Methods for Isolating Canine Mast Cell Progenitors, Melanocytes, and Keratinocytes
Type of DegreeMaster's Thesis
General Veterinary Medicine
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One of the most difficult aspects of employing precision medicine in the treatment of cancer is determining the best targets to treat within the tumor. A trademark of cancers is that, while two patients may present similar tumors of the same tissue, even at the same stages of development, they will likely not respond in the same way to identical treatment. This issue is common throughout all types of cancers and presents a significant hurdle in creating effective treatment regimens for patients. A novel approach to this problem is to sequence the transcript of the tumor to identify genes whose expression or regulation are altered in tumor. This approach requires normal cell transcriptomes as a comparator for the tumor transcriptome. In this study, I attempted to delelope protocols to isolate the normal cells corresponding to three tumors, melanoma, mast cell tumor, and squamous cell carcinomas. Melanoma is a significant tumor of dogs that typically occurs in the oral mucosa. Normal melanocytes from canine melanoma patients are needed to compare transcriptomes with malignant melanomas using deep sequencing, thereby enabling more precise selection of therapeutic targets. Epidermal melanocytes represent a population of approximately 3-7% of cell types throughout the skin, making them a difficult target to isolate. Here, two potential methods for the isolation of pure canine melanocytes from normal skin were evaluated. Melanocytes were enriched from oral mucosa in an impure population with contaminating keratinocytes. Mast cells are thought to be multi-functional master cells with involvement in histamine-based allergic reactions, wound healing, tissue remodeling and innate immunity. They are associated with the tumor microenvironment as well as pathologies such as anaphylaxis, allergic rhinitis, infantile asthma and others. Mast cells, which originate in bone marrow as hematopoietic stem cells, are found in nearly every tissue of the body except the central nervous system and the retina of the eye. After release from the bone marrow, they circulate in the blood as uncommitted, CD117+, CD34+, FcεRI+ and CD90- mast cell progenitors (MCp) before being recruited to peripheral tissues where they mature. Malignant transformation of mast cells results in a mast cell tumor, which is a common tumor of dogs. Canine mast cell tumors are most commonly found in the skin, but may also occur in the liver, intestine, spleen and elsewhere. Circulating MCp’s represent a population of approximately 0.1-0.5% of a cell types in blood, making them a difficult target to isolate. Herein two methods for isolating a CD117+ cell population from whole blood are described. The first involves magnetic activated cell sorting (MACS) to deplete CD90+ cells and enrich for CD117+ cells followed by flow cytometric analysis. This method did not produce an enriched population of CD117+ cells. The second uses MACS to deplete CD90+ cells first, followed by fluorescence activated cell sorting (FACS) to isolate CD117+ cells for downstream use in single cell sequencing. This method produced a CD90 depleted CD117+ population of potential mast cell progenitors. Keratinocytes make up the majority of the cutaneous epithelium. They are both the concrete foundation and the bricks that comprise the wall defined by the skin. Squamous cell and basal cell carcinomas are common cancers of the skin that frequently affect humans and dogs. Therefore, a technical objective was to evaluate a method to obtain normal keratinocytes from canine skin. Using methods similar to those used for melanocytes we were able to isolate keratinocytes with a few contaminating melanocytes. However these contaminating cells will likely be insignificant background in sequencing data. Ultimately, I was able to develop protocols for both mast cells and keratinocytes. In addition to the ability to generate usable sequencing data from these cells for our cancer studies, the development of these approaches will also provide other scientists with a means to obtain these cells to further study all aspects of their biology.