Defining Gene Functions in Tumorigenesis by Ex vivo Ablation of Floxed Alleles in Malignant Peripheral Nerve Sheath Tumor Cells

This protocol provides an alternative in vivo method to study the tumor growth role of an embryonic lethal gene knockout. The advantage of this method is that gene knockouts that are normally embryonic lethal can be performed in tumors derived from a genetically engineered mouse model to study their loss of function effects in vivo. This technique can be applied to diseases that require studying the in vivo role of embryonic essential genes unattainable from traditional gene knockout mouse models.

Generating the animal model takes time and empirical testing is required to determine the optimal MOI for adenoviral infection to ensure that appropriate recombination is achieved. To begin with, identify the mouse with the desired genotype. When a tumor-bearing mouse reaches its humane endpoint, euthanize the mouse and use a sterile scalpel knife to remove the tumor.

Dissect the tumor into three sections in bread loaf-style cuts using a scalpel knife to generate the tissue segments for formalin fixation paraffin embedding early passage culture generation and flash freezing for analytical analyses. Stain a five-micrometer thick formalin-fixed, paraffin-embedded tissue section with hematoxylin and eosin. Once a pathologist confirms the tumor, perform the immunohistochemical staining to confirm the tumor diagnosis.

Place the freshly-collected malignant peripheral nerve sheath tumor tissue in 10 milliliters of ice-cold sterile PBS on ice, and then transfer it to a sterile work area to establish early passage cultures. Mince the tumor tissue into two to four-millimeter pieces and triturate eight to 10 times in a 10-square centimeter treated tissue culture dish containing 10 milliliters of growth medium. Culture these tissues in high glucose DMEM-10 growth medium supplemented with 10 nanomolar neuregulin-1 beta and two micromolar forskolin.

Seed the early passaged tumor cells at a density of 1.5 times 10 to the sixth cells per one 10-square centimeter treated tissue culture dish containing DMEM-10 growth medium. After 12 to 16 hours, wash the adherent cultures with two to four milliliters of DPBS, and infect the cells with adenovirus at approximately 400 plaque-forming units per cell in 10 milliliters of serum-free DMEM. After 48 hours of the infection, briefly check the cells for EGFP signal on a fluorescence microscope to ensure efficient infection with approximately 50 to 100%positive cells, and then FACS sort the EGFP-positive infected cells.

After FACS sorting, let the cells recover in a tissue culture incubator for at least 24 to 48 hours, and then prepare the cells for in vitro cell-based analyses or in vivo grafting. Confirm Erbb4 deletion by performing PCR using genomic DNA isolated from a portion of the sorted EGFP-positive cells. Perform the proliferation assays over the next seven days on sorted tumor cells plated in a 96-well plate using an image-based automated cytometer.

Stain the cells with hooks and propidium iodide dyes and capture the images of the cells using an automated plate reader to count the number of live and dead cells in each well. Isolate total RNA from sorted tumor cells using standard acid guanidinium phenol and chloroform-based method. Open the software to perform the RNA sequence alignment following the program-specific steps using the default settings.

Select the analysis method, RNA seek, and then select the mouse reference genome. Upload the BED file if provided one by the sequencing core. Upload the FASTQ sequencing files and assign unique replicate names to the files.

Group replicate the FASTQ files, and designate the files to a replicate set. For the statistical and normalization analysis to identify the differential gene expression signals with robust statistical power, open the analysis software and select the GFP FASTQ files as the control data set. Next, select DESeq2 as the statistical and normalization method and start the assembly and analysis.

Use the gene ontology data sets integrated into the open access functional enrichment analysis tools to perform the functional enrichment analysis on the identified statistically significant Erbb4-mediated DEG to determine the biological and pathway significance of Erbb4 gene loss. Inject the tumor cells into the sciatic nerve of the anesthetized mouse to assess the in vivo allograft growth potential in post-infected cells. Once the tumor reaches the required size, dissect the tumor from a euthanized mouse in two-three sections as demonstrated previously.

Fix one tissue section in 4%Paraformaldehyde overnight, and then embed the fixed section in the paraffin. After making five-micrometer thick sections from the FFPE tissue, mount the section on the slides and confirm the presence of tumor cells in the grafted tissue using H&E and immunostaining as demonstrated previously. To confirm in vivo Erbb4 expression differences between the two experimental conditions, stain the formalin-fixed, paraffin-embedded tissue with Erbb4-specific antibodies.

The transduction of the malignant peripheral nerve sheath tumor tissue cells with adenovirus was analyzed. The EGFP expressing cells were detected using fluorescence microscopy, and the total number of cells was determined using phase-contrast microscopy. After the Cre-mediated gene ablation, depending on the transfection efficiency, the PCR genotyping showed a complete gene knockout and a heterogeneous population of induced and uninduced cells compared to the control transduction.

The characteristic histopathology found in the orthotopic allografts of malignant peripheral nerve sheath tumor tissues compared to the tumors derived from the original genetically-engineered animal models demonstrated a difference in the cell morphology. The Erbb4 ablation decreased cellular density and cellular proliferation in adeno5-CMV-Cre-ablated cells control adeno5-CMV-GFP-transduced cells. The expression of Erbb4 in the tumor cells transduced with adeno5-CMV-Cre, or EGFP virus, was decreased in the result in adeno5-CMV-Cre transduced allografts compared to the control adeno5-CMV-EGFP-treated allograft tumors.

The vascular density was assessed via immunoreactivity detection for CD31, and the representative results showed markedly reduced vascular density in allografts of adeno5-CMV-Cre-transduced cells compared to the control adeno5-CMV-EGFP-transduced cells. After gene ablation, the cells can be used for many different types of cell-based assays such as proliferation, migration, 3D growth, or microenvironment alterations, as well as for in vivo orthotopic injections.