Utilizing a novel zebrafish model of neuroblastoma to identify mechanisms of tumor regression
Mentor Name: Nicole Anderson
The clinical outcome for patients diagnosed with neuroblastoma (NB) span a spectrum from incurable progression to spontaneous regression. Children less than 12 months of age with metastasis are designated 4SNB, and 50% of 4SNB patients experience spontaneous regression without treatment. The adrenal gland is the most common site for primary disease in NB, but tumors can be in the head, neck, abdomen, or pelvis. A GWAS study identified a SNP within LMO1 that is a permissive allele for neuroblastoma formation and results in increased LMO1 expression. The MYCN_TT line is an aggressive zebrafish model of NB that highly expresses MYCN with brightly GFP-labeled tumor cells, and primary tumors exclusively arise from the interrenal gland (IRG, zebrafish adrenal gland). Our preliminary results indicate that MYCN_TT;LMO1 zebrafish form primary tumors that arise within the jaw region, as well as IRG. Histologically both jaw and IRG tumors in MYCN_TT;LMO1 zebrafish are composed of small, round, and undifferentiated neuroblasts. Additionally, both jaw and IRG tumors in MYCN_TT;LMO1 zebrafish stain positive for tyrosine hydroxylase, the marker of catecholamine synthesis. More than 60% of all jaw tumors completely regress by 15 weeks of age, whereas IRG-derived tumors persist over time. The microenvironment of the IRG (site of hematopoiesis) and jaw tumor (little immune infiltrate) are drastically different. We hypothesize that tumor regression is mediated by signals from the microenvironment that alter jaw tumor cells and make them permissive for regression. Goal 1: is to determine the cellular mechanism of regression in the jaw tumor of MYCN_TT;LMO1 zebrafish. To determine if regressing jaw tumors undergoing apoptotic or immune mediated cell death: MYCN_TT;LMO1 jaw tumor being maintained (stable size) vs that are beginning to regress (shrinking in size). Briefly, fish will be collected fixed, embedded, demineralized (EDTA), frozen, and sectioned. Frozen sections containing tumor cells will be stained with DAPI and apoptosis marker activated caspase 3 (AC3). Slides will be imaged by fluorescent microscopy dying cells (AC3+) enumerated (n=8, rep. 3x). Alternating slides will be stained for H&E and examined for the presence of immune infiltrate. Goal 2: to determine the molecular mechanisms of regression in the jaw tumor of MYCN_TT;LMO1 by bulk RNAseq. The jaw tumor (progressed, maintained, or regressed) and the IRG tumor will be harvested for RNA and transcriptionally profiled by bulk RNAseq (n=3 per status/location). Genes that are highly dysregulated in regressing tumors will be further validated by qPCR and additional animals.
