Mouse Model Of Bladder Cancer Research Articles

Rosiglitazone and trametinib exhibit potent anti-tumor activity in a mouse model of muscle invasive bladder cancer

Muscle invasive bladder cancers (BCs) can be divided into 2 major subgroups-basal/squamous (BASQ) tumors and luminal tumors. Since Pparg has low or undetectable expression in BASQ tumors, we tested the effects of rosiglitazone, Pparg agonist, in a mouse model of BASQ BC. We find that rosiglitazone reduces proliferation while treatment with rosiglitazone plus trametinib, a MEK inhibitor, induces apoptosis and reduces tumor volume by 91% after 1 month. Rosiglitazone and trametinib also induce a shift from BASQ to luminal differentiation in tumors, which our analysis suggests is mediated by retinoid signaling, a pathway known to drive the luminal differentiation program. Our data suggest that rosiglitazone, trametinib, and retinoids, which are all FDA approved, may be clinically active in BASQ tumors in patients.

Insight into Fructose-to-Sucrose Ratio as the Potential Target of Urinalysis in Bladder Cancer.

Bladder cancer usually has been diagnosed in elderly patients as it stays asymptomatic until it presents. Current detection methods for bladder cancer cannot be considered as an adequate screening strategy due to their high invasiveness and low sensitivity. However, there remains uncertainty about targets with high sensitivity and specificity for non-invasive bladder cancer examination. Our study aims to investigate the actionable non-invasive screening biomarkers in bladder cancer. Here, we employed scRNA-seq to explore the crucial biological processes for bladder cancer development. We then utilized bidirectional Mendelian randomization (MR) analysis to explore the bidirectional causal relationship between ATP-associated metabolites in urine and bladder cancer. Lastly, we used a BBN-induced mouse model of bladder cancer to validate the crucial gene identified by scRNA-seq and MR analysis. We found that (1) the ATP metabolism process plays a critical role in bladder cancer development; (2) there is a bidirectional and negative causal relationship between fructose-to-sucrose ratio in urine and the risk of bladder cancer; and (3) the higher expression of TPI1, a critical gene in the fructose metabolism pathway, was validated in BBN-induced bladder tumors. Our results reveal that fructose-to-sucrose ratio can serve as a potential target of urinalysis in bladder cancer.

Development of Traceable Mouse Models of Advanced and Metastatic Bladder Cancer.

Bladder cancer (BC) is the fourth most common cancer in men, with a poor patient prognosis for advanced disease. The poor survival of patients with muscle-invasive bladder cancer (MIBC) and metastatic status emphasizes the urgent need to develop new therapies. Lacking in the field of BC is the availability of relevant advanced BC mouse models, especially metastatic ones, that accurately recapitulate the complexities of human pathology to test and study new therapeutic strategies. Addressing this need, we developed a traceable mouse model of BC that expresses tumor-associated antigens within the context of advanced muscle-invasive BC. This novel system was achieved through the deletion of the tp53 and pten genes, alongside the incorporation of the fusion construct of Firefly luciferase (Luc) and the SIYRYYGL (SIY) T-cell antigen. We validate that the presence of the transgene did not impact on the development of the tumors while allowing us to measure tumor progression by bioluminescence. We show that the transgene did not influence the composition of the immune tumor microenvironment. More importantly, we report that this model was unresponsive to anti-PD-1 treatment, as in the majority of patients with BC. We also develop a new model based on the orthotopic injection of BC clonal cell lines derived from our first model. We demonstrate that this new model invades the muscle layer and has a metastasis development rate of 83%. The advantage of this model is that we can visualize tumor growth and metastasis development in vivo. These mouse models' characteristics, displaying many similarities with the human pathology, provide a valuable tool for tracking tumor progression, metastasis spread in vivo, and treatment resistance, as well as exploring fundamental and translational aspects of BC biology. This work contributes to the improvement in the landscape of mouse models of advanced BC for testing new therapeutic strategies.

Intravesical local administration of pembrolizumab for treatment of bladder cancer: A novel strategy to minimize toxicity.

e14687 Background: A major hallmark of bladder cancer (BCa) is a higher treatment success rate than prostate cancer following stimulation of innate anti-tumor immune response either by intravesical BCG or to FDA approved intravenous anti-PD1 checkpoint antibody (pembrolizumab) approved for BCG resistant BCa since 2021. However, injected pembrolizumab is characterized by a markedly higher incidence (66%) of serious adverse events than the median duration of complete response of ~40% for non-muscle invasive BCa (NMIBC) and 15-20% for invasive BCa. Since the toxicity (fatigue, Pneumonitis and diarrhea) of injected pembrolizumab is linked to systemic breakdown of T-cell mediated immune surveillance we enquired whether analogous to BCG, pembrolizumab can be administered intravesically to safely induce a localized anti-tumor response in mouse model of BCa. Methods: Six weeks old female mice of B6D2F1 strain (n=10) were ad libitum fed carcinogen, N-butyl-N-4-hydroxybutyl nitrosamine (BBN) in drinking water (0.05% w/v) for 12 weeks. The presence of BBN evoked tumor was ascertained by unenhanced T2 weighted MRI at week 11 prior to single intravesical dosing by 24G transurethral catheter under isoflurane anesthesia of 0.1mL vehicle (sterile water) with or without 0.3mg of Pembrolizumab (Lipella Pharmaceuticals). 6 weeks later, T2 weighted MRI was repeated together with T1 mapping by ICE-MRI prior to organ harvest. Results: BBN feeding evoked multiclonal tumors (red*) of comparable size on T2 weighted MRI at week 11 but repeat MRI 7 weeks after treatment revealed that tumor progression and perivesical invasion noted in vehicle group was arrested in pembrolizumab group. Longest dimension of BBN tumor in vehicle treated mice was ≥ 1mm longer (p<0.05) than in pembrolizumab treated mice. T1 mapping revealed thickened bladder wall missed by T2 MRI. Conclusions: These findings on single intravesical dose of pembrolizumab (10mg/kg) in mice are consistent with human study on multiple intravesical dosing of BCG plus pembrolizumab (1-2mg/kg) in BCG resistant NMIBC patients. Intravesical pembrolizumab mirrors the limited systemic uptake of radiolabelled antibodies together with substantially higher ingress into tumor foci. The tumor selective ingress for BCa immunotherapy without any toxicity will be enhanced with Lipella Pharmaceutical’s liposomal delivery of Pembrolizumab.

Abstract A011: Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition

Abstract Introduction: Epithelial changes can extend beyond the boundaries of an incipient tumor, leading to cancer recurrence even after effective excision of the primary tumor. This ‘field cancerization’ and consequent high recurrence rate is especially pronounced in bladder cancer. The purpose of this study is to characterize the urothelium in the context of ‘field cancerization’ in a mouse model of bladder cancer as well as in patients with bladder cancer and to determine if reversal of the field effect will prevent tumor formation. Results: We investigated field cancerization by single cell transcriptomics (scRNA-Seq) in a murine (male FVB mice) bladder carcinogenesis model using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and found that prior to overt tumor formation urothelial cells throughout the entire bladder take on a strongly basal phenotype, characterized by reduced expression of differentiation markers (i.e. Upk1a, Upk2, and Upk3a) and increased expression of basal markers (i.e. Krt14, Krt6a, Krt5). Using monocle, we applied single cells from a BBN treated mouse bladder library to a pseudotime trajectory generated from a control mouse bladder library in order to unbiasedly characterize urothelial differentiation in the non-tumor urothelium of BBN treated mouse bladders. scRNA-Seq analysis of non-tumor urothelium from bladder cancer patients revealed a similar ‘basalization’ among non-tumor urothelial cells. In order to apply our single cell findings to bulk RNA-seq data, we developed a basal index from differentially expressed genes in basal clusters versus non-basal clusters from healthy control human urothelium single cell libraries, and we applied this basal index to non-tumor bladder samples from the The Cancer Genome Atlas (TCGA). We found that among a small subset of patients with paired tumor and non-tumor urothelium in the bladder TCGA dataset, the basal index, when applied to the non-tumor urothelium, was prognostic of survival. Finally, we found that basalization in BBN treated mice could be reversed pharmacologically by epidermal growth factor (EGF) pathway antagonism using erlotinib and trametinib and that over a 4-month time course this reversal dramatically decreased tumor incidence and improved survival, suggesting a new, more effective approach using mechanism-based cancer drugs for the prevention of bladder cancer recurrence in patients. Conclusions: The non-tumor urothelium in BBN treated mouse bladders, prior to overt tumor formation, demonstrates a basal phenotype which we characterize with scRNA-Seq and pseudotime analysis. Similarly, in patients with bladder cancer we observe that the non-tumor urothelium also takes on a basal phenotype. This ‘basalization’ of the non-tumor urothelium may potentially be prognostic, and pharmacologic reversal of the basal phenotype in the non-tumor urothelium using EGF pathway inhibitors prevents tumors in BBN treated mice. Citation Format: Aaron M. Kershner, Kris B. Prado, Bernhard M. Kiss, Karim Mrouj, Lolita Penland, Wan-Jin Lu, Jiyun Choi, Edward Diaz, Rovin Lachmansingh, Bertha Chen, Chia-Sui Kao, Joseph C. Liao, Philip A. Beachy. Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition [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 A011.

Abstract A010: Cancer associated fibroblast dependency in a novel immunocompetent mouse model of bladder cancer

Abstract Introduction: The relationship between genotype and phenotype in targeted therapy has proven to be crucial for the successful use of most kinase inhibitors. In bladder cancer, the PI3K/AKT/mTOR pathway is widely activated by mutation but untargeted. Our study focuses on developing an immunocompetent, CRISPR-edited bladder cancer mouse model that mirrors common PI3K/AKT/mTOR pathway mutations. We established effective genotype-kinase inhibitor pairings using CRISPR-edited organoids and cancer associated fibroblasts (CAFs) to create a pro-tumorigenic environment in vivo. Methods and Results: Urothelial cells were isolated from the bladder of Rosa26 Lox-Stop-Lox-Cas9/GFP transgenic mice and grown as organoids in matrigel. These organoids were co-edited for Trp53 and Rb1 KO, and further editing of the Pten gene resulted in a nine-fold increase in sensitivity to the kinase inhibitor ipatasertib. Despite successful initial engraftment into mouse bladder, these edited cells regressed before tumor formation, and subcutaneous injection did not yield subcutaneous tumors. An alternate model featuring a 9p21 deletion with Trp53 KO also displayed similar regression post-engraftment into bladder or subcutaneous injections. However, co-injection with a previously established CAF line subcutaneously yielded palpable subcutaneous tumors that enlarged for 12 weeks. We then established a BBN bladder-tumor-derived CAF line, leading to the formation of tumors with squamous differentiation upon intramural co-injection with edited organoids. These models displayed immune-excluded architecture and a predominance of M2 over M1 macrophages. We also aimed to enhance the model's adaptability by introducing common PI3K/AKT/mTOR mutations, specifically TSC1/TSC2 KO, and Pik3ca hotspots, into our 9p21/Trp53 KO model. While TSC1/TSC2 KO lines exhibited elevated P-S6K signaling, they displayed resistance to Torin 1, Voxtalisib, and Everolimus—kinase inhibitors targeting mTORC1/2. In parallel, we employed CRISPR-mediated homologous recombination to successfully introduce a Pik3ca H1047R hotspot into the 9p21/Trp53 KO genotype, as evidenced by increased Phosphorylation of AKT compared to wild-type 9p21/Trp53 KO cell lines. This genotype resulted in approximately 80-fold selective increased sensitivity to pictilisib, a PI3K inhibitor compared to Pik3ca WT control. Conclusions: In this model, CAFs were required for successful engraftment of tumors into the bladder wall or subcutaneously. BUTU is a customizable model of bladder cancer and is suitable for direct targeting by kinase inhibitors while providing a platform for understanding the genotype-phenotype relationship in bladder cancer. We hope to use it as a tool to develop preclinical evidence on which to base precision medicine clinical trials. Citation Format: Philip H. Abbosh, Henkel Valentine. Cancer associated fibroblast dependency in a novel immunocompetent mouse model of 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 A010.

Abstract PR006: EG-70 (detalimogene voraplasmid), a novel, non-viral intravesical gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: Preclinical characterization and translation into the clinic

Abstract Background: EG-70 (detalimogene voraplasmid) is a novel, investigational, non-integrating, non-viral gene therapy specifically engineered to elicit local stimulation of anti-tumor immune response in the bladder and to drive durable efficacy in patients with BCG-unresponsive high-risk non-muscle-invasive bladder cancer (NMIBC), while mitigating the risk of systemic toxicities from immune stimulation. Previous preclinical data indicate that intravesical instillation (IVI) of EG-70 results in local expression of transgene products in murine and non-human primate models. Here we describe the latest preclinical data to support the mechanism of action of EG-70 and Phase 1 clinical study results for EG-70 in the treatment of BCG-unresponsive NMIBC. Methods: Preclinical: A murine surrogate formulation of EG-70 (mEG-70) was administered locally to the bladder by IVI in C57BL/6 mice. Efficacy was assessed in a luciferase-expressing MB49 model of orthotopic bladder cancer. Following confirmation of cancer cell implantation by in vivo imaging, mice received two weekly IVIs of mEG-70. Immune profiling was evaluated by assessing protein expression levels and cell compositions (via MSD, flow cytometry, and immunohistochemistry). Immune cell depletion studies were conducted using specific antibodies directed against CD4 or CD8 antigen to assess T-cell contribution to efficacy. Clinical: In a Phase 1/2 study in high-risk BCG-unresponsive NMIBC with carcinoma in situ (CIS) (LEGEND; NCT04752722), EG-70 was assessed in four separate dose groups. Results: Preclinical: In a mouse model of orthotopic bladder cancer, IVI of mEG-70 caused striking remodeling of the tumor microenvironment from an immunosuppressive phenotype to a pro-inflammatory milieu supportive of tumor clearance. This was demonstrated by a significant decrease in myeloid cells and IL-4 cytokine levels, an increase in NK-cells and T-cells, and an increase in pro-inflammatory cytokines in tumor tissue. Accordingly, administration of mEG-70 was associated with marked reduction in tumor burden and significant improvement of survival. The anti-tumor response was largely immune-mediated as demonstrated by the critical role of T-cells, durable clearance of tumors, and protection against subsequent tumorigenic re-challenge, both locally in the bladder as well as distally, after flank implantation. Clinical: Results from the Phase 1 portion of the LEGEND study show that IVI of EG-70 was well-tolerated in patients with high-risk BCG-unresponsive NMIBC with CIS across all dose levels, with an overall complete response rate of 73%. Conclusions: The latest preclinical findings demonstrate that EG-70 has the potential to deliver immunostimulatory payloads locally to the bladder. The mechanism of action described preclinically has been translated into the clinic, in the Phase 1 portion of the LEGEND study in patients with BCG-unresponsive NMIBC with CIS. Citation Format: Marie-Line Goulet, Shauna M Dauphinee, Daniel Veilleux, Kristine S Louis, David Lazure, Sarah Stevenson, Darius Bilimoria, Fazmina Zamzameer, Ximin Chen, Sébastien Sublemontier, Sahar Amirkhani, Carlos Fleet, Richard Bryce, Matthew Bui, Shreyas Joshi, Susan J. Kalota, Anthony Cheung, James Sullivan. EG-70 (detalimogene voraplasmid), a novel, non-viral intravesical gene therapy for BCG-unresponsive non-muscle-invasive bladder cancer: Preclinical characterization and translation into the clinic [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 PR006.

Silencing the Mitochondrial Gatekeeper VDAC1 as a Potential Treatment for Bladder Cancer.

The strategy for treating bladder cancer (BC) depends on whether there is muscle invasion or not, with the latter mostly treated with intravesical therapy, such as with bacillus Calmette-Guérin (BCG). However, BCG treatment is unsuccessful in 70% of patients, who are then subjected to radical cystectomy. Although immune-checkpoint inhibitors have been approved as a second-line therapy for a subset of BC patients, these have failed to meet primary endpoints in clinical trials. Thus, it is crucial to find a new treatment. The mitochondrial gatekeeper protein, the voltage-dependent anion channel 1 (VDAC1), mediates metabolic crosstalk between the mitochondria and cytosol and is involved in apoptosis. It is overexpressed in many cancer types, as shown here for BC, pointing to its significance in high-energy-demanding cancer cells. The BC cell lines UM-UC3 and HTB-5 express high VDAC1 levels compared to other cancer cell lines. VDAC1 silencing in these cells using siRNA that recognizes both human and mouse VDAC1 (si-m/hVDAC1-B) reduces cell viability, mitochondria membrane potential, and cellular ATP levels. Here, we used two BC mouse models: subcutaneous UM-UC3 cells and chemically induced BC using the carcinogen N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN). Subcutaneous UM-UC3-derived tumors treated with si-m/hVDAC1 showed inhibited tumor growth and reprogrammed metabolism, as reflected in the reduced expression of metabolism-related proteins, including Glut1, hexokinase, citrate synthase, complex-IV, and ATP synthase, suggesting reduced metabolic activity. Furthermore, si-m/hVDAC1-B reduced the expression levels of cancer-stem-cell-related proteins (cytokeratin-14, ALDH1a), modifying the tumor microenvironment, including decreased angiogenesis, extracellular matrix, tumor-associated macrophages, and inhibited epithelial-mesenchymal transition. The BBN-induced BC mouse model showed a clear carcinoma, with damaged bladder morphology and muscle-invasive tumors. Treatment with si-m/hVDAC1-B encapsulated in PLGA-PEI nanoparticles that were administered intravesically directly to the bladder showed a decreased tumor area and less bladder morphology destruction and muscle invasion. Overall, the obtained results point to the potential of si-m/hVDAC1-B as a possible therapeutic tool for treating bladder cancer.

Anti-Proliferative Effects of Lidocaine as an Autophagy Inducer in Bladder Cancer via Intravesical Instillation: In Vitro and Xenograft Mouse Model Experiments.

Lidocaine exerts potential anti-tumor effects on various cancer cell lines, and its intravesical instillation is considered safer than intravenous administration for bladder cancer. However, the mechanisms underlying its anti-tumor effects have not been fully elucidated. Here, we aimed to elucidate the anti-tumor molecular mechanisms of lidocaine in bladder cancer cells and a xenograft model to substantiate the efficacy of its intravesical administration. We investigated the anti-proliferative and autophagyinducing activities of lidocaine in Nara Bladder Tumor No. 2 (NBT-II) rat bladder carcinoma cells using cell viability, flow cytometry, a wound healing assay, and western blotting. We also established a xenograft mouse model of bladder cancer, and cancer growth was examined using in vivo bioluminescence imaging. Lidocaine decreased cell viability, induced G0/G1 phase cell cycle arrest, and inhibited cell migration partially via glycogen synthase kinase (GSK) 3β phosphorylation. Moreover, a combination of lidocaine and SB216763 (a GSK3β inhibitor) suppressed autophagy-related protein expression. Bafilomycin-A1 with lidocaine significantly enhanced microtubule-associated protein 1A/1B-light chain (LC3B) expression; however, it decreased LC3B expression in combination with 3-methyladenine compared to lidocaine alone. In the xenograft mouse model, the bladder cancer volume was reduced by lidocaine. Overall, lidocaine exerts anti-proliferative effects on bladder cancer via an autophagy-inducing mechanism.

Abstract 1436: Cancer associated fibroblast dependency in a model of bladder cancer

Abstract Introduction: The relationship between genotype and phenotype in targeted therapy has proven to be crucial for most kinase inhibitors. In bladder cancer, the PI3K/AKT/mTOR pathway is widely activated by mutation but untargeted. Our study focuses on developing an immunocompetent, CRISPR-edited bladder cancer mouse model that mirrors common PI3K/AKT/mTOR mutations. We aim to establish genotype-effective kinase inhibitor pairings using in vitro CRISPR-edited organoids and Cancer Associated Fibroblasts (CAFs) to create a pro-tumorigenic environment in vivo. Methods and Results: Urothelial cells were isolated from the bladder of Rosa26 Lox-Stop-Lox-Cas9/GFP transgenic mice to develop organoids. These organoids were co-edited for Trp53 and Rb1 KO, and further editing of the Pten gene resulted in a nine-fold increase in sensitivity to the kinase inhibitor ipatasertib. Despite successful engraftment into mouse bladder, these edited cells regressed before tumor formation, and intradermal injection did not yield subcutaneous tumors. An alternate model featuring a 9p21 deletion with Trp53 KO also displayed similar regression post-engraftment into bladder or skin. However, co-injection with skin-cancer-derived CAFs subcutaneously yielded palpable subcutaneous tumors that grew for 12 weeks. Enhancing orthotopic model fidelity, we established a BBN bladder-tumor-derived CAF line, leading to the formation of tumors with squamous differentiation upon intramural co-injection with edited organoids. These models displayed immune-excluded architecture and a predominance of M2 over M1 macrophages. Following our accomplishments in establishing bladder tumors, we aimed to enhance our model's adaptability by introducing common PI3K/AKT/mTOR mutations, specifically TSC1/TSC2 KO and Pik3ca hotspots, into our 9p21/Trp53 KO model. While TSC1/TSC2 KO lines exhibited elevated P-S6K signaling, they displayed resistance to Torin 1, Voxtalisib, and Everolimus—kinase inhibitors targeting mTORC1/2. In parallel, we employed CRISPR-mediated HDR to successfully introduce a Pik3ca H1047R hotspot into our 9p21/Trp53 KO model, as evidenced by increased Phosphorylation of AKT compared to wild-type 9p21/Trp53 KO cell lines. Conclusions: Our research establishes the crucial role of CAFs in bladder tumor formation. Our now established customizable model of bladder cancer is suitable for direct targeting by kinase inhibitors while providing a platform for understanding the genotype-phenotype relationship in bladder cancer. Citation Format: Henkel Valentine, Uttam Satyal, Laura Bukavina, Hassan Uddin, Yousttena Beyamin, Rhea Arya, Philip Abbosh. Cancer associated fibroblast dependency in a model of bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1436.

Abstract 5272: APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells

Abstract Introduction: Treatment approach combining cancer cell targeted therapy and immunotherapy is an effective strategy to trigger robust tumor-specific immune responses. The aim of this study is to determine the impact of APL-1202, a clinical-stage drug candidate (NCT04813107), on cellular stress-driven immunogenic outcomes in bladder cancer cells. Methods: RNA-seq analysis was performed using bladder cancer and endothelial cell lines (A56, J82, T24, HUVEC) treated with APL-1202. Key determinants of immunogenic cell death pathway were measured by cell-surface translocation of calreticulin (flow cytometry), extracellular ATP release assay (bioluminescence) and HMGB1 secretion (ELISA) in the supernatants of cancer cells after APL-1202 treatment. Effects on the innate immune effector function was measured by flow cytometry-based phagocytosis assay consisting of Far-Red dye-stained, pre-treated bladder cancer cell lines (UMUC3, T24) in coculture with THP1-derived macrophages/immature dendritic cells or human monocyte-derived dendritic cells. In vivo, tumor end-point immune cell analysis, tumor weight and mouse survival were compared in chemically induced (BBN: N-butyl-N-(4-hydroxybutyl)-nitrosamine) orthotopic bladder cancer mice treated with APL-1202 and anti-PD1 antibody. Results: Pathway analysis of RNA-seq data from APL-1202 treated cells suggested upregulation of cellular stress-related gene signatures (oxidative stress, autophagy, P53) and pro-inflammatory responses (IL-6, IL-8, HMGB1 signaling). Conversely, downregulation of cell cycle (E2F targets, G2-M checkpoint) and IL-10 signaling suggested increased genomic stress and inflammatory switching in the treated cancer cells. Acute exposure to APL-1202 increased cell-surface translocation of calreticulin as well as extracellular release of ATP and HMGB1 in the treated T24 and UMUC3 cells. Higher percentages of phagocytic activities were detected in both THP-1-derived macrophages/immature dendritic cells and human monocyte-derived dendritic cells after coculture with APL-1202 treated T24 and UMUC3 cells compared to coculture with DMSO treated control cells. Immune phenotyping in tumors derived from BBN bladder cancer mouse model revealed increased infiltration of NK cells and CD8+ T cells. Furthermore, APL-1202 enhanced the anti-tumor effects of a therapeutic anti-PD1 antibody in mice as reflected by significantly decreased tumor weights and increased survival in a combination group with APL-1202 treatment compared to the anti-PD-1 antibody alone treatment group. Conclusion: APL-1202 may improve the efficacy of immune-checkpoint blockade therapies in bladder cancer patients by enhancing cancer cell immunogenicity and subsequent anti-tumor effector immune cell processes, particularly in tumors with deficits in antigen-specific immunity. Citation Format: Rakesh BAM, Jingmin Guan, Neetha Nanoth Vellichirammal, Murli Manohar, David Mulholland, John Sfakianos, Qiaoling Sun, Zuoan Yi, Alice Chen. APL-1202 enhances anti-tumor immune response through induction of cellular stress signaling and immunogenic cell death pathway in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5272.

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.

Modulation of tumor microenvironment by targeting histone acetylation in bladder cancer

Alterations in the epigenetic machinery in both tumor and immune cells contribute to bladder cancer (BC) development, constituting a promising target as an alternative therapeutic option. Here, we have explored the effects of a novel histone deacetylase (HDAC) inhibitor CM-1758, alone or in combination with immune checkpoint inhibitors (ICI) in BC. We determined the antitumor effects of CM-1758 in various BC cell lines together with the induction of broad transcriptional changes, with focus on the epigenetic regulation of PD-L1. Using an immunocompetent syngeneic mouse model of metastatic BC, we studied the effects of CM-1758 alone or in combination with anti-PD-L1 not only on tumor cells, but also in the tumor microenvironment. In vitro, we found that CM-1758 has cytotoxic and cytostatic effects either by inducing apoptosis or cell cycle arrest in BC cells at low micromolar levels. PD-L1 is epigenetically regulated by histone acetylation marks and is induced after treatment with CM-1758. We also observed that treatment with CM-1758 led to an important delay in tumor growth and a higher CD8 + T cell tumor infiltration. Moreover, anti-PD-L1 alone or in combination with CM-1758 reprogramed macrophage differentiation towards a M1-like polarization state and increased of pro-inflammatory cytokines systemically, yielding potential further antitumor effects. Our results suggest the possibility of combining HDAC inhibitors with immunotherapies for the management of advanced metastatic BC.

MicroRNA-15a-5p suppresses hypoxia-induced tumor growth and chemoresistance in bladder cancer by binding to eIF5A2.

In various malignant tumors (including bladder cancer) poor prognosis is associated with hypoxia and therapeutic resistance. Evidence indicates that in bladder cancer, microRNAs (miRNAs) have vital functions in acquired drug resistance. However, the involvement of miRNAs in hypoxia-mediated bladder cancer doxorubicin (Dox) resistance is unknown. Herein, we showed that hypoxia and Dox treatment downregulated miR-15a-5p expression. Using UM-UC-3 and J82 bladder cancer cell lines and in vivo mouse models of bladder cancer, we confirmed that miR-15a-5p arrests tumor cell growth and Dox resistance in vitro and in vivo. Furthermore, we determined the interaction between miR-15a-5p and eukaryotic translation initiation factor 5A-2 (eIF5A2) using dual luciferase reporters and quantitative real-time reverse transcription polymerase chain reaction assays. We also showed that a miR-15a-5p agomir repressed EIF5A2 expression in bladder cancer cells, thereby inhibiting the epithelial-mesenchymal transition (EMT) induced by Dox or hypoxia. Moreover, ectopic expression of miR-15a-5p abrogated eIF5A2-mediated Dox resistance in bladder cancer cells. Collectively, these data indicated that hypoxia promotes tumor growth and chemoresistance through the HIF-1α/miR-15a-5p/eIFTA2/EMT pathway. This new finding not only has implications for improving our understanding of the Dox resistance process during bladder cancer progression but also indicates that the miR-15a-5p agomir is a promising tool to prevent Dox resistance in patients with bladder cancer.

CSF2 Impairs Nrf2 Signaling through the Akt/Mtor Pathway in the Development of Bladder Cancer.

Bladder Cancer (BCa) is one of the most common cancers of the urinary system. Colony-stimulating factor 2 (CSF2) is involved in many cancers, but not BCa. We investigated the effect of CSF2 on BCa in this study and the underlying molecular mechanisms. CSF2 mRNA levels in BCa were analyzed using the Cancer Genome Atlas (TCGA) database. Western blot was conducted to verify CSF2 expression in BCa tissue samples and cell lines. The effect of CSF2 on the growth of BCa cells was assessed by CCK8 and colony formation. To determine the migration and invasion capabilities of BCa cells, transwell analysis and wound healing assays were conducted. Next, western blot was used to explore the underlying mechanism. In the end, a xenografted BCa mouse model was established to examine the effects of CSF2 on tumorigenesis in vivo. Results showed that CSF2 mRNA was upregulated in BCa samples. Knocking down CSF2 significantly inhibited the proliferation and tumorigenesis of BCa cells in vitro and in vivo. Mechanism analysis revealed that CSF2 knockdown inhibited the proliferation and invasion of BCa cells via AKT/mTOR signaling. Based on these results, CSF2 promotes the proliferation and tumorigenesis of BCa.

Targeting DAD1 gene with CRISPR-Cas9 system transmucosally delivered by fluorinated polylysine nanoparticles for bladder cancer intravesical gene therapy.

Background: Intravesical chemotherapy is highly recommended after transurethral resection of bladder tumor for patients with bladder cancer (BCa). However, this localized adjuvant therapy has drawbacks of causing indiscriminate damage and inability to penetrate bladder mucosal. Methods: Fluorinated polylysine micelles (PLLF) were synthesized by reacting polylysine (PLL) with heptafluorobutyrate anhydride. Anti-apoptotic gene defender against cell death 1 (DAD1) was selected by different gene expression analysis between BCa patients and healthy individuals and identified by several biological function assays. The gene transfection ability of PLLF was verified by multiple in vitro and in vivo assays. The therapeutic efficiency of PLLF nanoparticles (NPs) targeting DAD1 were confirmed by intravesical administration using an orthotopic BCa mouse model. Results: Decorated with fluorinated chains, PLL can self-assemble to form NPs and condense plasmids with excellent gene transfection efficiency in vitro. Loading with the CRISPR-Cas9 system designed to target DAD1 (Cas9-sgDAD1), PLLF/Cas9-sgDAD1 NPs strongly inhibited the expression of DAD1 in BCa cells and induced BCa cell apoptosis through the MAPK signaling pathway. Furthermore, intravesical administration of PLLF/Cas9-sgDAD1 NPs resulted in significant therapeutic outcomes without systemic toxicity in vivo. Conclusion: The synthetized PLLF can transmucosally deliver the CRISPR-Cas9 system into orthotopic BCa tissues to improve intravesical instillation therapy for BCa. This work presents a new strategy for targeting DAD1 gene in the intravesical therapy for BCa with high potential for clinical applications.

Homologous Targeting and Adhesion Abilities of Cellular Microvesicles for Enhanced Intravesical Therapy of Bladder Cancer

AbstractBladder cancer is one of the most common clinical malignant tumors with a high recurrence rate after surgery. To improve the therapeutic effect, intravesical instillation of chemotherapeutic drugs is usually applied after surgery. However, due to the unique microenvironment in the bladder, the drug concentration is usually rapidly reduced with urinary excretion, leading to low accumulation in bladder cancer and poor therapeutic effect. Cellular microvesicles (MVs) have parental cell‐specific characteristics and hold homologous targeting and adhesion abilities to their parent cells, achieving efficient cell uptake in their parent cells. In this study, pirarubicin (THP), a clinically approved intravesical chemotherapeutic drug, is encapsulated into bladder cancer cell‐derived MVs to form THPMVs, which have high targeting and adhesion abilities to bladder cancer and facilitate intravesical therapy of bladder cancer. The results show THPMVs compared with free THP exhibited higher cellular uptake in MB49 bladder cancer cells and cause more significant apoptotic cells. After intravesical instillation into a bladder cancer mouse model, THPMVs can significantly accumulate in bladder cancer tissues, and cause an obvious reduction in bladder weights and volumes, exhibiting promising therapeutic effects. Homologous targeting and adhesion abilities of THPMVs greatly contribute to the intravesical therapy of bladder cancer.

Effective Antitumor Immunity Can Be Triggered by Targeting VISTA in Combination with a TLR3-Specific Adjuvant.

Resistance to anti-PD-1/PD-L1 treatment is often associated with accumulation of intratumoral inhibitory macrophages. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a nonredundant immune checkpoint that can induce both T-cell and myeloid-cell immunosuppression. In this study, we found that high levels of VISTA+ immune cells were associated with advanced stage bladder cancer and predicted poor survival in patients. A combination of high infiltration of VISTA+ immune cells and PD-L1+ immune cells or PD-1+ T cells predicted the worst survival. Flow cytometry and multiplex immunofluorescence analyses confirmed that VISTA expression was higher in macrophages than in T cells or neutrophils, and only VISTA+CD163+ macrophage density predicted poor prognosis in patients with bladder cancer. Toll-like receptor (TLR) agonists are known to trigger the innate immune response in macrophages. We found that the VISTA-specific mAb 13F3 augmented the ability of a TLR3-specific adjuvant to induce macrophage activation in vitro. In the MB49 syngeneic mouse model of bladder cancer, treatment with 13F3 curbed tumor growth and prolonged survival when combined with a TLR3-specific adjuvant. The combination treatment reduced the intratumoral frequency of CD206+ anti-inflammatory macrophages and levels of the immunosuppressive molecule TGFβ1, but it upregulated expression of immunostimulatory molecules (Ifna, Ifnb, and Trail) and increased the CD8+ T cell/regulatory T-cell ratio. These findings indicate that elevated VISTA expression in immune cells, particularly macrophages, is associated with an unfavorable prognosis in patients with bladder cancer and suggest that targeting VISTA in combination with a TLR3-specific adjuvant has translational potential.

Endothelial cells are a key target of IFN-g during response to combined PD-1/CTLA-4 ICB treatment in a mouse model of bladder cancer

To explore mechanisms of response to combined PD-1/CTLA-4 immune checkpoint blockade (ICB) treatment in individual cell types, we generated scRNA-seq using a mouse model of invasive urothelial carcinoma with three conditions: untreated tumor, treated tumor, and tumor treated after CD4+ Tcell depletion. After classifying tumor cells based on detection of somatic variants and assigning non-tumor cell types using SingleR, we performed differential expression analysis, overrepresentation analysis, and gene set enrichment analysis (GSEA) within each cell type. GSEA revealed that endothelial cells were enriched for upregulated IFN-g response genes when comparing treated cells to both untreated cells and cells treated after CD4+ Tcell depletion. Functional analysis showed that knocking out IFNgR1 in endothelial cells inhibited treatment response. Together, these results indicated that IFN-g signaling in endothelial cells is a key mediator of ICB induced anti-tumor activity.