A xenotransplantation mouse model to study physiology of the mammary gland from large mammals.

Although highly conserved in structure and function, many (patho)physiological processes of the mammary gland vary drastically between mammals, with mechanisms regulating these differences not well understood. Large mammals display variable lactation strategies and mammary cancer incidence, however, research into these variations is often limited to in vitro analysis due to logistical limitations. Validating a model with functional mammary xenografts from cryopreserved tissue fragments would allow for in vivo comparative analysis of mammary glands from large and/or rare mammals and would improve our understanding of postnatal development, lactation, and premalignancy across mammals. To this end, we generated functional mammary xenografts using mammary tissue fragments containing mammary stroma and parenchyma isolated via an antibody-independent approach from healthy, nulliparous equine and canine donor tissues to study these species in vivo. Cryopreserved mammary tissue fragments were xenotransplanted into de-epithelialized fat pads of immunodeficient mice and resulting xenografts were structurally and functionally assessed. Preimplantation of mammary stromal fibroblasts was performed to promote ductal morphogenesis. Xenografts recapitulated mammary lobule architecture and contained donor-derived stromal components. Mammatropic hormone stimulation resulted in (i) upregulation of lactation-associated genes, (ii) altered proliferation index, and (iii) morphological changes, indicating functionality. Preimplantation of mammary stromal fibroblasts did not promote ductal morphogenesis. This model presents the opportunity to study novel mechanisms regulating unique lactation strategies and mammary cancer induction in vivo. Due to the universal applicability of this approach, this model serves as proof-of-concept for developing mammary xenografts for in vivo analysis of virtually any mammals, including large and rare mammals.