MSTP in PhD Training
Current advisor: Todd A. Fehniger, MD, PhD
Undergraduate university: University of Maryland-College Park, 2019
Enrollment year: 2021
Developing a Cancer Immunotherapy: Human CAR-Memory-like Natural Killer Cells
During my first research rotation, I worked in the lab of Dr. David DeNardo. The focus of my project was to improve an in vitro organoid proliferation assay for Pancreatic Ductal Adenocarcinoma (PDAC). Patients with PDAC have one of the highest mortality rates among all cancer types with a 5-year survival rate of approximately 7.1%. This is due to PDAC’s highly fibro-inflammatory microenvironment and resistance to chemotherapy, radiation therapy, and immunotherapy. To address this, the DeNardo lab is investigating different therapies in combination that target the highly fibrotic tumor microenvironment. Specifically, an inhibitor against Focal Adhesion Kinase (FAK) in conjunction with Radiation Therapy (RT) has proven effective in diminishing the proliferation of PDAC cells and stabilizing disease progression. These studies were conducted in vitro using PDAC organoids as well as in vivo in a PDAC mouse model. Unfortunately, since PDAC cells proliferate quite rapidly, one of the in vitro proliferation assays based on Ki-67 expression showed close to 100% proliferation in all treatment conditions. Since Ki-67 labels all cells that have proliferated in the last 24-hours, we decided to use the proliferation marker BrdU that only labels cells that have undergone the S phase of the cell cycle. This would result in the labeling of fewer cells and thus improve the chances of detecting a difference in cell proliferation between treatment conditions if present. During my research rotation, I tested a new BrdU antibody and stained PDAC organoids via immunohistochemistry. I was able to optimize the BrdU staining conditions using ex vivo PDAC tumors and worked to optimize the staining with PDAC organoids which will allow for testing cell proliferation under the therapeutic treatment conditions.
During my second research rotation, I worked in the lab of Dr. Carl DeSelm studying Chimeric Antigen Receptors. Chimeric Antigen Receptors (CAR) have revolutionized our ability to harness the immune system to recognize and target tumor cells. Empowering cells of our innate immune system such as macrophages with a CAR will allow them to phagocytose and initiate an immune response against heterogenous tumors. During my lab rotation, I tested the efficacy of CAR-macrophages against tumor cells. Specifically, I studied whether these CAR-macrophages are able to recognize a surface antigen expressed on a mouse sarcoma tumor cell line and upregulate expression of CD40 ligand upon activation. Upregulation of CD40L would promote a more robust immune response. I conducted these experiments by first differentiating CD40L-CAR HoxB8 stem cells into macrophages using MCSF and then co-culturing them with the tumor cell line. Tumor cell proliferation, CAR-macrophage activation, as well as M2 macrophage repolarization were analyzed via flow cytometry. Additionally, live cell imaging and lattice light-sheet imaging was used to determine whether there is direct tumor cell killing and to visualize the cell-cell interactions.
During my third research rotation, I worked in the lab of Dr. Todd Fehniger studying Chimeric Antigen Receptors on human Memory-Like Natural Killer cells. During my lab rotation, I tested the efficacy of two types of human CAR-NK cells. Specifically, I studied whether CD123 CAR-ML-NK cells are able to recognize surface antigen expressed on a human Acute Myeloid Leukemia (AML) cell line and whether CD19 CAR-ML-NK cells have an enhanced response against a Follicular Lymphoma (FL) cell line. Activation and cytotoxicity of ML NK cells was studied using flow cytometry. Through engineering these CAR NK cells and performing expanded functional assays, I was able to determine the efficacy of CD123 and CD19 CAR-ML NK cells against AML and FL and propel this project forward to the next stages that may involve in vivo studies in humanized mice and the first in-human clinical trial.
For my PhD in the Fehniger lab, I am continuing develop powerful CAR ML NK cells against AML and FL and aim to link CAR anti-tumor potency to its mechanism of signaling within human NK cells.
Morina L, Jones ME, Oguz C, Kaplan MJ, Gangaplara A, Fitzhugh CD, Kanakry CG, Shevach EM, Buszko M. 2023 Co-expression of Foxp3 and Helios facilitates the identification of human T regulatory cells in health and disease. Front Immunol, 14():1114780. PMCID: PMC10282999