Human Muscle-invasive Bladder Cancer Research Articles

Isorhapontigenin inhibition of basal muscle-invasive bladder cancer attributed to its downregulation of SNHG1 and DNMT3b

BackgroundBladder cancer (BC) is among the most prevalent malignant urothelial tumors globally, yet the prognosis for patients with muscle-invasive bladder cancer (MIBC) remains dismal, with a very poor 5-year survival rate. Consequently, identifying more effective and less toxic chemotherapeutic alternatives is critical for enhancing clinical outcomes for BC patients. Isorhapontigenin (ISO), a novel stilbene isolated from a Gnetum found in certain provinces of China, has shown potential as an anticancer agent due to its diverse anticancer activities. Despite its promising profile, the specific anticancer effects of ISO on BC and the underlying mechanisms are still largely unexplored.MethodsThe anchorage-independent growth, migration and invasion of BC cells were assessed by soft agar and transwell invasion assays, respectively. The RNA levels of SOX2, miR-129 and SNHG1 were quantified by qRT-PCR, while the protein expression levels were validated through Western blotting. Furthermore, methylation-specific PCR was employed to assess the methylation status of the miR-129 promoter. Functional assays utilized siRNA knockdown, plasmid-mediated overexpression, and chemical inhibition approaches.ResultsOur study demonstrated that ISO treatment significantly reduced SNHG1 expression in a dose- and time-dependent manner in BC cells, leading to the inhibition of anchorage-independent growth and invasion in human basal MIBC cells. This effect was accompanied by the downregulation of MMP-2 and MMP-9 and the upregulation of the tumor suppressor PTEN. Further mechanistic investigations revealed that SOX2, a key upstream regulator of SNHG1, played a crucial role in mediating the ISO-induced transcriptional suppression of SNHG1. Additionally, we found that ISO treatment led to a decrease in DNMT3b protein levels, which in turn mediated the hypomethylation of the miR-129 promoter and the subsequent suppression of SOX2 mRNA 3’-UTR activity, highlighting a novel pathway through which ISO exerts its anticancer effects.ConclusionsCollectively, our study highlights the critical role of SNHG1 downregulation as well as its upstream DNMT3b/miR-129/SOX2 axis in mediating ISO anticancer activity. These findings not only elucidate the mechanism of action of ISO but also suggest novel targets for BC therapy.

Genome-wide analyses reveals an association between invasive urothelial carcinoma in the Shetland sheepdog and NIPAL1.

Naturally occurring canine invasive urinary carcinoma (iUC) closely resembles human muscle invasive bladder cancer in terms of histopathology, metastases, response to therapy, and low survival rate. The heterogeneous nature of the disease has led to the association of large numbers of risk loci in humans, however most are of small effect. There exists a need for new and accurate animal models of invasive bladder cancer. In dogs, distinct breeds show markedly different rates of iUC, thus presenting an opportunity to identify additional risk factors and overcome the locus heterogeneity encountered in human mapping studies. In the association study presented here, inclusive of 100 Shetland sheepdogs and 58 dogs of other breeds, we identify a homozygous protein altering point mutation within the NIPAL1 gene which increases risk by eight-fold (OR = 8.42, CI = 3.12-22.71), accounting for nearly 30% of iUC risk in the Shetland sheepdog. Inclusion of six additional loci accounts for most of the disease risk in the breed and explains nearly 75% of the phenotypes in this study. When combined with sequence data from tumors, we show that variation in the MAPK signaling pathway is an overarching cause of iUC susceptibility in dogs.

Abstract PR002: Sarcomatoid Histological Variants of Canine Muscle-Invasive Bladder Cancer: Transcription Factor Activation Highlights Pathways of Epithelial-Mesenchymal Transformation Similar to Humans

Abstract The histologic heterogeneity of human Muscle Invasive Bladder Cancer (MIBC) is one of the main contributing factors to poor treatment outcomes. Recent reports on MIBC histological variants have highlighted increased levels of transcription factors (TF) associated with epithelial-mesenchymal transformation (EMT) and epigenetic regulation in the sarcomatoid histology variant of MIBC. Dogs with spontaneously developing MIBC have been shown to display similar histological and molecular features, in addition to closely mimicking the clinical behavior of human MIBC. Here, we add to previously reported data on histological variants in canine MIBC by evaluating 21 tumor biopsies for variant histology and performing bulk RNAseq analyses on 3 organoid cell lines derived from one sarcomatoid subvariant and two conventional urothelial carcinoma (UC). MIBC biopsies from 21 dogs were reviewed for variant histology by a human urologic pathologist. Organoids derived from the urine of canine MIBC patients were cultured, preserved in RNA later, and sequenced for mRNA expression to identify TF associated with sarcomatoid variants versus conventional UC. All canine MIBC biopsies were classified as invasive, high-grade UC. Histologic variants were identified as glandular differentiation in 7/21, sarcomatoid differentiation in 3/21, squamous differentiation in 3/21, micropapillary carcinoma in 1/21 MIBC samples and small nested variants in 1/21 samples; with the rest of the tumors classified as conventional UC. Raw RNAseq analyses revealed no clear clustering of expression profiles between the three organoid cell lines, indicating distinct RNA expression profiles between the sarcomatoid variants and conventional UC of canine MIBC. To evaluate differences in the expression of TF targets between the organoid cell lines, pathway analyses and VIPER algorithms were used. Organoids derived from sarcomatoid variant UC showed increased expression of Snail familiy transcription repressor 1 (SNAI1) along with decreased expression of cadherin 1 (CDH1), indicating a signature of EMT, similar to that previously identified in human MIBC organoids with sarcomatoid variant histology. Canine MIBC organoids derived from conventional UC histology samples showed a TF profile associated with proliferation, metastasis, and progression, such as increased Zinc finger E-box binding homeobox 1 (ZEB1), E2F transcription factor 3 (E2F3), and centromere protein F (CENPF), as previously described in humans with high grade, invasive UC. In conclusion, the frequency of some histologic variants in canine MIBC, such as sarcomatoid UC, appear to be higher than reported in human patients. TF analyses of canine MIBC-derived organoid cell lines showed evidence of EMT, similar to the sarcomatoid variants of MIBC in humans. Organoids derived from canine MIBC could potentially serve as a valuable model for evaluating the efficacy of novel treatment modalities for the rare histologic variants of UC. Citation Format: Karin Allenspach, Mohamed Elbadawy, Hannah Nocholson, Christopher Zdyrski, John Cheville, Eugene Douglass, Elizabeth W. Howerth, Lilian Oliveira, Jonathan P. Mochel. Sarcomatoid Histological Variants of Canine Muscle-Invasive Bladder Cancer: Transcription Factor Activation Highlights Pathways of Epithelial-Mesenchymal Transformation Similar to Humans [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 PR002.

Abstract 6694: Gut microbial drivers of response to bladder-sparing therapy in human muscle-invasive bladder cancer enhance immunotherapy efficacy and limit immunosuppression following fecal microbiome transplantation in vivo

Abstract BACKGROUND: Radiation therapy (RT) is a bladder-sparing option for Muscle-Invasive Bladder Cancer (MIBC), yet encounters a 30% non-response rate, with half of the patients succumbing to metastasis. Preclinical models demonstrate enhanced antitumor responses with RT combined with PD-1/PD-L1 blockade but give limited insight into the determinants of success for such combination therapies (CT). The gut microbiome, pivotal in conditioning local and peripheral immunity, influences PD-1-based immunotherapy efficacy in various cancers. Immune profiling indicates heightened systemic and antitumor immunity in responders with a favorable gut microbiome, enriched in Akkermansia muciniphila, Bifidobacterium, and Faecalibacterium. Oral gavage of immunogenic bacteria further enhances combined RT and anti-PD-L1 therapy. We thus aim to 1) elucidate the role of patients’ microbiome in shaping anti-tumor immune responses to CT, and 2) use its composition as a predictive factor for CT success. METHODS: We performed shotgun metagenomics on microbial DNA from fecal materials of MIBC RT responders (R, n=26) and non-responders (NR, n=11). Fecal homogenates from donors were gavaged into germ-free (GF) mice. Three weeks post-gavage, MB49 tumor cells were subcutaneously delivered to mice, randomized into four groups: control, anti-PD-L1, RT, and RT + anti-PD-L1. Tumors were harvested seven days post-treatment for single-cell RNA-seq and TCR sequencing (10X Genomics), while stool samples were collected weekly for 16S sequencing. RESULTS: Significant gut microbial disparities were observed between R and NR MIBC patients, with particular enrichment in known promoters of anti-PD-L1 response, such as Phocaeicola dorei in R. Transplantation of R microbiome into GF mice resulted in significant increases in predicted survival, tumor growth delay, and counts of effector CD4+ T cell infiltration in the RT+PD-L1 arm. Conversely, NR microbiome transplantation correlated with increased neutrophil and Treg infiltration, abrogating the survival tumor growth delay benefits of the combination arm. Spatial tissue analyses revealed higher neutrophil densities around CD8+ T cells in all NR arms. Single-cell RNA-seq of tumor-infiltrating immune cells unveiled skewed CD4 and CD8 polarization between R and NR in vivo batches. SIGNIFICANCE: To our knowledge, this is the first study to use FMT as a modulator of response in the context of radiation therapy combinations in MIBC. Our results highlight that the gut microbiome alone may condition a favorable immune terrain for combinatorial therapies involving radiation. This offers strong predictive value for personalized therapeutic approaches in selecting patients who will benefit the most from bladder-sparing therapies. Citation Format: Eva Michaud, Sabina Fehric, Cynthia Faubert, Bertrand Routy, Irah King, José Joao Mansure, Wassim Kassouf. Gut microbial drivers of response to bladder-sparing therapy in human muscle-invasive bladder cancer enhance immunotherapy efficacy and limit immunosuppression following fecal microbiome transplantation in vivo [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 6694.

Activity of antibody-drug conjugates (ADCs) with radiation in preclinical bladder cancer models.

664 Background: Antibody-drug conjugates (ADCs) are a novel class of therapeutics that combine a tumor cell targeting antibody with a cytotoxic payload. Two ADCs are currently approved in the US for treatment of advanced bladder cancer: enfortumab vedotin (EV) and sacituzumab govitecan (SG). Radiation therapy (RT) plays a central role in trimodality therapy (TMT), a curative treatment approach for muscle-invasive bladder cancer (MIBC). However, the biological activity of ADCs combined with RT in preclinical MIBC models has not been reported. Methods: We use a molecularly annotated panel of human MIBC preclinical models to test the impact of RT on expression of ADC targets and define the combined activity of EV and SG with RT. We also test the in vivo activity of combining ADCs with image-guided, fractionated RT in bladder cancer flank xenograft mouse models. Results: Radiation has variable impact on expression levels of nectin-4 and trop-2, the targets of EV and SG, respectively, as determined by immunoblot, immunofluorescence microscopy, and immunohistochemistry (IHC). Whereas modest dose-dependent increases in nectin-4 and/or trop-2 levels were observed in some models, no significant changes were observed in other models. Importantly, RT did not lead to a significant decrease in nectin-4 or trop-2 expression in any of the models. EV and SG showed additive or synergistic cell killing when combined with RT across preclinical models in vitro. Combining EV or SG with fractionated RT in vivo was well-tolerated, showed improved tumor control, and prolonged survival in bladder cancer flank xenograft mouse models compared to either ADC or RT alone. Conclusions: ADCs demonstrate combination activity with RT across a panel of molecularly diverse bladder cancer preclinical models. These studies provide preclinical data supporting clinical trials to investigate the safety and efficacy of combining ADCs with RT as a bladder-preserving therapy for MIBC.

Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

Abstract 2509: Similar variant histology and immune cell infiltration in canine and human muscle-invasive bladder cancer

Abstract Introduction: Muscle-Invasive Bladder Cancer (MIBC) is associated with a poor overall survival rate. One important factor that contributes to poor treatment outcomes is the histologic heterogeneity of MIBC tumors, which limits the translational value of some rodent models. As opposed to mice, dogs with spontaneously occurring bladder cancer have been shown to present with similar clinical behavior as human MIBC. In addition, canines have an intact immune system, which could provide for an ideal pre-clinical study population to investigate the efficacy of immunotherapy. In this study, we evaluated canine MIBC tumor biopsies for their similarities to human MIBC with regards to histology and immune cell infiltration. Materials & Methods: Nine canine MIBC tumor samples were provided by the ISU pathology service and evaluated by a human uropathologist. Canine MIBC tumors were phenotypically characterized using either immunofluorescence (IF) or RNA in situ hybridization (RNA-ISH), including CD44, KRT7 and FOXA1, as well as specific markers characterizing the immune cell infiltrate, such as CD163 (tumor-associated macrophages), CD8a (cytotoxic T-lymphocytes), PD-L1 (immune-checkpoint protein) and TGFβ2 (cancer-associated fibroblasts). Results: Histologically, all canine MIBCs were classified as invasive, high-grade urothelial carcinomas (UC) with identical morphology to human MIBC. Interestingly, previously described histological variants of human MIBC tumors were also observed within the canine MIBC samples, such as glandular differentiation in 3/9 canine MIBC samples; epithelial-mesenchymal transition with sarcomatoid differentiation 1/9, squamous differentiation in 2/9 and micropapillary carcinoma in 1/9, all of which have been associated with poor clinical outcomes in human patients. IF was strongly positive for KRT7, FOXA1 and CD44, and immune cell markers CD163 and TGFβ2. However, CD8a was only weakly expressed (5/9) or negative, and PD-L1 was only positive in 4/9 canine MIBC biopsies. Conclusions: Canine MIBC tumors showed significant heterogeneity with highly similar histological variant morphologies to those reported in humans with MIBC. Furthermore, phenotypic characterization of immune cell infiltrates and checkpoint proteins suggest that the tumors were typically immunologically “cold”. These data further establish the utility of the spontaneous canine model of MIBC for pre-clinical investigation of novel therapeutic drug candidates, including immunotherapies. Citation Format: Karin Allenspach, John Cheville, Hannah Wickham, Christopher Zdyrski, Vojtech Gabriel, Basant Ahmed, Mei-Jyun Ciou, Fabrice Lucien, Igor Frank, Olufemi Fasina, Pablo Pineyro, Johnathan P. Mochel. Similar variant histology and immune cell infiltration in canine and human muscle-invasive bladder 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 2509.

Abstract 4141: Effects of exercise on tumorigenesis, immune system, and the gut microbiome in a bladder carcinogen mouse model

Abstract Recent studies have begun to demonstrate the therapeutic capacity of physical exercise on cancer treatment outcomes. While this research suggests exercise may improve health in cancer patients, the specific mechanisms by which exercise influences tumor progression remain largely unknown. We hypothesized that exercise alters the gut microbiome and promotes immune cell activation in relevant immune sites. Therefore, we utilized BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine), a carcinogen that mimics human muscle invasive bladder cancer conditions, and voluntary exercise wheels to determine the effect of exercise on bladder carcinogenesis, gut microbiome, and the immune system. Ultrasound imaging revealed that the average tumor volume of female exercising mice was 11.9% of the total bladder volume at time of sacrifice, while the tumor volume of their non-exercising counterparts was 56.4%, a difference determined to be significant using a two-tailed t-test with p-value of 0.05. A similar trend was seen in male mice, though the difference between groups was not significant. Immune cell analysis did not show significant variation in cytotoxic T cell, helper T cell, macrophage, or dendritic cell populations between exercising and non-exercising mice in spleen, colon, or bladder tissues as measured by FACS. Standardization of the amount exercised, as evaluated by expression of myosin and actin levels in the tibialis anterior muscle, and sequencing of the gut microbiome are in progress and could better indicate the mechanism of exercise-induced tumor suppression. Citation Format: Joseph P. LaMorte, Brittany Tian, Rashida Ginwala, Laura Bukavina, Philip Abbosh. Effects of exercise on tumorigenesis, immune system, and the gut microbiome in a bladder carcinogen mouse model. [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 4141.

Fibronectin-1: A Predictive Immunotherapy Response Biomarker for Muscle‑Invasive Bladder Cancer.

Muscle-invasive bladder cancer (MIBC) is characterized as bladder tumors that infiltrate into the muscle layer, along with multiple metastasis and poor prognosis. Numerous research studies have been performed to identify the underlying clinical and pathological alterations that occur. However, few studies have revealed the molecular mechanism of its progression based upon the immunotherapy response. Our present study was designed to identify biomarkers which may predict the immunotherapy response by investigating the tumor microenvironment (TME) in MIBC. The transcriptome and clinical data of MIBC patients were obtained and analyzed with R version 4.0.3 (POSIT Software, Boston, MA, USA) ESTIMATE package. Differentially expressed immune-related genes (DEIRGs) were identified and further analyzed via the protein-protein interaction network (PPI). Meanwhile, univariate Cox analysis was utilized to screen out the prognostic DEIRGs (PDEIRGs). Then, the PPI core gene was matched with PDEIRGs to obtain the target gene-fibronectin-1 (FN1). Human MIBC and control tissues were collected and FN1 was measured with Quantitative Reverse Transcription PCR (qRT-PCR) and Western-Blot. Finally, the relationship between FN1 expression level and MIBC was validated through survival, univariate Cox, multivariate Cox, Gene Set Enrichment Analysis (GSEA) and correlation analysis of tumor infiltrating immune cells. TME DEIRGs were identified and the target gene FN1 was obtained. The higher expression of FN1 was confirmed in MIBC tissues via bioinformatics analysis, qRT-PCR and Western-Blot. Moreover, higher FN1 expression correlated with reduced survival time and FN1 expression was further favorably correlated with clinic-pathological features (grade, TNM stage, invasion, lymphatic and distant metastasis). Additionally, the genes in the high FN1 expression group were mainly enriched in immune-related activities and macrophage M2, T cell CD4, T cell CD8 and T cell follicular helper cells were correlated with FN1. Finally, it was observed that FN1 was closely related to key immune checkpoints. FN1 was identified as a novel and independent prognostic factor for MIBC. Our data also suggests FN1 can predict MIBC patients' response to immune checkpoints inhibitors.

Structural or functional defects of PTEN in urothelial cells lacking P53 drive basal/squamous-subtype muscle-invasive bladder cancer

Muscle-invasive bladder cancer (MIBC) exhibits strong inter- and intra-tumor heterogeneity that affects biological behaviors, therapeutic responses, and prognoses. Mutations that activate RTK-RAS-PI3K and inactivate P19-P53-P21 coexist in 60–70% of MIBC. By time-controlled ablation of Tp53 and Pten, singly or combined, in adult mouse urothelium, we found that Tp53 loss alone produced no abnormality. While Pten loss elicited hyperplasia, it synergized with Tp53 loss to trigger 100% penetrant MIBC that exhibited basal/squamous features that resembled its human counterpart. Furthermore, PTEN was inactivated in human MIBC cell lines and specimens primarily by hyperphosphorylation of the C-terminus. Mutated or tailless PTEN incapable of C-terminal phosphorylation demonstrated increased inhibition of proliferation and invasion than full-length PTEN in cultured MIBC cells. In xenograft and transgenic mice, tailless PTEN, but not full-length PTEN, prevented further growth in established tumors. Collectively, deficiencies of both PTEN and P53 drive basal/squamous subtype MIBC. PTEN is inactivated by C-terminal hyperphosphorylation, and this modification may serve as a biomarker for subtyping MIBC and predicting tumor progression. Tailless PTEN is a potential molecular therapeutic for tumors, such as bladder cancer (BC), that can be readily accessed.

Loss of Cxcr2 in Myeloid Cells Promotes Tumour Progression and T Cell Infiltration in Invasive Bladder Cancer.

CXCR2 is a chemokine receptor expressed in myeloid cells, including neutrophils and macrophages. Pharmacological inhibition of CXCR2 has been shown to sensitize tumours to immune checkpoint inhibitor immunotherapies in some cancer types. To investigate the effects of CXCR2 loss in regulation of tumour-infiltrating myeloid cells and their relationship to lymphocytes during bladder tumorigenesis. Urothelial pathogenesis and immune contexture was investigated in an OH-BBN model of invasive bladder cancer with Cxcr2 deleted in myeloid cells (LysMCre Cxcr2 flox/flox ). CXCR2 gene alterations and expression in human muscle invasive bladder cancer were analysed in The Cancer Genome Atlas. Urothelial tumour pathogenesis was significantly increased upon Cxcr2 deletion compared to wildtype mice. This was associated with a suppression of myeloid cell infiltration in Cxcr2-deleted bladders shortly after the carcinogen induction. Interestingly, following a transient increase of macrophages at the outset of tumour formation, an increase in T cell infiltration was observed in Cxcr2-deleted tumours. The increased tumour burden in the Cxcr2-deleted bladder was largely independent of T cells and the status of immune suppression. The Cxcr2-deleted mouse model reflected the low CXCR2 mRNA range in human bladder cancer, which showed poor overall survival. In contrast to previous reports of increased CXCR2 signalling associated with disease progression and poor prognosis, CXCR2 was protective against bladder cancer during tumour initiation. This is likely due to a suppression of acute inflammation. The strategy for sensitizing checkpoint immunotherapy by CXCR2 inhibition in bladder cancer may benefit from an examination of immune suppressive status.

Abstract 6032: Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice

Abstract Despite recent advances in molecular profiling of bladder cancer, molecular mechanisms for urothelial carcinogenesis have not fully understood. It is partly because the lack of faithful disease models that accurately recapitulate human bladder cancer. In our previous study, we have analyzed mutation landscape of chemical-induced mouse bladder cancer and human bladder cancer by exome-sequence and found that some components of histone methyltransferase complex like Kmt2c, Kmt2d, Kdm6a were highly prevalent as common mutations and associated with Trp53. Additionally, the downregulation of tumor suppressor gene PTEN is known in human muscle invasive bladder cancer (MIBC). We also investigated cell of origin of chemical-induced mouse bladder cancer using lineage tracing method and found that Krt5 expressing cells with Trp53 alteration efficiently give a rise of high-grade MIBC with squamous differentiation. Based on these results, we have tried to make MIBC mouse model using genetically engineered mouse in which Cas9 and target gene mutation express specifically in Krt5 expressing cells by Cre-LoxP system, combined with CRISPR/Cas9 gene editing technique and 3D organoid culture system. Eight-week-old mice harboring Krt5CreERT2/+; LSL-Cas9; LSL-Trp53+/R172H were used in this study. After introducing gene recombination by administration of tamoxifen, we generated organoids from bladder epithelial cells. For further gene editing, we infected adeno-associated virus with sgPten and sgMll3 to the organoids, which successfully edited the target genes. There seemed to be no significant differences in growth of the organoids in culture between the wild-type and gene-edited organoids. However, gene-edited organoids showed tumorigenesis in vivo when inoculated in subcutaneous space, renal subcapsular space or bladder submucosa of athymic mice, whereas wild-type organoids did not show tumorigenesis in vivo. Histologically, the organoid-derived tumors showed morphological similarity to human urothelial carcinoma with squamous differentiation and intra-tumoral heterogeneity. Furthermore, tumor derived organoid has a potential of tumor development in immune-competent mice. These tumors also maintain squamous phenotype and tumor heterogeneity. These findings suggest that the technique is a promising approach toward the establishment of a useful model of human bladder cancer for the elucidation of bladder carcinogenesis including driver genes. In addition, we think our syngeneic mouse model may be useful tool for further research of tumor immunity and immune checkpoint inhibitor drugs. Citation Format: Akihiro Hamada, Yuki Kita, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Takeshi Sano, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6032.

Abstract 5285: Antitumor and mechanism of selective serotonin reuptake inhibitor fluoxetine on muscle invasive bladder cancer

Abstract Purpose/Introduction: Bladder cancer can be broadly divided into two type, including non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). Among them, MIBC has been recognized as hard to treatment type of cancer for many decades. Therefore, to develop a new treatment strategy for MIBC is urgently needed. Transcription factor nuclear factor-kappaB (NF-κB) plays important role in various cancer progression, including hepatocellular carcinoma (HCC), lung adenocarcinoma (LUAD), and MIBC. Thus, NF-κB may be a potential target to suppress the progression of MIBC. Fluoxetine is FDA approval antidepressant agent, belongs to selective serotonin reuptake inhibitor (SSRI). Interestingly, recent study indicated that Fluoxetine inhibited NF-κB and induced apoptosis in the HCC both in vivo and in vitro. However, whether Fluoxetine may regulate NF-κB and inhibit MIBC tumor progression is remaining unknown. Therefore, the aim of present study is to verify the treatment efficacy and underlying mechanism of fluoxetine on MIBC. Methods: Human MIBC cells TSGH8301 and T24 cells and mice bearing with MIBC MB49 cells was used in this study. Cytotoxicity, apoptosis, and metastasis ability of cells after fluoxetine treatment was validated through MTT assay, cleaved caspase-3, -8, -9 activation using flow cytometry and transwell migration/invasion assay, respectively. In in vivo study murine MB49 cells were subcutaneous inoculated into right flank of C57B6/J mice and treated with fluoxetine for 15 days. Result: Fluoxetine markedly induced cytotoxicity, cleaved caspase-3, -8, -9 expression, and inhibited migration and invasion in both TSGH8301 and T24 cells. Western blotting and reporter gene assay indicated that fluoxetine may suppress the phosphorylation and activity of NF-κB. In in vivo results, fluoxetine inhibited tumor growth and the expression of NF-κB within tumor. Tumor immunohistochemistry (IHC) staining results proved that the apoptosis marker such as Caspase-3, -8, -9 were all increased by fluoxetine. AST and ALT value that represent as liver function showed no significant difference between vehicle group and fluoxetine treatment group. Body weight and H&E results also proved the safety of fluoxetine usage. Conclusion: Fluoxetine induced cytotoxicity via both extrinsic/intrinsic apoptotic pathway and inhibited metastasis ability as well. Fluoxetine may also suppress the phosphorylation and activity NF-κB in vitro and in vivo. In sum, we suggested that fluoxetine may effectively suppress tumor progression via inactivate NF-κB signaling pathway. Citation Format: Zhao-Lin Tan, Yuan Chang, I-Tsang Chiang, Fei-Ting Hsu, Fei-Ting Hsu. Antitumor and mechanism of selective serotonin reuptake inhibitor fluoxetine on muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5285.

Abstract 3092: Urine-derived urinary carcinoma organoids: A novel tool for providing new insights into human and canine bladder cancer treatment

Abstract The outcome of patients with Muscle Invasive Bladder Cancer (MIBC) is highly variable with many patients experiencing progressive disease despite multimodal therapy. A key variable that contributes to poor patient outcomes lies in our inability to predict therapeutic response due to the molecular heterogeneity of MIBC tumors. There is, therefore, a significant unmet clinical need to accurately predict effective drug combinations and therapeutic doses in individual patients with MIBC. Patient-derived tumor organoids (PDOs) have the potential to serve as an ex vivo model of urothelial carcinoma (UC) capable of functionally predicting patient-specific treatment responses. However, current culture procedures typically rely on the expansion of UC organoids from resected tumors or biopsy specimens, which is inherently invasive and hence limits clinical application. We hypothesized that culture of MIBC PDOs can be achieved from urine samples, as is the case in dogs with MIBC, since histopathological features, tumor heterogeneity and subtypes, metastatic activity, and gene expression profiles of canine MIBC mimic those of human MIBC. Herein, we successfully cultured MIBC organoids from both human and canine patients using first-catch urine. Briefly, after centrifuging urine samples to pellet cells, the cells were washed with basal culture media containing FBS and Pen/Strep before being cultured in canine or human culture media containing essential growth factors based on their species of origin. Paraffin-embedded samples were used for molecular characterization by RNA in situ hybridization or immunohistochemistry. Organoids from canine MIBC patients were analyzed for expression of the urothelial differentiation marker (FOXA1) associated with high grade bladder cancer, CD44, a non-kinase transmembrane glycoprotein expressed in UC cancer stem cells, and CK7, an epithelial cell marker that is upregulated in neoplastic UC cells. FOXA1 expression was undetectable in canine MIBC, although CD44 expression was enhanced compared to healthy dog bladder organoids. All canine MIBC organoids had significantly enhanced CK7 expression. Similarly, in human-derived UC organoids, GATA binding protein 3 (GATA3), transcription factor paired box 8 (Pax8), and tumor protein p63 were utilized as markers, with a Pax8-/p63+/GATA3+ profile indicating UC origin of the organoids. Functional response to chemotherapy (cisplatin and gemcitabine) was further assessed using cell viability assay in canine and human UC organoids. Our data suggest that organoids cultured from the urine of patients with MIBC can recapitulate organ structures and maintain marker expression profiles of the original tumor tissues. 3D organoids may provide novel insights to predict therapeutic response to chemotherapy and serve as early diagnostic biomarkers of bladder cancer in both canine and human patients. Citation Format: Dipak Kumar Sahoo, Allison P. Mosichuk, Fabrice Lucien, Igor Frank, John C. Cheville, Karin Allenspach, Jonathan P. Mochel. Urine-derived urinary carcinoma organoids: A novel tool for providing new insights into human and canine bladder cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3092.

A Preclinical Study to Repurpose Spironolactone for Enhancing Chemotherapy Response in Bladder Cancer.

Neoadjuvant chemotherapy (NAC) followed by radical cystectomy is the standard-of-care for patients with muscle-invasive bladder cancer (MIBC). Defects in nucleotide excision repair (NER) are associated with improved responses to NAC. Excision Repair Cross-Complementation group 3 (ERCC3) is a key component of NER process. No NER inhibitors are available for treating patients with bladder cancer. We have developed an ex vivo cell-based assay of 6-4 pyrimidine-pyrimidinone (6-4PP) removal as a surrogate measure of NER capacity in human bladder cancer cell lines. The protein expression of ERCC3 was examined in human MIBC specimens and cell lines. Small molecule inhibitors were screened for NER inhibition in bladder cancer cell lines. Spironolactone was identified as a potent NER inhibitor. Combined effects of spironolactone with chemo-drugs were evaluated in vitro and in vivo. The efficacy between platinum and spironolactone on cytotoxicity was determined by combination index. A correlation between NER capacity and cisplatin sensitivity was demonstrated in a series of bladder cancer cell lines. Further, siRNA-mediated knockdown of ERCC3 abrogated NER capacity and enhanced cisplatin cytotoxicity. Spironolactone inhibited ERCC3 protein expression, abrogated NER capacity, and increased platinum-induced cytotoxicity in bladder cancer cells in vivo and in patient-derived organoids. Moreover, spironolactone exhibited the potential synergism effects with other clinical chemotherapy regimens in bladder cancer cell lines. Our data support the notion of repurposing spironolactone for improving the chemotherapy response of NAC in patients with MIBC. Further clinical trials are warranted to determine the safety and efficacy of spironolactone in combination with chemotherapy.

High ORAI3 expression correlates with good prognosis in human muscle-invasive bladder cancer

The involvement of store-operated calcium channels (SOCCs) in tumor initiation and metastatic dissemination has been extensively studied, but how its member ORAI3 influences tumor progression is still elusive. The present study aimed to evaluate the prognostic value of ORAI3 expression and examine the correlation between ORAI3 expression and immune cell infiltration within the tumor microenvironment (TME) in human muscle-invasive bladder cancer (MIBC). We examined the expression profile of ORAI3 in MIBC using data from two databases; analyzed the correlation between ORAI3 expression and patient survival; explored cellular pathways related to ORAI3 expression by Gene Set Enrichment Analysis (GSEA); and predicted potential drugs using Connectivity Map (CMap). ORAI3 was significantly lower expressed in tumor mass compared to normal samples in MIBC, with a higher level of methylation at the promoter region in tumor than in normal tissue, indicating that ORAI3 is suppressed during cancer progression. Survival analysis showed that higher expression of ORAI3 correlated with good prognosis in MIBC. GSEA demonstrated that ORAI3 expression inversely correlated with cell differentiation, development and gene silencing, with differential expression of genes involved in epidermal and keratinocyte differentiation pathways and inflammatory responses. RNA sequencing of an ORAI3-silenced human bladder cancer cell line (T24 cells) corroborated enhancement of pro-neoplastic pathways in absence of ORAI3. Western blottingMoreover, ORAI3 facilitated the recruitment of Th17 cells and natural killer cells, whereas hampered Th2 and macrophage infiltration. Our results revealed 4 molecules with potential to be beneficial as adjuvant drugs in MIBC treatment. We concluded that high ORAI3 expression correlates with increased survival in human MIBC.

The effect of hypoxia on PD-L1 expression in bladder cancer

IntroductionRecent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease.Materials and methodsThree human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA.ResultsWhen T24 cells were seeded at < 70% confluence, there was decreased PD-L1 protein (p = 0.009) and mRNA (p < 0.001) expression after culture in 0.1% oxygen. PD-L1 protein expression decreased in both 0.1% oxygen and 1% oxygen in a panel of muscle-invasive bladder cancer cells: T24 (p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p < 0.001) and mRNA (p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p < 0.001) cohorts, and between hypoxia and IFNγ signature scores (p < 0.001 for both).ConclusionTumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.

Detrimental Effect of Various Preparations of the Human Amniotic Membrane Homogenate on the 2D and 3D Bladder Cancer In vitro Models.

Despite being among the ten most common cancers with high recurrence rates worldwide, there have been no major breakthroughs in the standard treatment options for bladder cancer in recent years. The use of a human amniotic membrane (hAM) to treat cancer is one of the promising ideas that have emerged in recent years. This study aimed to investigate the anticancer activity of hAM homogenate on 2D and 3D cancer models. We evaluated the effects of hAM homogenates on the human muscle invasive bladder cancer urothelial (T24) cells, papillary cancer urothelial (RT4) cells and normal porcine urothelial (NPU) cells as well as on human mammary gland non-tumorigenic (MCF10a) cells and low-metastatic breast cancer (MCF7) cells. After 24 h, we observed a gradual detachment of cancerous cells from the culture surface, while the hAM homogenate did not affect the normal cells. The most pronounced effect hAM homogenate had on bladder cancer cells; however, the potency of their detachment was dependent on the treatment protocol and the preparation of hAM homogenate. We demonstrated that hAM homogenate significantly decreased the adhesion, growth, and proliferation of human bladder invasive and papillary cancer urothelial cells and did not affect normal urothelial cells even in 7-day treatment. By using light and electron microscopy we showed that hAM homogenate disrupted the architecture of 2D and 3D bladder cancer models. The information provided by our study highlights the detrimental effect of hAM homogenate on bladder cancer cells and strengthens the idea of the potential clinical application of hAM for bladder cancer treatment.

BBN-driven urinary bladder cancer mouse model.

Around 3% of new cancer diagnoses and 2% of all cancer deaths every year are caused by urinary bladder cancer (BC). This indicates a great need for intensive studying of BC by using different approaches including indispensable mice models. The most common preclinical mouse model of bladder carcinogenesis relies on the use of a nitrosamine compound, N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) which causes high-grade, invasive tumors in the urinary bladder. BBN-induced bladder cancer in mice recapitulates the histology and manifests genetic alterations similar to human muscle-invasive bladder cancer. Here we present a detailed protocol for the induction of BC in mice which is based on the administration of 0.05%-0.1% BBN in drinking water. Six-to-eight-week-old mice are treated orally with BBN for 12weeks and tumors are expected 8weeks after the termination of BBN regimen. Histopathologic examination of the lesions should be routinely assessed after hematoxylin and eosin staining by an experienced pathologist and it can vary from urothelial dysplasia to invasive bladder cancer with glandular and squamous divergent differentiation, the incidence of which might depend on the mouse strain, gender, BBN concentration and the timeline of the protocol. Utilizing half of the urinary bladder tissue for the isolation and analysis of RNA, DNA and proteins provides a comprehensive insight into the biology of BC and reduces the number of mice per study. Finally, the successful use of the BC model can facilitate fundamental biomedical discoveries leading to novel diagnostic and therapeutic approaches with clinical benefits.

Abstract A28: Deregulation of KMT2D promotes squamous differentiation in a mouse model of muscle-invasive bladder cancer

Abstract Cell differentiation and plasticity is precisely controlled by specific gene expression programs coordinate with chromatin remodeling, which are often deregulated during tumorigenesis. While divergent histopathologic differentiation in bladder cancer is prevalent and may affect clinical outcome, the underlying mechanisms are not well understood. Here, we aim to elucidate mechanisms for alteration of the histopathologic and molecular features of muscle-invasive bladder cancer (MIBC) by interrogating the functional role of chromatin-remodeling genes using relevant genetically engineered mouse models (GEMMs). High frequency of mutations in chromatin-remodeling genes encoding COMPASS-like chromatin-remodeling complex members has been identified in human MIBC. These genes include “writers” that create histone post-translational modification, such as the lysine methyltransferase KMT2D (MLL2/4), and “erasers” that remove these modifications, such as the lysine demethylase KDM6A (UTX). We have generated GEMMs that enable bladder-specific conditional inactivation of individual chromatin-remodeling genes in combination with loss of function of Trp53 and Pten, which leads to MIBC. Interestingly, bladder tumors from GEMMs having inactivation of Kmt2d along with Trp53 and Pten (e.g., Kmt2d;Trp53;Pten mice) develop a variant histologic phenotype that is not observed in tumors from either the Trp53;Pten or Kdm6a;Trp53;Pten mice. Specifically, these Kmt2d-inactivated tumors share histopathologic features in common with human urothelial carcinoma that display squamous differentiation. Furthermore, expression profiling of these Kmt2d;Trp53;Pten bladder tumors identified a unique set of differentially expressed genes that are enriched for genes representing squamous differentiation in other nonbladder cancers. Our findings suggest that deregulated Kmt2d expression promotes squamous differentiation in MIBC, at least in context of Trp53;pten coinactivation. Citation Format: Lijie Rong, Cory Abate-Shen. Deregulation of KMT2D promotes squamous differentiation in a mouse model of muscle-invasive 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 A28.