Abstract
Objective
To evaluate the RNA and protein expression of hypoxia-inducible factor (HIF-1α) in canine urothelial carcinoma (UC) compared to normal canine urinary bladder tissue.
Methods
Dogs with normal urinary bladder tissue were enrolled at the time of euthanasia with the tissue obtained via necropsy within 1 hour after death. The high-grade UC tissue was collected via necropsy or cystoscopically utilizing a resectoscope. Dogs in the UC group were excluded if they were treated with chemotherapy or radiation therapy prior to tissue collection. Immunohistochemistry was performed on all tissues to evaluate intracytoplasmic and intranuclear immunoreactivity of HIF-1α using a semiquantitative immunoreactivity score (IRS). Ribonucleic acid sequencing was also performed to evaluate the expression of HIF-1α in normal urinary bladders and canine UC.
Results
10 dogs with high-grade UC and 10 dogs with normal urinary bladder tissue were enrolled. The median intracytoplasmic HIF-1α in the UC group was mild in intensity with a low percentage of positive cells (median IRS, 1; range, 0 to 2). The control dogs had similar intracytoplasmic HIF-1α expression (median IRS, 1; range, 0 to 1). The difference in RNA expression of HIF-1α between groups was not significant (1.3-fold change).
Conclusions
This study did not identify any differential RNA or protein expression of HIF-1α between normal urinary bladder tissue and UC in dogs.
Clinical Relevance
HIF-1α is not differentially expressed in canine UC, but further exploration is necessary to evaluate if other proteins associated with hypoxia and angiogenesis could play a role in tumor growth and chemotherapy resistance in canine UC.
Urinary bladder cancer accounts for approximately 1.5% to 2% of all reported malignancies in dogs, and more than 40,000 dogs are affected by this cancer annually.1 Urothelial carcinoma (UC) is the most common form of urinary bladder cancer in dogs.1,2 Systemic medical therapy with cyclooxygenase inhibitors and chemotherapy are the mainstays of treatment due to the tumor location not being amenable to surgical excision.1 Radiation therapy has also been used in the treatment of UC in dogs.3 Depending on tumor stage and treatment, the median survival for dogs with UC treated with medical therapy has ranged from 152 to 531 days across multiple studies.1 To aid in treatment selection and novel therapy development, there is interest in determining predictive factors associated with response to therapy and survival.
Tumor microenvironment plays an important role in supporting the proliferation of cancer cells and can mediate resistance to chemotherapy.4 In malignant tumors, rapid growth and recruitment of new blood vessels leads to hypoxic regions in the tumor, which in turn lead to activation of genes that enhance cell survival, namely the hypoxia-inducible factor 1α (HIF-1α).4,5 Increased tumor expression of HIF-1α has been negatively correlated with survival in certain tumors in people.6–12 Urothelial carcinoma with increased HIF-1α protein expression in people tends to be more aggressive and resistant to chemotherapy and is therefore associated with a shorter survival time.6–8,11 Additionally, serum HIF-1α was found to be associated with metastasis and primary tumor staging in people with UC.13 Conversely, another study14 identified that HIF-1α protein expression was inversely correlated with tumor stage and microvessel density.
Protein expression of HIF-1α is also altered in some canine cancers.15–18 There was a significantly higher expression of HIF-1α in canine mammary adenocarcinomas compared to adenomas, allowing for aid in the differentiation of tumor type, grade, and vascularity.15,16 Additionally, HIF-1α protein expression has been associated with high-grade osteosarcoma and shorter disease-free interval.17 To the investigators’ knowledge, no studies have evaluated the expression of HIF-1α in canine UC. If HIF-1α expression is increased in canine UC, this could offer a valuable translational research opportunity for investigating treatment response and survival in human muscle invasive UC.
In people, UC is a heterogenous disease, and prognostic features such as pathologic stage and grade fail to fully predict outcomes or tumor behavior.19 Currently, standard clinical staging using the Tumor, Node, Metastasis Classification has been correlated with prognosis for dogs with UC, and other prognostic information includes tumor location within the urinary bladder and heterogeneity of the tumor on ultrasound.20,21 There is interest in identifying other potential biomarkers for tumor classification. Hypoxia-inducible factor 1α has not been previously evaluated in dogs with UC and has the potential to aid in tumor characterization, prediction of therapeutic response, and estimation of prognosis or survival.
The aim of the present study was to evaluate HIF-1α in canine UC. Protein expression and differential genetic expression of HIF-1α in canine UC were compared to normal canine bladder tissue using immunohistochemistry (IHC) and RNA sequencing. We hypothesized that IHC expression of HIF-1α would be significantly increased in canine UC when compared to normal canine bladder tissue. We further hypothesized that genes encoding for HIF-1α would be upregulated in canine UC when compared to normal canine bladder tissue.
Methods
Recruitment of dogs
Dogs were prospectively enrolled into the study and stratified into 2 groups: UC and control dogs. Dogs were initially enrolled in the UC group if there was a urinary bladder or urethral mass that was suspected or confirmed to be UC, and continuation in the study depended on histopathologic and immunohistochemical confirmation once samples were collected. Dogs in the UC group were excluded if they had received cytotoxic chemotherapy or radiation therapy for this tumor prior to enrollment. Previous or current use of NSAID medications did not exclude patients from study participation. Age, breed, weight, current medications, and duration of clinical signs were obtained for all dogs in the UC group. Prior medical records were reviewed when available. To ensure adequate tissue collection, sample collection was performed via postmortem examination within 60 minutes of death or, if antemortem, by cystoscopy using a resectoscope.
Inclusion criteria for the control group included any dog over 8 years of age presenting for euthanasia to the study institution. Dogs were excluded from the control group if there were any reported lower urinary tract clinical signs (ie, polyuria, pollakiuria, stranguria, or hematuria) or history of lower urinary tract disease (ie, bacterial cystitis, prostatitis, or urocystoliths) within 30 days of presentation. Additionally, if after sample collection any urinary bladder pathology was identified on histopathology, this dog was subsequently excluded from the control group. Age, breed, weight, and reason for euthanasia were collected from all dogs in the control group. Current medications and previous medical records were reviewed for these dogs when available.
This study was exempt from a unique IACUC protocol as tissues were collected after the dogs died or when aliquots of tissues removed during diagnostic cystoscopy were performed as part of the standard of care. All owners of dogs enrolled in both groups gave written or witnessed verbal consent for the collection of samples through the respective hospital’s informed consent form associated with the proposed procedure (euthanasia or cystoscopy).
Sample collection
For samples collected in patients being euthanized, samples were collected within 60 minutes of death and prior to the body being placed in a freezer. To collect these samples, a traditional surgical approach to the urinary bladder was performed. The urinary bladder was palpated for any gross abnormalities. The urethra at the level of the pelvic brim was isolated and transected to allow removal of the entire bladder. A longitudinal incision was made through 1 side of the urinary bladder wall from the transected urethra to the bladder apex, with the bladder then fanned out to expose the bladder mucosa. In the UC group, the region of greatest gross tumor burden was visualized and divided into roughly 4 equal sections, ensuring that tumor was included in all sections. For normal bladder tissue, the entire bladder was divided into roughly 4 equal sections for collection.
Dogs in the UC group undergoing cystoscopy using a resectoscope were enrolled at Friendship Hospital for Animals (Washington, DC) and North Carolina State University Small Animal Hospital (Raleigh, NC). At both locations, the dogs with suspected UC underwent general anesthesia, and cystoscopy was performed in a routine manner. Once the mass was identified on cystoscopy via a resectoscope, the electrical wire loop with monopolar electrocautery was used to collect multiple sections of the tissue.
Approximately half of the tissue collected by either method was placed into 10% neutral-buffered formalin and stored at room temperature. The other half of the tissue was immersed in TRIzol (Invitrogen) in a tissue grinder to homogenize the samples and prepare for RNA extraction. After grinding the tissue, the tissue fragments in TRIzol were placed in a cryovial and stored at −80 °C. If samples were obtained from institutions other than the primary institution (Purdue University), all samples were shipped overnight with the TRIzol samples on dry ice. TRIzol samples were immediately stored at −80 °C upon arrival at the study institution.
Histology and IHC
Formalin-fixed samples were processed routinely and embedded in paraffin wax. Histopathology was performed by a single board-certified veterinary anatomic pathologist (MFS). Histopathology was used to confirm normal bladder tissue without evidence of cystitis in the control group as well as to confirm and grade of UC in the UC group. Urothelial carcinoma was further confirmed using uroplakin III IHC (Anti-Uroplakin III [AU1]; Progen), with uroplakin III–positive labeling confirmed by the same pathologist (MFS).
On all included tissues, IHC for HIF-1α was performed by the Purdue Histology Laboratory utilizing a primary antibody (Anti-HIF-1α antibody [H1alpha67]; Abcam) and using the protocol previously published for evaluating HIF-1α immunoreactivity in canine mammary tumors.16 The antibodies were utilized at different concentrations to optimize IHC results under the guidance of a board-certified veterinary anatomic pathologist (MFS). To quantify the IHC reaction, a semiquantitative immunoreactivity score (IRS) was used to evaluate the immunohistochemical labeling of canine UC and healthy urinary bladder tissue (Table 1). This IRS was adapted from the 1 previously used in canine mammary tumors.15 Scores were assigned based on the percentage of positive cells and intensity of labeling. The product of these 2 scores was used to assign an IRS of 0, 1, 2, or 3. These can be further interpreted as negative, low, intermediate, and high, respectively. Intracytoplasmic and intranuclear labeling were evaluated and scored using the IRS. A permeabilizing treatment of Triton X-100 (Sigma-Aldrich) was performed to allow for nuclear labeling to be assessed. Renal tubules and mammary carcinoma cells were utilized as positive controls. Negative reagent controls were performed on every tumor sample. Negative controls were performed by omitting only the primary antibody for HIF-1α.
A semiquantitative immunoreactivity score (IRS) was used to evaluate the immunohistochemical labeling of canine urothelial carcinoma (UC) and healthy urinary bladder tissue using a primary antibody against hypoxia-inducible factor (HIF-1α).
| POPC | IOL | Calculated IRS (IRS = POPC X IOL) |
|---|---|---|
| 0: No labeling | 0: No labeling | 0: Total of score of 0 |
| 1: ≤ 10% | 1: Mild labeling | 1: Total score ≤ 3 |
| 2: ≤ 50% | 2: Moderate labeling | 2: Total score ≤ 6 |
| 3: ≤ 80% | 3: Intense labeling | 3: Total score ≤ 12 |
| 4: > 80% |
IOL = Intensity of labeling. POPC = Percentage of positive cells.
Scores were assigned based on the POPC and IOL. The product of these 2 scores was used to assign an IRS of 0, 1, 2, or 3. These can be further interpreted as negative, low, intermediate, and high, respectively.
RNA sequencing
TRIzol samples were submitted in batch to Azenta Life Sciences. Next-generation RNA sequencing was performed with an output of 20 million copies. The RNA sequencing data were analyzed in collaboration with the Purdue University Werling Comparative Oncology Research Center. The genetic material was aligned with the current canine reference genome, CanFam4.0. In addition to evaluating HIF-1α expression, we used gene ontology (GO) analysis to evaluate gene groups involved in hypoxia response.
Statistical analysis
Descriptive statistics were utilized for evaluating signalment, method of sample collection, medications, and reason for euthanasia. A Fisher exact test was utilized to evaluate whether the IRS was significantly different between the control group and UC group. A P value of less than .05 was considered statistically significant. Ribonucleic acid sequencing analysis was performed using Strand NGS (Azenta Life Sciences) to detect differential gene expression by applying edge-R on trimmed mean of M values normalized data. The genetic material was normalized to CanFam4.0. A fold change ≥ 2 was considered significant (corrected P value < .05).
Results
Twelve dogs were initially enrolled in the control group. Two dogs were excluded due to cystitis noted on histopathology. The median age was 11 years old (range, 8 to 15 years). The dogs were euthanized for various reasons, including quality-of-life concerns (n = 4), inability to walk (n = 1), lymphoma (n = 1), neurologic disease (n = 1), respiratory distress (n = 1), hypercalcemia with weight loss (n = 1), and megaesophagus (n = 1). One patient died while hospitalized, whereas the remainder were euthanized. No dogs in the control group were reported to receive corticosteroids or NSAIDs.
Ten dogs were enrolled in the UC group. All were confirmed to have high-grade UC on histopathology with positive uroplakin-III immunoreactivity. The median age was 10 years old (range, 8 to 13 years). Six samples were collected via necropsy, whereas 4 were collected via cystoscopy with a resectoscope. Six of the 10 dogs in the UC group were receiving NSAIDs, including carprofen (n = 3), piroxicam (n = 2), and grapiprant (n = 1). Other medications given to dogs in the UC group included gabapentin (n = 2), trazodone (n = 1), prazosin (n = 1), lactulose (n = 1), and amoxicillin (n = 1). Signalment, type of NSAID prescribed, and method of collection for each dog in the UC group and control group can be found in Supplementary Table S1.
The median intracytoplasmic HIF-1α immunoreactivity was mild in both groups (Figure 1; Table 2). Both groups had a median IRS score of 1 (UC range, 0 to 1; control range, 0 to 2; P = .32). There was no intranuclear labeling in the UC group, whereas 1 dog had positive intranuclear labeling in the control group.
A and B—Examples of immunoreactivity score grading in urothelial carcinoma. A—Grade 1 (mild) cytoplasmic labeling. B—Grade 2 (moderate) cytoplasmic labeling. Scale bar = 50 μm.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0391
The intracytoplasmic IRSs for HIF-1α were assigned to each dog in the canine UC group and control group.
| UC group | Control group | ||
|---|---|---|---|
| Dog | Score | Dog | Score |
| UC1 | 1 | N2 | 1 |
| UC2 | 1 | N3 | 0 |
| UC3 | 0 | N4 | 1 |
| UC4 | 0 | N5 | 1 |
| UC5 | 1 | N6 | 1 |
| UC6 | 2 | N7 | 1 |
| UC7 | 2 | N8 | 1 |
| UC8 | 1 | N9 | 1 |
| UC9 | 0 | N11 | 1 |
| UC10 | 0 | N12 | 1 |
| Median: 1 | Median: 1 | ||
IRSs were assigned numbers between 0 and 3, in which the scores represent the product of the POPC and IOL. Intracytoplasmic IRS for HIF-1α was not significantly different between the UC group and the control group (P = .32).
Ribonucleic acid sequencing was performed to evaluate the expression of HIF-1α between the UC and control groups. The principal component analysis showed appropriate variation within the dataset of the 2 groups. There was more genetic variability identified within the UC group than the control group (Figures 2 and 3). Over 5,000 differentially expressed genes were identified when comparing groups. Specifically, HIF-1α RNA expression was not significantly different between groups (fold change, 1.3; corrected P = .19). Gene ontology analysis related to cellular response to hypoxia (GO:0071456) and general response to hypoxia (GO:0001666) showed 25 of the 49 genes and 22 of 62 genes in these group were differentially expressed, respectively, indicating that hypoxia genes other than HIF-1α may be playing a larger role in canine UC.
Principal component analysis showing RNA sequencing variance for the first and second principal components (PC1 and PC2, respectively) between control group and urothelial carcinoma group.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0391
Heat map representing differentially expressed genes between the control and urothelial carcinoma group. The scale for downregulated and upregulated genes is from −1 to 1.
Citation: American Journal of Veterinary Research 2025; 10.2460/ajvr.24.12.0391
Discussion
Tumors have hypoxic areas that require the activation of genes, such as HIF-1α, to enhance survival and allow for continued growth. Hypoxia-inducible factor 1α is a major transcriptional factor activated in some cancerous cells.5 The majority of human UC studies8–11 evaluating HIF-1α show that increased tumor expression is associated with shorter survival times and chemotherapy resistance. Canine UC is often utilized as an animal model for human invasive UC due to its similar histological morphology and biological behavior.22,23 However, in the present study, there was no difference in protein and RNA expression of HIF-1α between canine UC and normal urinary bladder tissue.
Notably, 1 study14 of UC in people identified an inverse relationship between tumor stage and grade in that lower HIF-1α IRSs were found in higher-grade tumors. This study by Fus et al,14 compared to other studies of UC in people, had a more equitable distribution of tumor stages and grades. Other studies8–10 that identified a more linear relationship between HIF-1α expression and UC focused primarily on low-grade tumors. Fus et al14 theorized that the inverse relationship identified in their population of high-grade UC tumors was that HIF-1α may not be necessary for the urothelial cells to acquire a more aggressive phenotype or that there is a loss of HIF-1α expression due to additional genetic alterations associated with progression of the disease.14 In the present study, all dogs in the UC group had high-grade UC. There were no dogs with low-grade UC. The lack of difference in HIF-1α expression between control and UC tissue may indicate that HIF-1α does not contribute meaningfully to canine UC developing an aggressive phenotype. However, a larger sample size consisting of canine UC of varying grades will be important to determine if HIF-1α or angiogenic factors may have a role in tumor growth or progression.
The present study may have also benefited from standardizing the approach to sample collection. The duration and severity of clinical signs varied in the UC group due to the combination of samples collected post- and antemortem. Six of the 10 dogs had samples collected at the time of euthanasia, which indicates more severe clinical signs and advanced stage of disease, whereas 4 of the 10 dogs had samples collected via cystoscopy at the time of diagnosis of their disease. While this variability is representative of a clinical environment, the combination of tissues collected at the time of diagnosis versus the time of death may represent different tumor microenvironments due to disease progression, which could be more meaningfully investigated with a larger-scale study and more standardized inclusion criteria.
Nonsteroidal anti-inflammatory drug therapy in dogs with UC could have impacted HIF-1α expression. Nonsteroidal anti-inflammatory drugs are an accessible treatment in veterinary medicine for dogs with suspected or confirmed UC. Nonsteroidal anti-inflammatory drug use in this study did not exclude the enrollment of dogs with UC due to postmortem collection being the most necessary to obtain sufficient tissue for assessment and the challenges and ethical implications of identifying treatment-naïve dogs at the time of euthanasia. Seven of 10 dogs in the UC group were receiving NSAIDs at the time of tissue collection. Nonsteroidal anti-inflammatory drugs can inhibit angiogenesis by reducing the expression of VEGF, leading to decreased HIF-1α expression.24–26 In dogs not receiving NSAIDs in the UC group (n = 3 of 10), the intracytoplasmic IRS ranged from low to intermediate, with a median value of 2 (range, 1 to 2). In contrast, dogs receiving NSAIDs had a low intracytoplasmic IRS, with a median value of 1 (range, 0 to 1). The low sample size in each group prevented further statistical comparison of these 2 groups. Further studies with a larger sample size that includes more treatment-naïve dogs could evaluate the effect on NSAIDs on HIF-1α and angiogenesis in dogs with UC.
Only 1 dog in the control group had intranuclear immunoreactivity for HIF-1α. Notably, this dog was euthanized due to a hemoabdomen secondary to a splenic mass. The hypoxia induced by the hemoabdomen could explain the positive intranuclear labeling for HIF-1α in this sample. No dogs in the UC group had intranuclear HIF-1α immunoreactivity. In the study15 evaluating HIF-1α in canine mammary tumors, intranuclear HIF-1α labeling was considered positive. Intracytoplasmic labeling was not considered significant in the mammary tumor study. Hypoxia-inducible factor 1α is a major transcriptional factor, with the nucleus being the main site of action. It is degraded in the cytoplasm in normal conditions. The activity and significance of intracytoplasmic HIF-1α labeling is unclear but may indicate decreased degradation of this protein.
Ribonucleic acid sequencing did not identify any difference in the genetic regulation of HIF-1α in canine UC compared to normal bladder tissue. This, along with the lack of positive protein expression by IHC, suggests that the role of HIF-1α in canine UC is minimal. However, there are a multitude of genes related to hypoxia that were not evaluated in this initial investigation. The scope of the current study was focused on HIF-1α due to extrapolation from other studies in people and dogs, but additional research is warranted to investigate other hypoxia-related pathways and biomarkers. These could include hypoxia-upregulated 1 gene and phosphatase and tensin homolog-induced kinase 1. Both of these genes have been found to be dysregulated in human bladder tumors. Given the similarities between human and canine UC, these genes or other hypoxia pathways may be playing a role in prognosis and chemotherapy resistance in canine UC.27–31 Further review of RNA sequencing, including GO analysis, would allow the evaluation of multiple markers involved in specific metabolic pathways, like those associated with hypoxia, angiogenesis, or cellular defense.
This study suggests that HIF-1α expression is not increased in canine UC. Based on tumor behavior, hypoxia is likely to still be a contributor to tumorigenesis and growth, so additional markers of hypoxia are warranted for future investigation.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors would like to acknowledge the Werling Comparative Oncology Research Center for their expertise in obtaining samples for immunohistochemistry and RNA sequencing. They would also like to acknowledge Dr. Eva Kao for her contributions to sample collections.
Disclosures
Dr. Moore is a member of the JAVMA Scientific Review Board, but was not involved in the editorial evaluation of or decision to accept this article for publication.
No AI-assisted technologies were used in the composition of this manuscript.
Funding
The authors received the Veterinary Clinical Sciences Faculty Pilot Research grant from Purdue University that allowed for financial support to perform and publish this research.
ORCID
N. H. Gibbs https://orcid.org/0000-0002-2780-9366
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