Orthotopic Bladder Cancer Model Research Articles

Abstract A021: The role of MGAT5 in sex-dependent regulation of CD8+ T cells in muscle-invasive bladder cancer

Abstract Biological sex significantly affects bladder cancer incidence and outcome. Although men have much greater incidence of bladder cancer than women, females tend to be diagnosed with more advanced tumors and have worse outcomes. The mechanisms driving these differences are likely multifactorial, with biological factors playing an important role, however, much of the biology underlying these differences is unknown. The immune system is a critical regulator of cancer biology, and immune features have been associated with bladder cancer grade, stage, progression, and response to therapies. Sex dramatically affects the immune system including anti-tumor immunity. However, how sex and immunity intersect to affect clinical outcomes in bladder cancer patients remains unclear. CD8+ T cells are a key part of anti-tumor immune responses and the importance of inhibitory receptors such as programmed cell death-1 (PD-1), is now well established as a mechanism restricting T cell responses in MIBC. However, there are a variety of other regulatory pathways that restrict their anti-tumor function, which have yet to be investigated. One such pathway involves alterations in protein glycosylation, which can affect T cell function at many levels. The activity of the glycosyltransferase N-acetylglucosaminyltransferase V (MGAT5), responsible for complex N-glycan branching, has been shown to regulate T cell function in autoimmunity and chronic viral infections, however, its role in regulating anti-tumor CD8+ T cell responses has yet to be explored. We assessed the activity of MGAT5 in circulating and tumor-infiltrating CD8+ T cells in mice using a panel of cell lines in a syngeneic orthotopic model of muscle-invasive bladder cancer (MIBC). We found that in all tumor models, MGAT5 activity was upregulated in tumor infiltrating CD8+ T cells compared with circulating cells. Furthermore, using high parameter flow cytometry, we assessed the activity of MGAT5 in CD8+ T cells from mice with bladder cancer induced by the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), and in patients with MIBC. In both mice and humans, we found that sex affects MGAT5 activity in memory CD8+ T cells, with females exhibiting decreased MGAT5 activity in these cells compared to males. The functional significance of altering MGAT5 activity in T cells was also assessed in vivo. To do this, chimeric mice were generated by mixed bone-marrow transplant resulting in mice with loss of Mgat5 in T cells. Bladder cancer was then induced using BBN, and morbidity (defined as either 10% weight loss in one week, 10% weight loss post-BBN, or humane endpoint), as a proxy for mortality, was assessed over time. In this model, male mice were protected from BBN-induced morbidity compared to female mice, and this protection was abrogated by homozygous or heterozygous loss of Mgat5 in T cells. Overall, our data suggest that sex-dependent regulation of MGAT5 activity in CD8+ T cells may be an important underlying factor affecting outcomes in bladder cancer. Citation Format: Morgan E. Roberts, Igor Moskalev, Peter C. Black. The role of MGAT5 in sex-dependent regulation of CD8+ T cells in muscle-invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A021.

Targeted Delivery of Catalase and Photosensitizer Ce6 by a Tumor-Specific Aptamer Is Effective against Bladder Cancer In Vivo.

Photodynamic therapy (PDT) is often applied in a clinical setting to treat bladder cancer. However, current photosensitizers report drawbacks such as low efficacy, low selectivity, and numerous side effects, which have limited the clinical values of PDT for bladder cancer. Previously, we developed the first bladder cancer-specific aptamer that can selectively bind to and be internalized by bladder tumor cells versus normal uroepithelium cells. Here, we use an aptamer-based drug delivery system to deliver photosensitizer chlorine e6 (Ce6) into bladder tumor cells. In addition to Ce6, we also incorporate catalase into the drug complex to increase local oxygen levels in the tumor tissue. Compared with free Ce6, an aptamer-guided DNA nanotrain (NT) loaded with Ce6 and catalase (NT-Catalase-Ce6) can specifically recognize bladder cancer cells, produce oxygen locally, induce ROS in tumor cells, and cause mitochondrial apoptosis. In an orthotopic mouse model of bladder cancer, the intravesical instillation of NT-Catalase-Ce6 exhibits faster drug internalization and a longer drug retention time in tumor tissue compared with that in normal urothelium. Moreover, our modified PDT significantly inhibits tumor growth with fewer side effects such as cystitis than free Ce6. This aptamer-based photosensitizer delivery system can therefore improve the selectivity and efficacy and reduce the side effects of PDT treatment in mouse models of bladder cancer, bearing a great translational value for bladder cancer intravesical therapy.

Urease-powered nanobots for radionuclide bladder cancer therapy

Bladder cancer treatment via intravesical drug administration achieves reasonable survival rates but suffers from low therapeutic efficacy. To address the latter, self-propelled nanoparticles or nanobots have been proposed, taking advantage of their enhanced diffusion and mixing capabilities in urine when compared with conventional drugs or passive nanoparticles. However, the translational capabilities of nanobots in treating bladder cancer are underexplored. Here, we tested radiolabelled mesoporous silica-based urease-powered nanobots in an orthotopic mouse model of bladder cancer. In vivo and ex vivo results demonstrated enhanced nanobot accumulation at the tumour site, with an eightfold increase revealed by positron emission tomography in vivo. Label-free optical contrast based on polarization-dependent scattered light-sheet microscopy of cleared bladders confirmed tumour penetration by nanobots ex vivo. Treating tumour-bearing mice with intravesically administered radio-iodinated nanobots for radionuclide therapy resulted in a tumour size reduction of about 90%, positioning nanobots as efficient delivery nanosystems for bladder cancer therapy.

Stool Microbiome Signature Associated with Response to Neoadjuvant Pembrolizumab in Patients with Muscle-invasive Bladder Cancer

Neoadjuvant pembrolizumab has been shown to be a valid treatment for patients affected by muscle-invasive bladder cancer (MIBC), as demonstrated in the PURE-01 clinical trial (NCT02736266). Among the tumor-extrinsic factors influencing immunotherapy efficacy, extensive data highlighted that the microbiome is a central player in immune-mediated anticancer activity. This report aimed to investigate the composition and role of stool microbiome in patients enrolled in the PURE-01 clinical trial. An orthotopic animal model of bladder cancer (MB49-Luc) was used to support some of the findings from human data. An analysis of stool microbiome before pembrolizumab was conducted for 42 patients, of whom 23 showed a pathologic response. The information in the preclinical model of orthotopic bladder cancer treated with anti–PD-1 antibody or control isotype was validated. Linear discriminant analysis effect size and linear models were used to identify the bacterial taxa enriched in either responders or nonresponders. The identified taxa were also tested for their association with event-free survival (EFS). Survival at 31 d after tumor instillation was used as the study endpoint in the preclinical model. Responders and nonresponders emerged to differ in terms of enrichment for 16 bacterial taxa. Of these, the genus Sutterella was enriched in responders, while the species Ruminococcus bromii was enriched in nonresponders. The negative impact of R. bromii on anti–PD-1 antibody activity was also observed in the preclinical model. EFS and survival of the preclinical model showed a negative role of R. bromii. We found different stool bacterial taxa associated with the response or lack of response to neoadjuvant pembrolizumab. Moreover, we provided experimental data about the negative role of R. bromii on immunotherapy response. Further studies are needed to externally validate our findings and provide mechanistic insights about the host-pathogen interactions in MIBC. Patient summaryUsing prepembrolizumab stool samples collected from patients enrolled in the PURE-01 clinical trials, we identified some bacterial taxa that were enriched in patients who either responded or did not respond to immunotherapy. Using an animal model of bladder cancer, we gathered further evidence of the negative impact of the Ruminococcus bromii on immunotherapy efficacy. Further studies are needed to confirm the current findings and test the utility of these bacteria as predictive markers of immunotherapy response.

Enhanced urease-driven nanobots for radionuclide-based bladder cancer therapy

Bladder cancer treatment with intravenous medication delivery has reasonable survival rates but modest therapeutic efficacy. Taking advantage of their superior diffusion and mixing capacities in urine compared to traditional pharmaceuticals or passive nanoparticles, self-propelled nanoparticles, also known as nanobots, have been proposed as a solution to the latter issue. Nonetheless, nothing is known about how well nanobots can translate into bladder cancer treatments. Here, urease-powered, radiolabelled mesoporous silica-based nanobots were evaluated in an orthotopic animal model of bladder cancer. Both in vivo and ex vivo findings showed increased accumulation of nanobots at the tumor location; positron emission tomography in vivo showed an eight-fold increase. The ex vivo tumor penetration by nanobots was validated by label-free optical contrast based on polarization-dependent scattered light-sheet microscopy of cleared bladders. Intravesically treatment was given to tumor-bearing mice. About 90% less tumor size was observed when radio-iodinated nanobots were used for radionuclide therapy, establishing nanobots as effective delivery nanosystems for bladder cancer treatment.

A HER2-targeted Antibody-Drug Conjugate, RC48-ADC, Exerted Promising Antitumor Efficacy and Safety with Intravesical Instillation in Preclinical Models of Bladder Cancer.

More than half of non-muscle-invasive bladder cancer (NMIBC) patients eventually relapse even if treated with surgery and BCG without optional bladder-preserving therapy. This study aims to investigate the antitumor activity and safety of a HER2-targeted antibody-drug conjugate, RC48-ADC, intravesical instillation for NMIBC treatment. In this preclinical study, itis revealed that human epidermal growth factor receptor 2 (HER2) expression scores of 1+, 2+, and 3+ are recorded for 16.7%, 56.2%, and 14.6% of NMIBC cases. The antitumor effect of RC48-ADC is positively correlated with HER2 expression in bladder cancer (BCa) cell lines and organoid models. Furthermore, RC48-ADC is revealed to exert its antitumor effect by inducing G2/M arrest and caspase-dependent apoptosis. In an orthotopic BCa model, tumor growth is significantly inhibited by intravesical instillation of RC48-ADC versus disitamab, monomethyl auristatin E, epirubicin, or phosphate-buffered saline control. The potential toxicity of intravesical RC48-ADC is also assessed by dose escalation in normal nude mice and revealed that administration of RC48-ADC by intravesical instillation is safe within the range of effective therapeutic doses. Taken together, RC48-ADC demonstrates promising antitumor effects and safety with intravesical administration in multiple preclinical models. These findings provide a rational for clinical trials of intravesical RC48-ADC in NMIBC patients.

Targeting myeloid-derived suppressor cells with gemcitabine to enhance efficacy of adoptive cell therapy in bladder cancer.

New therapeutics in development for bladder cancer need to address the recalcitrant nature of the disease. Intravesical adoptive cell therapy (ACT) with tumor infiltrating lymphocytes (TIL) can potentially induce durable responses in bladder cancer while maximizing T cells at the tumor site. T cells infused into the bladder directly encounter immunosuppressive populations, such as myeloid derived suppressor cells (MDSCs), that can attenuate T cell responses. Intravesical instillation of gemcitabine can be used as a lymphodepleting agent to precondition the bladder microenvironment for infused T cell products. Urine samples from bladder cancer patients and healthy donors were analyzed by flow cytometry and cytometric bead array for immune profiling and cytokine quantification. MDSCs were isolated from the urine and cocultured with stimulated T cells to assess effects on proliferation. An orthotopic murine model of bladder cancer was established using the MB49-OVA cell line and immune profiling was performed. MDSCs from tumor-bearing mice were cocultured with OT-I splenocytes to assess T cell proliferation. Mice received intravesical instillation of gemcitabine and depletion of immune cells was measured via flow cytometry. Bladder tumor growth of mice treated with intravesical gemcitabine, OT-I transgenic T cells, or combination was monitored via ultrasound measurement. In comparison to healthy donors, urine specimen from bladder cancer patients show high levels of MDSCs and cytokines associated with myeloid chemotaxis, T cell chemotaxis, and inflammation. T cells isolated from healthy donors were less proliferative when cocultured with MDSCs from the urine. Orthotopic murine bladder tumors also presented with high levels of MDSCs along with enrichment of cytokines found in the patient urine samples. MDSCs isolated from spleens of tumor-bearing mice exerted suppressive effects on the proliferation of OT-I T cells. Intravesical instillation of gemcitabine reduced overall immune cells, MDSCs, and T cells in orthotopic bladder tumors. Combination treatment with gemcitabine and OT-I T cells resulted in sustained anti-tumor responses in comparison to monotherapy treatments. MDSCs are enriched within the microenvironment of bladder tumors and are suppressive to T cells. Gemcitabine can be used to lymphodeplete bladder tumors and precondition the microenvironment for intravesical ACT.

Hyaluronic Acid-Conjugated Fluorescent Probe-Shielded Polydopamine Nanomedicines for Targeted Imaging and Chemotherapy of Bladder Cancer.

Bladder cancer is one of the most common malignancies in the urinary system, with high risk of recurrence and progression. However, the difficulty in detecting small tumor lesions and the lack of selectivity of intravesical treatment seriously affect the prognosis of patients with bladder cancer. In the present work, a nanoparticle-based delivery system with tumor targeting, high biocompatibility, simple preparation, and the ability to synergize imaging and therapy was fabricated. Specifically, this nanosystem consisted of the core of doxorubicin (DOX)-loaded polydopamine nanoparticles (PDD NPs) and the shell of hyaluronic acid (HA)-conjugated IR780 (HA-IR780). The HA-IR780-covered PDD NPs (HR-PDD NPs) demonstrated tumor targeting and visualization both in vitro and in vivo with properties of promoted cancer cell endocytosis and lysosomal escape, efficiently delivering drugs to the target site and exerting a killing effect on tumor cells. Encouragingly, intravesical instillation of HR-PDD NPs improved drug retention in the bladder and promoted its accumulation in tumor tissue, resulting in better tumor proliferation inhibition and apoptosis in an orthotopic bladder cancer model in rats. This study provides a promising strategy for the diagnosis and therapy of bladder cancer.

ZEB1-mediated biogenesis of circNIPBL sustains the metastasis of bladder cancer via Wnt/β-catenin pathway

BackgroundCircular RNAs (circRNAs) circularized by back-splicing of pre-mRNA are widely expressed and affected the proliferation, invasion and metastasis of bladder cancer (BCa). However, the mechanism underlying circRNA biogenesis in mediating the distant metastasis of BCa still unexplored.MethodsRNA sequencing data between BCa and normal adjacent tissues was applied to identify the differentially expressed circRNAs. The functions of circNIPBL in BCa were investigated via a series of biochemical experiments. The Clinical significance of circNIPBL was examined in a cohort of larger BCa tissues.ResultsIn the present study, we identified a novel circRNA (hsa_circ_0001472), circNIPBL, which was significantly upregulated and had great influence on the poor prognosis of patients with BCa. Functionally, circNIPBL promotes BCa metastasis in vitro and in vivo. Mechanistically, circNIPBL upregulate the expression of Wnt5a and activated the Wnt/β-catenin signaling pathway via directly sponged miR-16-2-3p, leading to the upregulation of ZEB1, which triggers the EMT of BCa. Moreover, we revealed that ZEB1 interacted with the flanking introns of exons 2–9 on NIPBL pre-mRNA to trigger circNIPBL biogenesis, thus forming a positive feedback loop. Importantly, circNIPBL overexpression significantly facilitated the distant metastasis of BCa in the orthotopic bladder cancer model, while silencing ZEB1 remarkably blocked the effects of metastasis induced by circNIPBL overexpression.ConclusionsOur study highlights that circNIPBL-induced Wnt signaling pathway activation triggers ZEB1-mediated circNIPBL biogenesis, which forms a positive feedback loop via the circNIPBL/miR-16-2-3p/Wnt5a/ZEB1 axis, supporting circNIPBL as a novel therapeutic target and potential biomarker for BCa patients.Graphical

Abstract 2634: AMB302/GR1017, an antibody-drug conjugate (ADC) with topoisomerase 1 inhibitor shows therapeutic potency in orthotopic glioblastoma PDX and bladder cancer models with FGFR3-TACC3 fusion

Abstract Background: FGFR3-TACC3 (F3-T3) fusion leads to FGFR3 kinase activation constitutively and acts as a driver mutation in several solid tumors. AMB302/GR1017 is a novel FGFR3 targeting ADC that was developed using the intelligent Ligase-Dependent Conjugation (iLDC) technologies from GeneQuantum (GQ) that provide high homogeneity, excellent druggability and high linker stability, and contains a topoisomerase 1 inhibitor as a payload. Based on the preclinical characterization, AMB302/GR1017 shows impressive anti-tumor activities against glioblastoma (GBM) and bladder cancer (BC) models with either FGFR3 amplification or F3-T3 fusion, and demonstrates the potential as a first-in-class FGFR3 ADC against FGFR3 active solid tumor indications. Methods: in vitro anti-tumor effects and mechanism of action for AMB302/GR1017 were assessed on patients-derived cells with F3-T3 using a 3D-spheroid high throughput assay. in vivo anti-tumor effect of AMB302/GR1017 were assessed on F3-T3 fusion GBM orthotopic PDX model and several FGFR3+ or F3-T3 fusion BC models Results: AMB302/GR1017 was generated by linking FGFR3 targeting antibody (AimedBio) with TopoIx (GQ), a next generation Topoisomerase 1 inhibitor via a cleavable linker using the iLDC technologies. In a 3D-spheroid high throughput assay system, AMB302/GR1017 showed significant anti-tumor activity against glioblastoma patients-derived cells (PDCs) in F3-T3 dependent manner, which is superior to chemical conjugate applying the same antibody and Dxd payload. Also, AMB302/GR1017 prolonged the survival of GBM orthotopic PDX models with F3-T3 fusion by 200 % and achieved complete tumor regression in RT112 BC model with F3-T3 fusion. AMB302/GR1017 treatments were well-tolerated up to 200 mg/kg in a mouse safety study. Conclusion: AMB302/GR1017 showed robust anti-tumor efficacies in F3-T3 fusion and FGFR3 overexpression models derived from GBM and BC in vitro and in vivo. In addition, AMB302/GR1017 was well tolerated with no adverse effects in rodent model. Our data suggest AMB302/GR1017 has a potential therapeutic option as a first-in-class FGFR3 targeting ADC for GBM, BC, and other solid tumors with FGFR3 overexpression or alterations. Citation Format: Byeongkwi Min, Lily Shi, Hye Jin Kim, Sangwoo Kim, Beibei Fan, Cao Lv, Yajun Sun, Nam-Gu Her, Paul Song, Gang Qin, Do-Hyun Nam. AMB302/GR1017, an antibody-drug conjugate (ADC) with topoisomerase 1 inhibitor shows therapeutic potency in orthotopic glioblastoma PDX and bladder cancer models with FGFR3-TACC3 fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2634.

Abstract 1068: A precision CRISPR approach to target structural variant junctions in cancer

Abstract Background: Structural variants are common in cancer that drive tumor formation and typically occur early in carcinogenesis. Despite being a common and well-known feature of cancer, this cancer hallmark has not yet been broadly explored therapeutically in the clinic. We present a novel precision-cancer approach, KLIPP, which consists of guide RNAs that target cancer-specific structural variant junctions (SVJs) to nucleate two parts of dCas9-conjugated endonuclease, Fok1, leading to its activation and induction of toxic DSBs. Furthermore, this approach reduced tumor growth in an orthotopic bladder cancer model in mice. Methods and Materials: HCT116 colon cancer cells and UMUC3 bladder cancer cells were engineered to express combinations of SVJ-targeting sgRNAs and express Fok1-dCas9 under doxycycline (dox) regulation. The HCT116 and UMUC3 cells were treated with dox for 24 hours and assessed for DSBs at SVJs by γH2AX foci assay and γH2AX ChIP-PCR. The cells were assessed for cell death using flow cytometry measuring sub-G1 DNA content, the clonogenic survival assay (12 days), and PARP1 and caspase cleavage (after 48 h) using Western blot. Tumor growth of orthotopic xenografts of UMUC3 cells expressing Fok1-dCas9 alone or with 2 pairs of SVJ-targeting sgRNAs were assessed weekly after addition of dox to the drinking water. Bioluminescence assessment of tumor growth was monitored weekly. Results: Expression of SVJ-targeting sgRNA and the induction of Fok1-dCas9 by dox for 24 h in HCT116 colon cancer and UMUC3 bladder cancer cells, resulted in one or two γH2AX foci per cell. Using ChIP-PCR, the identity of the site of the DSB was validated to occur at the targeted SVJ. Furthermore, dox treatment for 12 days caused >50% reduced clonogenic survival that was augmented by the DNA-PK inhibitor NU7441. Further, we observed >50% induction of apoptosis when assessed by subG1 DNA content cells and PARP1 cleavage. When UMUC3 cancer cells were grown orthotopically in mice, activation of Fok1-dCas9 and together with two pairs of SVJ-targeting sgRNAs led to significantly reduced tumor growth with 7/11 mice showing no signs of tumor. Conclusions: We have developed a precision CRISPR approach based on the homodimerization of Fok1 endonuclease at sites of cancer specific SVJs to specifically induce toxic DSBs in cancer cells. Here we show that this approach led to efficient targeting of cancer cells both in cell culture and in vivo. We are currently testing the efficacy of the KLIPP approach in preclinical models of bladder and ovarian cancers using lipid nanoparticle delivery techniques. On successfully implementation of this approach, it will be transformative for cancer therapy. Further, KLIPP will be generated in a personalized way for each patient in a rapid and cost-effective way. Citation Format: Radhika Suhas Hulbatte, Huibin Yang, Natalie Gratsch, Alan Kellher, Phillip L. Palmbos, Mats Ljungman. A precision CRISPR approach to target structural variant junctions in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1068.

Intravesical oncolytic virotherapy and immunotherapy for non-muscle-invasive bladder cancer mouse model.

To test if intravesical instillation of both an anti-programmed cell death protein 1 (PD-1) inhibitor and an oncolytic reovirus would demonstrate a greater effect than either treatment alone, as non-muscle-invasive bladder cancer that is refractory to intravesical bacillus Calmette-Guérin can be treated by systemic anti-PD-1 immunotherapy and we previously demonstrated improved overall survival (OS) with six once-weekly instillations of intravesical anti-PD-1 in a murine model. Using an orthotopic syngeneic C3H murine model of MBT2 urothelial bladder cancer, groups of 10 mice were compared between no treatment, intravesical anti-PD-1, intravesical oncolytic reovirus, or intravesical reovirus + anti-PD-1. A single intravesical treatment session was given. The primary outcome was OS, and the secondary outcomes included long-term immunity and tumour-immune profile. With a median follow-up of 9 months, all mice that received no treatment died with a median survival of 41 days, while the comparison median OS was not reached for reovirus (hazard ratio [HR] 14.4, 95% confidence interval [CI] 3.9-32.6; P < 0.001), anti-PD-1 (HR 28.4, 95% CI 7.0-115.9; P < 0.001), and reovirus + anti-PD-1 (HR 28.4, 95% CI 7.0-115.9; P < 0.001). Monotherapy with anti-PD-1 or reovirus demonstrated no significant differences in survival (P = 0.067). Mass cytometry showed that reovirus + anti-PD-1 treatment enriched monocytes and decreased myeloid-derived suppressor cells, generating an immuno-responsive tumour microenvironment. Depletion of CD8+ T cells eliminated the survival advantage provided by the intravesical treatment. Treatment of murine orthotopic bladder tumours with a single instillation of intravesical reovirus, anti-PD-1 antibody, or the combination confers superior survival compared to controls. Tumour-immune microenvironment differences indicated myeloid-derived suppressor cells and CD8+ T cells mediate the treatment response.

PD25-06 COMBINATION TREATMENT WITH INTRAVESICAL INTERFERON GENE THERAPY AND PAN-ERBB RECEPTOR FAMILY BLOCKER IMPROVES SURVIVAL IN MICE WITH BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023PD25-06 COMBINATION TREATMENT WITH INTRAVESICAL INTERFERON GENE THERAPY AND PAN-ERBB RECEPTOR FAMILY BLOCKER IMPROVES SURVIVAL IN MICE WITH BLADDER CANCER Akshay Sood, Jan Rudzinski, Alberto Martini, Come Tholomier, Sharada Mokkapati, and Colin Dinney Akshay SoodAkshay Sood More articles by this author , Jan RudzinskiJan Rudzinski More articles by this author , Alberto MartiniAlberto Martini More articles by this author , Come TholomierCome Tholomier More articles by this author , Sharada MokkapatiSharada Mokkapati More articles by this author , and Colin DinneyColin Dinney More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003303.06AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recently, intravesical interferon (IFNα) gene therapy has demonstrated success in treatment of BCG-unresponsive NMIBC in a large, randomized, phase-III trial. While the results have been promising, there is room for improvement. One strategy to improve treatment efficacy and durability may be to identify actionable resistance mechanisms and employ targeted combination therapies. METHODS: We performed end-tumor RNA-seq analysis of MB49 mice tumors treated with IFNα gene therapy to identify potential mechanisms of therapy resistance – ErbB pathway was discovered as a potential target (Figure 1a). We therefore tested combination treatment with ErbB pathway blocker and IFNα in MB49 cells using lentiviral IFNα (LV-IFNα) with/without Afatinib in vitro. Next, in vivo studies were conducted in the MB49 orthotopic mice bladder cancer model: mice were randomized in to 5 treatment groups (n=10 each), saline (Ctrl), LV-Ctrl, Afatinib monotherapy, LV-IFNα monotherapy, and the experimental LV-IFNα + Afatinib combination therapy. Overall survival was assessed. RESULTS: Combination therapy significantly reduced the viability of MB49 cells in vitro compared to any of the other treatment conditions (mean relative ATPase activity for combination treatment at 72 h 4%, compared to 100%, 26%, and 28% for Ctrl, LV-IFNα, and Afatinib, p<0.001; Figure 1b). This effect on cell viability appeared to be driven by a combination of additive cytostatic and cytolytic effects (Figure 1c-d). The combination treatment also decreased cell migration (mean migrated cells/10x field on Boyden chamber assay: 92.3 for combination therapy and 631.0, 600.4, and 270.3 for Ctrl, LV-IFNα, and Afatinib, p<0.001; Figure 1e). Finally, in vivo studies demonstrated improved OS with combination therapy (median OS 49 d in the combination group vs 15, 29, and 26 d in the Ctrl, LV-IFNα, and Afatinib groups, Log-rank p<0.001; Figure 1f). No mice in the combination group died of drug toxicity. CONCLUSIONS: Our preliminary investigations suggest that ErbB pathway may be a clinically meaningful resistance mechanism, which when targeted alongside IFNα gene therapy may improve overall treatment efficacy. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e731 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akshay Sood More articles by this author Jan Rudzinski More articles by this author Alberto Martini More articles by this author Come Tholomier More articles by this author Sharada Mokkapati More articles by this author Colin Dinney More articles by this author Expand All Advertisement PDF downloadLoading ...

Melatonin Inhibits EMT in Bladder Cancer by Targeting Autophagy.

Melatonin, a naturally biosynthesized molecule secreted by the pineal gland, exhibits antitumor activities against several different types of cancer. The mechanisms of action of melatonin against tumor progression involve cellular apoptosis, antimetastatic activity, antioxidant and mutagenic effects, antiangiogenic activity, and the restoration of cancer immune surveillance. Melatonin has anticancer activity when administered alone or in combination with standard chemotherapeutic agents, with measurable improvements seen in the clinical endpoints of tumor regression and patient survival. However, scant clinical evidence supports the use of melatonin in bladder cancer treatment. Our study has found that melatonin treatment suppresses the bladder cancer cell migratory ability by inhibiting the epithelial-mesenchymal transition (EMT) process, which appears to be linked to melatonin-induced decreases in bladder cancer cell autophagy. Finally, an evaluation of in vivo melatonin-induced antitumor effects in an orthotopic animal model of bladder cancer indicated that melatonin treatment slightly prolonged the survival of tumor-bearing mice. Our study offers novel insights into the use of melatonin in bladder cancer treatment.

Preliminary efficacy of [90Y]DOTA-biotin-avidin radiotherapy against non-muscle invasive bladder cancer.

Bladder cancer represents 3% of all new cancer diagnoses per year. We propose intravesical radionuclide therapy using the β-emitter 90Y linked to DOTA-biotin-avidin ([90Y]DBA) to deliver short-range radiation against non-muscle invasive bladder cancer (NMIBC). Image-guided biodistribution of intravesical DBA was investigated in an animal model by radiolabeling DBA with the 68Ga and dynamic microPET imaging following intravesical infusion of [68Ga]DBA for up to 4h and post-necropsy γ-counting of organs. The antitumor activity of [90Y]DBA was investigated using an orthotopic MB49 murine bladder cancer model. Mice were injected with luciferase-expressing MB49 cells and treated via intravesical administration with 9.2MBq of [90Y]DBA or unlabeled DBA 3days after the tumor implantation. Bioluminescence imaging was conducted after tumor implantation to monitor the bladder tumor growth. In addition, we investigated the effects of [90Y]DBA radiation on urothelial histology with immunohistochemistry analysis of bladder morphology. Our results demonstrated that DBA is contained in the bladder for up to 4h after intravesical infusion. A single dose of [90Y]DBA radiation treatment significantly reduced growth of MB49 bladder carcinoma. Attaching 90Y-DOTA-biotin to avidin prevents its re-absorption into the blood and distribution throughout the rest of the body. Furthermore, immunohistochemistry demonstrated that [90Y]DBA radiation treatment did not cause short-term damage to urotheliumat day 10, which appeared similar to the normal urothelium of healthy mice. Our data demonstrates the potential of intravesical [90Y]DBA as a treatment for non-muscle invasive bladder cancer.

Single cell sequencing reveals that CD39 inhibition mediates changes to the tumor microenvironment

Single-cell sequencing technologies have noteworthily improved our understanding of the genetic map and molecular characteristics of bladder cancer (BC). Here we identify CD39 as a potential therapeutic target for BC via single-cell transcriptome analysis. In a subcutaneous tumor model and orthotopic bladder cancer model, inhibition of CD39 (CD39i) by sodium polyoxotungstate is able to limit the growth of BC and improve the overall survival of tumor-bearing mice. Via single cell RNA sequencing, we find that CD39i increase the intratumor NK cells, conventional type 1 dendritic cells (cDC1) and CD8 + T cells and decrease the Treg abundance. The antitumor effect and reprogramming of the tumor microenvironment are blockaded in both the NK cells depletion model and the cDC1-deficient Batf3−/− model. In addition, a significant synergistic effect is observed between CD39i and cisplatin, but the CD39i + anti-PD-L1 (or anti-PD1) strategy does not show any synergistic effects in the BC model. Our results confirm that CD39 is a potential target for the immune therapy of BC.

Tumor-targeting albumin nanoparticles as an efficacious drug delivery system and potential diagnostic tool in non-muscle-invasive bladder cancer therapy

Current intravesical chemotherapy for non-muscle invasive bladder cancer (NMIBC) has limited efficacy due to loss of the instilled agent from urine voiding and the agent's lack of specificity for the tumors. We developed a nanocarrier (txCD47-HNP, ∼100 nm) based on human serum albumin conjugated with a peptide that targets the cluster of differentiation 47 receptor overexpressed on bladder cancer (BC) cells. The IC50 of gemcitabine elaidate (GEM) loaded in the txCD47-HNP was almost an order of magnitude lower than that of free GEM. In a mouse orthotopic BC model, GEM loaded in txCD47-HNP effectively reduced the tumor burden. Tumor cells in BC patients' urine can also be targeted by fluorescence-labeled txCD47-HNP resulting in >83 % of the cells exhibiting fluorescence. Thus, txCD47-HNP can potentially be a theranostic agent in NMIBC management by serving as a targeted drug delivery vehicle as well as an alternative to urine cytology.

Establishment of an optimized orthotopic bladder cancer model in mice

BackgroundBladder cancer (BC) is one of the most common malignancies of the genitourinary system. Animal models offer an important tool to explore tumour initiation, progression, and therapeutic mechanisms. Our aim is to construct an optimized orthotopic BC model which is predictable, reproducible, and convenient.MethodsThe optimized orthotopic BC model was constructed in male C57BL/6 mice utilizing microsyringes to inoculate them with a murine BC cell line (MB49). Anesthetised mice were inoculated with an MB49 cell suspension (10 µL) at approximately 5 × 106/mL. The whole process of modelling was observed and monitored every 3 days for 21 days utilizing HE staining and transabdominal ultrasonography (TUS).ResultsIn this study, the model showed excellent success rates for tumour formation (96.67%) and metastatic rate (89.66%). Compared to the control group (sham operation), mice in the modelling group had serous cachexia, visible haematuresis and weight loss (all P < 0.05). The lungs, liver, ureter and kidneys were found to have tumour metastasis. Moreover, the average survival time (19.73 ± 1.69 d) of modelling mice was significantly shorter than that of the control mice (P < 0.05), which remained alive.ConclusionOur study established a method using microsyringes to inject murine BC cells into the bladder wall, creating a stable transplantable BC model in mice.

Bacteria-inspired transformable nanoparticle targets and covers residual tumor against bladder cancer recurrence

The regrowth of residual tumor left by incomplete surgery leads to bladder cancer recurrence. Postoperative intravesical instillation in clinic shows unsatisfactory recurrence inhibition owing to rapid drug emptying and non-specific distribution. Here, inspired by intrinsic bacterial adhesion towards exposed wounds through fibronectin binding peptide, we develop a bacteria-inspired peptide-based nanoparticle that targets exposed fibronectin on the surgical bed with residual tumor. Upon binding, the nanoparticle simultaneously transforms into fibrous coating as drug depots for long-term release of encapsulated doxorubicin. The transformable nanoparticle retains 8.5-fold higher doxorubicin concentration than free one in bladders of live mice 3 days post-instillation. Reasonably, this formulation obviously suppresses tumor regrowth in an incomplete tumor resection model. Compared to the clinical doxorubicin treatment, it reduces the recurrence rate from 71% to 29% in a murine orthotopic bladder cancer model. This bacteria-inspired biomaterial provides a promising intravesical treatment but also opportunities for clinical management on cancer recurrence.

Mannose-targeting Concanavalin A-Epirubicin Conjugate for Targeted Intravesical Chemotherapy of Bladder Cancer.

Intravesical instillation of chemotherapeutic drugs such as epirubicin (EPI) is routinely used to prevent tumor recurrence and progression after transurethral resection of bladder tumor. However, the lack of tumor selectivity often causes severe damage to normal bladder urothelium leading to intolerable side effects. Here, we analyzed abnormal changes in glycosylation in bladder cancer and identified mannose as the most aberrantly expressed glycan on the surface of bladder cancer cell lines and human bladder tumor tissues. We then constructed a lectin-drug conjugate by linking concanavalin A (ConA) - a lectin that specifically binds to mannose, with EPI through a pH-sensitive linker. This ConA-EPI conjugate conferred EPI with mannose-targeting ability and selectively internalized cancer cells in vitro. This conjugate showed selective cytotoxicity to cancer cells in vitro and better antitumor activity in an orthotopic mouse model of bladder cancer. Our lectin-drug conjugation strategy makes targeted intravesical chemotherapy of bladder cancer possible.