VISTA Is a Diagnostic Biomarker and Immunotherapy Target of Aggressive Feline Mammary Carcinoma Subtypes

Feline mammary carcinoma (FMC) is a common neoplasia, showing aggressive clinicopathological features, without viable therapeutic options. The study of tumor microenvironment has gained importance, due to the ability to control tumor progression by regulating the immune response. Considering the lack of knowledge, feline serum VISTA levels from cats with mammary carcinoma were compared with healthy controls, and with serum levels of PD-1/PD-L1, CTLA-4, LAG-3, IL-6, and TNF-α. In parallel, VISTA tumor expression was evaluated in FMC samples. The obtained data revealed that serum VISTA levels were significantly higher in cats presenting HER2-positive (p = 0.0025) or triple-negative subtypes (p = 0.0019), with higher serum levels in luminal A (p = 0.0025) correlated to the presence of metastasis (p = 0.0471). Furthermore, in HER2-positive or triple-negative tumors, correlations were obtained between serum VISTA levels and the serum levels of the above-mentioned molecules. In tumors, VISTA expression revealed a stronger intensity in cancer cells, when compared to TILs (p < 0.0001). Stratifying the samples by subtypes, a higher number of VISTA-positive TILs was observed in the HER2-positive subtype, compared with triple-negative tumors (p = 0.0138). In conclusion, results support the development of therapeutic strategies for HER2-positive and triple-negative FMC subtypes, reinforcing the use of cats as a human oncology model.

Emerging Biomarkers and Targeted Therapies in Feline Mammary Carcinoma

Feline mammary carcinoma (FMC) is a common aggressive malignancy with a low survival rate that lacks viable therapeutic options beyond mastectomy. Recently, increasing efforts have been made to understand the molecular mechanisms underlying FMC development, using the knowledge gained from studies on human breast cancer to discover new diagnostic and prognostic biomarkers, thus reinforcing the utility of the cat as a cancer model. In this article, we review the current knowledge on FMC pathogenesis, biomarkers, and prognosis factors and offer new insights into novel therapeutic options for HER2-positive and triple-negative FMC subtypes.

High Drug Resistance in Feline Mammary Carcinoma Cell Line (FMCm) and Comparison with Human Breast Cancer Cell Line (MCF-7)

Drug repurposing and drug combination are important therapeutic approaches in cancer therapy. Drug repurposing aims to give new indications to drugs, rather than the original indication, whereas drug combination presupposes that the effect that is obtained should be more beneficial than the effect obtained by the individual drugs. Previously, drug repurposing and the combination of different drugs was evaluated in our research group against human breast cancer cells (MCF-7 cells). Our results demonstrated that the response obtained through the combination of drugs, when compared with the single drugs, led to more synergic responses. Therefore, using potential drugs for repurposing, combined with a reference drug in breast cancer (5-Fluorouracil), was the major aim of this project, but for the first time using the feline mammary carcinoma cell line, FMCm. Surprisingly, the feline neoplastic cells demonstrated considerable resistance to the drugs tested in isolation, and the combination was not effective, which contrasted with the obtained MCF-7 cells’ response.

The implication of autoantibodies in early diagnosis and monitoring of plasmonic photothermal therapy in the treatment of feline mammary carcinoma

Feline mammary carcinoma (FMC) shows great similarities to human breast cancer in the cellular and molecular levels. So, in cats as in humans, the role of immune responses is indicated to detect and follow up the development of tumors. As a new breast cancer therapeutic approach, Plasmonic Photothermal Therapy (PPTT) is an effective localized treatment for canine and feline mammary-carcinoma. Its systemic effect has not been inquired yet and needs many studies to hypothesis how the PPTT eradicates tumor cells. In this study, it is the first time to detect (P53, PCNA, MUC-1, and C-MYC) feline autoantibodies (AAbs), study the relationship between PCNA AAbs and mammary-tumors, and investigate the effect of PPTT on the humoral immune response of cats with mammary-carcinoma through detection of AAbs level before, during, and after the treatment. The four-AAbs panel was evaluated in serum of normal and clinically diagnosed cats with mammary tumors using Enzyme-Linked Immunosorbent Assay. The panel showed 100% specificity and 93.7% sensitivity to mammary tumors. The panel was evaluated in PPTT monotherapy, mastectomy monotherapy, and combination therapy. PPTT monotherapy decreased AAbs level significantly while mastectomy monotherapy and combination therapy had a nonsignificant effect on AAbs level.

HER2-Targeted Immunotherapy and Combined Protocols Showed Promising Antiproliferative Effects in Feline Mammary Carcinoma Cell-Based Models

Feline mammary carcinoma (FMC) is a highly prevalent tumor, showing aggressive clinicopathological features, with HER2-positive being the most frequent subtype. While, in human breast cancer, the use of anti-HER2 monoclonal antibodies (mAbs) is common, acting by blocking the extracellular domain (ECD) of the HER2 protein and by inducing cell apoptosis, scarce information is available on use these immunoagents in FMC. Thus, the antiproliferative effects of two mAbs (trastuzumab and pertuzumab), of an antibody–drug conjugate compound (T-DM1) and of combined treatments with a tyrosine kinase inhibitor (lapatinib) were evaluated on three FMC cell lines (CAT-MT, FMCm and FMCp). In parallel, the DNA sequence of the her2 ECD (subdomains II and IV) was analyzed in 40 clinical samples of FMC, in order to identify mutations, which can lead to antibody resistance or be used as prognostic biomarkers. Results obtained revealed a strong antiproliferative effect in all feline cell lines, and a synergistic response was observed when combined therapies were performed. Additionally, the mutations found were not described as inducing resistance to therapy in breast cancer patients. Altogether, our results suggested that anti-HER2 mAbs could become useful in the treatment of FMC, particularly, if combined with lapatinib, since drug-resistance seems to be rare.

Quantification of the collagen fibers of healthy and neoplastic mammary tissue in cats by the Picrossirius Red histochemical method

Although feline mammary carcinoma is not the most prevalent among the species, its aggressive behavior represents a low life expectancy, compared with most undifferentiated types of breast cancer. Tissue stiffness induced by the accumulation of collagen fibers is related to a risk factor for carcinogenesis in healthy women and aggressiveness in those with breast cancer, which can also occur in cats. The objective of this work is to identify the relationship between stromal collagen density and aggressiveness of mammary carcinoma in cats, according to the peripheral and central tissue distribution by the Picrossirius Red histochemical method. Image.J® and MatLab® software were used for digital image processing. The mean values of kurtosis and entropy attributes were ​​grouped into a control group, and low and high-grade carcinoma groups, analyzed with one-way ANOVA and Bonferroni’s multiple comparison test (p <0.01). Interpretation of stromal dynamics is important to evaluate both central and peripheral locations. According to entropy, there was a significant increase in the peripheral density in the carcinoma groups in relation to the control group, which can be justified by blood support. The same can be said of the central region, with a significant gain in collagen fibers from the tumors, indicated by kurtosis. The results suggest the presence of increases in stromal density in mammary carcinomas of cats, regardless of their graduation, and occurring in both regions.

Serum and Tissue Expression Levels of Leptin and Leptin Receptor Are Putative Markers of Specific Feline Mammary Carcinoma Subtypes

Obesity is an established risk factor for breast cancer in post-menopausal women, being associated with elevated serum levels of leptin. Although overweight is a common condition in cat, the role of leptin and its receptor in feline mammary carcinoma remains unsettled. In this study, serum leptin and leptin receptor (ObR) levels were investigated in 58 cats with mammary carcinoma and compared with those of healthy animals, as were the expression levels of leptin and ObR in tumor tissues. The results showed that the Free Leptin Index is significantly decreased in cats with mammary carcinoma (p = 0.0006), particularly in those with luminal B and HER2-positive tumors, and that these animals also present significantly lower serum leptin levels (p &lt; 0.0001 and p &lt; 0.005, respectively). Interestingly, ulcerating tumors (p = 0.0005) and shorter disease-free survival (p = 0.0217) were associated to serum leptin levels above 4.17 pg/mL. In contrast, elevated serum ObR levels were found in all cats with mammary carcinoma (p &lt; 0.0001), with levels above 16.89 ng/mL being associated with smaller tumors (p = 0.0118), estrogen receptor negative status (p = 0.0291) and increased serum levels of CTLA-4 (p = 0.0056), TNF-α (p = 0.0025), PD-1 (p = 0.0023), and PD-L1 (p = 0.0002). In tumor samples, leptin is overexpressed in luminal B and triple-negative carcinomas (p = 0.0046), whereas ObR is found to be overexpressed in luminal B tumors (p = 0.0425). Altogether, our results support the hypothesis that serum levels of leptin and ObR can be used as biomarkers of specific feline mammary carcinoma subtypes, and suggests the use of leptin antagonists as a therapeutic tool, reinforcing the utility of the cat as a cancer model.

The Implication of Autoantibodies in Early Diagnosis and Monitoring of Plasmonic Photothermal Therapy in the Treatment of Feline Mammary Carcinoma

BackgroundLast year’s plasmonic photothermal therapy (PPTT) achieved great success in the treatment of feline mammary carcinoma and there is a demand for developing an easy, cheap, rapid, sensitive, and specific tool for early diagnosis and monitoring of treatment response.MethodsA panel of four autoantibodies (P53, PCNA, MUC-1, and C-MYC) was evaluated in serum of 11 normal cats and 34 cats diagnosed with mammary tumors before, during, and after the treatment using Enzyme-Linked Immunosorbent Assay (ELISA) assay. Out of the 34 cats, 15 did not receive any treatment, ten cats were treated with PPTT, three cats were treated by mastectomy, and six cats were treated with a combination of PPTT and surgery. ResultsThe panel showed high specificity and sensitivity to mammary tumors. The panel also was efficient in the evaluation of PPTT treatment response as their values decreased significantly after three months from the end of treatment. During the PPTT treatment course, the panel values increased within a month before the appearance of secondary tumors and/or metastasis but this increase was not statistically significant. As a result of the short follow-up period in this study, the studied panel was not valid in early diagnosis of primary and/or recurrent tumors nor the evaluation of treatment response of surgery alone or combination therapy. Cats treated with PPTT monotherapy showed the highest survival rate followed by cats treated with combination therapy then cats treated with mastectomy.ConclusionThe studied autoantibodies panel can be used to evaluate PPTT treatment response after three months of the end of treatment and may be efficient in early diagnosis of primary tumors, secondary tumors, recurrence, or metastasis.Trial registrationAll animals were handled in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care and Office of Laboratory Animal Welfare guidelines. All animal experiments were approved by the Institutional Animal Care and Use Committee (CU-IACUC) Cairo University (code: CU II F 9 16). The animal experiments were carried out after the owner's permission. Written informed consent was provided by each cat owner for the treatments.