Genomic Engineering of Oral Keratinocytes to Establish In Vitro Oral Potentially Malignant Disease Models as a Platform for Treatment Investigation

Area of Science:

• Oral oncology
• Cancer cell biology
• Genomic engineering

Background:

• Oral potentially malignant disorders (OPMDs) represent precancerous cells in the oral cavity.
• Malignant transformation of OPMDs is currently not preventable, highlighting the need for novel therapeutic strategies.
• Dysplasia may occur without visual manifestation in tumor-adjacent tissue.

Purpose of the Study:

• To generate oral precancer culture models for investigating therapeutic interventions.
• To assess the efficacy of small molecule inhibitors in novel oral precancer cell lines.
• To evaluate the feasibility of CRISPR/Cas9 editing and immortalization techniques in oral keratinocytes.

Main Methods:

• Established oral keratinocyte culture methods were combined with CRISPR/Cas9 gene editing.
• Mutations in CDKN2A and TP53 were introduced, alongside telomerase activation, to achieve prolonged proliferation.
• The methodology was applied to normal oral keratinocytes, tumor-adjacent biopsies, and oral leukoplakia biopsies.

Main Results:

• Culturing and genomic engineering were highly efficient for normal and tumor-adjacent oral keratinocytes, but not for oral leukoplakia.
• CDKN2A knockout combined with telomerase activation or TP53 knockout was essential for immortalization.
• Immortalized cell lines exhibited increased sensitivity to small molecule inhibitors compared to normal keratinocytes.

Conclusions:

• The developed cell culturing method shows limited success with oral leukoplakia biopsies.
• Genomic engineering facilitates prolonged culturing of oral leukoplakia-derived keratinocytes but introduces acquired genetic changes.
• Further investigation is needed to confirm the in vivo fidelity of these immortalized cell lines.