Environmental issues like exposure to air pollution or pesticides and their link with cancer development are at the forefront of current public health research. Animals have long been recognized as sentinels for environmental pollutants that can be hazardous for human health. Interestingly, and despite the fact that domestic animals like horses and dogs share comparable habitats, and thus, are exposed to similar environmental risk factors as humans, their susceptibility to develop certain types of cancer, like mammary cancer, differs greatly. The overarching goal of this research is to use a comparative species approach to identify the responses of horses (a species resistant to mammary cancer) and dogs (a species susceptible to mammary cancer) to environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) that are linked with increased breast cancer risk. Published work from our lab has shown that equine mammary cells undergo apoptosis while canine mammary cells continue to proliferate when exposed to 7,12 dimethylbenz[a]-anthracene (DMBA), a synthetic PAH derivate commonly used to induce mammary cancer in rodents. These data led to our hypothesis that mammary cancer-proof mammals eliminate DNA-damaged cells through apoptosis, while damaged cells from mammary cancer-susceptible mammals are repaired and persist, allowing for the accumulation of potential malignant mutations. However, it is difficult to test this hypothesis in vivo due to logistical challenges of treating horses and dogs with DMBA. To address this, we established a mammary xenograft model by transplanting mammary tissue fragments from equine or canine donors into the cleared mammary fat pads of immunodeficient mice. We confirmed that these transplanted tissue fragments are proliferative, hormone-responsive, and recapitulate the architecture and function of the donor gland. We now propose to use this novel model to assess the effects of DMBA on equine and canine mammary xenografts to further evaluate mammary cancer resistance and susceptibility mechanisms in vivo. Following DMBA treatment, the mammary xenografts will be assessed for histopathological features, DNA damage, and necrotic/mitotic rate. Activation of apoptotic and tumor-associated pathways will be assessed using a spatial transcriptomics approach, which maintains spatial context of the tissue while providing complete transcriptomic data. Successful outcome of this proposal will (i) improve our understanding of histopathological responses of mammary glands from sentinel species to DMBA and (ii) corroborate whether increased apoptosis is indeed a mechanism driving mammary cancer resistance in equine mammary glands. Evaluation of this potentially conserved mechanism of mammary cancer resistance in response to PAH exposure will help define important regulatory mechanisms that can ultimately result in the identification of biomarkers of exposure and breast cancer risk.