Murine Bladder Research Articles

Isoprostanes evoke contraction of the murine and human detrusor muscle via activation of the thromboxane prostanoid TP receptor and Rho kinase.

Local or systemic inflammation can severely impair urinary bladder functions and contribute to the development of voiding disorders in millions of people worldwide. Isoprostanes are inflammatory lipid mediators that are upregulated in the blood and urine by oxidative stress and may potentially induce detrusor overactivity. The aim of the present study was to investigate the effects and signal transduction of isoprostanes in human and murine urinary bladders in order to provide potential pharmacological targets in detrusor overactivity. Contraction force was measured with a myograph in murine and human urinary bladder smooth muscle (UBSM) ex vivo. Isoprostane 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in the murine UBSM, which was abolished in mice deficient in the thromboxane prostanoid (TP) receptor. The responses remained unaltered after removal of the mucosa or incubation with tetrodotoxin. Smooth muscle-specific deletion of Gα12/13 protein or inhibition of Rho kinase by Y-27632 decreased the contractions. In Gαq/11-knockout mice, responses were reduced and in the presence of Y-27632 abolished completely. In human UBSM, the TP agonist U-46619 evoked dose-dependent contractions. Neither atropine nor the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid decreased the effect, indicating that TP receptors directly mediate detrusor muscle contraction. 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in the human UBSM, and these responses were abolished by the TP antagonist SQ-29548 and were decreased by Y-27632. Our results indicate that isoprostanes evoke contraction in murine and human urinary bladders, an effect mediated by the TP receptor. The G12/13-Rho-Rho kinase pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target in detrusor overactivity.NEW & NOTEWORTHY Voiding disorders affect millions of people worldwide. Inflammation can impair urinary bladder functions and contribute to the development of detrusor overactivity. The effects and signal transduction of inflammatory lipid mediator isoprostanes were studied in human and murine urinary bladders ex vivo. We found that isoprostanes evoke contraction, an effect mediated by thromboxane prostanoid receptors. The G12/13-Rho-Rho kinase signaling pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target.

Identification of novel bladder sensory GPCRs.

Sensory GPCRs such as olfactory receptors (ORs), taste receptors (TRs), and opsins (OPNs) are now known to play important physiological roles beyond their traditional sensory organs. Here, we systematically investigate the expression of sensory GPCRs in the urinary bladder for the first time. We find that the murine bladder expresses 16 ORs, 7 TRs, and 3 OPNs. We additionally explore the ectopic expression of these GPCRs in tissues beyond the bladder, as well as the localization within the bladder. In future work, understanding the functional roles of these bladder sensory GPCRs may shed light on novel mechanisms which modulate bladder function in health and disease.

Mitochondrial heat shock protein mortalin as potential target for therapies based on oxidative stress.

Treatments based on production of reactive oxygen species for bladder cancer such as photodynamic therapy (PDT) have been marginalized due to low specificity and the existence of resistance mainly associated with the up-regulation of Heat Shock Proteins (HSPs). To overcome these barriers, the establishment of strategies combining PDTs with HSP inhibitors may be promising and the identification of HSPs involved with oxidative stress from bladder tumors in animal models represents a key step in this direction. Thus, the present study aims to identify cytosolic and mitochondrial HSPs up expressed in murine bladder tumors and in the urothelial carcinoma cell line MB49 by qRT-PCR screening, and to analyze the importance of the activity of the HSPs associated with oxidative stress protection in the survival of the MB49 using strategy of inhibition in vitro. Results showed that both tumor tissues and MB49 cells in culture had significant overexpression of the mitochondrial HSPA9 (mortalin) and HSP60 mRNAs, while the cytosolic HSP90 was overexpressed only in the tumor. The effect of mortalin in the MB49 cells survival under oxidative stress was evaluated in vitro in presence of the specific inhibitor MKT-077 and H2O2. The findings showed that MB49 viability was permanently reduced by the MKT-077 in a dose-dependent manner by inducing apoptosis or necrosis, mainly under oxidative stress conditions. Results suggest that mortalin is preferentially expressed in the MB49 cancer model and plays a key role in tumoral survival, especially under oxidative stress, making this HSP a potential target for an alternative treatment combining PDT with HSP inhibitors.

Evaluation of Loading Strategies to Improve Tumor Uptake of Gemcitabine in a Murine Orthotopic Bladder Cancer Model Using Ultrasound and Microbubbles

In this study we compared three different microbubble-based approaches to the delivery of a widely used chemotherapy drug, gemcitabine: (i) co-administration of gemcitabine and microbubbles (Gem+MB); (ii) conjugates of microbubbles and gemcitabine-loaded liposomes (GemlipoMB); and (iii) microbubbles with gemcitabine directly bound to their surfaces (GembioMB). Both in vitro and in vivo investigations were carried out, respectively, in the RT112 bladder cancer cell line and in a murine orthotopic muscle-invasive bladder cancer model. The in vitro (in vivo) ultrasound exposure conditions were a 1 (1.1) MHz centre frequency, 0.07 (1.0) MPa peak negative pressure, 3000 (20,000) cycles and 100 (0.5) Hz pulse repetition frequency. Ultrasound exposure produced no significant increase in drug uptake either in vitro or in vivo compared with the drug-only control for co-administered gemcitabine and microbubbles. In vivo, GemlipoMB prolonged the plasma circulation time of gemcitabine, but only GembioMB produced a statistically significant increase in cleaved caspase 3 expression in the tumor, indicative of gemcitabine-induced apoptosis.

Serial transplantation unmasks galectin-9 contribution to tumor immune escape in the MB49 murine model

Mechanisms of tumor immune escape are quite diverse and require specific approaches for their exploration in syngeneic tumor models. In several human malignancies, galectin-9 (gal-9) is suspected to contribute to the immune escape. However, in contrast with what has been done for the infiltrating cells, the contribution of gal-9 produced by malignant cells has never been demonstrated in an animal model. Therefore, we derived isogenic clones—either positive or negative for gal-9—from the MB49 murine bladder carcinoma cell line. A progressive and consistent reduction of tumor growth was observed when gal-9-KO cells were subjected to serial transplantations into syngeneic mice. In contrast, tumor growth was unaffected during parallel serial transplantations into nude mice, thus linking tumor inhibition to the enhancement of the immune response against gal-9-KO tumors. This stronger immune response was at least in part explained by changing patterns of response to interferon-γ. One consistent change was a more abundant production of CXCL10, a major inflammatory factor whose production is often induced by interferon-γ. Overall, these observations demonstrate for the first time that serial transplantation into syngeneic mice can be a valuable experimental approach for the exploration of novel mechanisms of tumor immune escape.

Bladder tumor metabolic alterations in response to IFNα gene therapy.

468 Background: Intravesical interferon-alpha (IFNα) gene therapy with Nadofaragene firadenovec has shown clinical efficacy in patients with non-muscle invasive bladder cancer (NMIBC) in a phase III clinical trial, highlighting the therapeutic potential of this approach in a disease with significant unmet clinical need. Optimizing the clinical efficacy of IFNα gene therapy requires an understanding of the underlying therapeutic mechanisms. Here, we investigate the impact of IFNα gene therapy on tumor metabolism using in vitro and orthotopic murine preclinical models and clinical trial data to elucidate mechanisms of tumor resistance and identify predictive biomarkers. Methods: In vitro murine bladder cancer cell lines treated with recombinant IFNα (rIFNα) and lentiviral IFNα (LV-IFNα) were analyzed by whole-transcriptome sequencing, glucose uptake, and lactate production. Preclinical murine bladder cancer models were treated with LV-IFNα (orthotopic tumor model) or Poly(I:C) (flank tumor model), a potent IFN inducer. Disease response was monitored by in vivo real-time luciferase imaging. Tumors were harvested and whole-transcriptome sequencing performed to assess effects of IFNα therapy on tumor metabolism and lipidomics. Lipidomic profiling was performed on patient urine samples from a phase II clinical trial of intravesical Nadofaragene firadenovec (7 clinical responders and 6 non-responders) to assess for clinically-relevant differences in lipid metabolism. Results: Following IFNα therapy in vitro and in murine orthotopic bladder cancer models, we identified downregulation of genes involved in fatty acid synthesis and upregulation of genes involved in glycolysis by whole-transcriptome sequencing. This was confirmed by higher glucose uptake and lactate production by IFNα-treated cells in vitro. These findings were recapitulated in whole-transcriptome sequencing data of human bladder tumors treated with intravesical Nadofaragene firadenovec. Lipidomics performed on murine MB49 tumors treated with poly(I:C) identified 79 upregulated lipids, including phosphotidyl choline, spingomyelin and phosphatidyl ethanolamine, and 12 downregulated lipids, notably the cardiolipin class. Lipidomics performed on patient urine samples collected pre- and post-treatment with intravesical Nadofaragene firadenovec detected >592 lipids with distinct expression profiles differentiating clinical responders and non-responders at both timepoints. Conclusions: We describe novel modulation of glucose and lipid metabolism by bladder tumor cells in response to IFNα gene therapy. These metabolic changes were reproducible across in vitro, in vivo and clinical trial studies and improve our mechanistic understanding of IFNα gene therapy, identify tumor escape pathways targetable with combination therapy regimens, and identify a new class of biomarkers for predicting clinical response of NMIBC to IFNα gene therapy.

Achieving highly efficient gene transfer to the bladder by increasing the molecular weight of polymer-based nanoparticles

Short dwell-time and poor penetration of the bladder permeability barrier (BPB) are the main obstacles to intravesical treatments for bladder diseases, and is evidenced by the lack of such therapeutic options on the market. Herein, we demonstrate that by finely tuning the molecular weight of our cationic polymer mucoadhesive nanoparticles, we enhanced our gene transfer, leading to improved adherence and penetrance through the BPB in a safe and efficient manner. Specifically, increasing the polymer molecular weight from 45 kDa to 83 kDa enhanced luciferase plasmid transfer to the healthy murine bladder, leading to 1.35 ng/g luciferase protein expression in the urothelium and lamina propria regions. The relatively higher molecular weight polymer (83 kDa) did not induce morphologic changes or inflammatory responses in the bladder. This approach of altering polymer molecular weight for prolonging gene transfer residence time and deeper penetration through the BPB could be the basis for the design of future gene therapies for bladder diseases.

Transcriptomic analysis reveals that the small protein MgtS contributes to the virulence of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the most common pathogen causing urinary tract infections (UTIs). The pathogenesis of UPEC relies on the formation of intracellular bacterial communities (IBCs) after invading bladder epithelial cells (BECs). In this study, the gene expression profiles of UPEC after invading BECs were comprehensively analyzed using RNA sequencing to reveal potential virulence-related genes. The small protein MgtS, which is transcriptionally upregulated in BECs, was further investigated. It was found that MgtS contributed positively to UPEC invasion of BECs and colonization in murine bladders. A two-component regulatory system, PhoPQ was confirmed as a direct activator of mgtS expression in BECs, and magnesium limitation is proposed as a host cue for the activation. This study provides the first comprehensive analysis of the transcriptome profile of UPEC during its intra-BECs life, revealing a new virulence-associated gene and its regulatory mechanism.

Mechanism of Genetically-Regulated Response to Bacillus- Calmette Guérin Immunotherapy for Bladder Cancer


 
 
 
 In this study, we investigated host response to Bacillus-Calmette Guérin (BCG) immunotherapy using the murine MM45T bladder tumor cell line. BALB/c (Bcgs allele) and BALB/c.CD2 (CD2) (Bcgr allele) mice were seeded subcutaneously with MM45T cells. Treatment began on day 7 with intra-tumor injections of BCG. Significant decreases in tumor growth in BALB/c (p < 0.05) but not in the CD2 (p = NS) mice were observed 6, 10 and 14 days following treatment. Investigation of the mechanism(s) underlining the difference in the response between the two strains of mice revealed key points, namely: (i) Intraperitoneal injection of BCG led to a significant increase (p < 0.05) in the number of cells recovered 14 days later by peritoneal lavage (in million cells: 5.1 ± 0.7 vs 9.5 ± 0.3 in BALB/c and 5.3 ± 0.4 vs 7.0 ± 0.4 in CD2, PBS vs BCG). (ii) Compositional analysis of the lavages following BCG injection indicated a significant increase in the lymphocyte/macrophage ratio in BALB/c mice as compared to CD2 mice (p < 0.05). (iii) Activated peritoneal macrophage production of nitric oxide (NO) was significantly lower (p < 0.05) in BALB/c than in CD2 (26.8 ± 1.6 vs 38.3 ± 0.7 uM of nitrite respectively) when challenged with lipopolysaccharide. (iv) No difference in the in vitro cytotoxicity against MM45T cells was observed between BALB/c and CD2 macrophages. Our results suggest that differences in the composition of monocytes infiltrating the site of BCG treatment and in the release of substances such as NO by macrophages may be key determinants of the response to BCG immunotherapy for bladder cancer.
 
 
 

Prostate-Specific Deletion of Cdh1 Induces Murine Prostatic Inflammation and Bladder Overactivity.

Benign prostatic hyperplasia (BPH) is an age-related debilitating prostatic disease that is frequently associated with prostatic inflammation and bothersome lower urinary tract symptoms (LUTS). Animal models have shown that formalin- and bacterial-induced prostatic inflammation can induce bladder dysfunction; however, the underlying mechanisms contributing to prostatic inflammation in BPH and bladder dysfunction are not clear. We previously reported that E-cadherin expression in BPH is downregulated in hyperplastic nodules compared with expression in adjacent normal tissues. Here, we explored the potential consequences of prostatic E-cadherin downregulation on the prostate and bladder in vivo using an inducible murine model of prostate luminal epithelial-specific deletion of Cdh1. The prostate-specific antigen (PSA)-CreERT2 transgenic mouse strain expressing tamoxifen-inducible CreERT2 recombinase driven by a 6-kb human PSA promoter/enhancer was crossed with the B6.129-Cdh1tm2Kem/J mouse to generate bigenic PSA-CreERT2/Cdh1-/- mice. Deletion of E-cadherin was induced by transient administration of tamoxifen when mice reached sexual maturity (7 weeks of age). At 21 to 23 weeks of age, the prostate, bladder, and prostatic urethra were examined histologically, and bladder function was assessed using void spot assays and cystometry. Mice with Cdh1 deletion had increased prostatic inflammation, prostatic epithelial hyperplasia, and stromal changes at 21 to 23 weeks of age, as well as changes in bladder voiding function compared with age-matched controls. Thus, loss of E-cadherin in the murine prostate could result in prostatic defects that are characteristic of BPH and LUTS, suggesting that E-cadherin downregulation could be a driving force in human BPH development and progression.

Combination of novel intravesical xenogeneic urothelial cell immunotherapy and chemotherapy enhances anti-tumor efficacy in preclinical murine bladder tumor models

BackgroundImmune checkpoint inhibitors induce robust and durable responses in advanced bladder cancer (BC), but only for a subset of patients. Xenovaccination has been proposed as an effective immunotherapeutic approach to induce anti-tumor immunity. Thus, we proposed a novel intravesical xenogeneic urothelial cell immunotherapy strategy to treat advanced BC based on the hypothesis that implanted xenogeneic urothelial cells not only provoke xeno-rejection immune responses but also elicit bystander anti-tumor immunity.MethodsMouse advanced bladder cancer models were treated with vehicle control, intravesical xenogeneic urothelial cells, cisplatin + gemcitabine, or the combination and assessed for tumor responses to treatments. Tumors and spleens samples were collected for immunohistological staining, cellular and molecular analysis assessed by antibody staining, ELISA, cytotoxicity, and flow cytometry, respectively.ResultsThe combination treatment of xenogeneic urothelial cell immunotherapy with chemotherapy was more efficacious than either single therapy to extend survival time in MBT-2 graft bladder tumor model and to suppress tumor progression in murine carcinogen BBN-induced bladder tumor model. The single-cell immunotherapy and combined therapy increased more tumor-infiltrating immune cells in MBT-2 graft tumors compared to vehicle control and chemotherapy treatment groups. The activated T-cell proliferation, cytokine production, and cytotoxicity capacities were also higher in mice with xenogeneic urothelial cell immunotherapy and combination treatments.ConclusionsOur results suggest the potential for a novel xenogeneic urothelial cell-based immunotherapy alone and synergy with chemotherapy in the combination therapy. Therefore, our study supports developing xenogeneic urothelial cells as an immunotherapeutic agent in combination with chemotherapy for BC treatment.

Optimizing Sequence of PD-L1 Immune-Checkpoint Inhibitors and Radiation Therapy in Bladder Cancer

BACKGROUND: New bladder preserving strategies are needed for muscle invasive bladder cancer (MIBC). Combined therapy of immune-checkpoint inhibitors and radiation was shown to have synergistic antitumoral effects in preclinical studies. OBJECTIVES: We aim to evaluate whether the sequence of administration of this combined therapy impacts antitumoral response. METHODS: We developed an in-vivo syngeneic MIBC mouse model where murine bladder cancer cells (MB49) were injected subcutaneously in the right flank of C57BL/6 mice. Mice were then randomized to the following treatments: control, anti-programmed cell death ligand 1 (PD-L1) alone, radiation alone (XRT) consisting of 6.25 Gy x2 fractions, concurrent anti-PD-L1 with XRT, neoadjuvant anti-PD-L1 followed by XRT, or XRT followed by adjuvant anti-PD-L1 therapy. Tumor growth, survival, and rate of response were analyzed. RESULTS: Total of 60 mice were randomized. One-way analysis of variance showed statistically significant difference in tumor growth rate across the treatment arms (p = 0.029). Importantly, timing of immunotherapy (neoadjuvant, concurrent, or adjuvant) did not alter either tumor growth or survival (p > 0.05). The rate of response was also similar in each combination arm (p > 0.05). CONCLUSION: Combining anti-PD-L1 immunotherapy and radiation therapy offers optimal antitumoral responses. Timing of immunotherapy (neoadjuvant, concurrent, or adjuvant) does not appear to affect outcomes. Whether the toxicity profile differs across various sequential deliveries of combination therapy requires further evaluation.

Relevance of iNOS expression in tumor growth and maintenance of cancer stem cells in a bladder cancer model.

The expression of inducible nitric oxide (NO) synthase (iNOS) in human bladder cancer (BC) is a poor prognostic factor associated with invasion and tumor recurrence. Here, we evaluated the relevance of iNOS expression in BC progression and in cancer stem cell (CSC) maintenance in a murine BC model. Also, iNOS expression and CSC markers were analyzed in human BC samples. iNOS inhibitors (L-NAME or 1400W) or shRNA were used on murine BC model with different iNOS expressions and invasiveness grades: MB49 (iNOS+, non-muscle invasive (NMI)) and MB49-I (iNOS++, muscle invasive (MI)), in order to analyzed cell proliferation, tumor growth, angiogenesis, number of CSC, and pluripotential marker expression. iNOS, SOX2, Oct4, and Nanog expressions were also analyzed in human BC samples by qPCR and immunohistochemistry. iNOS inhibtion reduced parameters associated with tumor progression and reduced the number of CSC, wich resulted higher in MB49-I than in MB49, in concordance with the higher expression of SOX2, Oct4, and Nanog. The expression of SOX2 was notoriously diminished, when iNOS was inhibited only in the MI cell line. Similar results were observed in human samples, where MI tumors expressed higher levels of iNOS and pluripotential genes, in comparison to NMI tumors with a positive correlation between those and iNOS, suggesting that iNOS expression is associated with CSC. iNOS plays an important role in BC progression and CSC maintenance. Its inhibition could be a potential therapeutic target to eradicate CSC, responsible fortumor recurrences. KEY MESSAGES: • iNOS expression is involved in bladder tumor development, growth, and angiogenesis. • iNOS expression is involved in bladder cancer stem cell generation and maintenance, playing an important role regulating their self-renewal capacity, especially in muscle invasive murine bladder cancer cells. • iNOS expression is higher in human muscle invasive tumors, in association with a high expression of pluripotential genes, especially of SOX2.

Combination Bacillus Calmette-Guérin and indoleamine 2,3-dioxygenase 1 inhibitor therapy of murine orthotopic bladder cancer.

Non-muscle invasive bladder cancer (NMIBC) can be managed initially with transurethral resection of a bladder tumor. In carcinoma in situ (CIS) or papillary high-grade NMIBC patients, further intravesical Bacillus Calmette-Guérin (BCG) is recommended for the prevention of recurrences. About 20% of patients suffer from BCG intolerance thereby limiting its use; up to 40% are BCG-refractory and face tumor recurrence despite initial BCG induction; finally there are BCG-resistant patients who respond to neither initial nor repeated BCG instillations [1].

Abstract 5553: The effect of PLAG on the PD-L1 immune-checkpoint drug therapy in the MB49 bladder cancer syngeneic model

Abstract Background: The development of immune-checkpoint drugs, a fourth-generation anticancer drug, is a significant breakthrough in anticancer research. Cancer therapy using immune-checkpoint drugs are still enlarged. To improve the efficacy of immune-checkpoint drugs, modulation of the tumor microenvironment is essentially required. In this experiment, we examined the elevated anti-cancer efficacy of 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol (PLAG), which has been demonstrated to attenuate tumor infiltrated neutrophils (TINs) in the tumor, along with the PD-L1 immune checkpoint inhibitor treatment. Methods: The syngeneic model was used (n=6) to investigate the enhanced anti-tumor effect of PD-L1 antibody with the addition of PLAG. MB49 murine bladder cancer cells were implanted into the C57BL/6 mice subcutaneously and bred for 5 weeks. After a week from tumor implantation, PLAG at different dosages (25/50/100 mpk) were daily administered orally for another 4 weeks with or without 5 mpk PD-L1 antibody (10F.9G2). PD-L1 antibody was delivered via IP injection twice a week. Tumor growth was calculated in 3-day intervals. Neutrophil chemotaxis-related chemokines and neutrophil-to-lymphocyte rate (NLR) were also evaluated with a 2-week interval. The degree of neutrophil infiltration in the tumor on the day of sacrifice and the degree of infiltration between lymphocytes and neutrophils were analyzed. Results: The PLAG treatment groups demonstrated that the tumor burden decreased in a concentration-dependent manner. In 50 and 100 mpk of PLAG treated mice, the tumor burden was decreased to a significant value compared to a positive control (p < 0.05). In the group treated with PD-L1 antibody alone, the growth rate of the tumor decreased until about 2 weeks. In the group treated simultaneously with PLAG and PD-L1 antibody, the growth of the tumor was significantly reduced compared to the group treated with PD-L1 antibody alone. As a result of calculating neutrophils and lymphocytes every two weeks, NLR level in the group treated with PLAG and PD-L1 was significantly decreased compared to that treated with PD-L1 antibody alone. Besides, the number of TINs were effectively reduced by PLAG treatment alone. The interruption of tumor growth and reduced chemokine secretion were also observed in the PLAG treated groups. In summary, our data suggest that PLAG provides an enhanced PD-L1 antibody effect on the regression of tumor burden in the syngeneic mice model via reducing the number of TINs. Conclusion: PLAG may be utilized for improving the efficacy of PD-L1 antibody on reducing the tumor burden at the devastating tumor microenvironment. Citation Format: Guen Tae Kim, Sun Young Yoon, Ji Sun Park, Ki-Young Sohn, Jae Wha Kim. The effect of PLAG on the PD-L1 immune-checkpoint drug therapy in the MB49 bladder cancer syngeneic model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5553.

Abstract 5150: MIF-2 in bladder cancer: potential therapeutic target

Abstract Macrophage migration inhibitory factor (MIF1) is a pro-tumorigenic cytokine overexpressed in multiple cancers. Our previous data in a carcinogen (BBN) induced murine bladder cancer (BCa) model indicates genetic depletion of MIF1 induces stage arrest. Pharmacological inhibition of MIF1 with the inhibitor CPSI-1306 reduces tumor stage and bladder weights. These data indicate MIF1 is a therapeutic target in BCa. However, a second protein with significant homology to MIF1 was recently identified and termed MIF2. Both MIF1 and MIF2 bind and activate their receptor CD74 yielding significant functional homology and thus, targeting both proteins may be more efficacious. We hypothesize MIF2 recapitulates the pro-tumorigenic effects of MIF1 in BCa in a CD74 dependent manner. We assessed MIF1 and MIF2 by ELISA in serum from human patients with BCa and MIF1/2 expression were evaluated in TCGA. WT mice (C57Bl/6 background), MIF-/-, and CD74-/- mice were treated with 0.05% BBN for 16 weeks to induce BCa, and then switched to water. Tumors were allowed to develop until 20 weeks post initiation of BBN, a time frame consistent with muscle invasive tumor development. Some animals were given either vehicle, or a dual MIF1/MIF2 inhibitor 4-IPP (80mg/kg i.p. daily) for 4 weeks after cessation of BBN. We assessed bladder weights as a surrogate for tumor weight, tumor stage and immunohistochemistry for proliferation, angiogenesis and inflammation. In vitro cell proliferation was measured using the hexosaminidase assay and tautomerase activity was measured using two separate enzymatic assays for MIF tautomerase activity. MIF1 and MIF2 mRNA levels were elevated in TCGA. Analysis of serum from BCa patients indicated increased MIF1 and MIF2 in BCa patients, although this didn't reach statistical significance. 4-IPP treated C57Bl/6 animals had reductions in bladder weight, although tumor staging was similar between groups. 4-IPP treatment had no effect on bladder weights or tumor stage in CD74-/- mice supporting a role for CD74 mediated MIF signaling. To more fully assess the role of MIF2 directly, MIF1-/- mice were treated with 4-IPP to solely block MIF2 4-IPP treatment lead to major reductions in tumor stage, tumor incidence and bladder weight indicating inhibition of MIF2 may be more effective when MIF1 is chronically absent. Inflammation and proliferation trended lower, but did not reach statistical significance. MIF1 depletion did not affect MIF2 mRNA levels in bladder, heart or liver. In vitro treatment with 4-IPP substantially reduced proliferation of two high grade BCa cell lines (HTB-5, HTB-9) at concentrations consistent with doses found to inhibit recombinant MIF activity. These data indicate MIF1/2 enhance BCa tumorigenesis through activation of CD74. Combined MIF1/MIF2 may improve therapeutic targeting of MIF, especially during chronic MIF1 inhibition. Citation Format: Ganeshkumar Rajendran, Benjamin L. Woolbright, Erika Abbott, Austin Martin, Katie Dennis, John Arthur Taylor. MIF-2 in bladder cancer: potential therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5150.

Abstract A01: Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for the treatment of bladder cancer

Abstract Bladder cancer remains a leading cause of death and one of the most expensive cancers to treat in the United States. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) is a personalized therapeutic approach that has the potential to treat bladder cancer at every stage of disease. We have previously demonstrated the ability to expand tumor reactive T cells from transurethral resection, radical cystectomy, and metastatic lymph node tumor specimens collected from bladder cancer patients. In this study, our goal is to investigate different delivery approaches of ACT in vivo to determine the best strategy to decrease tumor burden and advise clinical trial design. Experiments were performed using MB49 murine bladder cancer cells transfected to express ovalbumin (MB49OVA). MB49OVA cells are recognized by OVA-specific OT-I T cells as determined by IFN-gamma production in vitro. Our methods include both subcutaneous and orthotopic bladder tumor models. In vivo, we first tested systemic delivery of OT-I T cells (5.0 × 10e6) by tail vein injection after lymphodepletion with a regimen of cyclophosphamide followed by fludarabine in mice bearing subcutaneous MB49OVA tumors. ACT-treated mice experienced significant tumor regression (p=0.02), improved survival (median survival PBS=47 days, ACT=undefined, p=0.01), and increased tumor infiltration of CD3+, CD4+, and CD8+ T cells. OT-I T cells as well as endogenous OVA-specific CD8+ T cells were detectable in the spleens and tumors of treated mice. Next we tested the efficacy of intravesical delivery of OT-I T cells in mice bearing orthotopic MB49OVA tumors, without prior lymphodepletion. OT-I T cells (4.0 × 10e6) were delivered directly to the bladder via catherization and tumor growth was monitored weekly via ultrasound imaging. Infiltration of CellTrace Violet-labeled OT-I T cells into orthotopic tumors was detected after 3 hours of incubation by flow cytometry analysis of resected tumor. Orthotopic bladder tumor growth was significantly abrogated in ACT-treated mice compared with PBS control-treated mice (p=0.001) and improved survival was observed (median survival PBS=35 days, ACT=undefined). An increase in CD8+ T-cell infiltration was measured in tumors of mice at day 60 post treatment. In order to evaluate the efficacy of TIL transfer in these bladder tumor models, T cells infiltrating subcutaneous MB49OVA tumors were isolated and cultured in vitro in media containing IL-2. After 1 week, bladder TIL demonstrated reactivity against MB49OVA and MB49 parental cells as determined by IFN-gamma production. Future work will implement the adoptive transfer of MB49 TIL for the treatment of orthotopic bladder tumors alone or in combination with anti-PD-L1 therapy. Taken together, these data suggest that ACT using bladder tumor-specific T cells has the potential to reduce bladder tumor burden when given either systemically or intravesically directly into the bladder. Citation Format: Brittany L. Bunch, Jennifer Morse, Sarah Asby, Michael Poch, Shari Pilon-Thomas. Adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) for the treatment of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A01.

Abstract A12: Bladder cancer-derived extracellular vesicles and immune activation

Abstract Background: Bladder tumors occur on the luminal surface of the bladder, and cancer cells and cellular components are shed in urine, which permits the isolation of an enriched population of bladder cancer-specific extracellular vesicles (EVs). EVs contain proteins, miRNA, mRNA, and cellular metabolites, which may serve as stimulators of immune cells. The objective of this study was to evaluate the immune potential of tumor-derived EVs (TEVs) produced on exposure to BCG using a murine bladder cancer cell line and immune cells. Methods: TEVs were isolated by differential high-speed centrifugation of cell culture supernatants. They were confirmed to be extracellular vesicles by electron microscopy and size measurement using nanoparticle tracking analysis. Unstimulated murine bladder cancer cells (MB49) were used to prepare TEV1. The cells were then exposed to BCG Tokyo (MOI 4) and TEVs were isolated after 2 hours (TEV2). BCG was then washed off and the cells were allowed to grow in complete media for 2 days before isolation of the third sample, TEV3. EVs from BCG (BEV) were collected to serve as a control. Bone marrow-derived cells were obtained from C57BL/6J mice to generate dendritic cells (DC) and neutrophils. These cells were untreated or treated with 1ug of TEVs or BEV or BCG (MOI 5) for 24 hours. The supernatants were analyzed for cytokine production and the DCs and neutrophils were analyzed for activation markers by flow cytometry. The institutional animal care and use committee approved the use of mice. Results: DCs treated with TEV2 had significantly higher expression of CD86 (at similar levels when DCs were treated with BCG) and MHC2, both of which enhanced the DC's capacity to present antigens to T cells, when compared to TEV1 or TEV3. While neutrophils induced by BCG expressed significantly higher levels of costimulatory molecules CD80, CD83, CD86, and MHC2, TEV2 induced significant expression of CD80 and CD86. BCG caused a massive release of proinflammatory cytokines (IL6, TNFa, and IL12p70) and the anti-inflammatory cytokine IL10. Cytokine production induced by TEV2 was not as high as that induced by direct BCG stimulation, but it was significantly higher than that produced by untreated cells or TEV1 and TEV3. Unlike BCG, TEV2 did not significantly stimulate IL10 production. Conclusion: Our results demonstrate that tumor cells treated with BCG for 2 hours produce EVs that are able to cause significant modulation of DCs and neutrophils. TEVs generated during BCG immunotherapy have immunostimulatory potential. Therefore, TEVs represent a promising source of material to increase immune activation and efficacy of BCG therapy. This could reduce the number of instillations and adverse effects associated with BCG therapy. Citation Format: Sin Mun Tham, Kesavan Esuvaranathan, Ratha Mahendran. Bladder cancer-derived extracellular vesicles and immune activation [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A12.

Abstract A17: Gene expression changes associated with metastasis in mice with bladder cancer

Abstract Background: Prognosis for bladder cancer is dependent on both tumor stage and grade. In patients with non-muscle invasive bladder cancer, the tumor invariably recurs and may progress. Patients who subsequently develop metastatic disease or present with it have a worse prognosis. The purpose of this study was to detect and find differences in patterns of gene expression between the primary tumor and common metastatic sites in a mouse model of bladder cancer to identify possible triggering events that result in metastasis. Methods: Five- to six-week-old female C57BL/6 mice were orthotopically implanted with MB49-PSA (murine bladder cancer cells secreting human prostate-specific antigen as a tumor marker). The institutional animal care and use committee approved the use of mice. At termination (up to 78 days after tumor implantation), bladders, kidneys, lymph nodes, and lungs were snap frozen and RNA was extracted by homogenizing the tissue in Trizol. Tumor presence in the tissues was confirmed by PSA gene expression, which was quantified by real-time PCR. Gene expression level profiling of the transcriptome was analyzed via RNA sequencing of samples from 4 bladders, 4 kidneys, 3 lymph nodes, and 2 lungs. Gene expression of a select group of genes was verified by real-time PCR with a bigger population of RNA samples. Results: Common differentially expressed genes were found between bladder and kidney (42 genes), bladder and lung (54 genes), and bladder and lymph nodes (198 genes). Bioinformatics software Panther and String were used to assign the genes into 6 pathways. These were morphogenesis of a branching epithelium; positive regulation of transcription from RNA polymerase II promoter; cell differentiation; negative regulation of epithelial to mesenchymal transition; epithelial cell differentiation; and transcriptional mis-regulation in cancer. Expression of paired-box 8 (Pax8), a transcription factor involved in embryonic development, was significantly higher in the kidneys compared to the other tissues. This suggests that overexpression of Pax8 may facilitate malignant development but may not be required for later stages of cell differentiation. Nanog, also a transcription regulator, was significantly higher in the kidneys when compared to bladders and was elevated in the lungs as well. It promotes epithelial-mesenchymal transition, resulting in metastasis; thus, increased expression of Nanog in cancer cells may indicate tumor aggressiveness. In the literature, Pax8 is expressed in noninvasive bladder tumors but not in normal bladder tissue in humans and Nanog expression is high in bladder tumors. Conclusion: Pax8 and Nanog expression indicates that the murine model of bladder cancer with metastatic disease is similar to that observed in patients. Thus, this model could be used to evaluate methods of blocking progression. Citation Format: Sin Mun Tham, Stefanie Alicia Fung, Kesavan Esuvaranathan, Ratha Mahendran. Gene expression changes associated with metastasis in mice with bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A17.

MO064TISSUE-RESIDENT B CELLS DETERMINE SUSCEPTIBILITY TO URINARY TRACT INFECTION BY ORCHESTRATING MACROPHAGE POLARISATION

Abstract Background Urinary tract infection (UTI) is an important clinical problem. More than half of women and 1 in 10 men will be affected during their lifetime. Many of these affect the lower urinary tract but recurrent pyelonephritis can lead to scarring and chronic kidney disease. There is an increasing appreciation that tissue-resident immune cells, such as macrophages, play an important role in defence against infection, but only little is known about B lymphocytes in this context. Here we sought to address the question of whether B cells reside in the kidney and bladder in homeostasis and to determine their phenotype and contribution to local organ immunity. Methods and Results Using intravenous labelling and parabiosis, we identified a population of bona-fide self-renewing, tissue-resident B cells that included non-naïve and innate-like CD5+ B-1 cells, in murine kidneys and urinary bladder (but also in liver and lung). The size and phenotype of this B cell subset was influenced by genetic background, age, and microbiome, with an expanded population evident after co-housing with pet-store mice. Although kidney B cells had less diverse Igh repertoire compared to blood, their seeding was largely independent of their B-cell receptor specificity. In human kidneys we found a similar enrichment for non-naïve B cells compared to blood and spleen. Using two strains of genetically modified mice with higher (PI3KδE1020K-B) or lower (μMT-) numbers of tissue-resident B cells, we tested the function of these cells during UTI. Surprisingly, the number of tissue-resident B cells inversely correlated with bacterial clearance. We found that these B cells were spatially co-localised with kidney macrophages and skewed their polarization towards an anti-inflammatory M2 phenotype, leading to reduced anti-microbial responses. This effect was, at least in part, driven via IL-10. Conclusion In conclusion, our data identify a critical role for tissue-resident B cells in modulating local immunity in the urinary tract, determining the inflammatory ‘set-point’ of resident and recruited myeloid cells, with important clinical implications for the use of B-cell depleting therapies and conditions such as infection, transplant rejection, fibrosis or autoimmunity. Graphical Abstract