Ovarian cancer (OvCa) is the most lethal gynecologic malignancy, with two-thirds of patients having late-stage disease (II-IV) at diagnosis. Improved diagnosis and therapies are needed, yet preclinical animal models for ovarian cancer research have primarily been restricted to rodents, for data on which can fail to translate to the clinic. Thus, there is currently a need for a large animal OvCa model. Therefore, we sought to determine if pigs, being more similar to humans in terms of anatomy and physiology, would be a viable preclinical animal model for OvCa. We injected human OSPC-ARK1 cells, a chemotherapy-resistant primary ovarian serous papillary carcinoma cell line, into the neck muscle and ear tissue of four severe combined immune deficient (SCID) and two non-SCID pigs housed in novel biocontainment facilities to study the ability of human OvCa cells to form tumors in a xenotransplantation model. Tumors developed in ear tissue of three SCID pigs, while two SCID pigs developed tumors in neck tissue; no tumors were detected in non-SCID control pigs. All tumor masses were confirmed microscopically as ovarian carcinomas. The carcinomas in SCID pigs were morphologically similar to the original ovarian carcinoma and had the same immunohistochemical phenotype based on expression of Claudin 3, Claudin 4, Cytokeratin 7, p16, and EMA. Confirmation that OSPC-ARK1 cells form carcinomas in SCID pigs substantiates further development of orthotopic models of OvCa in pigs.

Severe combined immunodeficiency (SCID) is defined by the lack of an adaptive immune system. Mutations causing SCID are found naturally in humans, mice, horses, dogs, and recently in pigs, with the serendipitous discovery of the Iowa State University SCID pigs. As research models, SCID animals are naturally tolerant of xenotransplantation and offer valuable insight into research areas such as regenerative medicine, cancer therapy, as well as immune cell signaling mechanisms. Large-animal biomedical models, particularly pigs, are increasingly essential to advance the efficacy and safety of novel regenerative therapies on human disease. Thus, there is a need to create practical approaches to maintain hygienic severe immunocompromised porcine models for exploratory medical research. Such research often requires stable genetic lines for replication and survival of healthy SCID animals for months post-treatment. A further hurdle in the development of the ISU SCID pig as a biomedical model involved the establishment of facilities and protocols necessary to obtain clean SPF piglets from the conventional pig farm on which they were discovered. A colony of homozygous SCID boars and SPF carrier sows has been created and maintained through selective breeding, bone marrow transplants, innovative husbandry techniques, and the development of biocontainment facilities.

Background: Severe combined immunodeficient (SCID) pigs are an emerging animal model being developed for biomedical and regenerative medicine research. SCID pigs can successfully engraft human-induced pluripotent stem cells and cancer cell lines. The development of a humanized SCID pig through xenotransplantation of human hematopoietic stem cells (HSCs) would be a further demonstration of the value of such a large animal SCID model. Xenotransplantation success with HSCs into non-obese diabetic (NOD)-derived SCID mice is dependent on the ability of NOD mouse signal regulatory protein alpha (SIRPA) to bind human CD47, inducing higher phagocytic tolerance than other mouse strains. Therefore, we investigated whether porcine SIRPA binds human CD47 in the context of developing a humanized SCID pig.Methods: Peripheral blood mononuclear cells (PBMCs) were collected from SCID and non-SCID pigs. Flow cytometry was used to assess whether porcine monocytes could bind to human CD47. Porcine monocytes were isolated from PBMCs and were subjected to phagocytosis assays with pig, human, and mouse red blood cell (RBC) targets. Blocking phagocytosis assays were performed by incubating human RBCs with anti-human CD47 blocking antibody B6H12, non-blocking antibody 2D3, and nonspecific IgG1 antibody and exposing to human or porcine monocytes. Results:We found that porcine SIRPA binds to human CD47 in vitro by flow cytometric assays. Additionally, phagocytosis assays were performed, and we found that porcine monocytes phagocytose human and porcine RBCs at significantly lower levels than mouse RBCs. When human RBCs were preincubated with CD47 antibodies B6H12 or 2D3, phagocytosis was induced only after B6H12 incubation, indicating the lower phagocytic activity of porcine monocytes with human cells requires interaction between porcine SIRPA and human CD47. AUTH O R CO NTR I B UTI O N S ANB designed and performed experiments, analyzed data, and wrote the manuscript; JEC, CLL, EJP, and TKE designed experiments, reviewed data, and edited manuscript; EJP performed statistical analysis; SEC genotyped piglets, managed SCID pig colony, and helped with sample collection; and CKT designed experiments, reviewed data, and edited manuscript. O RCI D Adeline N. Boettcher

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T − B − NK − SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (ARTEMIS) −/− genetic background. We confirmed ART −/− IL2RG −/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we successfully performed a bone marrow transplant on one ART −/− IL2RG −/Y male SCID pig with bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34 + selected cord blood cells into the fetal ART −/− IL2RG −/Y SCID pigs. At birth, human CD45 + CD3ε + cells were detected in cord and peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.

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