Abstract
Background/Aim: Nuclear matrix protein-22 (NMP-22) is widely used in human medicine as a prognostic and diagnostic tool for urothelial carcinoma (UC). In addition, the use of urinary exosomes as a liquid biopsy tool is emerging for the diagnosis of certain types of cancer in human medicine. This study aimed to investigate the change in urinary exosomal NMP-22 for the diagnosis of UC in dogs. Patients and Methods: Among canine patients who visited the veterinary hospital, urine was collected from those whose owners provided consent. A total of 23 dogs (UC group, n=6; control group, n=17) were included in the analysis. After exosomes were isolated from the urine, NMP-22 was measured using enzyme-linked immunosorbent assay. Results: In the UC group, the expression of NMP-22 in urinary exosomes was significantly higher than that in non-UC groups (p<0.0001). Conclusion: NMP-22 is significantly increased in exosomes in the urine of dogs diagnosed with UC, suggesting that urinary exosome NMP-22 can be considered as one of the liquid biopsy tools for diagnosing UC in dogs.
Urothelial carcinoma (UC), also known as transitional cell carcinoma (TCC) is the most common cancer of the canine urinary tract (1, 2). Clinical signs, radiographs, abdominal ultrasound, computed tomography (CT) scans, and cytology from urine sediment are widely used to diagnose UC (2, 3). Direct cytology of the mass is associated with the risk of seeding through the needle tract. Therefore, the gold standard for diagnosing UC is through solid tissue biopsy obtained by cystoscopy, surgery, or in some cases with a urinary catheter; however, it usually requires general anesthesia or sedation and is limited by several factors, such as the size of the tumor, operator-dependence, and invasiveness (2-5). To overcome these issues, minimally invasive liquid biopsies have emerged in recent studies and clinics (5).
Nuclear matrix protein-22 (NMP-22) is a nuclear protein, which is a urothelial specific knub interrelated marker and associated with chromatid regulation and cell separation during replication (6-8). NMP-22 is expressed in small amounts in healthy people but can be found in higher than normal amounts in the urine of patients with some types of cancer, including bladder cancer (6, 8, 9). Therefore, the NMP-22 bladder cancer enzyme-linked immunosorbent assay (ELISA) and NMP-22 BladderChek test (Alere Scarborough, Inc., Waltham, MA, USA) have been approved by the Food and Drug Administration for diagnosing bladder cancer and are now widely being used in human medicine (10). In addition, many studies (7, 11-13) have reported the diagnostic value of the NMP-22 test in detecting UC and its comparisons with other diagnostic methods have also been studied (14, 15).
Exosomes are membrane-bound extracellular vesicles (EVs) ranging in diameter from approximately 30 to 150 nm (16). Exosomes are found in almost all body fluids, such as urine, saliva, and blood (16-18). They contain nucleic acids, lipids, proteins, and metabolites, reflecting the cell type of origin and the altered physiological and pathological states of their parental cell (17, 19, 20). In addition, exosomes are stable because of their lipid bilayer and resistant to enzymes and urinary acidity (5, 21). Therefore, exosomes are a potential diagnostic sample for liquid biopsies, and the use of urinary exosomes as diagnostic and prognostic biomarkers of UC is currently being investigated (17, 21-23). However, research on this topic in veterinary medicine is limited, and additional research is required for the use of urinary exosomes for diagnostic purposes in companion animals with UC.
In this study, we aimed to investigate the clinical usefulness of urinary exosomal NMP-22 for the diagnosis of UC in dogs.
Patients and Methods
Patient selection and sample collection. This study was conducted at the Veterinary Medical Teaching Hospital of the College of Veterinary Medicine, Seoul National University, in 2022. Twenty-nine client-owned dogs of various breeds, age, and sex were selected for this study. The patients visited the hospital for blood donation, diagnosis, and regular health checkups. This study was approved by the Institutional Animal Care and Use Committee (IACUC) of the SNU (approval number SNU-220816-2).
Voided mid-stream urine samples were collected in sterile urine cups and handled with sterile gloves. Urine specific gravity (USG), dip sticks, and cytology were performed on all urine samples. Samples with evidence of bacterial cystitis, proteinuria, leukocyturia, or hematuria were excluded.
Collected urine samples were centrifuged at 200 × g for 15 min at 25°C, and the supernatant urine was collected. Some supernatant urine samples were stored at −80°C and defrosted before ELISA analysis.
Isolation of exosomes. The other supernatant urine samples were transferred to a sterile tube, and the appropriate volume of ExoQuick-CG Exosome Precipitation Solution (System Biosciences, Palo Alto, CA, USA) was added in strict accordance with the manufacturer’s instructions and mixed well by inverting the tube. Samples were then refrigerated at 4°C at least 12 h for incubation, and the tubes were not rotated or handled during the incubation period. All samples were incubated for the same time (15 h) and exosomes were extracted according to the manufacturer’s instructions.
Biomarker measurements. NMP-22 levels were measured using a canine NMP-22 commercial ELISA kit (MyBioSource, San Diego, CA, USA) according to the manufacturer’s protocol. CD-63 levels were measured using canine CD-63 antigen commercial ELISA kit (MyBioSource). All samples were run in duplicate, and the mean values were used. Total protein concentration was measured using bicinchoninic acid assays.
Statistical analysis. All statistical analyses were performed using commercially available statistical software GraphPad Prism software (version 9.3.1, GraphPad Inc., San Diego, CA, USA). One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to compare the UC and non-UC groups. The results are presented as the mean±standard deviation (SD). A p-value <0.05 indicated a statistically significant difference.
Results
Patient data. Twenty-three client-owned dogs were included in the study. Six were diagnosed with UC, and the other 17 were non-UC. The signals for each group are presented in Table I. The UC patients were of the following breeds: Chihuahua (n=2), Poodle (n=2), Maltese (n=1), and Yorkshire terrier (n=1). In addition, 17 non-UC patients belonged to the following breeds: Shih Tzu [3], Labrador Retriever [2], Spitz [2], Alaskan Malamute, Bichon Frise, Doberman Pinscher, German Hunting Terrier, German Shepherd, Golden Retriever, Mongrel, Poodle, Siberian Husky, and standard poodle (for each, n=1). The median age was 12.3 years (11-15 years) in UC group, and 10.4 years (3-21 years) in the non-UC group.
Signalment and characteristics of six dogs with urothelial carcinoma (UC), 6 UC suspected dogs, and 17 non-UC dogs.
In the UC group, three patients had lesions in the urinary bladder, while the other two had lesions in the prostate. One patient had lesions in both the urinary bladder and prostate. In patients with urinary bladder lesions, the lesions were located in the trigone region (n=1), body (n=3), and neck (n=2). In the non-UC group, all 17 dogs revealed no lesions or clinical signs related to the urinary tract.
USG, NMP-22, and CD63 concentration in urine. Voided midstream urine samples were collected from sterile urine cups. All samples went through urinalysis including urine specific gravity, dip stick and cytology, such as direct and indirect smear. Patients with evidence of UTI were excluded from the study. The median USG in UC group was 1.020 (1-1.038), and 1.036 (1.011-1.049) in non-UC group (Figure 1A). No significant differences were observed between the two groups. The median NMP-22 level in the urine of the UC group was 1.925 (0.940-8.400) ng/ml, and 0.300 (0.110-0.730) ng/ml in the non-UC group. In the UC group, the urine NMP-22 concentration was significantly higher than that in the non-UC groups (p<0.005; Figure 1B). CD63 concentration in the urine samples of all the groups was not significantly different (Figure 1C).
Boxplots of urine specific gravity, nuclear matrix protein 22 (NMP-22), and CD63 of dogs with urothelial carcinoma (UC) and non-UC. (A) There was no significant difference in urine specific gravity (USG) between dogs with UC and non-UC. (B) The urine NMP-22 concentration was significantly higher than that in the non-UC group. (C) There was no significant difference in CD63 level between dogs with UC and non-UC.
Expression of NMP-22 in exosomes derived from urine of all groups. The median NMP-22 level in the urine of the UC group was 1.345 (0.800-2.830) ng/ml and 0.220 (0.110-1.560) ng/ml in the non-UC group. In the UC group, the expression of NMP-22 in exosomes derived from urine was significantly higher than that in the non-UC groups (p<0.0001).
Discussion
In this study, we investigated the change in urinary exosomal NMP-22 levels in canine UC. This is the first study to investigate the comparison of urinary exosomal NMP-22 in canine UC patients and healthy dogs. Urine NMP-22 concentration was significantly higher in the UC group than that in the non-UC group (Figure 1B). Furthermore, the expression of NMP-22 in urinary exosomes was significantly higher than that in the non-UC group (Figure 2).
Boxplots of expression of NMP-22 in the exosomes derived from urine of dogs with urothelial carcinoma (UC) and non-UC. Expression of NMP-22 in the exosomes was significantly higher in UC group than that in the non-UC group.
Although a gold standard diagnosis for UC is through solid tissue biopsy with cystoscopy or surgery, because of the invasiveness of the procedure and size of the mass and general anesthesia or sedation requirement, solid tissue biopsy is not always possible (1, 2). Direct cytology of masses and traumatic catheterization are also used; however, owing to several limitations, other minimally invasive diagnostic tools are now emerging (12, 24, 25). Cytokeratins 8 and 18, which are expressed in transitional cell carcinoma, were recently evaluated in a pilot trial in patients with bladder cancer (26). Additionally, evaluation of terbium and uromium chelates for detection of urinary bladder cancer showed the highest specificity compared to cytology (27). However, further trials using larger patient samples are needed and this has not been studied in veterinary medicine. In particular, somatic mutations in the canine BRAF (cBRAF) gene have been identified in several canine cancers, including a large proportion of canine UC; therefore, the presence of V595E mutation in urine has been widely studied and used in veterinary medicine. However, owing to the high cost, long inspection, and transportation period, and large sample size required, the BRAF test is limited to patients with urinary tract lesions (28, 29). Therefore, there is an urgent need for minimally invasive, cost-effective, and rapid diagnostic tests for canine UC. To solve these, additional studies on diagnostic markers are needed.
NMP-22 is a nuclear matrix protein that is involved in DNA recombination, replication, RNA transcription, and mitosis. Increased apoptotic and necrotic potential of urothelial tumor cells results in release of NMP-22 into urine, and therefore, it is found in higher than normal amounts (up to 70-80 times) in the urine of patients with some types of cancer, including bladder cancer (30). Previous studies in human medicine have compared the diagnostic efficacy of urine-based NMP-22 Bladder Cancer ELISA and NMP22 BladderChek test. The sensitivity and specificity vary among studies because of different patient populations, preselected patients, assay performance, and the use of different cut-off values (30).
The sensitivity of ELISA for primary bladder cancer detection ranges from 44-100%, with a specificity between 60-95% (30, 31). In addition, ELISA has several disadvantages and limitations, including cut-off values, laboratory work requirements, and examiner-dependent results. Therefore, the NMP-22 BladderChek test is widely used in human medicine. A systematic review and meta-analysis of the NMP-22 BladderChek test revealed that its overall sensitivity and specificity were 56% (52-59%) and 88% (87-89%) (30, 32). Owing to several factors influencing the results, such as UTI, proteinuria, and leukocyturia there are several limitations to be overcome (33).
In veterinary medicine, NMP-22 has not been well studied or used for the diagnosis of UC. There are no set cut-off values, sensitivity, or specificity of NMP-22 concentrations in urine samples from dogs. Therefore, we aimed to compare NMP-22 concentrations in UC, and non-UC dogs in this study.
Since exosomes play a key role in tumorigenesis, progression, and metastasis through transfer of carcinogenic molecules, studies on exosomal miRNAs, mRNA, and protein expression in patients with UC are now emerging in human medicine (21-23, 34). Exosomal lymph node metastasis-associated transcript 2 from bladder cancer cells are known to induce lymphangiogenesis and lymphatic metastasis (35). In addition, urinary exosomes from patients with bladder cancer contain higher levels of EGF-like repeats and discoidin I-like domain-3, which promotes tumor progression of endothelial and urothelial cell angiogenesis and migration (36). In addition, urinary exosomes possess oncogenic properties, including promotion of cell migration and angiogenesis and blockade of apoptosis (37). Studies on exosomal miRNA, mRNA, and protein expression in urine samples from patients with canine UC are limited for several reasons, including the study population, exclusion criteria, and laboratory work. Therefore, considering previous and this study’s results, determining NMP-22 levels in urinary exosomes could be used as an additional diagnostic method for canine UC.
This study had several limitations. The total study population and the number of patients with UC was relatively small; therefore, a follow-up study with a large number of patients should be conducted to confirm the usefulness of urinary exosomal NMP-22. Furthermore, we used the ExoQuick-CG technique to isolate exosomes from urine samples, which requires less starting material and no ultracentrifugation. However, this process requires an overnight incubation period, which increases the risk of contamination. Nevertheless, there are only a few studies (38-40) on the role of exosomes in canine tumors and this study can serve as the basis for developing a minimally invasive, rapid, and cost-effective diagnostic tool using exosomes derived from urine.
Conclusion
Urinary exosomal NMP-22 levels were significantly elevated in the urine of dogs with UC. These results support the use of urine exosomes as a diagnostic tool in dogs with UC.
Footnotes
Authors’ Contributions
JHS: Conception, design, acquisition, analysis, and interpretation of data, Writing draft. JHA: Conception, design, acquisition, analysis, and interpretation of data, Writing and revising draft. SMP: Interpretation of data, Revising draft. GHL: Interpretation of data, Revising draft. KWS: Interpretation of data, Revising draft. HYY: Conception and interpretation of data. Project administration and supervision, Writing, editing, and revising draft.
Conflicts of Interest
The Authors have no conflicts of interest to report in relation to this study.
Funding
This study was supported by 2023 Research Grant from Kangwon National University and partially supported by the Research Institute for Veterinary Science, Seoul National University, Seoul, Republic of Korea.
- Received September 6, 2023.
- Revision received September 20, 2023.
- Accepted September 21, 2023.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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