Protein Disulfide Isomerase-Enriched Extracellular Vesicles from Bladder Cancer Cells Support Tumor Survival and Malignant Transformation in the Bladder

Protein Disulfide Isomerase-Enriched Extracellular Vesicles from Bladder Cancer Cells Support Tumor Survival and Malignant Transformation in the Bladder | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Protein Disulfide Isomerase-Enriched Extracellular Vesicles from Bladder Cancer Cells Support Tumor Survival and Malignant Transformation in the Bladder Yi-Fen Lee, Chia-Hao Wu, Kit Yuen, Ryan Molony, Christopher Silvers, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4425743/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Apr, 2025 Read the published version in Oncogene → Version 1 posted 11 You are reading this latest preprint version Abstract 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 (PDI) 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 PDI was downregulated. These cells maintained homeostatic PDI levels through the EV-mediated release of PDI. Chronic exposure of urothelial cells to these PDI-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 PDI 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 PDI and ER stress. Together, these data suggest that cancer cells selectively sort oxidized PDI 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. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Over 70% of newly diagnosed bladder cancer (BC) patients have non-muscle invasive BC (NMIBC) confined to the urothelium and underlying lamina propria ( 1 , 2 ). NMIBC patients face high rates of recurrence, with two-thirds experiencing tumor recurrence within five years, and up to 88% within 15 years ( 3 ). One proposed explanation for these high rates of recurrence involves the cancer field effect wherein pre-malignant cells are predisposed to tumor development, potentially contributing to the multi-chronotropic and multifocal nature of recurrent NMIBC ( 4 ). How this occurs remains poorly understood, with some work supporting the stepwise accumulation of genetic alterations that ultimately result in tumor formation, whereas other studies suggest clonal expansion from a single common precursor as the major mechanism of recurrent tumor growth ( 5 – 7 ). Field cancerization may drive tumorigenesis regardless of the exact nature of these genetic transformation events. A comprehensive genetic analysis of bladder cancer samples using datasets from The Cancer Genome Atlas (TCGA) project identified multiple genomic alterations, suggesting that these progressive tumors are heterogeneous and can result from a permissive oncogenic environment in the whole bladder ( 8 ). Those tumors can recur anywhere in the bladder and may or may not share similar histology with the primary tumors ( 9 ), further supporting the theory that the entire bladder is permissive to tumorigenesis. The induction of endoplasmic reticulum (ER) stress responses can be cytoprotective ( 10 ), with unfolded protein accumulation within the ER lumen triggering a coordinated unfolded protein response (UPR) that can restore ER homeostasis ( 11 ). However, insufficient or sustained ER stress responses result in pathologic alterations that lead to oncogenesis ( 12 – 14 ). Indeed, ER stress and UPR induction are common features of human cancers ( 15 ). Highly proliferative tumors are exposed to several intrinsic and extrinsic stressors ( 16 ), potentially explaining the enhanced UPR signaling activity in these cells as a survival strategy that enables them to better tolerate exposure to stressful environments. The pronounced reliance of many cancer cells on the UPR has prompted interest in targeting this pathway as a form of anti-cancer therapy ( 15 ). However, the advancement of these strategies beyond the preclinical stage has been hampered by concerns regarding off-target effects ( 17 ), prompting a need for more in-depth studies of the molecular machinery governing the UPR in specific cancers. Members of the protein disulfide isomerase (PDI) family, including the canonical PDIA1 encoded by P4HB , are molecular chaperones and thiol-disulfide oxidoreductases abundantly present within the ER lumen ( 18 , 19 ). PDI can be phosphorylated and activated in response to UPR induction ( 20 ), and it functions in part by catalyzing disulfide bond formation and isomerization to help alleviate this bottleneck in the oxidative protein folding process ( 18 , 19 ). Oxidized PDI can effectively donate its disulfide bond to an unfolded or misfolded protein by accepting electrons from the thiol groups of these polypeptides such that they can fold into an appropriate conformation and form proper disulfide bonds ( 21 ). The reduced form of PDI can then recycle to the catalytically active oxidized state by passing these electrons to ER oxidoreductin 1 (ERO1), which, in turn, generates hydrogen peroxide (H 2 O 2 ) that can contribute to oxidative stress ( 21 , 22 ). P4HB upregulation has been reported in bladder tumors and linked to worse pathological staging, overall survival, and recurrence-free survival in patients, with corresponding overexpression in BC cell lines supporting tumor cell proliferation and invasion ( 23 , 24 ). P4HB knockdown can sensitize BC cells to gemcitabine ( 25 ), and it is a platinum-resistance-related gene in BC patients such that the knockout of this PDI-coding gene sensitizes bladder tumor cells to platinum-based treatment ( 26 ). While these prior data support an important pro-tumorigenic role for PDI in BC, relatively little remains known of the precise mechanisms whereby levels of PDI expression and activity ultimately shape malignant outcomes. Strikingly, extant data suggest that PDI can function as a dual-edged sword such that it can support cancer growth by activating the PERK branch of the UPR pathway to facilitate tumor proliferation and survival ( 27 , 28 ), whereas its excessive induction of NADPH oxidase activity can result in deleterious levels of reactive oxygen species (ROS) production and cell death ( 29 ). It may thus be incumbent on tumor cells to maintain levels of PDI activity sufficient to adapt to ER stress and maintain proteostasis while mitigating the potential for lethal oxidative stress associated with unrestrained ROS generation. Extracellular vesicles (EVs) are small membrane-enclosed structures released from cells that enable the intercellular transmission of macromolecular cargoes. We and others have demonstrated that certain cargo proteins present within tumor-derived EVs can promote tumorigenesis ( 15 , 30 – 32 ). We have further found that BC cell-derived EVs can drive UPR induction and oncogenic transformation in recipient urothelial cells ( 33 ). This, coupled with the potential need for tumor cells to regulate PDI activity and attendant oxidative stress within a tolerable range conducive to proliferation and drug resistance, raises the possibility that EVs may provide a release valve to manage levels of intracellular stress. Indeed, EV-mediated relief of excessive ER stress has been proposed in developmental settings ( 34 ), and may be co-opted by tumor cells, indirectly exposing non-malignant recipient cells to this stress in the process. The goal of this study was to explore the role of PDI as a regulator of BC malignancy, with a focus on the maintenance of homeostatic PDI activity, the role of EVs in this context, and the ability of PDI-containing EVs to induce normal urothelial cell transformation through the use of loss-of-function and rescue approaches. We further explored the potential relevance of PDI expression to NMIBC recurrence through a retrospective tissue microarray-based analysis to gain direct insight into the potential clinical relevance of these mechanisms. Results Reduced PDI expression promotes bladder cancer cell survival under elevated ER stress. In an effort to begin exploring the role that PDI plays in cancer cells, we used short hairpin RNA (shRNA) constructs to knock down PDI in TCCSUP BC cells (Fig. 1 A). To probe the relationship between PDI expression and the survival of tumor cells in the presence or absence of ER stress, tunicamycin (140 nM) was used to treat these tumor cells. While tunicamycin significantly increased the frequency of propidium iodide (PI)-positive TCCSUP cells expressing the scramble control shRNA, it had no impact on the frequency of PI-positive cells in which PDI had been knocked down (Fig. 1 B). Consistently, PDI knockdown reduced the degree of tunicamycin-induced caspase-3 activation in these BC cells (Fig. 1 C) while abrogating the ability of tunicamycin to compromise colony formation in a clonogenic assay (Fig. 1 D). Similar effects were also observed in the J82 BC cell line (Supplementary Fig. S1 ). These results suggest that elevated levels of ER stress compromise the survival of BC cells, while the silencing of PDI can restore the tolerance of these malignant cells to this form of stress. Given that ROS production is a byproduct of PDI activity, it is possible that high levels of ER stress may expose BC cells to increased oxidative stress, thereby contributing to the induction of apoptotic death. To test this possibility, H 2 O 2 production was analyzed, revealing that PDI knockdown was sufficient to significantly reduce basal H 2 O 2 levels while also markedly suppressing tunicamycin-induced production thereof (Fig. 1 E). In line with these results, tunicamycin significantly induced the upregulation of oxidative stress-related genes ( NFE2L2, NQO1, GCLC ) in control TCCSUP cells, whereas it failed to do so in these cells following PDI knockdown (Fig. 1 F). Moreover, tunicamycin significantly reduced the GSH/GSSG ratio in scramble control TCCSUP cells, whereas it had no impact on this ratio following PDI silencing (Fig. 1 G). Together, these data support a potential model wherein PDI-mediated ROS production can, under conditions of elevated ER stress, compromise redox homeostasis within BC cells, thereby inducing their apoptotic death. As higher levels of basal caspase-3 activity were observed following PDI knockdown (Fig. 1 C), however, this protein may play an important pro-survival role under conditions of reduced ER stress, suggesting that tumor cells need to carefully calibrate their intracellular PDI supply to maintain viability. Bladder cancer cells mediate PDI homeostasis through EV release EV-mediated release has been proposed as an important mechanism through which cells can eliminate any undesirable molecules ( 35 , 36 ). We thus speculated that BC cells may leverage secreted EVs as a means of maintaining intracellular PDI levels within a tolerable range that balances the beneficial effects of this protein against its potential to induce excessive oxidative stress. Strikingly, tunicamycin treatment markedly increased the levels of PDI found in EVs isolated from TCCSUP cells, whereas it had no impact on PDI cargo levels within EVs isolated from non-transformed SV-HUC urothelial cells (Fig. 2 A). This finding was further supported by the observation that PDI colocalized with the exosome/multivesicular body marker TSG101 in tunicamycin-treated TCCSUP and J82 BC cells, while this colocalization was not apparent in SV-HUC cells (Fig. 2 B). A reduced thiol quantification approach was further used to examine the redox composition of PDI within these cells and EVs. The majority of PDI detected within both TCCSUP and SV-HUC cells was present in the oxidized (oxidoreductase-active) form, but the frequency of reduced PDI rose significantly in TCCSUP but not in SV-HUC cells in response to tunicamycin treatment (Fig. 2 C). Interestingly, we found that the majority of PDI found in TCCSUP cell-derived EVs was present in the oxidized form (89.2%; Fig. 2 D). We thus posited that tumor cells may disfavor oxidative protein folding under excessively high levels of ER stress, packaging oxidized PDI into EVs such that it can be exported from the cell to preserve viability and mitigate oxidative stress. In further support of this model, we found that TCCSUP cells released significantly more EVs at baseline as compared to SV-HUC cells, while tunicamycin treatment significantly increased the release of EVs from these cells (Fig. 2 E). As tunicamycin also triggered higher levels of EV release in non-transformed SV-HUC cells, such stress-induced EV secretion may be general strategy by which cells can adapt to ER stress, with PDI packaging into these EVs being a particularly beneficial pro-survival mechanism engaged by BC cells. PDI-enriched BC-derived EVs induce ROS, DNA damage, and colony formation in recipient urothelial cells We have previously demonstrated that TCCSUP cell-derived EVs can promote the malignant transformation of SV-HUC cells together with the induction of ROS and DNA damage within these recipient cells ( 33 ). We thus sought to determine whether BC-derived EV-borne PDI plays a role in this context. To that end, we isolated EVs from TCCSUPs transfected with shPDI or scramble control constructs, confirming that PDI levels within EVs from cells in which PDI had been knocked down were markedly reduced (Fig. 3 A). A 2’, 7’-dichlorodihydrofluorescein diacetate (DCFDA) flow cytometry approach revealed that treatment with these shPDI EVs for 24 h induced significantly lower levels of ROS within non-transformed SV-HUC cells as compared to scramble control EV treatment (Fig. 3 B). In line with this result, immunofluorescent γH2AX straining revealed a significant reduction in DNA damage levels following shPDI EV treatment relative to scramble control EV treatment (Fig. 3 C). To directly establish the impact of BC EV-derived PDI on urothelial cell transformation, SV-HUC cells were continuously treated with shPDI or scramble control EVs for 13 weeks. Following a 5-week recovery period, a soft agar colony formation assay was used to assess the tumorigenic potential of these cells, revealing that while scramble EV treatment significantly increased the colony formation rate consistent with malignant transformation, shPDI EV treatment significantly reduced this colony formation rate to vehicle control levels (Fig. 3 D). To ensure result specificity, we used an extrusion approach described previously to restore recombinant PDI (rPDI) to the prepared shPDI cancer EVs ( 37 ). Control EVs were instead prepared by extruding scramble and shPDI EVs with PBS, with Western blotting confirming the successful restoration of PDI to shPDI EVs at levels comparable to those in the parental EVs (Fig. 4 A) (Supplementary Fig. S2 ). These EVs were then used to treat non-transformed SV-HUC cells as above, revealing that rPDI extrusion restored the ability of shPDI EVs to induce ROS production and to promote NFE2L2 expression in recipient SV-HUC cells (Fig. 4 B, C). PDI restoration similarly increased the levels of DNA damage induced by shPDI EV treatment (Fig. 4 D). Importantly, rPDI extrision restored the ability of shPDI EVs to promote the malignant transformation of SV-HUC cells over a 13-week treatment period (Fig. 4 E). To test whether PDI is required as an oncoprotein to maintain tumorigenicity within the resultant transformed SV-HUC cells, it was knocked down in these cells via shRNA, which had no impact on anchorage-independent growth of these cells (Fig. 4 F). Together, these data support the central role for PDI as a driver of the EV-induced malignant transformation of recipient urothelial cells following its release from tumor cells, but that PDI is not required to maintain malignancy once it has been established. PDI-enriched EV-transformed cells exhibit patterns of ROS-induced DNA damage Given our observation that PDI is essential for the cancer EV-induced malignant transformation of SV-HUC cells and for the induction of ROS and DNA damage in these cells, we then examined somatic mutation patterns in these transformed cells. Overall, the transformed cells exhibited a higher mutational burden with a somatic mutation prevalence of 8.2 per megabase, a 10.8% increase over parental cells (Fig. 5 A). Moreover, the transformed SV-HUC cells harbored more unique nonsynonymous mutations and more genes impacted by these unique nonsynonymous variants as compared to parental non-transformed SV-HUC cells (Fig. 5 B, Supplementary Table 1). Analyses of single base substitutions (SBS) revealed similar patterns in both cell lines (Fig. 5 C), suggesting that the unique variants found in the transformed SV-HUC cells are likely the result of cancer EV treatment and all six SBS classes were increased in the transformed cells, with the greatest relative increase being seen in G:C to T:A transversions (Fig. 5 D), the type of substitution most characteristic of ROS-induced DNA alterations ( 38 ). Numerous somatic mutational signatures and variant classes have been associated with different types of cancer and are understood to be the result of distinct mutational processes. We next performed strict signature refitting of the genomic variant data from the parental and transformed SV-HUC cells and identified eight previously defined SBS signatures found in the Catalog of Somatic Mutations in Cancer (COSMIC) ( 39 ). The signatures most prominently enhanced in the transformed cells relative to the parental cells were SBS18, SBS85, and SBS40 (cosine similarity > 0.51) (Fig. 5 E, Supplementary Fig. S3A). Notably, COSMIC signature SBS18 has been proposed to result from ROS-induced DNA damage ( 40 ). Taken together, the mutational patterns seen in these transformed cells are consistent with oxidative DNA damage in urothelial cells following cancer EV exposure, suggesting that tumor-derived PDI plays a central role in this process. Interestingly, COSMIC signature SBS85 (Supplementary Fig. S3B), reported to be caused by AID/APOBEC activity, which is one of the major sources of mutations in BC ( 41 ), is characterized by a concentration of variants in the T > A and T > C classes. This finding suggests that, besides elevating ROS levels in recipient cells, cancer EVs may also cause DNA mutations through other mechanisms. High levels of PDI expression in non-muscle invasive bladder cancer patient tumor tissue predicts a higher risk of recurrence Our in vitro studies indicated that BC cells release PDI packaged in EVs when subjected to high levels of ER stress, and the uptake of these PDI-enriched EVs by non-transformed cells was sufficient to drive their malignant. As any secondary tumors that arise through this mechanism in vivo may be diagnosed as recurrent tumors, these data suggest that PDI may offer value as a prognostic biomarker to predict BC recurrence. While a majority of MIBC patients will ultimately undergo radical cystectomy, NMIBC is characterized by frequent recurrences requiring surveillance cystoscopies as there is no current reliable marker currently available to monitor for recurrence and/or progression. To explore the utility of PDI in this context, we constructed a tissue microarray (TMA) consisting of 121 NMIBC patients who did (n = 55) or did not (n = 67) develop recurrence (Supplementary Table 2). We analyzed the BiP expression levels in the tissue samples comprising this TMA as an approach to estimating ER stress levels in these tumor cells, in addition to assessing PDI expression. Significantly higher levels of BiP and PDI were detected in tumor tissues from patients who experienced BC recurrence (Fig. 6 A, B). We also quantified the PDI positive area and intensity within BiP negative regions to demonstrate levels of non-ER residential PDI that are likely to be secreted to the extracellular environment. Interestingly, the tumors of patients with recurrent disease had higher levels of both PDI positive area and accumulation in the non-ER cytosolic regions (Fig. 6 C). Importantly, we also found that higher levels of both BiP and PDI expression were associated with significantly reduced patient recurrence-free survival (Fig. 6 D). These data suggest that tissues that are under higher levels of ER stress and PDI expression have a higher risk of recurrence, demonstrating that the PDI status of tumors offers potential as a marker for the prediction of NMIBC patient recurrence. Discussion The high rates of BC recurrence underscore the need to devise new approaches for identifying patients who are more likely to develop recurrent disease and mitigating this risk. Our data suggest that BC cells utilize EVs as a means of ensuring their ongoing survival under conditions of ER stress through the export of oxidized PDI. The incidental uptake of these PDI-enriched EVs by normal urothelial cells ultimately leads to their malignant transformation through a potential field cancerization mechanism (Fig. 7 ). While these results offer insight into the mechanisms that may underlie BC recurrence and suggest that levels of PDI and ER stress in tumor tissues are valuable biomarkers for predicting NMIBC recurrence risk, there are several important topics related to our findings that warrant further discussion. A range of neoplasia-related conditions including reduced genomic stability, the accumulation of mutations, increased protein production and secretion, and exposure to hypoxic or nutrient-deprived microenvironmental conditions can compromise proteostasis within tumor cells ( 15 ). ER stress response and UPR induction can thus preserve tumor cell viability under these challenging conditions ( 10 , 11 , 16 ), with UPR-induced oxidized PDI activation providing support to help restore appropriate protein folding ( 18 – 20 ). In this study, we found that BC cells exhibited elevated ER stress levels under basal conditions. However, subjecting them to further ER stress triggered apoptotic death that could be mitigated by knocking down PDI, thereby alleviating oxidative stress when PDI levels were reduced. This suggests that the maintenance of PDI homeostasis is vital for the survival of BC cells owing to the unique characteristics of this enzyme. Most cellular compartments maintain a reducing environment, and oxidized proteins are usually unstable in the cytosol ( 42 , 43 ). To prevent hyperoxidation and maintain ER homeostasis under conditions of UPR induction when levels of PDI activity are elevated, BC cells must limit the levels of oxidized PDI within the ER, balancing proteostasis against oxidative injury. We found that under ER stress, BC cells released high levels of oxidized PDI within EVs, thereby mitigating the ROS production and consequent cell death, ultimately promoting BC cell survival. This finding is highly innovative and aligns well with the observation that, while cells continuously shed EVs at steady state, multivesicular body formation and EV release are enhanced under conditions of ER stress or oxidative stress ( 44 , 45 ), supporting this process of EV-mediated oxidized PDI export as a stress relief mechanism ( 34 ). The mechanisms that underlie the packaging of oxidized PDI into EVs by BC cells warrant further clarification. While the loading of protein cargos within EVs may be partially stochastic, ubiquitination, palmitoylation, and other post-translational modifications can facilitate the sorting of certain proteins into EVs ( 46 , 47 ). It remains to be determined whether these modifications are relevant for PDI loading into EVs and whether the oxidized form of PDI is preferentially packaged into EVs under varying levels of ER stress. Assessing these factors will help to better elucidate the directed nature of this cargo loading process. Strikingly, we found that PDI-enriched EVs derived from BC cells promoted the malignant transformation of normal urothelial cells, potentially through ROS-induced DNA damage mediated by the oxidized PDI present within these vesicles or the induction of UPR activity within recipient cells. Indeed, we have previously demonstrated the ability of BC-derived EVs to induce UPR activity and malignant transformation in recipient urothelial cells ( 33 ), and both persistent UPR and oxidative stress exposure can readily drive tumorigenesis ( 12 – 14 ), suggesting that the delivery of PDI to the urothelium may predispose this field to the future development of recurrent bladder tumors. In addition to their effects on tumorigenesis and BC recurrence, these PDI-enriched EVs may also serve as pivotal regulators of various malignant processes directly within the tumor microenvironment. For instance, intracellular PDI plays a vital role in the synthesis of type I collagen, a core component of the extracellular matrix in tumors ( 48 , 49 ), whereas extracellular PDI directly activates integrins through the promotion of thiol-disulfide exchange ( 50 ). Integrin interactions with type I collagen can also facilitate the development of chemoresistance ( 51 ), promoting proliferative growth while protecting against apoptotic death. One report has also suggested a potential link between PDI and immune surveillance, with higher PDI levels potentially supporting immune evasion in breast cancer ( 52 ). Inhibition of PDI activity has shown therapeutic potential in reducing breast cancer cell adhesion and migration through the disruption of focal adhesion complex formation and associated phosphorylation events ( 53 ). Investigating whether EV-derived PDI can alter the composition of the tumor microenvironment, influence chemoresistance, or modulate immune-mediated detection of BC cells through these mechanisms may represent promising avenues for future research aimed at clarifying the broader role of PDI in bladder carcinogenesis. We found that both total PDI expression and non-ER-resident PDI levels were higher in those patients who developed recurrent disease. BiP expression was similarly associated with disease recurrence, underscoring the potential value of evaluating ER stress levels, PDI expression, and PDI localization as potential biomarkers of BC recurrence risk. While tumor and urothelial tissue collection are inherently invasive procedures poorly suited for routine monitoring, urine is an EV-rich biofluid that can be readily obtained from patients, providing an opportunity for noninvasive BC-related biomarker testing ( 54 , 55 ). Given our finding that BC cells release PDI-enriched EVs, this raises the urine EVs may offer diagnostic or prognostic utility when evaluating BC patients based on the levels of PDI or ER stress-related biomarkers present therein. However, prospective trials will be essential to evaluate this possibility, and the routine implementation of urine EV-based analyses will necessitate overcoming challenges related to the sensitivity, specificity, and standardization of these biomarkers ( 55 – 57 ). Our results also raise the question of whether therapeutic interventions targeting ER stress and/or PDI may afford benefits to BC patients. PDI inhibitors have been evaluated as promising anti-cancer treatments in patients with relapsed ovarian cancer and in various preclinical cancer models ( 58 – 60 ), although whether they can protect against field cancerization effects mediated by PDI-enriched EVs remains uncertain. Given that we found that lower PDI levels were beneficial to BC cell survival under high levels of ER stress, while ER stress loading has been advanced as an anti-cancer therapy ( 61 ), caution and individualized treatment planning would likely be vital for any interventional efforts targeting this regulatory axis. Together, our results offer new insights into the integral role that EVs play in the maintenance of BC cell homeostasis under conditions of ER stress, while highlighting PDI as an attractive biomarker and therapeutic that may directly contribute to the risk of BC recurrence. Materials and Methods Cell culture and EV isolation Cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in a humidified 37°C incubator under 5% CO 2 in media containing FB Essence (3100, Seradigm). For EV collection, cells were cultured in medium containing EV-depleted fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and penicillin-streptomycin (catalog no. 15140-122, Thermo Fisher Scientific) as described previously ( 62 ). Cell culture supernatants were processed immediately after collection by serial centrifugation at 400 × g for 10 min and 15,500 × g for 30 min to remove cells and debris and then stored at -80°C. EVs were isolated from thawed samples by ultracentrifugation performed twice at 200,000 × g for 70 min at 4°C, and the resulting pellets were resuspended in a small volume of DPBS. Aggregates were removed from the samples by another 15,500 × g centrifugation for 5 min. Final total protein concentrations in the samples were measured with a Micro BCA assay (catalog no. 23235, Thermo Fisher Scientific), and samples were stored at -80°C. Extrusion Initially, 250 µL of PBS containing 37.5 µg of shPDI TCCSUP EVs with or without 25 µg of recombinant PDI (catalog no. enz-262, Prospec) were extruded 10 times using an Avanti extruder set with 0.1 µm polycarbonate membrane filters (catalog no. 610023, Avanti, Alabaster, AL, USA). The mixture was then transferred to a 1.5 mL microcentrifuge tube (catalog no. 357448, Beckman Coulter) filled with 900 µL of PBS and subjected to a 2-hour ultracentrifugation step (Optoma MAX-XP ultracentrifuge, Beckman Coulter, Brea, CA, USA) in a TLA110 rotor at 100K × g at 4°C. After removing the supernatant, the EV pellet was resuspended in PBS. Whole genome sequencing Genomic DNA concentration was assessed with the Qubit Fluorometer (Thermo Fisher Scientific) and quality was assessed using the Agilent Tapestation (Agilent, Santa Clara, CA). The Illumina Nextera Flex kit (Illumina, San Diego, CA) was used for library construction per the manufacturer’s instructions. Briefly, 500 ng of gDNA was tagmented with Bead-Linked Transposome (BLT) beads while simultaneously adding Illumina sequencing primers. Tagmented genomic DNA was purified, followed by reduced-cycle (5-cycle) PCR amplification to add index and adapter sequences for sequencing. Sequence data was generated using Illumina's NovaSeq 6000 sequencer. Data were analyzed using MuSiCa (Mutational Signatures in Cancer) ( 63 ) and the MutationalPatterns R package ( 64 ). Tissue microarray We retrieved primary tumor specimens from 121 index cases (initial detection) of non-invasive (pTa) low-grade urothelial carcinoma obtained by transurethral resection performed at the University of Rochester Medical Center. These patients included 80 men and 41 women, with a mean age of 69.7 years (range: 46.5–93.1 years) at the time of surgery. All the sections were reviewed for confirmation of original diagnoses, according to the 2004 World Health Organization/International Society of Urological Pathology classification system for urothelial neoplasms ( 65 ). Appropriate approval from the Institutional Review Board was obtained prior to the construction and use of the TMA. Bladder TMAs were constructed from formalin-fixed paraffin-embedded specimens as previously described ( 66 ). Some cases in the initial TMA patient group were not represented in the first sections taken from the TMA paraffin blocks. Following de-paraffinization of TMA sections, antigen retrieval was performed in heated citrate buffer (Vector H-3300) for 30 min. Primary antibody incubation was conducted overnight at 4°C using anti-PDI (Cell Signaling 3501, 1:500) and anti-BiP (Santa Cruz sc-166490, 1:100) antibodies. Stained tissues were photographed at 40× magnification using a Leica DM5000 B microscope. Tumor regions in each photographed field were masked manually 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 ( 67 ). To assess the colocalization of two epitopes, the Manders overlap coefficients were determined using the JACoP plugin in ImageJ ( 68 ). Supplementary methods For further details regarding other assays conducted in this study, see the Supplementary Materials and Methods. Statistical analysis All experiments were performed using at least three biological repeats. Utilized statistical tests are indicated in the figure legends. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Statistical analyses were performed using GraphPad Prism 9.2.0 and the R statistical computing environment, version 4.0.3. Declarations Competing interests The authors declare no competing interests. Data availability statement The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Acknowledgments The authors thank the University of Rochester Medical Center Mass Spectrometry Resource Laboratory and Genomic Research Center for assisting in the proteomic and whole genome sequencing analyses. Author contributions CHW participated in study conception, experiment design, the acquisition, analysis, and interpretation of data, and drafting the manuscript. KLY participated in the analysis and interpretation of data. RDM participated in the analysis and interpretation of data, and drafting the manuscript. MMHA participated in data acquisition, analysis, and interpretation. CRS participated in data acquisition, analysis, and interpretation. EMM participated in study conception and editing of the manuscript. YFL participated in study conception, the design/supervision of experiments, funding acquisition, writing, and editing of the manuscript. All authors read and approved the final manuscript. References Messing EM, Madeb R, Young T, Gilchrist KW, Bram L, Greenberg EB, et al. Long-term outcome of hematuria home screening for bladder cancer in men. Cancer. 2006;107(9):2173-9. Scosyrev E, Noyes K, Feng C, Messing E. Sex and racial differences in bladder cancer presentation and mortality in the US. Cancer. 2009;115(1):68-74. Brandau S, Suttmann H. Thirty years of BCG immunotherapy for non-muscle invasive bladder cancer: a success story with room for improvement. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie. 2007;61(6):299-305. Czerniak B, Dinney C, McConkey D. Origins of bladder cancer. Annual Review of Pathology: Mechanisms of Disease. 2016;11:149-74. Jones TD, Wang M, Eble JN, MacLennan GT, Lopez-Beltran A, Zhang S, et al. Molecular evidence supporting field effect in urothelial carcinogenesis. Clin Cancer Res. 2005;11(18):6512-9. Cheng L, MacLennan GT, Pan CX, Jones TD, Moore CR, Zhang S, et al. Allelic loss of the active X chromosome during bladder carcinogenesis. Arch Pathol Lab Med. 2004;128(2):187-90. Cheng L, Jones TD, McCarthy RP, Eble JN, Wang M, MacLennan GT, et al. Molecular genetic evidence for a common clonal origin of urinary bladder small cell carcinoma and coexisting urothelial carcinoma. Am J Pathol. 2005;166(5):1533-9. Cancer Genome Atlas Research N. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature. 2014;507(7492):315-22. Cheng L, Davidson DD, Maclennan GT, Williamson SR, Zhang S, Koch MO, et al. The origins of urothelial carcinoma. Expert review of anticancer therapy. 2010;10(6):865-80. Ma Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer. 2004;4(12):966-77. Hetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nature reviews Molecular cell biology. 2012;13(2):89-102. Wang M, Kaufman RJ. The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat Rev Cancer. 2014;14(9):581-97. Clarke HJ, Chambers JE, Liniker E, Marciniak SJ. Endoplasmic reticulum stress in malignancy. Cancer cell. 2014;25(5):563-73. Chevet E, Hetz C, Samali A. Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis. Cancer discovery. 2015;5(6):586-97. Oakes SA. Endoplasmic reticulum stress signaling in cancer cells. The American journal of pathology. 2020;190(5):934-46. Urra H, Dufey E, Avril T, Chevet E, Hetz C. Endoplasmic Reticulum Stress and the Hallmarks of Cancer. Trends Cancer. 2016;2(5):252-62. Liang D, Khoonkari M, Avril T, Chevet E, Kruyt FA. The unfolded protein response as regulator of cancer stemness and differentiation: Mechanisms and implications for cancer therapy. Biochemical Pharmacology. 2021;192:114737. Wang CC, Tsou CL. Protein disulfide isomerase is both an enzyme and a chaperone. The FASEB journal. 1993;7(15):1515-7. Wang L, Wang X, Wang C-c. Protein disulfide–isomerase, a folding catalyst and a redox-regulated chaperone. Free Radical Biology and Medicine. 2015;83:305-13. Yu J, Li T, Liu Y, Wang X, Zhang J, Wang Xe, et al. Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress. The EMBO journal. 2020;39(10):e103841. Shergalis AG, Hu S, Bankhead III A, Neamati N. Role of the ERO1-PDI interaction in oxidative protein folding and disease. Pharmacology & therapeutics. 2020;210:107525. Zito E. ERO1: A protein disulfide oxidase and H2O2 producer. Free Radical Biology and Medicine. 2015;83:299-304. Lyu L, Xiang W, Zheng F, Huang T, Feng Y, Yuan J, et al. Significant prognostic value of the autophagy-related gene P4HB in bladder urothelial carcinoma. Frontiers in oncology. 2020;10:1613. Wu Y, Peng Y, Guan B, He A, Yang K, He S, et al. P4HB: A novel diagnostic and prognostic biomarker for bladder carcinoma. Oncology letters. 2021;21(2):1-. Wang X, Bai Y, Zhang F, Yang Y, Feng D, Li A, et al. Targeted inhibition of P4HB promotes cell sensitivity to gemcitabine in Urothelial carcinoma of the bladder. OncoTargets and therapy. 2020:9543-58. Xiong S, Li S, Zeng J, Nie J, Liu T, Liu X, et al. Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB. Frontiers in Immunology. 2023;14. Kranz P, Neumann F, Wolf A, Classen F, Pompsch M, Ocklenburg T, et al. PDI is an essential redox-sensitive activator of PERK during the unfolded protein response (UPR). Cell Death Dis. 2017;8(8):e2986. de APAM, Verissimo-Filho S, Guimaraes LL, Silva AC, Takiuti JT, Santos CX, et al. Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation. J Leukoc Biol. 2011;90(4):799-810. Fernandes DC, Manoel AH, Wosniak J, Jr., Laurindo FR. Protein disulfide isomerase overexpression in vascular smooth muscle cells induces spontaneous preemptive NADPH oxidase activation and Nox1 mRNA expression: effects of nitrosothiol exposure. Arch Biochem Biophys. 2009;484(2):197-204. Beckham CJ, Olsen J, Yin PN, Wu CH, Ting HJ, Hagen FK, et al. Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression. The Journal of urology. 2014;192(2):583-92. Silvers CR, Liu YR, Wu CH, Miyamoto H, Messing EM, Lee YF. Identification of extracellular vesicle-borne periostin as a feature of muscle-invasive bladder cancer. Oncotarget. 2016. Silvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF. Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. Oncotarget. 2017;8(53):91199-208. Wu C-H, Silvers CR, Messing EM, Lee Y-F. Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells. Journal of Biological Chemistry. 2019;294(9):3207-18. Fu B, Ma H, Zhang Dj, Wang L, Li Zq, Guo Zh, et al. Porcine oviductal extracellular vesicles facilitate early embryonic development via relief of endoplasmic reticulum stress. Cell Biology International. 2022;46(2):300-10. Baixauli F, López-Otín C, Mittelbrunn M. Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness. Front Immunol. 2014;5:403. Fan J, Kuai B, Wu G, Wu X, Chi B, Wang L, et al. Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export. Embo j. 2017;36(19):2870-86. Fu S, Wang Y, Xia X, Zheng JC. Exosome engineering: Current progress in cargo loading and targeted delivery. NanoImpact. 2020;20:100261. Yasui M, Kanemaru Y, Kamoshita N, Suzuki T, Arakawa T, Honma M. Tracing the fates of site-specifically introduced DNA adducts in the human genome. DNA Repair (Amst). 2014;15:11-20. Tate JG, Bamford S, Jubb HC, Sondka Z, Beare DM, Bindal N, et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Research. 2018;47(D1):D941-D7. Kucab JE, Zou X, Morganella S, Joel M, Nanda AS, Nagy E, et al. A Compendium of Mutational Signatures of Environmental Agents. Cell. 2019;177(4):821-36.e16. Glaser AP, Fantini D, Wang Y, Yu Y, Rimar KJ, Podojil JR, et al. APOBEC-mediated mutagenesis in urothelial carcinoma is associated with improved survival, mutations in DNA damage response genes, and immune response. Oncotarget. 2018;9(4):4537-48. Parakh S, Atkin JD. Novel roles for protein disulphide isomerase in disease states: a double edged sword? Front Cell Dev Biol. 2015;3:30. Frand AR, Cuozzo JW, Kaiser CA. Pathways for protein disulphide bond formation. Trends Cell Biol. 2000;10(5):203-10. Kanemoto S, Nitani R, Murakami T, Kaneko M, Asada R, Matsuhisa K, et al. Multivesicular body formation enhancement and exosome release during endoplasmic reticulum stress. Biochemical and biophysical research communications. 2016;480(2):166-72. Zhang W, Liu R, Chen Y, Wang M, Du J. Crosstalk between oxidative stress and exosomes. Oxidative Medicine and Cellular Longevity. 2022;2022. Chen Y, Zhao Y, Yin Y, Jia X, Mao L. Mechanism of cargo sorting into small extracellular vesicles. Bioengineered. 2021;12(1):8186-201. Waury K, Gogishvili D, Nieuwland R, Chatterjee M, Teunissen CE, Abeln S. Proteome encoded determinants of protein sorting into extracellular vesicles. Journal of Extracellular Biology. 2024;3(1):e120. Rauch F, Fahiminiya S, Majewski J, Carrot-Zhang J, Boudko S, Glorieux F, et al. Cole-Carpenter syndrome is caused by a heterozygous missense mutation in P4HB. Am J Hum Genet. 2015;96(3):425-31. Liang Y, Diehn M, Bollen AW, Israel MA, Gupta N. Type I collagen is overexpressed in medulloblastoma as a component of tumor microenvironment. J Neurooncol. 2008;86(2):133-41. Popielarski M, Ponamarczuk H, Stasiak M, Michalec L, Bednarek R, Studzian M, et al. The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11. Biochem Biophys Res Commun. 2018;495(2):1635-41. Baltes F, Pfeifer V, Silbermann K, Caspers J, Wantoch von Rekowski K, Schlesinger M, et al. β(1)-Integrin binding to collagen type 1 transmits breast cancer cells into chemoresistance by activating ABC efflux transporters. Biochim Biophys Acta Mol Cell Res. 2020;1867(5):118663. Alhammad R, Khunchai S, Tongmuang N, Limjindaporn T, Yenchitsomanus PT, Mutti L, et al. Protein disulfide isomerase A1 regulates breast cancer cell immunorecognition in a manner dependent on redox state. Oncology Reports. 2020;44(6):2406-18. Chen IH, Chang FR, Wu YC, Kung PH, Wu CC. 3,4-Methylenedioxy-β-nitrostyrene inhibits adhesion and migration of human triple-negative breast cancer cells by suppressing β1 integrin function and surface protein disulfide isomerase. Biochimie. 2015;110:81-92. Oeyen E, Hoekx L, De Wachter S, Baldewijns M, Ameye F, Mertens I. Bladder cancer diagnosis and follow-up: the current status and possible role of extracellular vesicles. International journal of molecular sciences. 2019;20(4):821. Oliveira MCd, Caires HR, Oliveira MJ, Fraga A, Vasconcelos MH, Ribeiro R. Urinary biomarkers in bladder cancer: where do we stand and potential role of extracellular vesicles. Cancers. 2020;12(6):1400. Liu Y-R, Ortiz-Bonilla CJ, Lee Y-F. Extracellular vesicles in bladder cancer: biomarkers and beyond. International journal of molecular sciences. 2018;19(9):2822. Choi J-Y, Kim S, Kwak H-B, Park D-H, Park J-H, Ryu J-S, et al. Extracellular vesicles as a source of urological biomarkers: lessons learned from advances and challenges in clinical applications to major diseases. International neurourology journal. 2017;21(2):83. Gelzinis JA, Szahaj MK, Bekendam RH, Wurl SE, Pantos MM, Verbetsky CA, et al. Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2023;37(5):e22914. Robinson RM, Reyes L, Duncan RM, Bian H, Strobel ED, Hyman SL, et al. Tuning isoform selectivity and bortezomib sensitivity with a new class of alkenyl indene PDI inhibitor. European journal of medicinal chemistry. 2020;186:111906. Powell LE, Foster PA. Protein disulphide isomerase inhibition as a potential cancer therapeutic strategy. Cancer medicine. 2021;10(8):2812-25. Kazama H, Hiramoto M, Miyahara K, Takano N, Miyazawa K. Designing an effective drug combination for ER stress loading in cancer therapy using a real-time monitoring system. Biochem Biophys Res Commun. 2018;501(1):286-92. Silvers CR, Liu YR, Wu CH, Miyamoto H, Messing EM, Lee YF. Identification of extracellular vesicle-borne periostin as a feature of muscle-invasive bladder cancer. Oncotarget. 2016;7(17):23335-45. Díaz-Gay M, Vila-Casadesús M, Franch-Expósito S, Hernández-Illán E, Lozano JJ, Castellví-Bel S. Mutational Signatures in Cancer (MuSiCa): a web application to implement mutational signatures analysis in cancer samples. BMC Bioinformatics. 2018;19(1):224. Blokzijl F, Janssen R, van Boxtel R, Cuppen E. MutationalPatterns: comprehensive genome-wide analysis of mutational processes. Genome Med. 2018;10(1):33. Miyamoto H, Miller JS, Fajardo DA, Lee TK, Netto GJ, Epstein JI. Non-invasive papillary urothelial neoplasms: the 2004 WHO/ISUP classification system. Pathol Int. 2010;60(1):1-8. Miyamoto H, Yao JL, Chaux A, Zheng Y, Hsu I, Izumi K, et al. Expression of androgen and oestrogen receptors and its prognostic significance in urothelial neoplasm of the urinary bladder. BJU Int. 2012;109(11):1716-26. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82. Bolte S, Cordelières FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 2006;224(Pt 3):213-32. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryMaterials0501.docx SupplementaryTable1AnnotatedDamagingVariants05011.xlsx Cite Share Download PDF Status: Published Journal Publication published 11 Apr, 2025 Read the published version in Oncogene → Version 1 posted Editorial decision: Reject after peer review 27 Aug, 2024 Review # 2 received at journal 23 Aug, 2024 Review # 3 received at journal 25 Jul, 2024 Reviewer # 3 agreed at journal 16 Jul, 2024 Reviewer # 2 agreed at journal 16 Jul, 2024 Review # 1 received at journal 01 Jul, 2024 Reviewer # 1 agreed at journal 17 Jun, 2024 Reviewers invited by journal 25 May, 2024 Submission checks completed at journal 16 May, 2024 Editor assigned by journal 15 May, 2024 First submitted to journal 15 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4425743","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":306754262,"identity":"a53c8104-a359-4648-b59b-3c02a0a09500","order_by":0,"name":"Yi-Fen Lee","email":"data:image/png;base64,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","orcid":"","institution":"University of Rochester","correspondingAuthor":true,"prefix":"","firstName":"Yi-Fen","middleName":"","lastName":"Lee","suffix":""},{"id":306754263,"identity":"6011ebb3-3bf0-40fc-82b4-c871fcd9cced","order_by":1,"name":"Chia-Hao Wu","email":"","orcid":"","institution":"University of Rochester","correspondingAuthor":false,"prefix":"","firstName":"Chia-Hao","middleName":"","lastName":"Wu","suffix":""},{"id":306754264,"identity":"4e73c884-802b-4ad5-8c15-74a748985d48","order_by":2,"name":"Kit Yuen","email":"","orcid":"","institution":"University of Rochester","correspondingAuthor":false,"prefix":"","firstName":"Kit","middleName":"","lastName":"Yuen","suffix":""},{"id":306754265,"identity":"fb8f5d8d-43ee-4f8f-aada-7745417e36c8","order_by":3,"name":"Ryan Molony","email":"","orcid":"","institution":"University of Rochester","correspondingAuthor":false,"prefix":"","firstName":"Ryan","middleName":"","lastName":"Molony","suffix":""},{"id":306754266,"identity":"5302cd8e-3548-4afe-8e4f-c4666b662038","order_by":4,"name":"Christopher Silvers","email":"","orcid":"","institution":"University of Rochester","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"","lastName":"Silvers","suffix":""},{"id":306754267,"identity":"174197cc-0fa6-4704-b578-36d97f429747","order_by":5,"name":"Akash Md. Mahmudul Hasan","email":"","orcid":"","institution":"University of Rochester","correspondingAuthor":false,"prefix":"","firstName":"Akash","middleName":"Md. Mahmudul","lastName":"H","suffix":"Md."},{"id":306754268,"identity":"b7702940-3c5d-40fc-bb3a-85cebc935ce1","order_by":6,"name":"Edward Messing","email":"","orcid":"","institution":"University of Rochester","correspondingAuthor":false,"prefix":"","firstName":"Edward","middleName":"","lastName":"Messing","suffix":""}],"badges":[],"createdAt":"2024-05-15 14:16:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4425743/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4425743/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41388-025-03380-6","type":"published","date":"2025-04-11T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58145012,"identity":"dba6ca81-330c-47ba-bd3c-99ed3fb53366","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":8453948,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eReduced PDI expression promotes bladder cancer cell survival under elevated ER stress. \u003c/strong\u003eA. Western blot analysis of PDI abundance in cell lysates derived from scramble control and PDI-targeting lentiviral shRNA transduced TCCSUP cells. B-C. TCCSUP cell death was measured by propidium iodide (PI) staining and quantified using flow cytometry (B). Apoptosis was assessed by Western blotting analyses of cleaved caspase-3 (C). D. TCCSUP cancer cell survival following tunicamycin treatment was tested in a clonogenic assay. E-G. Scramble control and shPDI TCCSUP cells were treated with or without tunicamycin (T) and examined to assess H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels (D), oxidative stress gene expression (E), and the GSH/GSSG ratio (F). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; One- or two-way ANOVAs.\u003c/p\u003e","description":"","filename":"Figure10510.png","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/608a9f6fb07c723654cff746.png"},{"id":58145018,"identity":"71b94ac8-064f-4b25-96af-42327dfda66f","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27970554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCancer cells mediate PDI homeostasis through EV release\u003c/strong\u003e. A. Western blot showing PDI levels in whole cell lysates and EVs derived from non-transformed SV-HUC and TCCSUP cancer cells with or without tunicamycin treatment. B. Immunofluorescence staining demonstrating PDI and TSG101 intensity and cellular localization in SV-HUC, TCCSUP, and J82 cancer cells with or without tunicamycin (Tuni/Tun) treatment. C. The percentage of reduced PDI in TCCSUP cancer cells (left) or SV-HUC non-transformed cells (right) with or without tunicamycin treatment as shown by reduced thiol quantification. D. Percentage of reduced and oxidized PDI in TCCSUP EVs as estimated by reduced thiol quantification. E. EV release kinetics for SVHUC and TCCSUP cells following Tunicamycin (140 nM) treatment for 36 hours. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; One- or two-way ANOVAs.\u003c/p\u003e","description":"","filename":"Figure20501.png","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/606d7ce3198eec33d0fc7e9f.png"},{"id":58145016,"identity":"9c889af9-5b7c-41a9-911d-7510489b9a42","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12603160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDI-enriched BC-derived EVs induce ROS, DNA damage, and colony formation in recipient urothelial cells.\u003c/strong\u003e A. Western blotting analysis of PDI abundance in EVs derived from scramble control and PDI-targeting lentiviral shRNA-transduced TCCSUP cells. B. ROS levels in SV-HUC cells following a 24-hour treatment with scramble control, shPDI TCCSUP EVs, or SV-HUC control EVs, as analyzed by DCFDA and flow cytometry assays. Data represent the fold change in the DCFDA histogram geometric mean relative to the PBS control. C. Representative images and quantification of DNA double-strand break in SV-HUC cells following an 18-hour treatment with scramble control or shPDI TCCSUP EVs, as assessed by γH2AX immunofluorescence staining. D. Representative images and quantification of the number of colonies in soft agar formed by SV-HUC cells following a 13-week treatment with scramble control or shPDI TCCSUP EVs and 5 weeks of regular culture for stabilization. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; One- or two-way ANOVAs.\u003c/p\u003e","description":"","filename":"Figure30501.png","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/947b01048aad13455eebc91f.png"},{"id":58145020,"identity":"90f0294b-c9ba-47c5-91b6-e02a5b9834ae","added_by":"auto","created_at":"2024-06-11 18:27:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":20650230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRestoration of PDI to shPDI EVs rescues their ability to increase cellular stress and anchorage-independent growth\u003c/strong\u003e. A. Western blot showing PDI levels in TCCSUP EVs following extrusion with recombinant PDI. B. ROS level in SV-HUC cells following treatment with EVs derived from scramble TCCSUP control cells, or from shPDI TCCSUP EVs extruded with rPDI or PBS control, as analyzed via DCFDA and flow cytometry assays. Data represent the fold change in the DCFDA histogram geometric mean relative to PBS control. C. \u003cem\u003eNFE2L2\u003c/em\u003e gene expression in SV-HUC cells treated with EVs derived from scramble TCCSUP control cells, or from shPDI TCCSUP EVs extruded with rPDI or PBS control. D. Representative images and quantification of DNA damage in SV-HUC cells following an 18-hour treatment with EVs derived from scramble TCCSUP control cells or from shPDI TCCSUP EVs extruded with rPDI or PBS control, as assessed by γH2AX immunofluorescence staining. E. Representative images and quantification of the colonies in soft agar formed by SV-HUC cells following a 13-week treatment with EVs derived from scramble TCCSUP control cells, or from shPDI TCCSUP EVs extruded with rPDI or PBS control. F. Representative images and quantification of the colonies in soft agar formed by transformed SV-HUC established by long-term cancer EV stimulation (parental), and by transformed SV-HUC transduced with scramble control (scramble) and PDI-targeting lentiviral shRNA (shPDI). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; One- or two-way ANOVAs.\u003c/p\u003e","description":"","filename":"Figure40501.png","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/0f3479ef2eabefe3ea5ec996.png"},{"id":58145013,"identity":"268a3deb-3063-455b-8c79-8d4e7e57cf10","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2509887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePDI-enriched EV-transformed cells exhibit patterns of ROS-induced DNA damage\u003c/strong\u003e. A. Quantification of genomic variants detected in non-transformed (parental) and long-term cancer EV-stimulated (transformed) SV-HUC cells by whole genome sequencing. B. Total numbers of unique variants present within coding regions (left) and the total number of genes affected (right) in parental and transformed cells as annotated using wAnnovar. C. The 96 trinucleotide mutational profiles that were found in parental and transformed SV-HUC cells. D. Fold increase in the six SBS classes in transformed SV-HUC cells as compared to the parental SV-HUC cells. E. Plot showing the change in the relative contribution of COSMIC SBS signatures detected in transformed vs. parental SV-HUC cells.\u003c/p\u003e","description":"","filename":"Figure50501.png","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/e7ed868822ad1f94dd580d60.png"},{"id":58145015,"identity":"966c4202-0553-4b44-ae03-e58e92603f53","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":19012864,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh levels of PDI expression in non-muscle invasive bladder cancer patient tumor tissue predicts a higher risk of recurrence. \u003c/strong\u003eA. Intensity of BiP (left) and PDI (right) immunofluorescence staining. Each dot represents the average values of a patient. B. Representative images of tumor tissue derived from patients with non-recurrent (left) and recurrent (right) NMIBC. PDI, BiP, and cell nuclei are respectively stained in green, red, and blue. Scale bars: 40 µm. C. Area (left) and fluorescence signal intensity (right) of PDI in BiP negative regions. D. Recurrence-free survival rates for patients with corresponding PDI (left) or BiP (right) expression. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01; One- or two-way ANOVAs.\u003c/p\u003e","description":"","filename":"Figure60501.png","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/ca79344d4489c54bba1610a1.png"},{"id":58145014,"identity":"ca0c7392-c626-40b0-a6a4-85635dbd1b38","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6042963,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA proposed model of the role of PDI and PDI-enriched EVs in bladder cancer cell survival and disease recurrence.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure70501.png","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/77ef1977aa4993f352dba6ed.png"},{"id":58145011,"identity":"63752524-3a46-45f3-ab35-2a84ad8595ee","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":493791,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/3c7cf491-d5b8-4c54-9d47-1a079399922e.pdf"},{"id":58145019,"identity":"56803c7c-e62c-4461-aa00-622550982fe8","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":668959,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials0501.docx","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/5e204e4bfe8a133b41604e6a.docx"},{"id":58145017,"identity":"35b78d3c-2dc1-4ccc-b5a8-ad7b0c6440c3","added_by":"auto","created_at":"2024-06-11 18:27:47","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":16835,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1AnnotatedDamagingVariants05011.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4425743/v1/708cca85d02492f36492496f.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Protein Disulfide Isomerase-Enriched Extracellular Vesicles from Bladder Cancer Cells Support Tumor Survival and Malignant Transformation in the Bladder","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOver 70% of newly diagnosed bladder cancer (BC) patients have non-muscle invasive BC (NMIBC) confined to the urothelium and underlying lamina propria (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). NMIBC patients face high rates of recurrence, with two-thirds experiencing tumor recurrence within five years, and up to 88% within 15 years (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). One proposed explanation for these high rates of recurrence involves the cancer field effect wherein pre-malignant cells are predisposed to tumor development, potentially contributing to the multi-chronotropic and multifocal nature of recurrent NMIBC (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). How this occurs remains poorly understood, with some work supporting the stepwise accumulation of genetic alterations that ultimately result in tumor formation, whereas other studies suggest clonal expansion from a single common precursor as the major mechanism of recurrent tumor growth (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Field cancerization may drive tumorigenesis regardless of the exact nature of these genetic transformation events. A comprehensive genetic analysis of bladder cancer samples using datasets from The Cancer Genome Atlas (TCGA) project identified multiple genomic alterations, suggesting that these progressive tumors are heterogeneous and can result from a permissive oncogenic environment in the whole bladder (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Those tumors can recur anywhere in the bladder and may or may not share similar histology with the primary tumors (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), further supporting the theory that the entire bladder is permissive to tumorigenesis.\u003c/p\u003e \u003cp\u003eThe induction of endoplasmic reticulum (ER) stress responses can be cytoprotective (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), with unfolded protein accumulation within the ER lumen triggering a coordinated unfolded protein response (UPR) that can restore ER homeostasis (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, insufficient or sustained ER stress responses result in pathologic alterations that lead to oncogenesis (\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Indeed, ER stress and UPR induction are common features of human cancers (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Highly proliferative tumors are exposed to several intrinsic and extrinsic stressors (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), potentially explaining the enhanced UPR signaling activity in these cells as a survival strategy that enables them to better tolerate exposure to stressful environments. The pronounced reliance of many cancer cells on the UPR has prompted interest in targeting this pathway as a form of anti-cancer therapy (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). However, the advancement of these strategies beyond the preclinical stage has been hampered by concerns regarding off-target effects (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), prompting a need for more in-depth studies of the molecular machinery governing the UPR in specific cancers.\u003c/p\u003e \u003cp\u003eMembers of the protein disulfide isomerase (PDI) family, including the canonical PDIA1 encoded by \u003cem\u003eP4HB\u003c/em\u003e, are molecular chaperones and thiol-disulfide oxidoreductases abundantly present within the ER lumen (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). PDI can be phosphorylated and activated in response to UPR induction (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e), and it functions in part by catalyzing disulfide bond formation and isomerization to help alleviate this bottleneck in the oxidative protein folding process (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Oxidized PDI can effectively donate its disulfide bond to an unfolded or misfolded protein by accepting electrons from the thiol groups of these polypeptides such that they can fold into an appropriate conformation and form proper disulfide bonds (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The reduced form of PDI can then recycle to the catalytically active oxidized state by passing these electrons to ER oxidoreductin 1 (ERO1), which, in turn, generates hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) that can contribute to oxidative stress (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). \u003cem\u003eP4HB\u003c/em\u003e upregulation has been reported in bladder tumors and linked to worse pathological staging, overall survival, and recurrence-free survival in patients, with corresponding overexpression in BC cell lines supporting tumor cell proliferation and invasion (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). \u003cem\u003eP4HB\u003c/em\u003e knockdown can sensitize BC cells to gemcitabine (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and it is a platinum-resistance-related gene in BC patients such that the knockout of this PDI-coding gene sensitizes bladder tumor cells to platinum-based treatment (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile these prior data support an important pro-tumorigenic role for PDI in BC, relatively little remains known of the precise mechanisms whereby levels of PDI expression and activity ultimately shape malignant outcomes. Strikingly, extant data suggest that PDI can function as a dual-edged sword such that it can support cancer growth by activating the PERK branch of the UPR pathway to facilitate tumor proliferation and survival (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), whereas its excessive induction of NADPH oxidase activity can result in deleterious levels of reactive oxygen species (ROS) production and cell death (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). It may thus be incumbent on tumor cells to maintain levels of PDI activity sufficient to adapt to ER stress and maintain proteostasis while mitigating the potential for lethal oxidative stress associated with unrestrained ROS generation.\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) are small membrane-enclosed structures released from cells that enable the intercellular transmission of macromolecular cargoes. We and others have demonstrated that certain cargo proteins present within tumor-derived EVs can promote tumorigenesis (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). We have further found that BC cell-derived EVs can drive UPR induction and oncogenic transformation in recipient urothelial cells (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). This, coupled with the potential need for tumor cells to regulate PDI activity and attendant oxidative stress within a tolerable range conducive to proliferation and drug resistance, raises the possibility that EVs may provide a release valve to manage levels of intracellular stress. Indeed, EV-mediated relief of excessive ER stress has been proposed in developmental settings (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), and may be co-opted by tumor cells, indirectly exposing non-malignant recipient cells to this stress in the process.\u003c/p\u003e \u003cp\u003eThe goal of this study was to explore the role of PDI as a regulator of BC malignancy, with a focus on the maintenance of homeostatic PDI activity, the role of EVs in this context, and the ability of PDI-containing EVs to induce normal urothelial cell transformation through the use of loss-of-function and rescue approaches. We further explored the potential relevance of PDI expression to NMIBC recurrence through a retrospective tissue microarray-based analysis to gain direct insight into the potential clinical relevance of these mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eReduced PDI expression promotes bladder cancer cell survival under elevated ER stress.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn an effort to begin exploring the role that PDI plays in cancer cells, we used short hairpin RNA (shRNA) constructs to knock down PDI in TCCSUP BC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). To probe the relationship between PDI expression and the survival of tumor cells in the presence or absence of ER stress, tunicamycin (140 nM) was used to treat these tumor cells. While tunicamycin significantly increased the frequency of propidium iodide (PI)-positive TCCSUP cells expressing the scramble control shRNA, it had no impact on the frequency of PI-positive cells in which PDI had been knocked down (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Consistently, PDI knockdown reduced the degree of tunicamycin-induced caspase-3 activation in these BC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) while abrogating the ability of tunicamycin to compromise colony formation in a clonogenic assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Similar effects were also observed in the J82 BC cell line (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results suggest that elevated levels of ER stress compromise the survival of BC cells, while the silencing of PDI can restore the tolerance of these malignant cells to this form of stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven that ROS production is a byproduct of PDI activity, it is possible that high levels of ER stress may expose BC cells to increased oxidative stress, thereby contributing to the induction of apoptotic death. To test this possibility, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production was analyzed, revealing that PDI knockdown was sufficient to significantly reduce basal H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels while also markedly suppressing tunicamycin-induced production thereof (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). In line with these results, tunicamycin significantly induced the upregulation of oxidative stress-related genes (\u003cem\u003eNFE2L2, NQO1, GCLC\u003c/em\u003e) in control TCCSUP cells, whereas it failed to do so in these cells following PDI knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Moreover, tunicamycin significantly reduced the GSH/GSSG ratio in scramble control TCCSUP cells, whereas it had no impact on this ratio following PDI silencing (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). Together, these data support a potential model wherein PDI-mediated ROS production can, under conditions of elevated ER stress, compromise redox homeostasis within BC cells, thereby inducing their apoptotic death. As higher levels of basal caspase-3 activity were observed following PDI knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), however, this protein may play an important pro-survival role under conditions of reduced ER stress, suggesting that tumor cells need to carefully calibrate their intracellular PDI supply to maintain viability.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBladder cancer cells mediate PDI homeostasis through EV release\u003c/h2\u003e \u003cp\u003eEV-mediated release has been proposed as an important mechanism through which cells can eliminate any undesirable molecules (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). We thus speculated that BC cells may leverage secreted EVs as a means of maintaining intracellular PDI levels within a tolerable range that balances the beneficial effects of this protein against its potential to induce excessive oxidative stress. Strikingly, tunicamycin treatment markedly increased the levels of PDI found in EVs isolated from TCCSUP cells, whereas it had no impact on PDI cargo levels within EVs isolated from non-transformed SV-HUC urothelial cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). This finding was further supported by the observation that PDI colocalized with the exosome/multivesicular body marker TSG101 in tunicamycin-treated TCCSUP and J82 BC cells, while this colocalization was not apparent in SV-HUC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). A reduced thiol quantification approach was further used to examine the redox composition of PDI within these cells and EVs. The majority of PDI detected within both TCCSUP and SV-HUC cells was present in the oxidized (oxidoreductase-active) form, but the frequency of reduced PDI rose significantly in TCCSUP but not in SV-HUC cells in response to tunicamycin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Interestingly, we found that the majority of PDI found in TCCSUP cell-derived EVs was present in the oxidized form (89.2%; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). We thus posited that tumor cells may disfavor oxidative protein folding under excessively high levels of ER stress, packaging oxidized PDI into EVs such that it can be exported from the cell to preserve viability and mitigate oxidative stress. In further support of this model, we found that TCCSUP cells released significantly more EVs at baseline as compared to SV-HUC cells, while tunicamycin treatment significantly increased the release of EVs from these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). As tunicamycin also triggered higher levels of EV release in non-transformed SV-HUC cells, such stress-induced EV secretion may be general strategy by which cells can adapt to ER stress, with PDI packaging into these EVs being a particularly beneficial pro-survival mechanism engaged by BC cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePDI-enriched BC-derived EVs induce ROS, DNA damage, and colony formation in recipient urothelial cells\u003c/h2\u003e \u003cp\u003eWe have previously demonstrated that TCCSUP cell-derived EVs can promote the malignant transformation of SV-HUC cells together with the induction of ROS and DNA damage within these recipient cells (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). We thus sought to determine whether BC-derived EV-borne PDI plays a role in this context. To that end, we isolated EVs from TCCSUPs transfected with shPDI or scramble control constructs, confirming that PDI levels within EVs from cells in which PDI had been knocked down were markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). A 2\u0026rsquo;, 7\u0026rsquo;-dichlorodihydrofluorescein diacetate (DCFDA) flow cytometry approach revealed that treatment with these shPDI EVs for 24 h induced significantly lower levels of ROS within non-transformed SV-HUC cells as compared to scramble control EV treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In line with this result, immunofluorescent γH2AX straining revealed a significant reduction in DNA damage levels following shPDI EV treatment relative to scramble control EV treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo directly establish the impact of BC EV-derived PDI on urothelial cell transformation, SV-HUC cells were continuously treated with shPDI or scramble control EVs for 13 weeks. Following a 5-week recovery period, a soft agar colony formation assay was used to assess the tumorigenic potential of these cells, revealing that while scramble EV treatment significantly increased the colony formation rate consistent with malignant transformation, shPDI EV treatment significantly reduced this colony formation rate to vehicle control levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eTo ensure result specificity, we used an extrusion approach described previously to restore recombinant PDI (rPDI) to the prepared shPDI cancer EVs (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Control EVs were instead prepared by extruding scramble and shPDI EVs with PBS, with Western blotting confirming the successful restoration of PDI to shPDI EVs at levels comparable to those in the parental EVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) (Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These EVs were then used to treat non-transformed SV-HUC cells as above, revealing that rPDI extrusion restored the ability of shPDI EVs to induce ROS production and to promote \u003cem\u003eNFE2L2\u003c/em\u003e expression in recipient SV-HUC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). PDI restoration similarly increased the levels of DNA damage induced by shPDI EV treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Importantly, rPDI extrision restored the ability of shPDI EVs to promote the malignant transformation of SV-HUC cells over a 13-week treatment period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To test whether PDI is required as an oncoprotein to maintain tumorigenicity within the resultant transformed SV-HUC cells, it was knocked down in these cells via shRNA, which had no impact on anchorage-independent growth of these cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Together, these data support the central role for PDI as a driver of the EV-induced malignant transformation of recipient urothelial cells following its release from tumor cells, but that PDI is not required to maintain malignancy once it has been established.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePDI-enriched EV-transformed cells exhibit patterns of ROS-induced DNA damage\u003c/h2\u003e \u003cp\u003eGiven our observation that PDI is essential for the cancer EV-induced malignant transformation of SV-HUC cells and for the induction of ROS and DNA damage in these cells, we then examined somatic mutation patterns in these transformed cells. Overall, the transformed cells exhibited a higher mutational burden with a somatic mutation prevalence of 8.2 per megabase, a 10.8% increase over parental cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Moreover, the transformed SV-HUC cells harbored more unique nonsynonymous mutations and more genes impacted by these unique nonsynonymous variants as compared to parental non-transformed SV-HUC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, Supplementary Table\u0026nbsp;1). Analyses of single base substitutions (SBS) revealed similar patterns in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), suggesting that the unique variants found in the transformed SV-HUC cells are likely the result of cancer EV treatment and all six SBS classes were increased in the transformed cells, with the greatest relative increase being seen in G:C to T:A transversions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), the type of substitution most characteristic of ROS-induced DNA alterations (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNumerous somatic mutational signatures and variant classes have been associated with different types of cancer and are understood to be the result of distinct mutational processes. We next performed strict signature refitting of the genomic variant data from the parental and transformed SV-HUC cells and identified eight previously defined SBS signatures found in the Catalog of Somatic Mutations in Cancer (COSMIC) (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The signatures most prominently enhanced in the transformed cells relative to the parental cells were SBS18, SBS85, and SBS40 (cosine similarity\u0026thinsp;\u0026gt;\u0026thinsp;0.51) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, Supplementary Fig. S3A). Notably, COSMIC signature SBS18 has been proposed to result from ROS-induced DNA damage (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Taken together, the mutational patterns seen in these transformed cells are consistent with oxidative DNA damage in urothelial cells following cancer EV exposure, suggesting that tumor-derived PDI plays a central role in this process.\u003c/p\u003e \u003cp\u003eInterestingly, COSMIC signature SBS85 (Supplementary Fig. S3B), reported to be caused by AID/APOBEC activity, which is one of the major sources of mutations in BC (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e), is characterized by a concentration of variants in the T\u0026thinsp;\u0026gt;\u0026thinsp;A and T\u0026thinsp;\u0026gt;\u0026thinsp;C classes. This finding suggests that, besides elevating ROS levels in recipient cells, cancer EVs may also cause DNA mutations through other mechanisms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHigh levels of PDI expression in non-muscle invasive bladder cancer patient tumor tissue predicts a higher risk of recurrence\u003c/b\u003e \u003c/p\u003e \u003cp\u003eOur \u003cem\u003ein vitro\u003c/em\u003e studies indicated that BC cells release PDI packaged in EVs when subjected to high levels of ER stress, and the uptake of these PDI-enriched EVs by non-transformed cells was sufficient to drive their malignant. As any secondary tumors that arise through this mechanism \u003cem\u003ein vivo\u003c/em\u003e may be diagnosed as recurrent tumors, these data suggest that PDI may offer value as a prognostic biomarker to predict BC recurrence. While a majority of MIBC patients will ultimately undergo radical cystectomy, NMIBC is characterized by frequent recurrences requiring surveillance cystoscopies as there is no current reliable marker currently available to monitor for recurrence and/or progression. To explore the utility of PDI in this context, we constructed a tissue microarray (TMA) consisting of 121 NMIBC patients who did (n\u0026thinsp;=\u0026thinsp;55) or did not (n\u0026thinsp;=\u0026thinsp;67) develop recurrence (Supplementary Table\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eWe analyzed the BiP expression levels in the tissue samples comprising this TMA as an approach to estimating ER stress levels in these tumor cells, in addition to assessing PDI expression. Significantly higher levels of BiP and PDI were detected in tumor tissues from patients who experienced BC recurrence (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). We also quantified the PDI positive area and intensity within BiP negative regions to demonstrate levels of non-ER residential PDI that are likely to be secreted to the extracellular environment. Interestingly, the tumors of patients with recurrent disease had higher levels of both PDI positive area and accumulation in the non-ER cytosolic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Importantly, we also found that higher levels of both BiP and PDI expression were associated with significantly reduced patient recurrence-free survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These data suggest that tissues that are under higher levels of ER stress and PDI expression have a higher risk of recurrence, demonstrating that the PDI status of tumors offers potential as a marker for the prediction of NMIBC patient recurrence.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe high rates of BC recurrence underscore the need to devise new approaches for identifying patients who are more likely to develop recurrent disease and mitigating this risk. Our data suggest that BC cells utilize EVs as a means of ensuring their ongoing survival under conditions of ER stress through the export of oxidized PDI. The incidental uptake of these PDI-enriched EVs by normal urothelial cells ultimately leads to their malignant transformation through a potential field cancerization mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). While these results offer insight into the mechanisms that may underlie BC recurrence and suggest that levels of PDI and ER stress in tumor tissues are valuable biomarkers for predicting NMIBC recurrence risk, there are several important topics related to our findings that warrant further discussion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA range of neoplasia-related conditions including reduced genomic stability, the accumulation of mutations, increased protein production and secretion, and exposure to hypoxic or nutrient-deprived microenvironmental conditions can compromise proteostasis within tumor cells (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). ER stress response and UPR induction can thus preserve tumor cell viability under these challenging conditions (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), with UPR-induced oxidized PDI activation providing support to help restore appropriate protein folding (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In this study, we found that BC cells exhibited elevated ER stress levels under basal conditions. However, subjecting them to further ER stress triggered apoptotic death that could be mitigated by knocking down PDI, thereby alleviating oxidative stress when PDI levels were reduced. This suggests that the maintenance of PDI homeostasis is vital for the survival of BC cells owing to the unique characteristics of this enzyme.\u003c/p\u003e \u003cp\u003eMost cellular compartments maintain a reducing environment, and oxidized proteins are usually unstable in the cytosol (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). To prevent hyperoxidation and maintain ER homeostasis under conditions of UPR induction when levels of PDI activity are elevated, BC cells must limit the levels of oxidized PDI within the ER, balancing proteostasis against oxidative injury. We found that under ER stress, BC cells released high levels of oxidized PDI within EVs, thereby mitigating the ROS production and consequent cell death, ultimately promoting BC cell survival. This finding is highly innovative and aligns well with the observation that, while cells continuously shed EVs at steady state, multivesicular body formation and EV release are enhanced under conditions of ER stress or oxidative stress (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e), supporting this process of EV-mediated oxidized PDI export as a stress relief mechanism (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe mechanisms that underlie the packaging of oxidized PDI into EVs by BC cells warrant further clarification. While the loading of protein cargos within EVs may be partially stochastic, ubiquitination, palmitoylation, and other post-translational modifications can facilitate the sorting of certain proteins into EVs (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). It remains to be determined whether these modifications are relevant for PDI loading into EVs and whether the oxidized form of PDI is preferentially packaged into EVs under varying levels of ER stress. Assessing these factors will help to better elucidate the directed nature of this cargo loading process.\u003c/p\u003e \u003cp\u003eStrikingly, we found that PDI-enriched EVs derived from BC cells promoted the malignant transformation of normal urothelial cells, potentially through ROS-induced DNA damage mediated by the oxidized PDI present within these vesicles or the induction of UPR activity within recipient cells. Indeed, we have previously demonstrated the ability of BC-derived EVs to induce UPR activity and malignant transformation in recipient urothelial cells (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), and both persistent UPR and oxidative stress exposure can readily drive tumorigenesis (\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), suggesting that the delivery of PDI to the urothelium may predispose this field to the future development of recurrent bladder tumors. In addition to their effects on tumorigenesis and BC recurrence, these PDI-enriched EVs may also serve as pivotal regulators of various malignant processes directly within the tumor microenvironment. For instance, intracellular PDI plays a vital role in the synthesis of type I collagen, a core component of the extracellular matrix in tumors (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e), whereas extracellular PDI directly activates integrins through the promotion of thiol-disulfide exchange (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Integrin interactions with type I collagen can also facilitate the development of chemoresistance (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), promoting proliferative growth while protecting against apoptotic death. One report has also suggested a potential link between PDI and immune surveillance, with higher PDI levels potentially supporting immune evasion in breast cancer (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). Inhibition of PDI activity has shown therapeutic potential in reducing breast cancer cell adhesion and migration through the disruption of focal adhesion complex formation and associated phosphorylation events (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Investigating whether EV-derived PDI can alter the composition of the tumor microenvironment, influence chemoresistance, or modulate immune-mediated detection of BC cells through these mechanisms may represent promising avenues for future research aimed at clarifying the broader role of PDI in bladder carcinogenesis.\u003c/p\u003e \u003cp\u003eWe found that both total PDI expression and non-ER-resident PDI levels were higher in those patients who developed recurrent disease. BiP expression was similarly associated with disease recurrence, underscoring the potential value of evaluating ER stress levels, PDI expression, and PDI localization as potential biomarkers of BC recurrence risk. While tumor and urothelial tissue collection are inherently invasive procedures poorly suited for routine monitoring, urine is an EV-rich biofluid that can be readily obtained from patients, providing an opportunity for noninvasive BC-related biomarker testing (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Given our finding that BC cells release PDI-enriched EVs, this raises the urine EVs may offer diagnostic or prognostic utility when evaluating BC patients based on the levels of PDI or ER stress-related biomarkers present therein. However, prospective trials will be essential to evaluate this possibility, and the routine implementation of urine EV-based analyses will necessitate overcoming challenges related to the sensitivity, specificity, and standardization of these biomarkers (\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). Our results also raise the question of whether therapeutic interventions targeting ER stress and/or PDI may afford benefits to BC patients. PDI inhibitors have been evaluated as promising anti-cancer treatments in patients with relapsed ovarian cancer and in various preclinical cancer models (\u003cspan additionalcitationids=\"CR59\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e), although whether they can protect against field cancerization effects mediated by PDI-enriched EVs remains uncertain. Given that we found that lower PDI levels were beneficial to BC cell survival under high levels of ER stress, while ER stress loading has been advanced as an anti-cancer therapy (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e), caution and individualized treatment planning would likely be vital for any interventional efforts targeting this regulatory axis.\u003c/p\u003e \u003cp\u003eTogether, our results offer new insights into the integral role that EVs play in the maintenance of BC cell homeostasis under conditions of ER stress, while highlighting PDI as an attractive biomarker and therapeutic that may directly contribute to the risk of BC recurrence.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and EV isolation\u003c/h2\u003e \u003cp\u003eCell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained in a humidified 37\u0026deg;C incubator under 5% CO\u003csub\u003e2\u003c/sub\u003e in media containing FB Essence (3100, Seradigm). For EV collection, cells were cultured in medium containing EV-depleted fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and penicillin-streptomycin (catalog no. 15140-122, Thermo Fisher Scientific) as described previously (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e). Cell culture supernatants were processed immediately after collection by serial centrifugation at 400 \u0026times; g for 10 min and 15,500 \u0026times; g for 30 min to remove cells and debris and then stored at -80\u0026deg;C. EVs were isolated from thawed samples by ultracentrifugation performed twice at 200,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 70 min at 4\u0026deg;C, and the resulting pellets were resuspended in a small volume of DPBS. Aggregates were removed from the samples by another 15,500 \u0026times; g centrifugation for 5 min. Final total protein concentrations in the samples were measured with a Micro BCA assay (catalog no. 23235, Thermo Fisher Scientific), and samples were stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExtrusion\u003c/h3\u003e\n\u003cp\u003eInitially, 250 \u0026micro;L of PBS containing 37.5 \u0026micro;g of shPDI TCCSUP EVs with or without 25 \u0026micro;g of recombinant PDI (catalog no. enz-262, Prospec) were extruded 10 times using an Avanti extruder set with 0.1 \u0026micro;m polycarbonate membrane filters (catalog no. 610023, Avanti, Alabaster, AL, USA). The mixture was then transferred to a 1.5 mL microcentrifuge tube (catalog no. 357448, Beckman Coulter) filled with 900 \u0026micro;L of PBS and subjected to a 2-hour ultracentrifugation step (Optoma MAX-XP ultracentrifuge, Beckman Coulter, Brea, CA, USA) in a TLA110 rotor at 100K \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C. After removing the supernatant, the EV pellet was resuspended in PBS.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eWhole genome sequencing\u003c/h2\u003e \u003cp\u003eGenomic DNA concentration was assessed with the Qubit Fluorometer (Thermo Fisher Scientific) and quality was assessed using the Agilent Tapestation (Agilent, Santa Clara, CA). The Illumina Nextera Flex kit (Illumina, San Diego, CA) was used for library construction per the manufacturer\u0026rsquo;s instructions. Briefly, 500 ng of gDNA was tagmented with Bead-Linked Transposome (BLT) beads while simultaneously adding Illumina sequencing primers. Tagmented genomic DNA was purified, followed by reduced-cycle (5-cycle) PCR amplification to add index and adapter sequences for sequencing. Sequence data was generated using Illumina's NovaSeq 6000 sequencer. Data were analyzed using MuSiCa (Mutational Signatures in Cancer) (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e) and the MutationalPatterns R package (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTissue microarray\u003c/h2\u003e \u003cp\u003eWe retrieved primary tumor specimens from 121 index cases (initial detection) of non-invasive (pTa) low-grade urothelial carcinoma obtained by transurethral resection performed at the University of Rochester Medical Center. These patients included 80 men and 41 women, with a mean age of 69.7 years (range: 46.5\u0026ndash;93.1 years) at the time of surgery. All the sections were reviewed for confirmation of original diagnoses, according to the 2004 World Health Organization/International Society of Urological Pathology classification system for urothelial neoplasms (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Appropriate approval from the Institutional Review Board was obtained prior to the construction and use of the TMA. Bladder TMAs were constructed from formalin-fixed paraffin-embedded specimens as previously described (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). Some cases in the initial TMA patient group were not represented in the first sections taken from the TMA paraffin blocks.\u003c/p\u003e \u003cp\u003eFollowing de-paraffinization of TMA sections, antigen retrieval was performed in heated citrate buffer (Vector H-3300) for 30 min. Primary antibody incubation was conducted overnight at 4\u0026deg;C using anti-PDI (Cell Signaling 3501, 1:500) and anti-BiP (Santa Cruz sc-166490, 1:100) antibodies. Stained tissues were photographed at 40\u0026times; magnification using a Leica DM5000 B microscope. Tumor regions in each photographed field were masked manually 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 (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). To assess the colocalization of two epitopes, the Manders overlap coefficients were determined using the JACoP plugin in ImageJ (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSupplementary methods\u003c/h2\u003e \u003cp\u003eFor further details regarding other assays conducted in this study, see the Supplementary Materials and Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed using at least three biological repeats. Utilized statistical tests are indicated in the figure legends. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. Statistical analyses were performed using GraphPad Prism 9.2.0 and the R statistical computing environment, version 4.0.3.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the University of Rochester Medical Center Mass Spectrometry Resource Laboratory and Genomic Research Center for assisting in the proteomic and whole genome sequencing analyses. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCHW participated in study conception, experiment design, the acquisition, analysis, and interpretation of data, and drafting the manuscript. KLY participated in the analysis and interpretation of data. RDM participated in the analysis and interpretation of data, and drafting the manuscript. \u0026nbsp;MMHA participated in data acquisition, analysis, and interpretation. CRS participated in data acquisition, analysis, and interpretation. EMM participated in study conception and editing of the manuscript. YFL participated in study conception, the design/supervision of experiments, funding acquisition, writing, and editing of the manuscript. \u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMessing EM, Madeb R, Young T, Gilchrist KW, Bram L, Greenberg EB, et al. Long-term outcome of hematuria home screening for bladder cancer in men. Cancer. 2006;107(9):2173-9.\u003c/li\u003e\n\u003cli\u003eScosyrev E, Noyes K, Feng C, Messing E. Sex and racial differences in bladder cancer presentation and mortality in the US. Cancer. 2009;115(1):68-74.\u003c/li\u003e\n\u003cli\u003eBrandau S, Suttmann H. Thirty years of BCG immunotherapy for non-muscle invasive bladder cancer: a success story with room for improvement. Biomedicine \u0026amp; pharmacotherapy = Biomedecine \u0026amp; pharmacotherapie. 2007;61(6):299-305.\u003c/li\u003e\n\u003cli\u003eCzerniak B, Dinney C, McConkey D. Origins of bladder cancer. Annual Review of Pathology: Mechanisms of Disease. 2016;11:149-74.\u003c/li\u003e\n\u003cli\u003eJones TD, Wang M, Eble JN, MacLennan GT, Lopez-Beltran A, Zhang S, et al. Molecular evidence supporting field effect in urothelial carcinogenesis. Clin Cancer Res. 2005;11(18):6512-9.\u003c/li\u003e\n\u003cli\u003eCheng L, MacLennan GT, Pan CX, Jones TD, Moore CR, Zhang S, et al. Allelic loss of the active X chromosome during bladder carcinogenesis. Arch Pathol Lab Med. 2004;128(2):187-90.\u003c/li\u003e\n\u003cli\u003eCheng L, Jones TD, McCarthy RP, Eble JN, Wang M, MacLennan GT, et al. Molecular genetic evidence for a common clonal origin of urinary bladder small cell carcinoma and coexisting urothelial carcinoma. Am J Pathol. 2005;166(5):1533-9.\u003c/li\u003e\n\u003cli\u003eCancer Genome Atlas Research N. Comprehensive molecular characterization of urothelial bladder carcinoma. Nature. 2014;507(7492):315-22.\u003c/li\u003e\n\u003cli\u003eCheng L, Davidson DD, Maclennan GT, Williamson SR, Zhang S, Koch MO, et al. The origins of urothelial carcinoma. Expert review of anticancer therapy. 2010;10(6):865-80.\u003c/li\u003e\n\u003cli\u003eMa Y, Hendershot LM. The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer. 2004;4(12):966-77.\u003c/li\u003e\n\u003cli\u003eHetz C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nature reviews Molecular cell biology. 2012;13(2):89-102.\u003c/li\u003e\n\u003cli\u003eWang M, Kaufman RJ. The impact of the endoplasmic reticulum protein-folding environment on cancer development. Nat Rev Cancer. 2014;14(9):581-97.\u003c/li\u003e\n\u003cli\u003eClarke HJ, Chambers JE, Liniker E, Marciniak SJ. Endoplasmic reticulum stress in malignancy. Cancer cell. 2014;25(5):563-73.\u003c/li\u003e\n\u003cli\u003eChevet E, Hetz C, Samali A. Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis. Cancer discovery. 2015;5(6):586-97.\u003c/li\u003e\n\u003cli\u003eOakes SA. Endoplasmic reticulum stress signaling in cancer cells. The American journal of pathology. 2020;190(5):934-46.\u003c/li\u003e\n\u003cli\u003eUrra H, Dufey E, Avril T, Chevet E, Hetz C. Endoplasmic Reticulum Stress and the Hallmarks of Cancer. Trends Cancer. 2016;2(5):252-62.\u003c/li\u003e\n\u003cli\u003eLiang D, Khoonkari M, Avril T, Chevet E, Kruyt FA. The unfolded protein response as regulator of cancer stemness and differentiation: Mechanisms and implications for cancer therapy. Biochemical Pharmacology. 2021;192:114737.\u003c/li\u003e\n\u003cli\u003eWang CC, Tsou CL. Protein disulfide isomerase is both an enzyme and a chaperone. The FASEB journal. 1993;7(15):1515-7.\u003c/li\u003e\n\u003cli\u003eWang L, Wang X, Wang C-c. Protein disulfide\u0026ndash;isomerase, a folding catalyst and a redox-regulated chaperone. Free Radical Biology and Medicine. 2015;83:305-13.\u003c/li\u003e\n\u003cli\u003eYu J, Li T, Liu Y, Wang X, Zhang J, Wang Xe, et al. Phosphorylation switches protein disulfide isomerase activity to maintain proteostasis and attenuate ER stress. The EMBO journal. 2020;39(10):e103841.\u003c/li\u003e\n\u003cli\u003eShergalis AG, Hu S, Bankhead III A, Neamati N. Role of the ERO1-PDI interaction in oxidative protein folding and disease. Pharmacology \u0026amp; therapeutics. 2020;210:107525.\u003c/li\u003e\n\u003cli\u003eZito E. ERO1: A protein disulfide oxidase and H2O2 producer. Free Radical Biology and Medicine. 2015;83:299-304.\u003c/li\u003e\n\u003cli\u003eLyu L, Xiang W, Zheng F, Huang T, Feng Y, Yuan J, et al. Significant prognostic value of the autophagy-related gene P4HB in bladder urothelial carcinoma. Frontiers in oncology. 2020;10:1613.\u003c/li\u003e\n\u003cli\u003eWu Y, Peng Y, Guan B, He A, Yang K, He S, et al. P4HB: A novel diagnostic and prognostic biomarker for bladder carcinoma. Oncology letters. 2021;21(2):1-.\u003c/li\u003e\n\u003cli\u003eWang X, Bai Y, Zhang F, Yang Y, Feng D, Li A, et al. Targeted inhibition of P4HB promotes cell sensitivity to gemcitabine in Urothelial carcinoma of the bladder. OncoTargets and therapy. 2020:9543-58.\u003c/li\u003e\n\u003cli\u003eXiong S, Li S, Zeng J, Nie J, Liu T, Liu X, et al. Deciphering the immunological and prognostic features of bladder cancer through platinum-resistance-related genes analysis and identifying potential therapeutic target P4HB. Frontiers in Immunology. 2023;14.\u003c/li\u003e\n\u003cli\u003eKranz P, Neumann F, Wolf A, Classen F, Pompsch M, Ocklenburg T, et al. PDI is an essential redox-sensitive activator of PERK during the unfolded protein response (UPR). Cell Death Dis. 2017;8(8):e2986.\u003c/li\u003e\n\u003cli\u003ede APAM, Verissimo-Filho S, Guimaraes LL, Silva AC, Takiuti JT, Santos CX, et al. Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation. J Leukoc Biol. 2011;90(4):799-810.\u003c/li\u003e\n\u003cli\u003eFernandes DC, Manoel AH, Wosniak J, Jr., Laurindo FR. Protein disulfide isomerase overexpression in vascular smooth muscle cells induces spontaneous preemptive NADPH oxidase activation and Nox1 mRNA expression: effects of nitrosothiol exposure. Arch Biochem Biophys. 2009;484(2):197-204.\u003c/li\u003e\n\u003cli\u003eBeckham CJ, Olsen J, Yin PN, Wu CH, Ting HJ, Hagen FK, et al. Bladder cancer exosomes contain EDIL-3/Del1 and facilitate cancer progression. The Journal of urology. 2014;192(2):583-92.\u003c/li\u003e\n\u003cli\u003eSilvers CR, Liu YR, Wu CH, Miyamoto H, Messing EM, Lee YF. Identification of extracellular vesicle-borne periostin as a feature of muscle-invasive bladder cancer. Oncotarget. 2016.\u003c/li\u003e\n\u003cli\u003eSilvers CR, Miyamoto H, Messing EM, Netto GJ, Lee YF. Characterization of urinary extracellular vesicle proteins in muscle-invasive bladder cancer. Oncotarget. 2017;8(53):91199-208.\u003c/li\u003e\n\u003cli\u003eWu C-H, Silvers CR, Messing EM, Lee Y-F. Bladder cancer extracellular vesicles drive tumorigenesis by inducing the unfolded protein response in endoplasmic reticulum of nonmalignant cells. Journal of Biological Chemistry. 2019;294(9):3207-18.\u003c/li\u003e\n\u003cli\u003eFu B, Ma H, Zhang Dj, Wang L, Li Zq, Guo Zh, et al. Porcine oviductal extracellular vesicles facilitate early embryonic development via relief of endoplasmic reticulum stress. Cell Biology International. 2022;46(2):300-10.\u003c/li\u003e\n\u003cli\u003eBaixauli F, L\u0026oacute;pez-Ot\u0026iacute;n C, Mittelbrunn M. Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness. Front Immunol. 2014;5:403.\u003c/li\u003e\n\u003cli\u003eFan J, Kuai B, Wu G, Wu X, Chi B, Wang L, et al. Exosome cofactor hMTR4 competes with export adaptor ALYREF to ensure balanced nuclear RNA pools for degradation and export. Embo j. 2017;36(19):2870-86.\u003c/li\u003e\n\u003cli\u003eFu S, Wang Y, Xia X, Zheng JC. Exosome engineering: Current progress in cargo loading and targeted delivery. NanoImpact. 2020;20:100261.\u003c/li\u003e\n\u003cli\u003eYasui M, Kanemaru Y, Kamoshita N, Suzuki T, Arakawa T, Honma M. Tracing the fates of site-specifically introduced DNA adducts in the human genome. DNA Repair (Amst). 2014;15:11-20.\u003c/li\u003e\n\u003cli\u003eTate JG, Bamford S, Jubb HC, Sondka Z, Beare DM, Bindal N, et al. COSMIC: the Catalogue Of Somatic Mutations In Cancer. Nucleic Acids Research. 2018;47(D1):D941-D7.\u003c/li\u003e\n\u003cli\u003eKucab JE, Zou X, Morganella S, Joel M, Nanda AS, Nagy E, et al. A Compendium of Mutational Signatures of Environmental Agents. Cell. 2019;177(4):821-36.e16.\u003c/li\u003e\n\u003cli\u003eGlaser AP, Fantini D, Wang Y, Yu Y, Rimar KJ, Podojil JR, et al. APOBEC-mediated mutagenesis in urothelial carcinoma is associated with improved survival, mutations in DNA damage response genes, and immune response. Oncotarget. 2018;9(4):4537-48.\u003c/li\u003e\n\u003cli\u003eParakh S, Atkin JD. Novel roles for protein disulphide isomerase in disease states: a double edged sword? Front Cell Dev Biol. 2015;3:30.\u003c/li\u003e\n\u003cli\u003eFrand AR, Cuozzo JW, Kaiser CA. Pathways for protein disulphide bond formation. Trends Cell Biol. 2000;10(5):203-10.\u003c/li\u003e\n\u003cli\u003eKanemoto S, Nitani R, Murakami T, Kaneko M, Asada R, Matsuhisa K, et al. Multivesicular body formation enhancement and exosome release during endoplasmic reticulum stress. Biochemical and biophysical research communications. 2016;480(2):166-72.\u003c/li\u003e\n\u003cli\u003eZhang W, Liu R, Chen Y, Wang M, Du J. Crosstalk between oxidative stress and exosomes. Oxidative Medicine and Cellular Longevity. 2022;2022.\u003c/li\u003e\n\u003cli\u003eChen Y, Zhao Y, Yin Y, Jia X, Mao L. Mechanism of cargo sorting into small extracellular vesicles. Bioengineered. 2021;12(1):8186-201.\u003c/li\u003e\n\u003cli\u003eWaury K, Gogishvili D, Nieuwland R, Chatterjee M, Teunissen CE, Abeln S. Proteome encoded determinants of protein sorting into extracellular vesicles. Journal of Extracellular Biology. 2024;3(1):e120.\u003c/li\u003e\n\u003cli\u003eRauch F, Fahiminiya S, Majewski J, Carrot-Zhang J, Boudko S, Glorieux F, et al. Cole-Carpenter syndrome is caused by a heterozygous missense mutation in P4HB. Am J Hum Genet. 2015;96(3):425-31.\u003c/li\u003e\n\u003cli\u003eLiang Y, Diehn M, Bollen AW, Israel MA, Gupta N. Type I collagen is overexpressed in medulloblastoma as a component of tumor microenvironment. J Neurooncol. 2008;86(2):133-41.\u003c/li\u003e\n\u003cli\u003ePopielarski M, Ponamarczuk H, Stasiak M, Michalec L, Bednarek R, Studzian M, et al. The role of Protein Disulfide Isomerase and thiol bonds modifications in activation of integrin subunit alpha11. Biochem Biophys Res Commun. 2018;495(2):1635-41.\u003c/li\u003e\n\u003cli\u003eBaltes F, Pfeifer V, Silbermann K, Caspers J, Wantoch von Rekowski K, Schlesinger M, et al. \u0026beta;(1)-Integrin binding to collagen type 1 transmits breast cancer cells into chemoresistance by activating ABC efflux transporters. Biochim Biophys Acta Mol Cell Res. 2020;1867(5):118663.\u003c/li\u003e\n\u003cli\u003eAlhammad R, Khunchai S, Tongmuang N, Limjindaporn T, Yenchitsomanus PT, Mutti L, et al. Protein disulfide isomerase A1 regulates breast cancer cell immunorecognition in a manner dependent on redox state. Oncology Reports. 2020;44(6):2406-18.\u003c/li\u003e\n\u003cli\u003eChen IH, Chang FR, Wu YC, Kung PH, Wu CC. 3,4-Methylenedioxy-\u0026beta;-nitrostyrene inhibits adhesion and migration of human triple-negative breast cancer cells by suppressing \u0026beta;1 integrin function and surface protein disulfide isomerase. Biochimie. 2015;110:81-92.\u003c/li\u003e\n\u003cli\u003eOeyen E, Hoekx L, De Wachter S, Baldewijns M, Ameye F, Mertens I. Bladder cancer diagnosis and follow-up: the current status and possible role of extracellular vesicles. International journal of molecular sciences. 2019;20(4):821.\u003c/li\u003e\n\u003cli\u003eOliveira MCd, Caires HR, Oliveira MJ, Fraga A, Vasconcelos MH, Ribeiro R. Urinary biomarkers in bladder cancer: where do we stand and potential role of extracellular vesicles. Cancers. 2020;12(6):1400.\u003c/li\u003e\n\u003cli\u003eLiu Y-R, Ortiz-Bonilla CJ, Lee Y-F. Extracellular vesicles in bladder cancer: biomarkers and beyond. International journal of molecular sciences. 2018;19(9):2822.\u003c/li\u003e\n\u003cli\u003eChoi J-Y, Kim S, Kwak H-B, Park D-H, Park J-H, Ryu J-S, et al. Extracellular vesicles as a source of urological biomarkers: lessons learned from advances and challenges in clinical applications to major diseases. International neurourology journal. 2017;21(2):83.\u003c/li\u003e\n\u003cli\u003eGelzinis JA, Szahaj MK, Bekendam RH, Wurl SE, Pantos MM, Verbetsky CA, et al. Targeting thiol isomerase activity with zafirlukast to treat ovarian cancer from the bench to clinic. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2023;37(5):e22914.\u003c/li\u003e\n\u003cli\u003eRobinson RM, Reyes L, Duncan RM, Bian H, Strobel ED, Hyman SL, et al. Tuning isoform selectivity and bortezomib sensitivity with a new class of alkenyl indene PDI inhibitor. European journal of medicinal chemistry. 2020;186:111906.\u003c/li\u003e\n\u003cli\u003ePowell LE, Foster PA. Protein disulphide isomerase inhibition as a potential cancer therapeutic strategy. Cancer medicine. 2021;10(8):2812-25.\u003c/li\u003e\n\u003cli\u003eKazama H, Hiramoto M, Miyahara K, Takano N, Miyazawa K. Designing an effective drug combination for ER stress loading in cancer therapy using a real-time monitoring system. Biochem Biophys Res Commun. 2018;501(1):286-92.\u003c/li\u003e\n\u003cli\u003eSilvers CR, Liu YR, Wu CH, Miyamoto H, Messing EM, Lee YF. Identification of extracellular vesicle-borne periostin as a feature of muscle-invasive bladder cancer. Oncotarget. 2016;7(17):23335-45.\u003c/li\u003e\n\u003cli\u003eD\u0026iacute;az-Gay M, Vila-Casades\u0026uacute;s M, Franch-Exp\u0026oacute;sito S, Hern\u0026aacute;ndez-Ill\u0026aacute;n E, Lozano JJ, Castellv\u0026iacute;-Bel S. Mutational Signatures in Cancer (MuSiCa): a web application to implement mutational signatures analysis in cancer samples. BMC Bioinformatics. 2018;19(1):224.\u003c/li\u003e\n\u003cli\u003eBlokzijl F, Janssen R, van Boxtel R, Cuppen E. MutationalPatterns: comprehensive genome-wide analysis of mutational processes. Genome Med. 2018;10(1):33.\u003c/li\u003e\n\u003cli\u003eMiyamoto H, Miller JS, Fajardo DA, Lee TK, Netto GJ, Epstein JI. Non-invasive papillary urothelial neoplasms: the 2004 WHO/ISUP classification system. Pathol Int. 2010;60(1):1-8.\u003c/li\u003e\n\u003cli\u003eMiyamoto H, Yao JL, Chaux A, Zheng Y, Hsu I, Izumi K, et al. Expression of androgen and oestrogen receptors and its prognostic significance in urothelial neoplasm of the urinary bladder. BJU Int. 2012;109(11):1716-26.\u003c/li\u003e\n\u003cli\u003eSchindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82.\u003c/li\u003e\n\u003cli\u003eBolte S, Cordeli\u0026egrave;res FP. A guided tour into subcellular colocalization analysis in light microscopy. J Microsc. 2006;224(Pt 3):213-32.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4425743/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4425743/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBladder 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 (PDI) 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 PDI was downregulated. These cells maintained homeostatic PDI levels through the EV-mediated release of PDI. Chronic exposure of urothelial cells to these PDI-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 PDI 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 PDI and ER stress. Together, these data suggest that cancer cells selectively sort oxidized PDI 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.\u003c/p\u003e","manuscriptTitle":"Protein Disulfide Isomerase-Enriched Extracellular Vesicles from Bladder Cancer Cells Support Tumor Survival and Malignant Transformation in the Bladder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 18:27:42","doi":"10.21203/rs.3.rs-4425743/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Reject after peer review","date":"2024-08-27T12:13:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-08-23T09:59:31+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-25T13:46:37+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-16T17:55:54+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-16T15:42:23+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-01T11:52:16+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-06-17T12:10:18+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-05-25T20:35:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-16T10:44:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-15T14:12:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Oncogene","date":"2024-05-15T14:12:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"oncogene","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"onc","sideBox":"Learn more about [Oncogene](http://www.nature.com/onc/)","snPcode":"41388","submissionUrl":"https://mts-onc.nature.com/cgi-bin/main.plex","title":"Oncogene","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6ad23212-0fd2-44a0-b5db-4eab25958c13","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-11T07:12:05+00:00","versionOfRecord":{"articleIdentity":"rs-4425743","link":"https://doi.org/10.1038/s41388-025-03380-6","journal":{"identity":"oncogene","isVorOnly":false,"title":"Oncogene"},"publishedOn":"2025-04-11 04:00:00","publishedOnDateReadable":"April 11th, 2025"},"versionCreatedAt":"2024-06-11 18:27:42","video":"","vorDoi":"10.1038/s41388-025-03380-6","vorDoiUrl":"https://doi.org/10.1038/s41388-025-03380-6","workflowStages":[]},"version":"v1","identity":"rs-4425743","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4425743","identity":"rs-4425743","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}