Canine Mammary Cancer Research Articles

An Abscopal Effect on Lung Metastases in Canine Mammary Cancer Patients Induced by Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Nanoparticles and Anti-Canine PD-1.

Neoadjuvant intratumoral (IT) therapy could amplify the weak responses to checkpoint blockade therapy observed in breast cancer (BC). In this study, we administered neoadjuvant IT anti-canine PD-1 therapy (IT acPD-1) alone or combined with IT cowpea mosaic virus therapy (IT CPMV/acPD-1) to companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. CMC patients treated weekly with acPD-1 (n = 3) or CPMV/acPD-1 (n = 3) for four weeks or with CPMV/acPD-1 (n = 3 patients not candidates for surgery) for up to 11 weeks did not experience immune-related adverse events. We found that acPD-1 and CPMV/acPD-1 injections resulted in tumor control and a reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of treated dogs. In two metastatic CMC patients, CPMV/acPD-1 treatments resulted in the control and reduction of established lung metastases. CPMV/acPD-1 treatments were associated with altered gene expression related to TLR1-4 signaling and complement pathways. These novel therapies could be effective for CMC patients. Owing to the extensive similarities between CMC and human BC, IT CPMV combined with approved anti-PD-1 therapies could be a novel and effective immunotherapy to treat local BC and suppress metastatic BC.

Bicalutamide Enhances Conventional Chemotherapy in In Vitro and In Vivo Assays Using Human and Canine Inflammatory Mammary Cancer Cell Lines.

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are highly aggressive neoplastic diseases that share numerous characteristics. In IBC and IMC, chemotherapy produces a limited pathological response and anti-androgen therapies have been of interest for breast cancer treatment. Therefore, the aim was to evaluate the effect of a therapy based on bicalutamide, a non-steroidal anti-androgen, with doxorubicin and docetaxel chemotherapy on cell proliferation, migration, tumor growth, and steroid-hormone secretion. An IMC-TN cell line, IPC-366, and an IBC-TN cell line, SUM149, were used. In vitro assays revealed that SUM149 exhibited greater sensitivity, reducing cell viability and migration with all tested drugs. In contrast, IPC-366 exhibited only significant in vitro reductions with docetaxel as a single agent or in different combinations. Decreased estrogen levels reduced in vitro tumor growth in both IMC and IBC. Curiously, doxorubicin resulted in low efficacy, especially in IMC. In addition, all drugs reduced the tumor volume in IBC and IMC by increasing intratumoral testosterone (T) levels, which have been related with reduced tumor progression. In conclusion, the addition of bicalutamide to doxorubicin and docetaxel combinations may represent a potential treatment for IMC and IBC.

Exploring Canine Mammary Cancer through Liquid Biopsy: Proteomic Profiling of Small Extracellular Vesicles.

(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.

Establishment of canine mammary gland tumor cell lines harboring PI3K/Akt activation as a therapeutic target

Canine mammary gland tumors (MGT) have a poor prognosis in intact female canines, posing a clinical challenge. This study aimed to establish novel canine mammary cancer cell lines from primary tumors and characterize their cellular and molecular features to find potential therapeutic drugs. The MGT cell lines demonstrated rapid cell proliferation and colony formation in an anchorage-independent manner. Vimentin and α-SMA levels were significantly elevated in MGT cell lines compared to normal canine kidney (MDCK) cells, while CDH1 expression was either significantly lower or not detected at all, based on quantitative real-time PCR (qRT-PCR) analysis. Functional annotation and enrichment analysis revealed that epithelial-mesenchymal transition (EMT) phenotypes and tumor-associated pathways, particularly the PI3K/Akt signaling pathway, were upregulated in MGT cells. BYL719 (Alpelisib), a PI3K inhibitor, was also examined for cytotoxicity on the MGT cell lines. The results show that BYL719 can significantly inhibit the proliferation of MGT cell lines in vitro. Overall, our findings suggest that the MGT cell lines may be valuable for future studies on the development, progression, metastasis, and management of tumors.

Anticancer Effects of Mitoquinone via Cell Cycle Arrest and Apoptosis in Canine Mammary Gland Tumor Cells.

Treating female canine mammary gland tumors is crucial owing to their propensity for rapid progression and metastasis, significantly impacting the overall health and well-being of dogs. Mitoquinone (MitoQ), an antioxidant, has shown promise in inhibiting the migration, invasion, and clonogenicity of human breast cancer cells. Thus, we investigated MitoQ's potential anticancer properties against canine mammary gland tumor cells, CMT-U27 and CF41.Mg. MitoQ markedly suppressed the proliferation and migration of both CMT-U27 and CF41.Mg cells and induced apoptotic cell death in a dose-dependent manner. Furthermore, treatment with MitoQ led to increased levels of pro-apoptotic proteins, including cleaved-caspase3, BAX, and phospho-p53. Cell cycle analysis revealed that MitoQ hindered cell progression in the G1 and S phases in CMT-U27 and CF41.Mg cells. These findings were supported using western blot analysis, demonstrating elevated levels of cleaved caspase-3, a hallmark of apoptosis, and decreased expression of cyclin-dependent kinase (CDK) 2 and cyclin D4, pivotal regulators of the cell cycle. In conclusion, MitoQ exhibits in vitro antitumor effects by inducing apoptosis and arresting the cell cycle in canine mammary gland tumors, suggesting its potential as a preventive or therapeutic agent against canine mammary cancer.

Interspecies similarities between canine and human mammary cancer

Interspecies similarities between canine and human mammary cancer

Canine Mammary Tumors: Classification, Biomarkers, Traditional and Personalized Therapies.

In recent years, many studies have focused their attention on the dog as a proper animal model for human cancer. In dogs, mammary tumors develop spontaneously, involving a complex interplay between tumor cells and the immune system and revealing several molecular and clinical similarities to human breast cancer. In this review, we summarized the major features of canine mammary tumor, risk factors, and the most important biomarkers used for diagnosis and treatment. Traditional therapy of mammary tumors in dogs includes surgery, which is the first choice, followed by chemotherapy, radiotherapy, or hormonal therapy. However, these therapeutic strategies may not always be sufficient on their own; advancements in understanding cancer mechanisms and the development of innovative treatments offer hope for improved outcomes for oncologic patients. There is still a growing interest in the use of personalized medicine, which should play an irreplaceable role in the research not only in human cancer therapy, but also in veterinary oncology. Moreover, immunotherapy may represent a novel and promising therapeutic option in canine mammary cancers. The study of novel therapeutic approaches is essential for future research in both human and veterinary oncology.

Evaluación del daño en el ADN inducido por quimioterapia metronómica en leucocitos de sangre periférica de pacientes caninos con cáncer de mama mediante el ensayo cometa alcalino

Mammary cancer is a disease that requires effective treatments. Conventional chemotherapy, while effective, often causes harmful side effects. In contrast, metronomic chemotherapy (mCHT), which involves the continuous administration of low doses of anticancer drugs, is presented as a less aggressive alternative. In this study, the genotoxic impact of treatment with Cyclophosphamide and Meloxicam under the mCHT approach was evaluated in ten canine (Canis lupus familiaris) patients with mammary carcinoma after undergoing mastectomy. The patients underwent monthly evaluations, including chest X–rays, blood tests, and the alkaline comet assay to measure genotoxic effects of the antineoplastic drugs. These results were compared with those of a group that received conventional chemotherapy. The results revealed that patients treated with mCHT experienced significantly lower levels of DNA damage compared to those who received conventional chemotherapy. Furthermore, DNA damage decreased over time during mCHT, suggesting that dogs may have developed tolerance to the treatment. Blood parameters remained stable in the mCHT–treated group, and X–rays showed no signs of recurrence or metastasis. All dogs survived during the one–year follow–up without mammary cancer recurrence. It is concluded that mCHT with Cyclophosphamide appears to be a less aggressive therapeutic option with a more favorable genotoxic profile in the treatment of mammary cancer in dogs.

C6 Ceramide Inhibits Canine Mammary Cancer Growth and Metastasis by Targeting EGR3 through JAK1/STAT3 Signaling

C6 Ceramide Inhibits Canine Mammary Cancer Growth and Metastasis by Targeting EGR3 through JAK1/STAT3 Signaling

Anti-cancer effects of DHP107 on canine mammary gland cancer examined through in-vitro and in-vivo mouse xenograft models

BackgroundCanine mammary gland cancer (CMGC) is a common neoplasm in intact bitches. However, the benefit of adjuvant chemotherapy is unclear. The aim of this study was to investigate the anti-proliferative effects of paclitaxel on CMGC in in-vitro and in-vivo settings.ResultsPaclitaxel dose-dependently inhibited viability and induced G2/M phase cell cycle arrest and apoptosis in both primary and metastatic CMGC cell lines (CIPp and CIPm). In animal experiments, the average tumour volume decreased significantly in proportion to the administered oral paclitaxel dose. By examining tumour tissue using a TUNEL assay and immunohistochemical staining with anti-CD31 as a marker of endothelial differentiation, respectively, it was confirmed that oral paclitaxel induced apoptosis and exerted an anti-angiogenetic effect in tumour tissues. Further, downregulation of cyclin D1 in tumour tissues suggested that oral paclitaxel induced cell cycle arrest in tumour tissues in-vivo.ConclusionsOur results suggest that paclitaxel may have anti-cancer effects on CMGC through cell cycle arrest, induction of apoptosis, and anti-angiogenesis. This study could provide a novel approach to treat CMGC.

Application of Newcastle Disease Virus in the Treatment of Human and Canine Mammary Cancer

Application of Newcastle Disease Virus in the Treatment of Human and Canine Mammary Cancer

Establishment and Characterization of a New Canine Mammary Cancer Cell Line CMT-N7: Implications for Comparative Oncology and Therapeutic Development

Establishment and Characterization of a New Canine Mammary Cancer Cell Line CMT-N7: Implications for Comparative Oncology and Therapeutic Development

Doxorubicin as a Potential Treatment Option in Canine Mammary Tumors.

Canine mammary tumors represent one of the leading malignant pathologies in female dogs, displaying the importance of efficient therapeutic findings, besides the golden-standard surgery, able to limit the development of the disease. Studies in human cancers demonstrated that Doxorubicin presents a good effect in different biological processes like apoptosis, autophagy, the cell cycle, cell invasion, and the epithelial-to-mesenchymal transition. This study followed the effects of Doxorubicin on two canine mammary cancer cell lines P114 and CMT-U27. Doxorubicin treatment in both cell lines shows an inhibitory effect in cell proliferation and an alteration in expression of the EMT-related genes. The obtained results provide valuable information for revealing the link between Doxorubicin, phenotypic changes, and proliferation dynamics in canine mammary tumor models.

Anti-tumor activity of a recombinant measles virus against canine lung cancer cells

Canine primary lung cancer with metastasis has a poor prognosis with no effective treatment. We previously generated a recombinant measles virus (MV) that lost binding affinity to a principal receptor, SLAM, to eliminate its virulence as a new cancer treatment strategy. The virus, rMV-SLAMblind, targets nectin-4, recently listed as a tumor marker, and exerts antitumor activity against nectin-4-positive canine mammary cancer and urinary bladder transitional cell carcinoma cells. However, the effectivity of rMV-SLAMblind for other types of canine cancers is still unknown. Here we evaluated the antitumor effect of rMV-SLAMblind to canine lung cancer. Nectin-4 is expressed on three canine lung cancer cell lines (CLAC, AZACL1, AZACL2) and rMV-SLAMblind was able to infect these cell lines. CLAC cells showed reduced cell viability after virus infection. In the CLAC xenograft nude mouse model, intratumoral administration of rMV-SLAMblind significantly suppressed tumor growth. In rMV-SLAMblind-treated mice, natural killer cells were activated, and Cxcl10 and Il12a levels were significantly increased in comparison with levels in the control group. In addition, the depletion of NK cells reduced the anti-tumor effect. To understand difference in efficacy among canine lung cancer cell lines, we compared virus growth and gene expression pattern after virus treatment in the three canine lung cancer cell lines; virus growth was highest in CLAC cells compared with the other cell lines and the induction of interferon (IFN)-beta and IFN-stimulated genes was at lower levels in CLAC cells. These results suggested that rMV-SLAMblind exhibits oncolytic effect against some canine lung cancer cells and the cellular response after the virus infection may influence its efficacy.

Canine Mammary Cancer: State of the Art and Future Perspectives.

Mammary cancer is the most frequently diagnosed neoplasia in women and non-spayed female dogs and is one of the leading causes of death in both species. Canines develop spontaneous mammary tumors that share a significant number of biological, clinical, pathological and molecular characteristics with human breast cancers. This review provides a detailed description of the histological, molecular and clinical aspects of mammary cancer in canines; it discusses risk factors and currently available diagnostic and treatment options, as well as remaining challenges and unanswered questions. The incidence of mammary tumors is highly variable and is impacted by biological, pathological, cultural and socioeconomic factors, including hormonal status, breed, advanced age, obesity and diet. Diagnosis is mainly based on histopathology, although several efforts have been made to establish a molecular classification of canine mammary tumors to widen the spectrum of treatment options, which today rely heavily on surgical removal of tumors. Lastly, standardization of clinical study protocols, development of canine-specific biological tools, establishment of adequate dog-specific disease biomarkers and identification of targets for the development of new therapies that could improve survival and have less adverse effects than chemotherapy are among the remaining challenges.

Therapeutic trial of fluvastatin in a cell line xenograft model of canine mammary gland cancer.

The Hippo signalling pathway is involved in breast cancer and canine mammary tumour (CMT). This study sought to evaluate the efficacy of fluvastatin on the Hippo pathway and its main effectors, YAP and TAZ, in vivo in a murine CMT cell line xenograft model. On treatment day 1, mice were divided into four groups: vehicle, fluvastatin, doxorubicin or a combination therapy. Tumour volumes were monitored with callipers and tissues harvested on day 28th of treatment. Histopathological examination of tumour tissues and major organs was performed as well as tumour evaluation of necrosis, apoptosis, cellular proliferation, expression of YAP, TAZ and the mRNA levels of four of their target genes (CTGF, CYR61, ANKRD1 and RHAMM2). Results showed a statistically significant variation in tumour volumes only for the combination therapy and final tumour weight only for the doxorubicin group compared to control. There was no significant difference in tumour necrosis, expression of CC3, ki-67, YAP and TAZ measured by immunohistochemistry and in the mRNA levels of the target genes. Unexpectedly, lung metastases were found in the control group (9) and not in the fluvastatin treated group (7). In addition, mass spectrometry-based quantification of fluvastatin reveals concentrations comparable to levels reported to exert therapeutic effects. This study shows that fluvastatin tumours concentration reached therapeutic levels without having an effect on the hippo pathway or various tumour parameters. Interestingly, only the control group had lung metastases. This study is the first to explore the repurposing of statins for cancer treatment in veterinary medicine.

Transcriptomics of Canine Inflammatory Mammary Cancer Treated with Empty Cowpea Mosaic Virus Implicates Neutrophils in Anti-Tumor Immunity.

Canine inflammatory mammary cancer (IMC) is a highly aggressive and lethal cancer in dogs serving as a valuable animal model for its human counterpart, inflammatory breast cancer (IBC), both lacking effective therapies. Intratumoral immunotherapy (IT-IT) with empty cowpea mosaic virus (eCPMV) nanoparticles has shown promising results, demonstrating a reduction in tumor size, longer survival rates, and improved quality of life. This study compares the transcriptomic profiles of tumor samples from female dogs with IMC receiving eCPMV IT-IT and medical therapy (MT) versus MT alone. Transcriptomic analyses, gene expression profiles, signaling pathways, and cell type profiling of immune cell populations in samples from four eCPMV-treated dogs with IMC and four dogs with IMC treated with MT were evaluated using NanoString Technologies using a canine immune-oncology panel. Comparative analyses revealed 34 differentially expressed genes between treated and untreated samples. Five genes (CXCL8, S100A9, CCL20, IL6, and PTGS2) involved in neutrophil recruitment and activation were upregulated in the treated samples, linked to the IL17-signaling pathway. Cell type profiling showed a significant increase in neutrophil populations in the tumor microenvironment after eCPMV treatment. These findings highlight the role of neutrophils in the anti-tumor response mediated by eCPMV IT-IT and suggest eCPMV as a novel therapeutic approach for IBC/IMC.

Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Induces Local and Systemic Antitumor Efficacy in Canine Mammary Cancer Patients.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

The Carnitine Palmitoyltransferase 1A Inhibitor Teglicar Shows Promising Antitumour Activity against Canine Mammary Cancer Cells by Inducing Apoptosis.

Canine mammary tumours (CMTs) are the most common cancer in intact female dogs. In addition to surgery, additional targeted and non-targeted therapies may offer survival benefits to these patients. Therefore, exploring new treatments for CMT is a promising area in veterinary oncology. CMT cells have an altered lipid metabolism and use the oxidation of fatty acids for their energy needs. Here we investigated the tumoricidal effects of teglicar, a reversible inhibitor of carnitine palmitoyl transferase 1A (CPT1A), the rate-limiting enzyme for fatty acid import into mitochondria, on two CMT cells, P114 and CMT-U229. Viability and apoptosis were examined in CMT cells using the crystal violet assay, trypan blue assay, and flow cytometry analysis. The expression of mediators of apoptosis signalling (e.g., caspase-9, caspase-8, and caspase-3) was assessed by quantitative real-time polymerase chain reaction and western blot analyses. Teglicar was able to decrease cell viability and induce apoptosis in P114 and CMT-U229 cells. At the molecular level, the effect of teglicar was associated with an upregulation of the mRNA expression levels of caspase-9, caspase-8, and caspase-3 and an increase in their protein levels. In summary, our results show that teglicar has a potential effect against CMTs through the induction of apoptotic cell death, making it a promising therapeutic agent against CMTs.

Deciphering Stromal Changes between Metastatic and Non-metastatic Canine Mammary Carcinomas

Cancer-associated stroma (CAS) is widely recognized to influence development and progression of epithelial tumours including breast cancer. Canine mammary tumours (CMTs) such as simple canine mammary carcinomas represent valuable models for human breast cancer also with respect to stromal reprogramming. However, it remains unclear whether and how CAS changes in metastatic tumours compared to non-metastatic ones. To characterize stromal changes between metastatic and non-metastatic CMTs and identify potential drivers of tumour progression, we analysed CAS and matched normal stroma from 16 non-metastatic and 15 metastatic CMTs by RNA-sequencing of microdissected FFPE tissue. We identified 1438 differentially regulated genes between CAS and normal stroma, supporting previous results demonstrating stromal reprogramming in CMTs to be comparable with CAS in human breast cancer and validating deregulation of pathways and genes associated with CAS. Using primary human fibroblasts activated by treatment with TGFβ, we demonstrate some of the strongest expression changes to be conserved in fibroblasts across species. Furthermore, we identify 132 differentially expressed genes between CAS from metastatic and non-metastatic tumours, with strong changes in pathways including chemotaxis, regulation of apoptosis, immune response and TGFβ signalling and validate deregulation of several targets using RT-qPCR. Finally, we identify specific upregulation of COL6A5, F5, GALNT3, CIT and MMP11 in metastatic CAS, suggesting high stromal expression of these targets to be linked to malignancy and metastasis of CMTs. In summary, our data present a resource supporting further research into stromal changes of the mammary gland in relation to metastasis with implications for both canine and human mammary cancer.