Development and characterization of an immunologically humanized and cancer xenograft model in pigs with severe combined immunodeficiency (SCID)
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Swine with severe combined immunodeficiency (SCID) are an emerging large animal model for biomedical research. There have been several SCID pig models described since our first discovery of naturally occurring SCID with mutations in Artemis (DCLRE1C) in 2012. SCID animals are particularly useful in biomedical research due to their lack of T, B, and sometimes NK cells. Absence of the adaptive immune system allows for human cell and tissue xenotransplantation into these SCID animals. The works described within this thesis are categorized under four main goals: (1) further characterization of the immune system of Art-/- SCID pigs, (2) development of a method for in utero injection of human stem cells, (3) in utero injection of human stem cells for the study of human immune cell engraftment within Art-/- IL2RG-/Y SCID pigs, and (4) development of an ovarian carcinoma model in SCID pigs.
Characterization of the SCID pig immune system is important so that researchers can understand how components of the system could impact their research model within the pigs. This thesis describes the characterization of porcine monocytes and their interaction with human cells, as well as “leaky” T cell development in SCID pigs. Firstly, we show that porcine SIRPA, which is expressed on myeloid cells, binds to the “self” protein, CD47, on human cells. Binding between porcine SIRPA and human CD47 prevents phagocytosis of human cells by porcine myeloid cells. Binding compatibility of these two proteins suggests that porcine monocyte phagocytosis of human cells would not be a barrier to human immune cell engraftment within SCID pigs. In addition, this thesis provides an initial investigation into “leaky” CD3ε+ T cells that develop in Art-/- SCID pigs. If functional, these T cells could negatively impact the ability of human cells to engraft within the pigs. I describe that there are small populations of leaky T cells within the blood and lymph nodes of Art-/- SCID pigs.
Another major goal described within this thesis is the development of in-house laparotomy surgical protocols to be utilized for the in utero injection of SCID pig fetuses with human hematopoietic stem cells. We performed two practice laparotomies on non-SCID litters and attempted to inject fetuses within the fetal liver or intraperitoneal space with saline and wire. We showed that our procedures were safe and successful, as we did not have incidence of abortion and 5 of 6 wire-injected piglets were liveborn. We were able to radiographically detect injected wire within the intraperitoneal space, and in one case, in the liver.
After developing the laparotomy procedures, we were able to utilize the surgery techniques to introduce human hematopoietic stem cells into fetal SCID pigs. Art-/- IL2RG-/Y pigs were generated from an Art-/- fetal fibroblast cell line via CRISPR/Cas9 mutagenesis. These Art-/- IL2RG-/Y SCID pigs have a T- B- NK- cellular phenotype, which is optimal for humanization studies, based on previous SCID mouse literature. SCID fetuses were injected with human hematopoietic stem cells utilizing developed laparotomy procedures. We detected human leukocytes cells within the blood, thymus, spleen, liver, and bone marrow of the injected Art-/- IL2RG-/Y pigs. More specifically, we detected human CD3ε+ cells within the blood, spleen, and thymus of these animals. These findings warrant further investigation to improve the reconstitution of human cells within SCID pigs.
Lastly, I describe the beginning stages of a model of ovarian cancer in SCID pigs. Human ovarian serous papillary carcinoma (OSPC)-ARK1 cells were injected into the ear and neck muscles to answer if this carcinoma could develop in an ectopic site within the SCID pig. We found that 3 out of 4 injected SCID pigs developed carcinomas in at least one injection site. OSPC-ARK1 tumors from SCID pigs, SCID mice, as well as the OSPC-ARK1 cell line and the original human tumor sample were stained with commonly used diagnostic markers: Claudin 3/4, Cytokeratin 7, p16, and EMA to assess if tumors that developed in SCID pigs resembled the human tumor. We found that expression of these proteins was highly similar between SCID pig and human carcinomas. The next step of this project is to develop an orthotopic model of ovarian cancer by injection of these cells into the ovary of SCID pigs. Development of such a model could be used for diagnostic imaging research.
In all, this work describes further characterization and development of biomedical models in SCID pigs with a focus on humanization and cancer modeling. The findings described here can be utilized to improve SCID pig models in future research.