Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

Although there are many therapies against cancer, they involve devastating side effects. Hence, we need to understand the nature of cancer cells and the unique markers that exist within these cells that allow them to evade the immune system. These cell properties could be exploited to our advantage. CD47 is a cell membrane receptor protein widely expressed in most cells and is a versatile and crucial target in the tumor microenvironment for creating novel therapeutic approaches for cancer treatment. However, few studies have examined CD47 in glioma. CD47 is found on the surface of multiple cell types, and it usually protects the cells from being removed by phagocytes. It has been found that most cancer cells have high CD47 expression that prevents them from being engulfed by macrophages or activated microglial cells, essentially acting as a “don't eat me signal.” A second protein expressed on cancer cells, calreticulin (CALR), facilitates cell removal by phagocytes, serving as an “eat me signal.” In this project, we compared CD47 expression in glioma cancer stem cells (CSCs), which are negative for Hoechst 33342 nuclear staining (H-), and non-stem glioma cells (Hoechst-positive, H+) of the C6 cell line derived from a rat astrocytoma. We examined the colocalization of CALR with CD47 in both C6 cell types using immunocytochemistry and compared CALR and CD47 gene expression reported in the NCI-60 database of multiple human cancers. We found a significant difference in CD47 expression, with more CD47 in the H+ cells than the H- cells, which could imply that GSCs are more susceptible to CD47 immunotherapy. The highest expression of CD47 ( 50% above signal range) appeared to be in exosomes related to both cell types. We found a positive correlation between CD47 and CALR distribution in the H+ cells (p = 0.0204) and in both H+ and H- cells combined (p = 0.0121), suggesting that the cells might protect themselves from CALR-induced phagocytosis by increasing CD47. We al (open full item for complete abstract)

Committee: Michael Geusz PhD (Committee Chair); Julia Halo PhD (Committee Member); Paul Morris PhD (Committee Member) Subjects: Biology; Medicine

Master of Science (MS), Bowling Green State University, 2023, Biological Sciences

An important mechanism that cancer cells use to survive adverse conditions is upregulating their unfolded protein response (UPR) to secrete more chaperone proteins including calreticulin (CALR). CALR is a resident endoplasmic reticulum protein that has various functions spanning from the nucleus, cytoplasm, and up to the cell membrane. Evidence indicates that cancer cells and especially cancer stem cells (CSCs) have adapted an elevated UPR causing CALR to be produced abundantly such that some of the protein relocates to the cell membrane. At the cell membrane CALR functions as an extracellular signal to immune cells that triggers removal of the cell by microglia and macrophages through phagocytosis. This project tested whether CSCs of gliomas have higher surface CALR expression than non-stem-like cancer cells that form most of the tumor. CSCs previously identified in the C6 rat glioma cell line were further characterized by immunocytochemistry. Based on previous reports from our lab and others, we identified the CSCs in C6 rat cell cultures along with immunofluorescence imaging of cell-surface CALR. Significantly higher surface CALR expression was present in CSCs within the cell clusters forming early tumorspheres. We also extracted RNA-Seq results from the IVY Glioblastoma Atlas Project database describing gene expression for CALR and genes coding for 25 proteins closely related by function to CALR. We compared this activity across five glioblastoma tumor structures and found elevated gene expression in the Pseudopalisading Cells Around Necrosis and Microvascular Proliferation. The known prevalence of CSCs in these tumor structures and the transcriptome data agree with our C6 stem cell results indicating elevated CALR in glioma CSCs. When we induced epithelial-mesenchymal transition (EMT) in C6 cells, an event leading to invasive CSCs, surface CALR was significantly elevated in the resulting cells. We also examined by immunofluorescence the expression of ATF4, a tr (open full item for complete abstract)

Committee: Michael Geusz Ph.D. (Committee Chair); Carol Heckman Ph.D. (Committee Member); Vipa Phuntumart Ph.D. (Committee Member) Subjects: Biology; Biomedical Research; Health; Immunology; Medicine; Neurosciences