These methods were used to develop the first zebrafish model of neuroblastoma metastasis. And to demonstrate that this model can faithfully recapitulate many features of metastasis seen in patients with high risk of neuroblastoma. The main advantage of this metastatic zebrafish model is the real-time in vivo imaging of tumor dissemination to better understand when, and possibly how metastasis occurs.
This technique isn't specific to neuroblastoma. It can be applied to zebrafish models of diverse types of cancers as long as the tumor cells can be identified and tracked leading to an array of possibilities. To begin, develop a heterozygous transgenic fish line overexpressing both MYCN and LMO1 by interbreeding MYCN and LMO1 transgenic lines.
At one day post fertilization, use a stereoscopic fluorescence microscope to sort the progeny of outcross for EGFP expression, which presents as EGFP positive points. After sorting, isolate screened embryos into separate Petri dishes and label dishes as MYCN positive or MYCN negative. Visualize and sort embryos of both groups for LMO1 expression three to four days post fertilization.
Look for red fluorescence spots in both the superior cervical ganglion and non PSNS dopaminergic neuronal cells especially in the oblongata medulla of the hindbrain. Perform the screening before embryos reach five days post fertilization. Then isolate sorted fish into four groups of different genotypes and label as MYCN only, LMO1 only, MYCN and LMO1 both and wild type.
Raise them in identical conditions according to standard protocols. Visualize tumors with a stereoscopic fluorescence microscope by gently flipping fish with a metal spatula on both lateral sides to view the tumor. After identification of the possible tumor-bearing fish, isolate them into a separate tank with appropriate labels which include the date of birth, date when the tumor was screened, and genotype.
At six weeks post fertilization, repeat previous steps to screen for tumor-bearing fish and non-tumor bearing fish to confirm the presence of tumors for previously-screened fish or identify new possible tumor-bearing fish. Look for the sustained or increased size of the fluorescence positive mass and confirmed tumor-bearing fish. After identifying tumor-bearing fish, monitor them biweekly for evidence of tumor cell migration, which presents as tiny EGFP or mCherry positive tumor masses far from the primary site of tumor genesis.
Isolate these fish into separate tanks as needed and label appropriately to indicate possible metastasis. After interpreting the heterozygous MYCN in LMO1 fish, and while sorting their progeny, EGFP MYCN expression was prominent in the non PSNS dopaminergic neuronal cells at one day post fertilization. In contrast, the LMO1 expression was prominent in both PSNS cells and non PSNS dopaminergic neuronal cells.
Fluorescent positive tumor masses were detected in tissues in organs, distant from the primary tumor site in the compound transgenic fish with overexpression of both MYCN and LMO1, but not in the transgenic fish with the expression of MYCN alone. H&E staining of the surgical sections show that the primary tumor arose from the interrenal gland region, the zebrafish equivalent of the human adrenal gland which is the most common site of primary disease in neuroblastoma patient. Tumor masses distant from the interrenal gland were detected in multiple regions, including the distal portion of the kidney, orbit, gill, spleen, and the inner wall of the atrial chamber of the heart.
The PSNS neuroblast lineage of tumor cells at the primary tumor and all the metastatic sites was confirmed. Significantly amplified amounts and increased thickness of PSR-stained collagen fibers were found in MYCN LMO1 tumors when compared to the tumors expressing MYCN alone. After using this procedure, drug screening and testing novel cancer therapies can be applied, which can potentially lead to the development of alternative agents to prevent and treat metastatic cancers.
