In recent years, the tumor microenvironment (TME) has been a research hotspot, as it is composed of distinct cellular and non-cellular elements that may influence the diagnosis, prognosis, and treatment of breast cancer patients. Cancer cells are able to escape immune control through an immunoediting process which depends on complex communication networks between immune and cancer cells. Thus, a better understanding of the immune cell infiltrate in the breast cancer microenvironment is crucial for the development of more effective therapeutic approaches. In this review article, we overview the different actors that orchestrate the complexity of the TME, including tumor infiltrating lymphocytes (TILs), natural killer cells, tumor infiltrating dendritic cells (TIDCs), tumor associated macrophages (TAMs), tumor associated neutrophils (TANs), cancer associated fibroblasts (CAFs), myeloid-derived suppressor cells (MDSCs), distinct pro-angiogenic factors and immune checkpoint biomarkers. Additionally, we summarize the recent advances in the TME of feline mammary carcinoma (FMC). FMC has been proposed as a reliable cancer model for the study of human breast cancer, as they share clinicopathological, histopathological and epidemiological features, as well as the pathways involved in cancer initiation and progression.

Tumor microenvironment has gained great relevance due to its ability to regulate distinct checkpoints mediators, orchestrating tumor progression. Serum programmed cell death protein-1 (PD-1) and programmed death ligand-1 (PD-L1) levels were compared with healthy controls and with serum cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and tumor necrosis factor-alpha (TNF-α) levels in order to understand the role of PD-1/PD-L1 axis in cats with mammary carcinoma. PD-1 and PD-L1 expression was evaluated in tumor-infiltrating lymphocytes (TILs) and cancer cells, as the presence of somatic mutations. Results showed that serum PD-1 and PD-L1 levels were significantly higher in cats with HER2-positive (p = 0.017; p = 0.032) and triple negative (TN) normal-like mammary carcinomas (p = 0.004; p = 0.015), showing a strong positive correlation between serum CTLA-4 and TNF-α levels. In tumors, PD-L1 expression in cancer cells was significantly higher in HER2-positive samples than in TN normal-like tumors (p = 0.010), as the percentage of PD-L1-positive TILs (p = 0.037). PD-L1 gene sequencing identified two heterozygous mutations in exon 4 (A245T; V252M) and one in exon 5 (T267S). In summary, results support the use of spontaneous feline mammary carcinoma as a model for human breast cancer and suggest that the development of monoclonal antibodies may be a therapeutic strategy.

Cytotoxic T lymphocyte associated antigen 4 (CTLA-4) serves an important role in breast cancer progression, which has led to the development of novel immunotherapies aimed at blocking tumor immune evasion. Although feline mammary carcinoma is increasingly recognized as a valuable cancer model, no studies on CTLA-4 function had been conducted in this species. The serum CTLA-4, TNF-α and IL-6 levels of 57 female cats with mammary carcinoma were determined by ELISA, and immunohistochemistry was performed to evaluate CTLA-4 and FoxP3 expression in tumor cells and interstitial lymphocytes. The results obtained show that serum CTLA-4 levels are increased in cats with mammary carcinoma (P = 0.022), showing an association with a number of clinicopathological features: smaller tumor size, P < 0.001; absence of tumor necrosis, P < 0.001; non-basal status, P < 0.02 and HER-2-positive status. Additionally, a strong positive correlation was found between serum CTLA-4 levels and serum TNF-α (R = 0.88, P < 0.001) and IL-6 levels (R = 0.72, P < 0.001). Concerning the CTLA-4 and FoxP3 expression, although detected in both interstitial lymphocytes and tumor cells, a positive association was found only between interstitial CTLA-4 and FoxP3 expressions (R = 0.387, P = 0.01), which is negatively associated with the serum CTLA-4 levels (P = 0.03). These findings provide a preliminary step in the characterization of immune profiles in feline mammary carcinoma, uncovering a molecular rationale for targeted therapy with CTLA-4 pathway inhibitors. Finally, by strengthening the hypothesis of an immunomodulatory role for this regulator, we further validate the utility of spontaneous feline mammary carcinoma as a model for human breast cancer.

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.

Feline mammary carcinoma (FMC) shares key molecular and clinicopathological features with human breast cancer. We have herein studied the inflammatory infiltrate of FMC in order to uncover potential therapeutic targets and prognostic markers. To this end, the expression of different markers (CD3, CD4, CD8, CD20, CD56, FoxP3, CD68 and CD163) was analyzed in total, stromal (s) and intratumoral (i) tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs), in 73 feline mammary carcinomas. The results revealed that higher percentages of sCD8⁺ TILs were associated with longer disease-free survival (p = 0.05) and overall survival (p = 0.021). Additionally, higher percentages of iCD4⁺ TILs correlated with positive lymph node status (p = 0.003), whereas CD163⁺ TAMs were associated with undifferentiated tumors (p = 0.013). In addition, sCD3⁺ (p = 0.033), sCD8⁺ (p = 0.044) and sCD68⁺ (p = 0.023) immune cells were enriched in triple negative normal-like carcinomas compared to other subtypes. Altogether, our results suggest that specific subsets of immune cells may play a major role in clinical outcome of cats with mammary carcinoma, resembling what has been reported in human breast cancer. These data further support the relevance of the feline model in breast cancer studies.

Vascular endothelial growth factor (VEGF-A) plays an essential role in tumor-associated angiogenesis, exerting its biological activity by binding and activating membrane receptors, as vascular endothelial growth factor receptor 1 and 2 (VEGFR-1, VEGFR-2). In this study, serum VEGF-A, VEGFR-1, and VEGFR-2 levels were quantified in 50 cats with mammary carcinoma and 14 healthy controls. The expression of these molecules in tumor-infiltrating lymphocytes (TILs) and in cancer cells was evaluated and compared with its serum levels. Results obtained showed that serum VEGF-A levels were significantly higher in cats with HER2-positive and Triple Negative (TN) Normal-Like subtypes, when compared to control group (p = 0.001, p = 0.020). Additionally, serum VEGFR-1 levels were significantly elevated in cats presenting luminal A, HER2-positive and TN Normal-Like tumors (p = 0.011, p = 0.048, p = 0.006), as serum VEGFR-2 levels (p = 0.010, p = 0.046, p = 0.005). Moreover, a positive interaction was found between the expression of VEGF-A, VEGFR-1, and VEGFR-2 in TILs and their serum levels (p = 0.002, p = 0.003, p = 0.003). In summary, these findings point to the usefulness of VEGF-A and its serum receptors assessment in clinical evaluation of cats with HER2-positive and TN Normal-Like tumors, suggesting that targeted therapies against these molecules may be effective for the treatment of these animals, as described in human breast cancer.