Canine Bladder Cancer Research Articles

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.

Characteristics and Applications of Canine In Vitro Models of Bladder Cancer in Veterinary Medicine: An Up-to-Date Mini Review.

Simple SummaryBladder cancer (BC) in dogs is often lethal at the time of diagnosis. Therefore, there is a constant need for novel research on improvements of its characterization and treatment. Due to high cost and limited number of available dog patients, in vitro models of canine BC have been increasingly used for the last 25 years. In the present article, we present existing in vitro models of canine BC, including available simple (two-dimensional) and more complex (three-dimensional) models.Bladder cancer (BC) constitutes approximately 2% of all spontaneously occurring cancers in dogs. It is characterized by a devastating clinical course in most cases, which emphasizes a constant need for the development of novel methods of disease characterization and treatment. Over the past years, advances in cell engineering have resulted in the development of various canine in vitro models of BC, emerging as complements for in vivo research. In this article, we aimed to review the available data on existing in vitro models of canine BC, focusing primarily on their characteristics, applications in veterinary medicine, as well as advantages and disadvantages. The most commonly used in vitro models of canine BC comprise immortalized cell lines grown as adherent monolayers. They provide an unlimited supply of research material, however, they do not faithfully reflect the conditions prevailing in vivo, since the spatial cellular interactions are lost. The importance of the three-dimensional (3D) features of solid tumors in relation to carcinogenesis or drug response process has resulted in the development of the first canine 3D models of BC available for in vitro research. So far, results obtained with in vitro and in vivo research should be interpreted together. With the constantly growing complexity of in vitro models of BC cancer, animal-based research might be reduced in the future.

RNA-seq of nine canine prostate cancer cell lines reveals diverse therapeutic target signatures

BackgroundCanine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount.MethodsThis study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin.ResultsIndependent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing.ConclusionComparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.

Combination therapy with cannabidiol and chemotherapeutics in canine urothelial carcinoma cells.

BackgroundCanine urothelial carcinoma is the most common form of canine bladder cancer. Treatment with chemotherapy has variable response rates leading to most dogs succumbing to their disease within a year. Cannabidiol is an emerging treatment within the field of oncology. In reported in vivo studies, cannabidiol has induced apoptosis, reduced cell migration, and acted as a chemotherapy sensitizer in various human tumor types. The aim of this study was to characterize the effects of cannabidiol on canine urothelial carcinoma cell viability and apoptosis as both a single agent and in combination with chemotherapy in vitro.ResultsCannabidiol reduced cell viability and induced apoptosis in canine urothelial cells as determined by crystal violet viability assay and annexin V/propidium iodide flow cytometry. Furthermore, combinations of cannabidiol with mitoxantrone and vinblastine chemotherapy yielded significantly reduced cell viability and increased apoptosis compared to single agent treatment alone. The drug interactions were deemed synergistic based on combination index calculations. Conversely, the combination of cannabidiol and carboplatin did not result in decreased cell viability and increased apoptosis compared to single agent treatment. Combination index calculations suggested an antagonistic interaction between these drugs. Finally, the combination of the non-steroidal anti-inflammatory drug piroxicam with cannabidiol did not significantly affect cell viability, although, some cell lines demonstrated decreased cell viability when mitoxantrone was combined with piroxicam.ConclusionsCannabidiol showed promising results as a single agent or in combination with mitoxantrone and vinblastine for treatment of canine urothelial carcinoma cells. Further studies are justified to investigate whether these results are translatable in vivo.

Characterizing the molecular and immune landscape of canine bladder cancer.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

Abstract PR05: Similarities in molecular subtypes and subtype immune patterns between naturally occurring canine and human invasive bladder cancer

Abstract Relevant animal models are needed to study treatment strategies related to the molecular subtypes of bladder cancer and the immune response between these subtypes. The purpose of the study was to characterize the molecular subtypes and immune features in naturally occurring canine invasive urothelial carcinoma (InvUC). Canine InvUC is already known to mimic the human condition in pathologic features, metastatic behavior, and chemotherapy response. Molecular subtyping could further increase the utilization of the canine model. RNA-seq data from canine InvUC (n=56) and normal canine bladder mucosa (n=4) were analyzed using Strand NGS (Strand Genomics, San Francisco, CA). Data were normalized (DESeq, TMM), and DEGs selected (2FC, pcorr <0.05) using DESeq2 and edge R, respectively. Following unsupervised clustering that identified two groups of tumors, supervised clustering was performed in which the data were interrogated using a panel of genes known to classify human bladder cancer subtypes (TCGA), as well as multiple gene panels that classify human bladder cancer as immune hot (infiltrated) or immune cold (noninfiltrated, immune-suppressor features). Infiltrating immune cells were observed in histologic sections with CD3 immunohistochemistry. Distinct luminal (n=28) and basal (n=28) subtypes were identified in the canine InvUC samples, with subclassifications emerging in both subtypes. There was anticipated heterogeneity between the samples, but of the 28 basal tumors, all had hot immune features, with 10 being exceptionally hot. Of the luminal tumors, 22 of 28 were notably immune cold. In conclusion, canine InvUC mimics human invasive bladder cancer in regard to molecular subtypes and immune patterns within subtypes. This expands the value of the canine bladder cancer model and offers opportunities to study strategies for individualized therapy and to develop strategies to enhance the tumor immune environment in order to improve treatment outcomes. Support: NIH/NCI P30CA023168 Supp (Knapp, Ratliff). This abstract is also being presented as Poster A26. Citation Format: Deepika Dhawan, Breann C. Sommer, José A. Ramos-Vara, Noah M. Hahn, Deborah W. Knapp. Similarities in molecular subtypes and subtype immune patterns between naturally occurring canine and human 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 PR05.

Characterizing the Molecular and Immune Landscape of Canine Bladder Cancer

Transitional cell carcinoma (TCC) is the most common bladder cancer in both humans and dogs. The majority of canine TCCs are invasive tumors of intermediate‐ to high‐grade at diagnosis and result in a median survival of less than a year. Approximately 20% of human TCCs are muscle‐invasive tumors, which frequently metastasize and have a poor prognosis. Human and canine TCCs share similarities in risk factors, histopathology, and sites of metastasis. Thus, naturally‐occurring canine TCC has the potential to serve as a model for the assessment of novel therapeutics which may inform treatment modalities in human TCC.In this study, we performed whole exome sequencing of 11 canine TCC tumors and three matched normal tissue samples, identifying 581 variants in known protein coding genes. Protein coding variants showed enrichment of KEGG pathways involved in PI3K‐Akt signaling, axon guidance, focal adhesion, and chemokine signaling. Variants were screened against the COSMIC Cancer Gene Census, identifying 37 variants in 27 cancer‐related genes. BRAF V to E missense mutations were identified in 4/11 samples and later confirmed via Sanger sequencing in 73% of samples (8/11). Other cancer genes mutated in at least 2 samples include LRP1B, CUL3, MSH2, and RNF213. Microarray analysis was utilized to characterize the transcriptome of canine TCC relative to normal bladder. 802 and 1,327 genes were up‐ and down‐regulated in tumor samples, respectively. Up‐regulated genes were enriched for cell cycle, p53 signaling, and metabolic KEGG pathways. Down‐regulated genes include members of focal adhesion, ECM‐receptor interaction, and PI3K‐Akt signaling pathways. Gene set enrichment analysis was performed, identifying four hallmark gene sets enriched in tumors: E2F targets, G2M checkpoint, mitotic spindle, and interferon alpha response.A major advantage of the dog model is the ability to evaluate novel immunotherapies in spontaneous tumors that develop under normal immunosurveilance. However, a basic understanding of the immune landscape of canine cancers, such as TCC, is required. Using immunohistochemical labeling, we quantified CD3+ T cells and MAC387+ myeloid cells in TCC tumors. CD3+ T cell counts were variable, exhibiting a bimodal distribution, ranging from 1 to 415 cells/mm2, suggesting immunologically “hot” and “cold” tumors. MAC387+ cell counts ranged from 3 to 613 cells/mm2 and did not correlate with CD3+ T cell counts. Clustering of tumors using expression of immune‐related genes (GO:0002376) revealed two distinct groups. Group I exhibited a strong type I interferon gene expression signature, showing enrichment of hallmark gene sets for interferon alpha and gamma responses. Group II exhibited enrichment of hallmark gene sets for G2M checkpoint and E2F targets. The type I interferon signature was applied to human muscle‐invasive bladder tumors in the TCGA database and was associated with a better disease‐free survival. Overall, the results of this study establish the utility of canine bladder cancer as a model for identifying novel therapeutics, specifically immunotherapies, for the treatment of human muscle‐invasive bladder cancer.Support or Funding InformationMorris Animal Foundation and Shipley Foundation

A novel, safe, fast and efficient treatment for Her2-positive and negative bladder cancer utilizing an EGF-anthrax toxin chimera.

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC50 < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF-anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.

An ultrasound based platform for image-guided radiotherapy in canine bladder cancer patients.

Background and purposeUltrasound (US) is a non-invasive, non-radiographic imaging technique with high spatial and temporal resolution that can be used for localizing soft-tissue structures and tumors in real-time during radiotherapy (RT) (inter- and intra-fraction). A comprehensive approach incorporating an in-house 3D-US system within RT is presented. This system is easier to adopt into existing treatment protocols than current US based systems, with the aim of providing millimeter intra-fraction alignment errors and sensitivity to track intra-fraction bladder movement.Materials and methodsAn in-house integrated US manipulator and platform was designed to relate the computed tomographic (CT) scanner, 3D-US and linear accelerator coordinate systems. An agar-based phantom with measured speed of sound and densities consistent with tissues surrounding the bladder was rotated (0–45°) and translated (up to 55 mm) relative to the US and CT coordinate systems to validate this device. After acquiring and integrating CT and US images into the treatment planning system, US-to-US and US-to-CT images were co-registered to re-align the phantom relative to the linear accelerator.ResultsStatistical errors from US-to-US registrations for various patient orientations ranged from 0.1 to 1.7 mm for x, y, and z translation components, and 0.0–1.1° for rotational components. Statistical errors from US-to-CT registrations were 0.3–1.2 mm for the x, y and z translational components and 0.1–2.5° for the rotational components.ConclusionsAn ultrasound-based platform was designed, constructed and tested on a CT/US tissue-equivalent phantom to track bladder displacement with a statistical uncertainty to correct and track inter- and intra-fractional displacements of the bladder during radiation treatments.

New Therapeutic Perspective for Bladder Cancer in Dogs: Toxicological and Clinical Effects of OncoTherad Nanostructured Immunotherapy

Bladder cancer (BC) comprehends around 2% of all spontaneously occurring cancers in dogs. BC treatment remains a challenge since recurrence and progression of the disease, as well as the pronounced side effects associated to the available therapeutic modalities are present. In this scenario, a new perspective is represented by OncoTherad nanostructured immunomodulator. Thus, the aims of this study were to characterize and to evaluate the efficacy and possible toxicological effects of OncoTherad intravesical immunotherapy in 6 dogs with BC. Our results demonstrated that before the first instillation of OncoTherad, all dogs presented irregular tumor mass, mixed echogenicity, and hyperechoic echotexture, with a mean tumor volume of 9.38 cm3. After 6 instillations of OncoTherad, the tumor mass reduced 62.34% of its volume in relation to the initial ultrasound. At the end of 24 instillations, the tumor mass reduced 84.54% of its volume. Hematuria was decreased throughout OncoTherad treatment, disappearing on average after the eighteenth application and not returning after the last application. OncoTherad treatment showed no signs of systemic toxicity at the proposed therapeutic dose. In conclusion, OncoTherad intravesical immunotherapy seems a safe and effective treatment option for spontaneous canine bladder cancer and may provide benefit for preventing tumor recurrence.

Detection of tyrosine kinase inhibitors-induced COX-2 expression in bladder cancer by fluorocoxib A.

Among challenges of targeted therapies is the activation of alternative pro-survival signaling pathways in cancer cells, resulting in an acquired drug resistance. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer cells, making it an attractive molecular target for the detection and treatment of cancer. Fluorocoxib A is an optical imaging agent that selectively targets COX-2. In this study, we evaluated the ability of fluorocoxib A to monitor the responses of bladder cancer to targeted therapies in vivo. The effects of several tyrosine kinase inhibitors (TKIs: axitinib, AB1010, toceranib, imatinib, erlotinib, gefitinib, imatinib, sorafenib, vandetanib, SP600125, UO126, and AZD 5438) on COX-2 expression were validated in ten human and canine bladder cancer cell lines (J82, RT4, T24, UM-UC-3, 5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#5Lilly) in vitro. The effects of TKIs on bladder cancer in vivo were evaluated using the COX-2-expressing K9TCC#5Lilly xenograft mouse model and detected by fluorocoxib A. The increased COX-2 expression was detected by all tested TKIs in at least one of the tested COX-2-expressing bladder cancer cell lines (5637, SW780, TCCSUP, K9TCC#1Lillie, K9TCC#2Dakota, and K9TCC#5Lilly) in vitro. In addition, fluorocoxib A uptake correlated with the AB1010- and imatinib-induced COX-2 expression in the K9TCC#5Lilly xenografts in vivo. In conclusion, these results indicate that fluorocoxib A could be used for the monitoring the early responses to targeted therapies in COX-2-expressing bladder cancer.

MP32-04 IMMUNE CHECKPOINT B7X (B7-H4/B7S1/VTCN1) IS HIGHLY EXPRESSED IN SPONTANEOUS CANINE BLADDER CANCER: THE FIRST REPORT AND ITS IMPLICATIONS IN A PRECLINICAL MODEL

INTRODUCTION AND OBJECTIVES:Canine bladder cancer provides a novel model system for the study of invasive human urothelial carcinoma. The immunobiology of canine tumors is largely unknown. We repor...

Targeting the ErbB/MAPK Signaling Cascade in Canine Bladder Cancer Cell Lines

Canine transitional cell carcinoma (TCC) of the bladder accounts for 2% of diagnosed canine cancers. Most TCCs are inoperable and unresponsive to traditional chemotherapy. Median survival time is typically less than a year for all treatments indicating a need for more effective therapies. Recent studies have discovered that approximately 70% of canine TCCs harbor mutations in the proto‐oncogene BRAF, a kinase involved in the mitogen‐activated protein kinase (MAPK) pathway that controls cell proliferation, differentiation, and apoptosis. BRAF mutations are present in several human cancers, most of which exhibit adaptive or intrinsic resistance to BRAF‐targeted inhibitors. The goals of this study were to (1) further characterize the role of mutant BRAF in canine TCC and (2) determine whether inhibition of the MAPK pathway alone or in combination with other gene targets may be an effective therapy for TCC treatment.Analysis of ERK1/2 phosphorylation following serum starvation indicates that TCC cell lines exhibit constitutive MAPK pathway activation independent of their BRAF mutation status. MAPK activity was further quantified using gene expression analysis of ten MAPK target genes, revealing that TCC cell lines have significantly higher MAPK pathway activity compared to other canine cancer cell lines. These data suggest a causative role for MAPK signaling in TCC pathogenesis. To determine whether the MAPK pathway could be a therapeutic target for TCC treatment we assessed the effect of BRAF and MEK inhibition on TCC cell proliferation and ERK1/2 phosphorylation. Four BRAF mutant human cell lines with varying degrees of sensitivity to BRAF‐targeted agents were used to determine the relative sensitivity of canine TCC cell lines. BRAF mutant TCC cell lines were sensitive to BRAF inhibition with the “paradox‐breaking” inhibitor PLX7904 (IC50: 0.2–1.2μM), but not vemurafenib (IC50: 7–21μM). Both BRAF wild type and mutant TCC cell lines were sensitive to MEK inhibition with selumetinib (IC50: 15–420nM) and trametinib (IC50: 0.4–8nM). ERK1/2 phosphorylation decreased after 6‐hour treatments with MAPK inhibitors, but rebounded by 24 hours suggesting the presence of resistance mechanisms. Microarray analysis indicated that the ErbB family of receptors and ligands are up‐regulated in TCC cell lines relative to other canine cancer cell lines (fold‐change &gt; 2, q &lt; 0.05). Combined BRAF and ErbB inhibition synergized in the BRAF mutant Bliley TCC cell line, while combined MEK and ErbB inhibition synergized in both Bliley and BRAF wild type Kinsey cells. These findings suggest that targeting ErbB receptors with MAPK inhibition is a potential therapy for canine TCC treatment. Additionally, our data indicate that canine TCC may serve as a naturally‐occurring model for the study of resistance mechanisms to MAPK inhibition in human cancers.Support or Funding InformationMorris Animal Foundation, Shipley University Chair in Comparative OncologyThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immune Checkpoint B7x (B7-H4/B7S1/VTCN1) is Over Expressed in Spontaneous Canine Bladder Cancer: The First Report and its Implications in a Preclinical Model.

Background:B7x (B7-H4/B7S1/VTCN1), an inhibitory immune checkpoint molecule is a potential therapeutic target owing to its immunosuppressive effect and well-known expression in cancers. Immune checkpoints in canine bladder cancer are largely undefined. Here, we report the first evaluation on expression of B7x in spontaneous canine invasive bladder cancer, a novel model system for the study of invasive human urothelial carcinoma.Objective:This work aims to study expression of immune checkpoint B7x in spontaneous canine invasive bladder cancer.Methods:RNA-seq analysis was performed to determine B7x expression in tumor versus normal bladder. Gene ontology (GO) study was used to explore the biological role of B7x. B7x protein expression was evaluated by immunohistochemistry (IHC). TCGA and GTEx were used to examine B7x expression in 599 human bladder urothelial carcinoma (BLCA).Results:RNA-seq analysis indicated 5.72 and 7.04 fold up regulation of B7x in tumors, using DESeq2 and edge R respectively (p < 0.00008). B7x was closely associated with immune processes in GO analysis. IHC results revealed 60% of cases as B7x positive. B7x intensity was scored as negative in 40% (n = 20/50), low in 24% (n = 12/50), medium in 14% (n = 7/50) and high in 22% (n = 11/50) of cases. In human BLCA, B7x expression was significantly associated with worse overall survival (p = 0.02).Conclusions:Our results suggest that B7x is over expressed in canine bladder cancer. Thus canine model can be vital in advancing the translational research on B7x, a new potential therapeutic target in human bladder cancer.

Cross-species analysis of the canine and human bladder cancer transcriptome and exome.

We investigated the correspondence between transcriptome and exome alterations in canine bladder cancer and the correspondence between these alterations and cancer-driving genes and transcriptional alterations in human bladder cancer. We profiled canine bladder tumors using mRNA-seq and exome-seq in order to investigate the similarity of transcriptional alterations in bladder cancer, in humans and canines, at the levels of gene functions, pathways, and cytogenetic regions. We found that the transcriptomes of canine and human bladder cancer are remarkably similar at the functional and pathway levels. We demonstrated that canine bladder cancer involves coordinated differential expression of genes within cytogenetic bands, and that these patterns are consistent with those seen in human bladder cancer. We found that genes that are mutated in canine bladder cancer are more likely to be transcriptionally downregulated than non-mutated genes, in the tumor. Finally we report three novel mutations (FAM133B, RAB3GAP2, and ANKRD52) for canine bladder cancer.

Homologous Mutation to Human BRAF V600E Is Common in Naturally Occurring Canine Bladder Cancer—Evidence for a Relevant Model System and Urine-Based Diagnostic Test

Targeted cancer therapies offer great clinical promise, but treatment resistance is common, and basic research aimed at overcoming this challenge is limited by reduced genomic and biologic complexity in artificially induced rodent tumors compared with their human counterparts. Animal models that more faithfully recapitulate genotype-specific human pathology could improve the predictive value of these investigations. Here, a newly identified animal model for oncogenic BRAF-driven cancers is described. With 20,000 new cases in the United States each year, canine invasive transitional cell carcinoma of the bladder (InvTCC) is a common, naturally occurring malignancy that shares significant histologic, biologic, and clinical phenotypes with human muscle invasive bladder cancer. In order to identify somatic drivers of canine InvTCC, the complete transcriptome for multiple tumors was determined by RNAseq. All tumors harbored a somatic mutation that is homologous to the human BRAF(V600E) mutation, and an identical mutation was present in 87% of 62 additional canine InvTCC tumors. The mutation was also detectable in the urine sediments of all dogs tested with mutation-positive tumors. Functional experiments suggest that, like human tumors, canine activating BRAF mutations potently stimulate the MAPK pathway. Cell lines with the mutation have elevated levels of phosphorylated MEK, compared with a line with wild-type BRAF. This effect can be diminished through application of the BRAF(V600E) inhibitor vemurafenib. These findings set the stage for canine InvTCC as a powerful system to evaluate BRAF-targeted therapies, as well as therapies designed to overcome resistance, which could enhance treatment of both human and canine cancers This study demonstrates the activating BRAF mutation (V600E), which is found in multiple human cancers, is a driver of canine InvTCC, and highlights a urine-based test for quick diagnosis.

Highlights of This Issue

Studies have shown that nitric oxide (NO) has both protumor and antitumor growth properties. Rabender and colleagues demonstrate that NO synthase (NOS) in tumor cells is uncoupled and instead of generating NO it synthesizes superoxide and peroxynitrite. Furthermore, recoupling NOS with the BH4 precursor, sepiapterin, is cytotoxic to tumor cells. These findings shed light on the conflicting results of NO signaling and reveal a critical switching mechanism for tumor progression. Synthetic BH4 is used to treat some phenylketonuria patients and diseases associated with endothelial dysfunction, suggesting a testable approach for correcting an abnormality of tumor metabolism and controlling tumor growth.Oral squamous cell carcinoma (OSCC) is a widespread form of cancer with poor survival rates. Progress in treatment strategies has been limited, and thus understanding dysregulated signals contributing to OSCC is required. Hiemer and colleagues show that the transcriptional regulators YAP and TAZ, downstream effectors of the Hippo pathway, drive protumorigenic signals in OSCC in vitro and in vivo. Further, comparing the YAP/TAZ-regulated gene expression changes with those identified in OSCC tissues by The Cancer Genome Atlas (TCGA) demonstrated a significant contribution of nuclear YAP/TAZ activity to OSCC onset and progression. This study reveals novel insight into the dysregulated signals contributing to this poorly understood cancer.Canine invasive transitional cell carcinoma of the bladder (InvTCC) is a common, naturally occurring malignancy that shares histologic, biologic, and clinical characteristics with human invasive bladder cancers. Decker and colleagues observed that a large majority of tested canine InvTCC tumors are driven by a canonical activating BRAF (V600E) mutation. Administration of vemurafenib, a clinical-grade BRAF inhibitor, demonstrably reduced growth of canine bladder cancer cell lines, highlighting both the clinical and translational potential of this animal model for studies of human BRAF-dependent tumors. Finally, a urine-based diagnostic test was developed, based on the BRAF mutation, which is highly effective in dogs.MicroRNA-21 (miR-21) is upregulated in many human malignancies and often correlates with poor clinical outcomes. Wagenaar and colleagues detected significant overexpression of miR-21 in two large independent cohorts of patients with hepatocellular carcinoma (HCC) and in HCC cell lines. Using potent anti–miR-21 compounds and genetic tools, they determined that miR-21 inhibition results in profound deregulation of multiple prosurvival pathways and leads to the suppression of HCC growth in vitro and in vivo. These results establish a critical role for miR-21 in maintaining tumorigenic phenotype in HCC and suggest that miR-21 represents an important target for pharmacologic inhibition in liver cancer.

Animal model of naturally occurring bladder cancer: characterization of four new canine transitional cell carcinoma cell lines.

BackgroundDevelopment and further characterization of animal models for human cancers is important for the improvement of cancer detection and therapy. Canine bladder cancer closely resembles human bladder cancer in many aspects. In this study, we isolated and characterized four primary transitional cell carcinoma (K9TCC) cell lines to be used for future in vitro validation of novel therapeutic agents for bladder cancer.MethodsFour K9TCC cell lines were established from naturally-occurring canine bladder cancers obtained from four dogs. Cell proliferation rates of K9TCC cells in vitro were characterized by doubling time. The expression profile of cell-cycle proteins, cytokeratin, E-cadherin, COX-2, PDGFR, VEGFR, and EGFR were evaluated by immunocytochemistry (ICC) and Western blotting (WB) analysis and compared with established human bladder TCC cell lines, T24 and UMUC-3. All tested K9TCC cell lines were assessed for tumorigenic behavior using athymic mice in vivo.ResultsFour established K9TCC cell lines: K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#4Molly, and K9TCC#5Lilly were confirmed to have an epithelial-cell origin by morphology analysis, cytokeratin, and E-cadherin expressions. The tested K9TCC cells expressed UPIa (a specific marker of the urothelial cells), COX-2, PDGFR, and EGFR; however they lacked the expression of VEGFR. All tested K9TCC cell lines confirmed a tumorigenic behavior in athymic mice with 100% tumor incidence.ConclusionsThe established K9TCC cell lines (K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#4Molly, and K9TCC#5Lilly) can be further utilized to assist in development of new target-specific imaging and therapeutic agents for canine and human bladder cancer.

Naturally occurring diseases in animals: contributions to translational medicine.

T he natural world brings to us an array of opportunities to understand the processes of life and disease. Naturally occurring or spontaneous diseases of animals allow us to gain important and fundamental knowledge of human disease and translational medicine. This edition of the ILAR Journal brings together a number of reviews that enlighten us about naturally occurring diseases of animals. These reviews are written in context to lessons learned about disease processes and translating this knowledge to the treatment of human disease. Among these articles are well-documented examples of animal diseases with causes ranging from genetic to infectious. Importantly, these natural animal models provide insight into the pathogenesis of human maladies and offer unique perspectives of how this information can be applied to intervention strategies in people. Naturally occurring cancers in dogs provide interesting parallels in our understanding of human cancers. Dr. Alvarez’s article on naturally occurring canine cancers provides a current view of the advantage of studying canine cancer to understand the genetic basis of human cancers. Although mouse models are clearly a preferred model for genetic manipulation throughout biomedical science, the canine naturally selected model provides opportunities to study the causes of cancer in context of the same environment as their owners. Dogs provide a novel model in this setting, in part, because of purposeful genetic breeding that amplifies phenotypes pleasing to humans. The reduced generation time of dogs and recent knowledge of the canine genome have accelerated the usefulness of dogs as models of human cancer. The canine cancer model is further focused on in the review by Drs. Fenger, London, and Kisseberth, who document the parallels between canine osteosarcoma and sarcomas in children. Osteosarcoma is the most common malignant bone cancer in children and dogs. Although conventional treatments have greatly improved the survival of children and dogs suffering from osteosarcoma, new treatments are required to further extend the success of therapies against this aggressive cancer. The authors point out the important parallel clinical and biological features of human and canine osteosarcoma. At the molecular level, the canine cancer parallels what we know about pediatric osteosarcoma, making the dog model particularly relevant in the development of new targeted drugs against the cancer. This theme continues in the review by Dr. Knapp and colleagues of bladder cancer in dogs and humans. Urinary bladder cancer affects thousands of human and canine patients each year. Naturally occurring bladder cancer in dogs has many similarities to invasive bladder cancer in humans at the cellular, molecular, and biological levels, making this naturally occurring model attractive to study new forms of therapy against this deadly cancer. This review illustrates the value of combining knowledge about canine bladder cancer with that of induced rodent models to help guide translational approaches to understanding invasive urothelial carcinoma of humans. Bladder cancer studies in dogs have the potential to define heritable and environmental risks for the human disease. In addition, recent clinical trials in dogs suffering from transitional cell carcinoma serve the dual purpose to improve the lives of pet dogs and to inform human clinical trials. Drs. Davis and Ostrander provide an insightful overview of the value of understanding naturally occurring cancers in dogs from a genomics perspective. With more than 70 million pure and mixed breed dogs in the United States, combined with the fact that cancer is a leading cause of death in this species, the value of understanding the genetics of dog cancers is dramatic. As knowledge of the canine genome and of specific mutations in their cancers expands, so too have opportunities to clarify the underlying causes of cancer in people suffering similar cancer types. Improvements in the care of dogs have paralleled their owner’s willingness to seek out new treatments for dog cancers. Through the careful evaluation of breed tendencies, combined with surveys of specific genetic mutations, researchers have provided a new avenue to understand the causes of cancer in both humans and dogs. This review illustrates that genomic analysis in pet dogs may offer new insights Michael D. Lairmore, DVM, PhD, is Professor and Dean at the University of California-Davis, School of VeterinaryMedicine in Davis, California. Chand Khanna, DVM, PhD, is Head of the Tumor and Metastasis Biology Section and Senior Investigator at the Center for Cancer Research in Bethesda, Maryland. Address correspondence and reprint requests to Michael D. Lairmore University of California-Davis, School of Veterinary Medicine, One Shields Avenue, Davis, California 95616 or mdlairmore@ucdavis.edu

Urinary bladder cancer in dogs, a naturally occurring model for cancer biology and drug development.

Each year more than 65,000 people are diagnosed with urinary bladder cancer, and more than 14,000 people die from the disease in the United States. Studies in relevant animal models are essential to improve the management of bladder cancer. Naturally occurring bladder cancer in dogs very closely mimics human invasive bladder cancer, specifically high-grade invasive transitional cell carcinoma (TCC; also referred to as invasive urothelial carcinoma) in cellular and molecular features; biological behavior, including sites and frequency of metastasis; and response to therapy. Canine bladder cancer complements experimentally induced rodent tumors in regard to animal models of bladder cancer. Results of cellular and molecular studies and -omics analyses in dogs are expected to lead to improved detection of TCC and preneoplastic lesions, earlier intervention, better prediction of patient outcome, and more effective TCC management overall. Studies in dogs are being used to help define heritable risks (through very strong breed-associated risk) and environment risks and to evaluate prevention and treatment approaches that benefit humans as well as dogs. Clinical treatment trials in pet dogs with TCC are considered a win-win scenario by clinician scientists and pet owners. The individual dog benefits from effective treatment, the results are expected to help other dogs, and the findings are expected to ultimately help humans with TCC. This article provides an overview of canine TCC, a summary of the similarities and differences between canine and human invasive TCC, and examples of the types of valuable translational research that can be done using dogs with naturally occurring TCC.