BME PhD Defense: Katelyn Mistretta: "Three-Dimensional Modeling of the NB Microenvironment for Immunotherapeutic Drug Testing "

PhD Dissertation Defense

"Three-Dimensional Modeling of the NB Microenvironment for Immunotherapeutic Drug Testing " 

Katelyn Mistretta

Thursday, May 16, 2024

Gateway Park, Room GP 1002

9:00am-10:00am
 

Abstract:Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It accounts for 50% of cancers diagnosed in infants less than 1 year old, and 10% of all pediatric cancer deaths in the United States. High-risk patients have a less than 50% 5-year survival rate with current treatment strategies. The complex tumor microenvironment of NB makes the development of treatment strategies for high-risk patients challenging. There is increasing evidence that Intratumoral immune suppression plays an important role in the progression and invasion of NB tumors. Natural killer (NK) cells present in the tumor microenvironment are less able to identify tumor specific antigens of NB cells, as signaling in the microenvironment can lead to shedding of NB surface markers that identify them as abnormal. Tumor associated macrophages (TAMs), are also known to exhibit phenotypic changes that encourage localized immunosuppression. High levels of TAMs in NB are associated with highly aggressive and invasive tumors. Previous in vitro studies of NB and immune cell co-culture rely on two-dimensional (2D) cell culture, which does not capture the complexity of the tumor microenvironment. Few 3D cancer models include components of the immune system, and no cancer-immune co-culture models for the study of NB have been developed previously. The work presented in this dissertation develops a preclinical 3D NB-immune co-culture model. This was achieved using SK-N-AS NB cells, NK-92 natural killer cells, and THP-1 derived macrophages, co-cultured on porous 3D silk scaffolds to provide tumor architecture. Initial work with conditioned media and indirect cell co-culturing showed changes in SK-N-AS gene expression associated with immunoregulatory signaling, and changes in NK-92 gene expression that are associated with reduced cytotoxicity. This motivated the development of a 3D direct co-culture system as conditioned media and indirect co-culture do not allow for direct cell-cell communication which is important for a more complete understanding of cellular behavior. For direct co-culturing, NB cells were seeded prior to immune cells to allow incorporation and deposition of extracellular matrix within the construct. Immune cells were then able to infiltrate into the silk scaffolds to achieve direct co-culture with SK-N-AS cells within the scaffold. Changes in THP-1 macrophage polarization were observed in 3D direct co-culture, as well as altered NK-92 cell protein secretion and cytotoxic activity. Preliminary testing of immunotherapeutics within the model was conducted on both NB-macrophage and NB-NK co-cultures, but the model demonstrated limited response to immunotherapeutics. In summary, this work demonstrates the use of a 3D scaffolded NB model with incorporated immune cells to examine the infiltration and formation of an immune privileged microenvironment, for use as a platform for immunotherapeutic drug testing. Future work should investigate the use of primary cells within this co-culture model, as they have the potential to recapitulate the behavior and response to therapeutics of native cancer and immune cells more closely. 

For a zoom link, please email kharrison@wpi.edu or jnorton@wpi.edu