Mammaglobin-A: A Potential Immunotherapy Target for Canine Mammary Cancer

Abstract

Canine mammary tumors are the most common neoplasms in unspayed, female canine patients. Currently, there is no universally accepted standard-of-care for chemotherapy or radiation after surgery. Therapies aimed at tumor-specific molecular targets are needed to enhance the efficacy and specificity of treatments for both canine and human mammary tumor patients. One of these molecular targets is Mammaglobin-A (MAM-A), a protein that is overexpressed in human breast tumors whose function is still unknown. MAM-A has been proven to be a molecular marker for the detection of metastatic breast cancer, and its tissue specificity and mammary tissue overexpression make this gene an excellent target for a breast cancer vaccine. Using a panel of five well-established canine mammary tumor (CMT) cell lines we demonstrated that MAM-A mRNA levels were higher in CMT12, CMT28 and CMT119 cell lines, whereas low to undetectable levels were observed in CMT27 and CMT47 cells, as well as in normal canine mammary epithelial cells (CMEC), respectively. MAM-A protein levels were also higher in CMT12, CMT28 and CMT119 compared to CMT27, CMT47 and CMEC. The canine anti-mammaglobin-A polyclonal antibody detected a protein of approximately 45 kDa that could be a MAM-A/lipophilin B complex or a MAM-A/lipophilin B dimer interacting with an unknown protein. Discordance between mRNA and protein was found in CMT27 and CMT47 cell lines and CMEC; they had measurable protein, but mRNA levels were low or undetectable measured by RT-PCR and qPCR. Expression of MAM-A was also evaluated by immunohistochemical analysis in 55 spontaneous canine mammary gland carcinomas; 52.72% of the cases had moderate to strong, multifocal, cytoplasmic immunolabeling for MAM-A that was not correlated with histologic subtype or grade. A few cases had membranous or luminal immunolabeling. Once we demonstrated that MAM-A was overexpressed in a panel of CMT cell lines and canine mammary neoplastic tissues, we developed and evaluated the efficacy of an autologous dendritic cell (DC) vaccine in unspayed, healthy, adult female dogs. Each dog received 3 intranodal injections at three-week intervals. The vaccine consisted of peripheral blood-derived DCs transfected with a vector encoding canine MAM-A and induced to mature with a cocktail containing TNF-α, IFN-γ and CpG for 24 hr. Mature and transfected DCs showed an upregulation of co-stimulatory molecules CD80, CD83 and CD86. The efficacy of the vaccine was evaluated by its ability to induce a cytotoxic T-cell response to CMT119 cells and secretion of IFN-γ by responder T cells in vitro. In vitro experiments demonstrated that mature DCs transfected with MAM-A elicited potent anti-tumor immune responses against CMT119 cells expressing MAM-A by stimulating the cytotoxic activity of responder T-cells. Additionally, an increase in MAM-A IFN-γ-producing T cells following vaccination was demonstrated. In general, the vaccine was well-tolerated with no adverse reactions. Taken together our data indicates that vaccination with autologous DCs transfected with MAM-A can evoke an anti-tumor immune response and supports the use of this vaccine as a novel therapy to treat canine mammary tumors.