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

Abstract

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.

Keywords: HER2; combined therapies; feline her2 mutations; feline mammary carcinoma; monoclonal antibodies; tyrosine kinase inhibitors.

Figure 1

Trastuzumab, pertuzumab and T-DM1 presented strong antiproliferative effects on feline mammary carcinoma cell lines. (A) CAT-MT cells exposure to trastuzumab allowed to obtain a maximum of 92.6% of cytotoxicity. (B) Cell lines exposure to pertuzumab presented a lower antiproliferative effect, with a maximum of 61.8% of cytotoxicity in the FMCm cells. (C) T-DM1 had the highest antiproliferative effect in the CAT-MT cells, with 94.0% of cytotoxicity. The experiments were performed in triplicates, in three independent assays.

Figure 2

Feline mammary carcinoma cell lines presented different HER2 expressions. (A) CAT-MT cell line was classified as HER2-positive (HER2 2+ score). (B) FMCm was classified with 1+ score, being considered slightly positive for HER2 expression. (C) FMCp cell line presented no HER2 signal (HER2-negative). (D) The human SKBR-3 cell line was classified as a HER2-overexpressing cell line (3+ score). 400× magnification.

Figure 3

Trastuzumab, pertuzumab and T-DM1 induce apoptosis in the feline cell lines. (A) CAT-MT and (B) FMCm cell lines presented an increase in cell apoptosis after drugs exposure, being the cells predominantly in late apoptosis phase. (C) In the FMCp cell line, cells occurred predominantly in early stage apoptosis. (D) The human SKBR-3 cell line had an equilibrated death pattern, between early and late stage apoptosis, with the cells mainly in late apoptosis phase, by exposure to T-DM1. The experiment was performed in triplicates and repeated simultaneously three times.

Figure 4

Combined treatments with trastuzumab plus pertuzumab showed synergistic antiproliferative effects in the carcinoma cell lines. (A) CAT-MT cells presented the highest cytotoxic response by the use of trastuzumab at 125 µg/mL plus pertuzumab at 2000 µg/mL, being possible to achieve 40.0% of cytotoxicity (** p < 0.01). (B) In FMCm cell line, the combination trastuzumab at 125 µg/mL plus pertuzumab at 2000 µg/mL was the one that allows to achieve the highest cytotoxic effect, 45.1% of cytotoxicity (*** p < 0.001). (C) In the FMCp cells a maximum cytotoxic effect of 28.8% using the combination trastuzumab at 500 µg/mL plus pertuzumab at 2000 µg/mL (* p < 0.05) was achieved. (D) Similarly to the feline cell lines, in the human SKBR-3 cell line synergistic antiproliferative effects were obtained, revealing all the combinations valuable. The experiments were performed in triplicates and repeated three separate times.

Figure 5

Combined treatments with trastuzumab plus lapatinib allowed to obtain a valuable synergistic antiproliferative effect in the carcinoma cell lines. (A) CAT-MT and (C) FMCp cells presented a significant increase in the cytotoxicity for all the concentrations tested (*** p < 0.001). (B) FMCm cells presented the highest increase in the cytotoxic effect by the combination trastuzumab at 125 µg/mL plus lapatinib at 7.26 µg/mL, being possible to achieve 37.4% of cytotoxicity (** p < 0.01). (D) In the human SKBR-3 cell line, all the combinations presented a synergistic antiproliferative effect (*** p < 0.001). The experiments were performed in triplicates and repeated three separated times.

Figure 6

The combination of pertuzumab with lapatinib showed an improved antiproliferative effect in all the carcinoma cell lines. (A) CAT-MT cells presented synergistic antiproliferative effects for all the conjugations tested, the highest cytotoxic response occurring by the use of pertuzumab at 250 µg/mL plus lapatinib at 7.26 µg/mL, with 76.8% of cytotoxicity (*** p < 0.001). (B) The FMCm cells achieved the highest synergist effect, by the use of pertuzumab at 250 µg/mL plus lapatinib at 7.26 µg/mL, with 47.8% of cytotoxicity (*** p < 0.001), while the combination of pertuzumab at 2000 µg/mL plus lapatinib at 7.26 µg/mL presented a smaller cytotoxic effect (** p < 0.01). (C) The combination of pertuzumab at 250 µg/mL plus lapatinib at 7.26 µg/mL, in the FMCp cells, allowed to obtain 53.7% of cytotoxicity (*** p < 0.001). (D) As control, the human SKBR-3 cell line presented a synergistic effect for all the conjugations tested (*** p < 0.001). The experiments were performed in triplicates and repeated three separated times.

Figure 7

Mutations identified in the feline her2 ECD, subdomains II and IV were not reported as induce resistance to therapy. All the mutations found occur in a low frequency (n = 1), being more common in exon 3, subdomain II (69.4%). Any of the described mutations were reported in breast cancer patients as induce therapeutic resistance.