In Vitro Establishment of a Genetically Engineered Murine Head and Neck Cancer Cell Line using an Adeno-Associated Virus-Cas9 System

This protocol enabled the development of head and neck cancer models with specific genomic operation, significantly impacting our understanding of the role of specific gene mutations in the process of neoplasia. The main advantage of this technique is the ease with which the cell line can be developed from virtually any organ. Demonstrating the procedure will be Manu Prasad a grad student from my laboratory.

Begin by using surgical scissors to harvest the tongue from a six week old male or female B6 transgenic mouse. Use a scalpel to mince the tissue into very small fragments and collect the pieces into a 15 milliliter tube containing 4.5 milliliters of RPMI plain medium without serum. Add 200 microliters of triple enzyme mix to the tube and incubate the sample at 37 degrees Celsius for 30 minutes tapping the tube every 10 minutes to enhance the enzymatic dissociation of the tissue.

At the end of the incubation, stop the reaction with 5%FBS in PBS and filter the cell suspension through a 70 micro meter mesh strainer to separate the cells from the larger tissue fragments. Collect the cells by centrifugation and re-suspend the pellet in three milliliters of complete medium. Then plate the cells in three milliliters of complete medium per 16 millimeter dish.

Transfer cell aggregates retained on top of the filter to a separate 16 millimeter culture dish and add three milliliters of complete medium. Keep both dishes in the incubator until distinct cell colonies are formed. After one week of culture, microscopically examine the primary cell cultures for the presence of fibroblast contamination, treating the cells with 25%trypsin in 02%EDTA at 37 degrees Celsius for one minute, and remove any fibroblasts.

On day 10 of culture use 25%trypsin in 02%EDTA for three to five minutes at 37 degrees Celsius to harvest the cells from the cell suspension or cell aggregate cultures and see two times 10 to the fifth primary cells in two milliliters of medium per well into each well of the 6-well plate. The next day transduce the cells with one times 10 to the 12 viral genome per milliliter and incubate the cells in the viral particle-containing medium for 48 hours at 37 degrees Celsius. At the end of the incubation, replace the culture supernatants with two milliliters of fresh complete medium per well and return the cells to the cell culture incubator.

After two weeks of culture expansion, seed the cells for validation and in vivo tumorigenic experiments. Using the AAV CAS9 vector, one times 10 to the 12 viral genomes per milliliter of virus have been determined to efficiently transform primary meth cells to tumorigenic maps using the protocol as demonstrated. The transduced cells Express Cre, CAS9 and Green Fluorescent Protein.

Injection of five times 10 to the fifth primary AAV transduced maps into the tongue, lip and skin of NOD SCID mice, results in tumor formation in these animals unlike the injection of primary maps, which do not induce tumors. The tumorigenic transformation of primary tongue epithelial cells can be confirmed using immunofluorescence and western blotting. Genomic analysis by deep sequencing of DNA, isolated from the transformed cells, confirms the effective gene editing and frameshift of the tumor protein 53 and APC genes by the AAV CAS9 system.

In addition, the transformed tongue cells efficiently form tumors in wild type C57 black six mice. Immunohistochemical analysis of the neoplastic tumor cells reveals cytoplasmic expression of E-Cadherin and Keratin 14, both epithelial tissue markers. When attempting this procedure, always remember that too much mincing or too much enzymatic reaction lead to reduced viability of the cells, leading to reduced virus transaction efficiency.

Using this procedure, the cell lines can be genetically modified to gain insight into tumorigenic and metastatic potential of cancer cells from any tissue of interest.