Canine Mammary Cancer Research Articles

High lysyl oxidase expression is an indicator of poor prognosis in dogs with cutaneous mast cell tumours.

Mast cell tumour (MCT) is one of the most frequent skin tumours in dogs. Due to their unpredictable biological behaviour, MCTs often cause several therapeutic frustrations, leading to investigation regarding prognostic markers. Lysyl oxidase (LOX) is an enzyme that promotes extracellular matrix stability and contributes to cell migration, angiogenesis and epithelial-mesenchymal transition. Its expression positively correlates with poor prognoses in several human and canine mammary cancers. The aim of this study was to characterise the immunohistochemical expression of LOX in MCT samples and compare it with histological grading and post-surgical survival. Twenty-six tumours were submitted to immunohistochemistry for LOX expression evaluation. All samples were positive for LOX, with variable percentages of cytoplasmic and nuclear positivity. Cytoplasmic positivity was significantly higher in high-grade MCTs (P = .0297). Our results indicate that high expression of cytoplasmic LOX in neoplastic mast cells is an indicator of poor prognosis for canine cutaneous MCTs.

Synthesis of structurally diverse derivatives of aconitine-type diterpenoid alkaloids and their anti-proliferative effects on canine breast cancer cells

As one of the most common malignancies in female dogs, no drugs have been developed specifically for the treatment of canine mammary carcinoma. In our previous study, a series of diterpenoid alkaloids derivatives were synthesized and exhibited good anti-proliferative activity in vitro against both normal and adriamycin-resistant human breast cancer cells lines. In this study, a series of structurally diverse aconitine-type alkaloids derivatives were also synthesized basing on the minimal modification principle, by modifying on A-ring, C-ring, D-ring, N-atom or salt formation on aconitine skeleton. Their anti-proliferative effects and mechanism on canine mammary cancer cells were investigated, exhibiting the importance of the substitution at A ring, the long chain ester at the C8, the hydroxyl group at the C13, the phenyl ring at the C14 and the N-ethyl group, while the methoxy group at the C1 and C16 showed little effect on the activity. The results of the proliferation, apoptosis and ultrastructure tests of the treated canine mammary carcinoma cells referred that the representative compound, aconitine linoleate (25) could block the cell cycle of canine mammary carcinoma cells in the G0/G1 phase, and exhibit the anti-proliferative effect by inducing apoptosis.

Treatment of mammary cancer with focused ultrasound: A pilot study in canine and feline patients

In recent years, veterinary medicine has expanded its practices beyond conventional methods, gradually integrating the Focused Ultrasound (FUS) technology in the care of companion animals like dogs and cats. The current study aimed to examine the feasibility and provide insights into the application of thermal FUS in canine and feline mammary cancer therapy. FUS was delivered by a 2-MHz single-element spherically focused ultrasonic transducer as integrated with an existing robotic positioning device. The functionality of the FUS system and sonication protocol in efficiently and safely ablating live tissue was initially validated in a rabbit thigh model in a laboratory environment. Nine (9) dogs and cats with superficial mammary cancer were recruited through a dedicated campaign according to specific safety criteria. The veterinary patients underwent FUS ablation followed by immediate surgical resection of the entire malignancy. Histopathology examination demonstrated well-defined regions of coagulative necrosis in all treated tumors with no off-target damage. Further study with a larger patient population is needed to confirm the current findings and demonstrate the safety and feasibility of complete FUS ablation of deep-seated tumors.

MTOR pathway as a potential therapeutic target for cancer stem cells in canine mammary carcinoma.

Mammary adenocarcinoma, the most common cancer in female dogs, often exhibits the lymph node and lung metastases and has a higher mortality rate. However, mammary adenocarcinoma has no established treatment, except early surgical excision. Canine mammary carcinoma has many common features with human mammary carcinoma, including clinical characteristics, heterogeneity, and genetic aberrations, making it an excellent spontaneous tumor model for human breast cancer. Diverse cancers comprised heterogeneous cell populations originating from cancer stem cells (CSCs) with self-renewal ability. Therefore, in addition to conventional therapy, therapeutic strategies targeting CSCs are essential for cancer eradication. The present study aimed to extract inhibitors of canine mammary CSCs that suppress their self-renewal ability. Sphere-formation assay, which evaluates self-renewal ability, was performed for the canine mammary cancer cell lines CTBp and CNMp. The spheres formed in this assay were used in inhibitor library screening, which identified various signaling pathways such as proteosome, stress inducer, and mammalian target of rapamycin (mTOR). The present study focused on the mTOR signaling pathway. Western blotting showed higher levels of phosphorylated mTOR in sphere-forming CTBp and CNMp cells than in adherent cells. Drug sensitivity examination using the mTOR inhibitors everolimus and temsirolimus revealed dose-dependent reductions in viability among both sphere-forming cells and adherent cells. Expression of phosphorylated mTOR in adherent and sphere-forming cells decreased by everolimus and temsirolimus treatment. In mice transplanted with CTBp-derived spheres, everolimus treatment significantly decreased tumor volume compared to control. These results reveal that the mTOR signaling pathway may be a potential to be a therapeutic target in both cancer cells and CSCs. Novel therapeutic strategies for canine mammary carcinoma are expected to benefit to human breast carcinoma as well.

Proof-of-Concept Study of an Alpha-Fetoprotein-Derived Peptide for the Management of Canine Mammary Cancer.

Novel, well-tolerated drugs are needed for the management of canine mammary cancer. Many of these cancers are promoted in their growth by estrogen. Alpha-fetoprotein (AFP) is a ubiquitous mammalian protein that has anti-estrogenic properties. AFPep (the anti-estrogenic site of AFP) has been developed into a readily synthesizable drug. AFPep has been shown to have anti-mammary cancer activity in several models of this disease, both in cell culture and in rodents. The purpose of the study reported herein was to determine the tolerability of AFPep in normal and tumor-bearing dogs. AFPep was given to dogs via both parenteral and oral routes in a single application and in repeated daily doses. Full clinical chemistry and hematology values were determined before and after drug administration. Blood levels of the drug were achieved in dogs that had been previously found to be oncostatic in rodents. No changes in clinical chemistry, hematology, and clinical behaviors were found in dogs following drug administration. The data support the further development of AFPep for clinical use against canine mammary cancer.

Histopathological and immunohistochemical analysis of predictive and prognostic markers in spontaneous canine mammary cancer

The study investigates the interspecies similarities between canine and human mammary cancer in the sense of innovative predictive and prognostic tumour markers. Surgical resection specimens with diagnosed spontaneous primary mammary cancer obtained from 100 female canine patients were included in this study. Expression of carbonic anhydrase IX (CAIX) enzyme and human epidermal growth factor receptor 2 (HER2) expression was evaluated immunohistochemically. The study was completed with investigation of Ki67 expression and proliferation with marker of myogenous differentiation. Histopathological grading was performed using the Nottingham/modified Bloom-Richardson system. As in humans, our analysis of canine mammary cancer has shown that CAIX positivity in tumour cells significantly correlates with higher levels of HER2 immunoreactivity (P = 0.001), and increased tumour grade (P < 0.001). The percentage of smooth muscle actin (SMA) positive cases was significantly higher (P = 0.002) in the group of mammary carcinomas with CAIX positivity compared to the tumours that were negative. Using antibody Ki67 proliferative activity was not significantly different between mammary tumours that were CAIX positive and CAIX negative. Canine mammary gland carcinomas may, therefore, represent valuable animal models for the study of hypoxic signaling pathways involved in mammary carcinogenesis in humans. Further research investigating this possibility is required.

Macrophage regulation of Wnt pathway in canine and murine mammary cancer cells

Interactions between various cell types in a tumor microenvironment (TME) are important for cancer progression and metastasis. Tumor-associated macrophages (TAMs) play a key role in this intratumoral dialogue regulating Wnt signaling pathway in cancer cells. Co-culture of canine and murine tumor cells with TAMs lead to inhibition of the canonical Wnt pathway and activation of the non-canonical Wnt pathway in tumor cells resulting in change in tumor cell phenotype and subsequent higher potential to invasion and metastasis. These molecular changes in cancer cells that are induced by TAMs resemble the quercetin activity. This natural product is a plant flavonoid from the group of polyphenols. Its biological function is inhibition of the canonical Wnt pathway. As quercetin supplementation is broadly discussed in cancer patients, findings of this study show that this compound should be used with caution, as it may enhance cancer spread.

Protein Expression of PI3K/AKT/mTOR Pathway Targets Validated by Gene Expression and its Correlation with Prognosis in Canine Mammary Cancer.

Mammary cancer is the main type of neoplasia in female dogs and is considered an adequate model for the biological and therapeutic study of cancer in women. The PIK3CA/AKT/mTOR pathway plays a central role in cellular homeostasis and is often dysregulated in cancer. The increased expression of PI3K protein in the literature is associated with a poor prognosis, and alterations in the PIK3CA gene can lead to changes in downstream pathways. Thus, the objective of this study was to validate the protein expression to confirm the gene expression of proteins belonging to the main pathway PI3K and PTEN, and their downstream pathways through ZEB1, ZEB2, HIF1A, VHL, CASP3 and PARP1 relating to prognosis in canine mammary cancer. For protein studies, the samples came from 58 female dogs with mammary neoplasia, immunohistochemistry was performed and its analysis by the histoscore method. For the genetic evaluation, the samples came from 13 patients, the DNA was extracted and the analysis for quantitative expression. Through immunohistochemistry, PI3K positivity was significantly associated with affected regional lymph node, distant metastasis, patients with HER2+, Triple Negative and Luminal B phenotypes, and the lowest survival rates. Through gene expression, we observed higher gene expression of ZEB2 and PARP1 both among patients who were alive and who died, which was not true for the expressions of PIK3CA and HIF1A. In conclusion, the data observed in this work are promising in the study of new molecular prognostic markers such as PI3K, ZEB2 and PARP1 for canine mammary cancer.

Cell adhesion molecules E-cadherin and CADM1 are differently expressed in canine inflammatory mammary cancer

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most aggressive and lethal types of mammary tumors with specific characteristics such as exacerbated angiogenesis, lymphangiogenesis and lymphangiotropism. E-cadherin expression is another specific feature of IBC not previously studied in canine IMC. In this study, the expression of E-cadherin and CADM1 (Cell Adhesion molecule 1) and their possible role as key molecules involved in the pathogenesis of IMC were immunohistochemically analyzed in 19 canine IMC and 15 grade III non-IMC cases. E-cadherin and CADM1 expression was higher in IMC cases (p = 0.002, p = 0.008, respectively). In the IMC group, E-cadherin cytoplasmic immunolabeling was more frequent (p = 0.035) and it was associated to the expression of the angiogenic and lymphangiogenic factors COX-2 (p = 0.009), VEGF-A (p = 0.031) and VEGF-D (p = 0.008). The differential mRNA expression between IMC and non-IMC was studied by microarray analysis in 6 cases. E-cadherin gene (CDH1) was not up-regulated in IMC cases at a transcriptional level; interestingly CADM1 was 7-fold upregulated. The differential expression of E-cadherin protein in IMC suggests a possible role of E-cadherin in the characteristic exacerbated angiogenesis and lymphangiogenesis and further support IMC as a natural model for the study of human IBC. Future studies in IBC and IMC including a broad panel of adhesion molecules are necessary to elucidate their role in the metastatic process and angiogenesis.

Stemness inhibition by (+)-JQ1 in canine and human mammary cancer cells revealed by machine learning

Stemness is a phenotype associated with cancer initiation and progression, malignancy, and therapeutic resistance, exhibiting particular molecular signatures. Targeting stemness has been proposed as a promising strategy against breast cancer stem cells that can play a key role in breast cancer progression, metastasis, and multiple drug resistance. Here, using a previously published one-class logistic regression machine learning algorithm (OCLR) built on pluripotent stem cells to predict stemness in human cancer samples, we provide the stemness index (mRNAsi) of different canine non-tumor and mammary cancer cells. Then, we confirmed that inhibition of BET proteins by (+)-JQ1 reduces stemness in a high mRNAsi canine cancer cell. Furthermore, using public data, we observed that (+)-JQ1 can also decrease stemness in human triple-negative breast cancer cells. Our work suggests that mRNAsi can be used to estimate stemness in different species and confirm epigenetic modulation by BET inhibition as a promising strategy for modulating the stemness phenotype in canine and human mammary cancer cells.

Hormonal Homologies between Canine Mammary Cancer and Human Breast Cancer in a Series of Cases.

Simple SummaryThere is worldwide interest in understanding the cancerous diseases that are causing increasing deaths in humans. In recent years, interest has grown in finding suitable models of different types of cancer in animals to lead the scientific community to a better understanding of the disease, in order to win the battle against cancer. The aim of this investigation was to compare breast cancer samples and canine mammary tumors from a hormonal point of view to validate the canine species as a model to study human breast cancer. There was a close similarity between premenopausal human breast cancer and canine mammary cancer in terms of hormonal receptors. In both species, all hormones assayed were increased in tumors compared to normal mammary gland samples. This research not only further supports canine mammary cancer as a spontaneous model for the study of human breast cancer but is also important in providing a deeper understanding of the hormonal pathogenesis of breast/mammary cancer in each independent species.The validity of spontaneous canine mammary cancer (CMC) as a natural model for the study of human breast cancer (HBC) from a hormonal point of view has never been thoroughly investigated. In this study, we analyzed the immunohistochemical expression of aromatase (Arom) and steroid receptors [estrogen receptor α (ER α), estrogen receptor β (ER β), progesterone receptor (PR) and androgen receptor (AR)] and intratumor steroid hormone levels of 17β-estradiol (E2), estrone sulfate (SO4E1), progesterone (P4), androstenedione (A4), dehydroepiandrosterone (DHEA), and testosterone (T) in 78 samples of mammary cancer—51 human breast cancer (HBC) and 27 canine mammary cancer (CMC)—and corresponding controls. Frequency of tumors expressing Arom, ERβ, PR, and AR was similar in both species, whereas ERα+ tumors were less frequent in the canine species. There was a closer similarity between premenopausal HBC and CMC. In HBC and CMC, all hormones assayed were increased in tumors compared to control samples. Intratumor androgen levels were similar in the two species, although levels of progesterone and estrogens were higher in the HBC samples than the CMC samples. Statistical associations among Arom, receptors, and hormones analyzed suggest that the major hormonal influence in both species is estrogenic through the ER, being the α isoform predominant in the human samples. Our findings further support CMC as a spontaneous model for the study of HBC, especially premenopausal HBC, although several differences, such as the more prevalent ERα immunoexpression and higher intratumor levels of estrogens and P4 in HBC, should be taken into account in comparative hormonal studies.

Epidemiology of canine mammary tumours on the Canary Archipelago in Spain

BackgroundMammary gland tumours are the most frequently diagnosed tumours in the female dogs but just a few studies have analysed their epidemiology. Therefore, we set out to describe the epidemiology of canine mammary cancer in the Canary Archipelago, Spain. We analysed a pathology tumour registry (PTR) and identified 7362 samples obtained from 5240 female dogs resident on the Canary Archipelago during an 18-year period (2003–2020). Using a case–control study design, we compared mammary tumour affected dogs with the Canarian canine population registry in order to elucidate the breed associations for these tumours.ResultsThe frequency of a diagnosis of mammary tumours relative to all tumour diagnoses in female dogs decreased during the study period from 62.7% to 48.9%. Contemporaneously, the proportion of dogs diagnosed with mammary tumours who were also neutered increased from 13.6% to 26.9%. There was a negative correlation (R = -0.84) between these changes. Additional findings were that: the proportion of female dogs diagnosed with multiple tumours increased by 23.5% and that the proportion of malignant tumours 89.2% diagnosed has remained stable through the period. Benign mammary tumours were diagnosed at younger ages (9.2 years old) than carcinomas (9.7 years old) and sarcomas (10.4 years old). Epithelial mammary tumours were diagnosed at younger ages in entire female dogs. Samoyed, Schnauzer, Poodle, German Pinscher and Cocker Spaniel were the breeds with the highest odds-ratios (OR) in comparison with the reference (crossbreeds) while Miniature Pinscher, American Staffordshire Terrier, English Pointer as well as some local breeds such as the Canary Warren Hound and the Majorero had the lowest ORs.ConclusionsThis study provides a description of the changing epidemiology of canine mammary cancer in the Canary Archipelago over the last two decades. We found high rates of CMT with a significant predominance of malignant tumours. Exact risk factors are uncertain, but a combination of environmental, regional socioeconomic affecting human and their pets, and animal management factors are likely to play a part. Specifically, neutering was negatively associated with the proportion of epithelial mammary gland tumours and breeds native to the region were at lower risk of mammary tumours. A deeper analysis of all these factors will facilitate a deeper understanding of the epidemiology of mammary gland tumours in both the canine and the human population.

Synthesis and Antiproliferative Activity of Novel Quercetin-1,2,3-Triazole Hybrids using the 1,3-Dipolar Cycloaddition (Click) Reaction

AbstractTwenty-three new quercetin-1,2,3-triazole hybrids were synthesized in good to quantitative yields via Cu(I)-catalyzed azide-alkyne cycloaddition reaction under microwave irradiation. These new hybrids contain a 1,4-disubstituted 1,2,3-triazole ring at the 3-OH position of quercetin whilst the remaining hydroxyl groups were either protected as methyl or benzyl groups or left unprotected. All the quercetin-1,2,3-triazole hybrids I–IV were evaluated against REM-134 canine mammary cancer cell line, which is used as a translational model for human breast cancer. These new analogues exhibit potent antiproliferative activity against this cancer cell line. Furthermore, the results show that some of the new quercetin-1,2,3-triazole hybrids have better activity than quercetin. Our best inhibitors displayed IC50 values in the range of 41–180 nM, and undoubtedly will have an important impact on the treatment of both canine and human breast cancer.

Establishment of a new cell line of canine inflammatory mammary cancer: IMC-118.

Canine inflammatory mammary cancer (IMC) has long been regarded as an attractive animal model for research into human inflammatory breast cancer (IBC), Although some canine mammary tumour cell lines corresponding to human mammary cancer cell lines have been established, there is still a need to supplement the canine mammary tumour cell bank. The goal of this study was to create a new type of IMC cell line. The primary tumour, IMC-118, was identified as IMC by pathology examination. Immunohistochemistry analysis revealed negative immunoreactivity to oestrogen receptor (ER), but positive immunoreactivity to progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2). Immunofluorescence (IF) analysis revealed that the IMC-118 cell line from this primary tumour was negative for ER but positive for PR and HER-2, and was also positive for epithelial and mesenchymal cell markers. This cell line was cultured stably for more than 50 passages and grew well after cryopreservation. In vivo, tumour masses and metastases in the lungs were discovered after inoculating the IMC-118 cells into the nude mice model. As a result, a novel canine IMC cell line, IMC-118, was effectively established, and could be employed as a promising model for immunotherapy and epithelial-mesenchymal transition mechanism of IMC research in both dogs and humans.

Non-Exosomal and Exosome-Derived miRNAs as Promising Biomarkers in Canine Mammary Cancer.

Canine mammary cancer (CMC), similar to human breast cancer (HBC) in many aspects, is the most common neoplasm associated with significant mortality in female dogs. Due to the limited therapy options, biomarkers are highly desirable for early clinical diagnosis or cancer progression monitoring. Since the discovery of microRNAs (miRNAs or miRs) as post-transcriptional gene regulators, they have become attractive biomarkers in oncological research. Except for intracellular miRNAs and cell-free miRNAs, exosome-derived miRNAs (exomiRs) have drawn much attention in recent years as biomarkers for cancer detection. Analysis of exosomes represents a non-invasive, pain-free, time- and money-saving alternative to conventional tissue biopsy. The purpose of this review is to provide a summary of miRNAs that come from non-exosomal sources (canine mammary tumor, mammary tumor cell lines or canine blood serum) and from exosomes as promising biomarkers of CMC based on the current literature. As is discussed, some of the miRNAs postulated as diagnostic or prognostic biomarkers in CMC were also altered in HBC (such as miR-21, miR-29b, miR-141, miR-429, miR-200c, miR-497, miR-210, miR-96, miR-18a, miR19b, miR-20b, miR-93, miR-101, miR-105a, miR-130a, miR-200c, miR-340, miR-486), which may be considered as potential disease-specific biomarkers in both CMC and HBC.

Neoadjuvant in situ vaccination with cowpea mosaic virus as a novel therapy against canine inflammatory mammary cancer

BackgroundInflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This...

Effect of major versus minor mastectomy on host immunity in canine mammary cancer

Surgical procedures can affect host immunity proportionally to the extent of surgical trauma. In cancer cases, surgery-induced immunosuppression can potentially promote tumour metastasis. The aim of this study was to investigate, in bitches with malignant mammary tumours, whether major surgery (total unilateral mastectomy or bilateral regional mastectomy) has a more negative effect than minor surgery (unilateral regional mastectomy) on components of host immunity. Twenty bitches with mammary cancer of clinical stage II or III were allocated to group A (minor surgery) or group B (major surgery) of 10 animals each receiving the same anaesthetic protocol for mastectomy. Immune cell measurements in blood [number of leukocytes, neutrophils, lymphocytes and platelets, and relative percentages of T-lymphocytes (CD3+) and their CD4+, CD8+ and CD5low+ subpopulations] were performed before anaesthesia (day 0) and on days 3 and 10 post-mastectomy. On day 3, leukocytes, neutrophils and platelets numbers were higher (p = 0.016, 0.032 and 0.017, respectively) in group B than in group A. For all 20 bitches, T-lymphocytes and the CD4+, CD5low+ T-cells were significantly decreased on day 3, but no significant differences were noted between groups. Minor mastectomy seemed to preserve innate immunity better than major mastectomy, but cellular immunity was rather equally affected.

ECPMV Neoadjuvant Immunotherapy Induced Neutrophilic Infiltration and Increased Survival in Canine Inflammatory Mammary Cancer

ECPMV Neoadjuvant Immunotherapy Induced Neutrophilic Infiltration and Increased Survival in Canine Inflammatory Mammary Cancer

Impact of Lymph Node Micrometastases and Isolated Tumour Cells in Canine Mammary Cancer Prognosis

Impact of Lymph Node Micrometastases and Isolated Tumour Cells in Canine Mammary Cancer Prognosis

Epigenetic alterations in canine mammary cancer.

In dogs, mammary cancer is the most common tumor type, especially in unspayed females. As in humans, this type of cancer has spontaneous development and is influenced by several risk factors, such as age and hormonal exposure in addition to genetic and epigenetic factors. Epigenetic mechanisms are responsible for gene expression modulation without alterations in the DNA sequence and include but are not limited to DNA methylation, histone modifications, and noncoding RNAs. Epigenetic patterns are known to influence a variety of biological mechanisms, such as cellular differentiation and development, and dysregulations of those patterns may result in several diseases, such as cancer. In this respect, this review summarizes the main findings concerning epigenetic alterations in canine mammary cancer, their relationship with the carcinogenic process, and their use as diagnostic and prognostic markers.