

The field cancerization effect has been proposed to explain bladder cancer's multifocal and recurrent nature, yet the mechanisms of this effect remain unknown. In this work, using cell biology, flow cytometry, and qPCR analyses, along with a xenograft mouse tumor model, we show that chronic exposure to tumor-derived extracellular vesicles (TEVs) results in the neoplastic transformation of nonmalignant human SV-HUC urothelial cells. Inhibition of EV uptake prevented this transformation. Transformed cells not only possessed several oncogenic properties, such as increased genome instability, loss of cell–cell contact inhibition, and invasiveness, but also displayed altered morphology and cell structures, such as an enlarged cytoplasm with disrupted endoplasmic reticulum (ER) alignment and the accumulation of smaller mitochondria. Exposure of SV-HUC cells to TEVs provoked the unfolded protein response in the endoplasmic reticulum (UPRER). Prolonged induction of UPRER signaling activated the survival branch of the UPRER pathway, in which cells had elevated expression of inositol-requiring enzyme 1 (IRE1), NF-κB, and the inflammatory cytokine leptin, and incurred loss of the pro-apoptotic protein C/EBP homologous protein (CHOP). More importantly, inhibition of ER stress by docosahexaenoic acid prevented TEV-induced transformation. We propose that TEVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress–induced unfolded protein response and inflammation. This study provides detailed insight into the mechanisms underlying the bladder cancer field effect and tumor recurrence.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2017MP65-02 CANCER EXTRACELLULAR VESICLES PROMOTE BLADDER TUMORIGENESIS BY INDUCING CHRONIC ENDOPLASMIC RETICULUM STRESS AND INFLAMMATION: A NOVEL MECHANISM FOR FIELD CANCERIZATION Chia-Hao Wu, Christopher Silvers, Edward Messing, and Yi-Fen Lee Chia-Hao WuChia-Hao Wu More articles by this author , Christopher SilversChristopher Silvers More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1996AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Cancer field effect describes the predisposition of a field of tissue with histological anomalies or molecular alterations to a high incidence of tumor initiation and has been proposed to explain bladder cancer's (BC's) multifocal and recurrence nature, yet its mechanisms remain unknown. Extracellular vesicles (EVs) are small, membrane-bound vesicles with functions in cell-cell communication. We hypothesize that EVs derived from BC cells transfer bioactive cargo to reprogram recipient cells in the field and induce malignant transformation. METHODS EVs from TCC-SUP, a BC line, were collected and purified. SV-HUC cells, a non-malignant immortalized urothelial line, were used as recipients. Tumorigenicity was determined by an in vitro anchorage-independent colony formation assay and an in vivo xenograft mouse model. Hallmarks of cancer such as loss of contact inhibition, genome instability, and invasion were studied. Molecular alterations in EV-transformed cells were assayed by qPCR and Western blot. Pro-inflammatory cytokines were compared by multiplex ELISA assays. RESULTS Bladder cancer EVs induced malignant transformation of SV-HUC cells in vitro and in vivo. Molecular profiling revealed abnormal levels of endoplasmic reticulum (ER) stress sensors/effectors and pro-inflammatory cytokines in transformed cells. Moreover, the expression of ER chaperone protein Grp78 and two ER resident sensors, PERK and IRE1, was induced. Pro-apoptotic ER stress effector CHOP was absent, suggesting cancer EVs promote tumorigenesis by activating tumor-promoting signals while inhibiting pro-apoptotic signals. CONCLUSIONS Our data support a novel mechanism whereby cancer EVs and their cargo molecules play key roles in the malignant field of BC. Our study reveals that cancer EVs promote malignant transformation of predisposed cells by inhibiting pro-apoptotic signals and activating tumor-promoting ER stress induced unfolded protein response and inflammation. This study provides insight into mechanism of BC's field effect and suggests EV as potential markers of disease recurrence and progression © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e853 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Chia-Hao Wu More articles by this author Christopher Silvers More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The mechanisms of bladder cancer progression are unknown, and new treatments and biomarkers are needed. Patient urinary extracellular vesicles (EVs) derive in part from bladder cancer cells and contain a specific protein cargo which may provide information about the disease. We conducted a proteomics study comparing EVs from the muscle-invasive bladder cancer (MIBC) cell line TCCSUP to EVs from normal urothelial line SVHUC. GO term analysis showed that TCCSUP EVs are enriched in proteins associated with the cell membrane, extracellular matrix, and inflammation and angiogenesis signaling pathways. Proteins characteristic of cancer EVs were further screened at the mRNA level in bladder cancer cell lines. In Western blots, three of six proteins examined showed greater than fifteenfold enrichment in patient urinary EVs compared to healthy volunteers (n = 6). Finally, we performed immunohistochemical staining of bladder tissue microarrays for three proteins of interest. One of them, transaldolase (TALDO1), is a nearly ubiquitous enzyme and normally thought to reside in the cytoplasm. To our surprise, nuclei were stained for transaldolase in 94% of MIBC tissue samples (n = 51). While cytoplasmic transaldolase was found in 89-90% of both normal urothelium (n = 79) and non-muscle-invasive samples (n = 71), the rate falls to 39% in MIBC samples (P < 0.001), and negative cytoplasmic staining was correlated with worse cancer-specific survival in MIBC patients (P = 0.008). The differential EV proteomics strategy reported here successfully identified a number of proteins associated with bladder cancer and points the way to future investigation.

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2017MP65-01 EXTRACELLULAR VESICLES AS A POSSIBLE MECHANISM FOR BACILLUS CALMETTE-GUÉRIN IMMUNOTHERAPY Carlos Ortiz-Bonilla, Christopher Silvers, Peng-Nien Yin, Edward Messing, and Yi-Fen Lee Carlos Ortiz-BonillaCarlos Ortiz-Bonilla More articles by this author , Christopher SilversChristopher Silvers More articles by this author , Peng-Nien YinPeng-Nien Yin More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1995AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical Bacillus Calmette-Guérin (BCG) immunotherapy has been used to treat non-muscle invasive bladder cancer (BC) for nearly 40 years but its underlying mechanism remains largely unknown. It is generally believed that BCG adheres to integrin ?5?1 in the urothelial lining by interacting with fibronectin, then triggers an immune response cascade. Extracellular vesicles (EVs), small membrane-bound vesicles, act as immune modulators by transferring molecular cargos to recipient cells. We hypothesize that EVs derived from BC cells play key roles in mediating BCG-induced anti-tumor host immune responses, and the patient derived EVs may serve as predictive biomarkers that can differentiate BCG responders from non-responders. METHODS BC cell lines, human T24 and murine MB49, and immortalized bladder SV-HUC cells were treated with 1-4x106 CFU/ml live BCG. After 12-72 hours, secreted EVs were isolated by serial ultracentrifugation and analyzed by Nanoparticle Tracking Analysis. Cell lysate and total RNA was collected for immuno-molecular profiling by quantitative PCR and Western blotting analyses. The alterations in EV secretion, gene and protein expression, and molecules from BC derived EVs in response to BCG were compared. Urinary EVs were collected and purified before and after BCG patients' 1st and 3rd BCG instillations and EV secretion profiles were compared. RESULTS In response to BCG, SV-HUC cells showed decreased EV secretion and their immuno- molecules were significantly reduced compared with EVs from naïve SV-HUC cells. In contrast, in BC cells the EV secretion rate was significantly induced by BCG as well as the expression of the key molecules in modulating immune response, such as MHC and co-stimulatory molecules at gene and protein levels. Critically, we found that BC cells, but not SV-HUC cells, released immuno-molecules containing EVs in response to BCG. Importantly, we found that urinary EV numbers were increased significantly after the 3rd BCG instillation in BCG responders, but not in BCG-non-responders in a pilot study. CONCLUSIONS We conclude that BCG treatment resulted in increased EV release from BC cells as well as in increased EVs in urine of BC patients. In addition, BCG induced expression of key immuno-modulatory molecules in BC cells and within the EVs. This up-regulated expression of immuno-molecules in response to BCG supports the hypothesis that EVs have a role in activating the immune system during BCG immunotherapy. The immunologically active EVs detected in patients' urine can be further explored as predictive biomarkers. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e852-e853 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Carlos Ortiz-Bonilla More articles by this author Christopher Silvers More articles by this author Peng-Nien Yin More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Muscle-invasive bladder cancer (MIBC) is an aggressive malignancy with high mortality, and heterogeneity in MIBC results in variable clinical outcomes, posing challenges for clinical management. Extracellular vesicles (EVs) derived from MIBC have been shown to promote cancer progression. EVs derived from bladder cell lines were subjected to proteomic analysis, and periostin was chosen for further characterization due to its stage-specific gene expression profile. Knockdown of periostin by RNA interference reduces invasiveness in vitro and produces a rounder morphology. Importantly, treating low grade BC cells with periostin-rich EVs promotes cell aggressiveness and activates ERK oncogenic signals, and periostin suppression reverses these effects. These data suggest that MIBC might transfer periostin in an EV-mediated paracrine manner to promote the disease. To determine the potential of periostin as a bladder cancer indicator, patient urinary EVs were examined and found to have markedly higher levels of periostin than controls. In addition, immunohistochemical staining of a bladder cancer tissue microarray revealed that the presence of periostin in MIBC cells is correlated with worse prognosis. In conclusion, periostin is a component of bladder cancer cells associated with poor clinical outcome, and EVs can transfer oncogenic molecules such as periostin to affect the tumor environment and promote cancer progression.

You have accessJournal of UrologyBladder Cancer: Basic Research IV1 Apr 2015MP68-14 EXTRACELLULAR VESICLES AS BIOMARKERS AND DIAGNOSTICS IN BLADDER CANCER Christopher Silvers, Chia-Hao Wu, Peng-Nien Yin, Yu-Ru Liu, Edward Messing, and Yi-Fen Lee Christopher SilversChristopher Silvers More articles by this author , Chia-Hao WuChia-Hao Wu More articles by this author , Peng-Nien YinPeng-Nien Yin More articles by this author , Yu-Ru LiuYu-Ru Liu More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2476AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Extracellular vesicles (EV) are small membrane vesicles secreted by most cell types. They are first found in reticulocytes and play important roles in cell-to-cell communication. Recent studies suggest that EV secreted by tumor cells containing tumor specific proteins and miRNAs. Previous study shows that EV purified from muscle invasive bladder cancer promote cancer progression. This study aims to characterize the genetic contents, such as proteins and nucleic acids and establish the bladder cancer specific EV biomarkers METHODS EV were purified from bladder cancers, immortalized bladder cells, and urine of healthy volunteers and muscle invasive bladder cancer patients. The protein contents were analyzed by mass spectrometry. To prioritize the proteins of interest, we performed gene expression profile analysis of proteins via public microarray repositories from NCBI Gene Expression Omnibus profile #GDS1479 (carcinoma in situ lesions of the urinary bladder) that consists of 5 categories: normal urothelium, Non-muscle invasive BC with cancer in situ (CIS), superficial TCC without CIS, Muscle invasive bladder cancer, and CIS. In addition to protein, we also performed miRNA expression profiling microarray on EV from high grade bladder cancer cells, and immortalized bladder cells (LC Sciences). RESULTS More than 400 proteins were found in bladder EV. Among them, we found 9 cancer-associated EV proteins that are particularly interesting because their gene expression levels are significantly elevated in cancer stages as compared to normal urothelium. Those proteins are subjected to further functional characterization via gene knockdown/overexpression strategies. The differences in miRNA expression were compared and differences were determined by a student T test (P<0.05). Results showed significant difference miRNAs between cancer cells vs normal cells. Some of cancer-specific miRNAs which have shown to play critical roles in tumorigenesis and cancer progression will be study priority CONCLUSIONS Our study enables us to establish a panel of EV protein and miRNA signatures that can be further developed into a urine-based method for rapid detection of stage-specific biomarkers which will be valuable for detecting and managing bladder cancer. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e863 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christopher Silvers More articles by this author Chia-Hao Wu More articles by this author Peng-Nien Yin More articles by this author Yu-Ru Liu More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 2015MP49-18 IDENTIFICATION OF EXTRACELLULAR VESICLE PERIOSTIN AS A URINARY BIOMARKER OF MUSCLE INVASIVE BLADDER CANCER Christopher Silvers, Miyamoto Hiroshi, Chia-Hao Wu, Edward Messing, and Yi-Fen Lee Christopher SilversChristopher Silvers More articles by this author , Miyamoto HiroshiMiyamoto Hiroshi More articles by this author , Chia-Hao WuChia-Hao Wu More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.2813AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Periostin is an extracellular matrix protein with roles in cell adhesion and motility and is overexpressed in several cancer types. Multiple microarray dataset analyses of patient tissues have shown periostin mRNA to be overexpressed in muscle-invasive bladder cancer (MIBC), but further investigation has been limited. Extracellular vesicles (EV) are 30-2000 nm membrane-bound structures released by a variety of cell types and bearing a select cargo of proteins and nucleic acids. Our objectives were to investigate functions of periostin in MIBC, the contribution of EV-borne periostin, and the potential of EV periostin as a urinary marker METHODS Multiple bladder cancer cell lines were screened by quantitative real-time PCR (qPCR) for periostin expression. Two highly expressing lines (TCC-SUP and J82) were selected for periostin suppression by RNA interference (shPOSTN). Single clone knockdowns were assessed for changes in morphology, motility, invasiveness, and tumorigenicity in nude mice. EV were isolated and assayed for their ability to affect recipient cells' behaviors. Bladder cancer tissue microarray was performed and periostin expression levels in patients' tumors were correlated to to clinical outcomes. EV were isolated from the urine of MIBC patients and healthy controls, and periostin levels were assayed by Western blot. RESULTS J82 and TCC-SUP cells migrate and invade rapidly and have an extended morphology with long invadopodia. Periostin knockdown significantly slowed migration and invasion and produced a more circular morphology with fewer invadopodia. EV derived from shPOSTN cells had reduced ability to promote migration in recipient cells. Periostin expression in patient MIBC epithelial cells was negatively correlated with progression-free survival (P<0.021) and disease-specific survival (P<0.002). Ten MIBC (pT2-pT4) patient urine EV samples had markedly higher levels of periostin than stage pT1 disease samples (P<0.033), post-TURBT non-MIBC patients (P<0.004), or healthy volunteers (P<0.00003). CONCLUSIONS Periostin plays a heretofore unrecognized role in MIBC. Its expression in bladder cancer epithelial cells is related to the acquisition of an invasive phenotype and may be a promising therapeutic target, and the ability of EV containing high levels of periostin to influence recipient cells invites further study. The easy detectability of periostin in urine EV suggests its suitability as a biomarker of MIBC progression and a possible alternative to expensive cystoscopic surveillance © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e609-e610 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Christopher Silvers More articles by this author Miyamoto Hiroshi More articles by this author Chia-Hao Wu More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Exsosomes are nano-vesicles secreted by most types of cells, and substantially increased secretion in cancer cells. Their role in tumorigenesis/cancer progression have been extensively studied but their role in regulating therapy response have been suggested without in depth investigation. We are particularly interested in exosome9s roles in cisplatin resistant, since cisplatin is one of the most widely used chemodrugs for many cancer types. To investigate the role of exosomes in cisplatin resistance in bladder cancer, we examined the cisplatin sensitivity among five bladder cancer cell lines. The expression levels of exosome associated genes that are involved in exosome biogenesis and secretion/internalization were examined by quantative PCR. Interestingly, cisplatin resistant bladder cancer cells express relative high level of exosome associated genes as compared to cisplatin sensitive cells. More importantly, cisplatin treatment induces the expression of exosome associated genes’ expression in cisplatin resistant cells, but not in sensitive cell. Among all those genes, we are particularly interested in Rab gene family, and mechanistic study of targeting those Rab proteins is undergoing. The correlative exosome genes profiles with cisplatin resistance suggested exosome production might facilitate cancer cells escaping from cisplatin-induced cell death; thereby, inhibition of exosome might sensitize cancer cells response to cisplatin. To test this, we have screened several compounds that are known to inhibit exosome biogenesis and uptake, and. found that inhibition of exosomes by dynasore + NSC23766, indeed can sensitize bladder cancer cells to cisplatin. In summary, we concluded that cancer exosome play a critical role of promoting cisplatin resistance in bladder cancer, and inhibition of exosomes sensitizes bladder cancer cell response to cisplain. The exosome related profiles potentially can serve as therapy biomarkers Citation Format: Chiahao Wu, Chris Silvers, Elizabeths Guancial, Jong-Wei Hsu, Edward Messing, Yi-Fen Lee. Cancer exosome promotes cisplatin resistance In bladder cancer and inhibition of exosome sensitizes bladder cancer cells to cisplatin chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5475. doi:10.1158/1538-7445.AM2015-5475

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2014MP28-05 EXOSOMES PROMOTE BLADDER CELL MALIGNANT TRANSFORMATION, A NEW MECHANISM FOR THE FILED EFFECT Chia-Hao Wu, Christopher Silvers, Christopher Silvers, Edward Messing, and Yi-Fen Lee Chia-Hao WuChia-Hao Wu More articles by this author , Christopher SilversChristopher Silvers More articles by this author , Christopher SilversChristopher Silvers More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.654AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Greater than 70% of newly diagnosed bladder cancers are non-muscle invasive and these tumors tend to recur throughout the bladder lining and are not strictly limited to previous sites of resection, suggesting that the entire bladder is made permissive for tumorigenesis. This phenomenon has been termed field effect change (FEC), and the contributing factors driving this FEC are not clear. Exosomes are small membrane vesicles ranging from 30 to 100 nm. They are secreted from numerous types of cells into the extracellular milieu and can be taken up by neighboring cells, which act as novel intercellular modes of communication. To reveal exosomes¡¦ roles in bladder tumorigenesis, we conducted a series of laboratory experiments. METHODS Immortalized bladder cells, SV-HUC, were used as a model system to evaluate exosome-induced cellular transformation. Exosomes were purified from high-grade bladder cancer cells, TCC-SUP and SV-HUC cells, following a series of steps of centrifugation. We screened the exosome intake inhibitors via monitoring PKH67-labeling exosomes internalization. The tumorigenicity was determined by soft agar assay and further confirmed by xenografted nude mice. DNA damages and cellular senescence, as the results of exosome-induced oncogenic insults, were determined by gamma-H2Ax and senescence-associated beta-gal stainings. The genes involving in the DNA damage response, tumor suppressive function and oncogenic pathways were examined by real-time quantification PCR. RESULTS Long-term exposure of SV-HUC to exosome- enriched environment induced SV-HUC malignant transformation, where exosome-treated cells gain anchorage-independent growth by soft agar assay, and PBS-treated cells did not. The tumorigenicity was confirmed in vivo by the xenografted nude mice model. In addition, these transformed SV-HUC cells also gain the function of mobility and invasiveness. Importantly, dynasore, the inhibitor that blocks exosome intake, reversed the exosome-induced SV-HUC malignant transformation. Genes that are involved in DNA damage response and tumor suppressive function were found to be reduced, and several oncogenes were elevated in the transformed SV-HUC cells as compared to parental SV-HUC cells. CONCLUSIONS These data, for the first time, demonstrate that exosomes play a role in the early stage of neoplasmic transformation and provide a novel mechanism to explain FEC. More importantly, deprivation of exosome intake resulted in diminished SV-HUC malignant transformation revealing the therapeutic potential of dynasore. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e297-e298 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Chia-Hao Wu More articles by this author Christopher Silvers More articles by this author Christopher Silvers More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...