Recent genomic analyses have identified cell death signalling and NF-kB pathways as molecular drivers in oral squamous cell carcinoma (OSCC), a major sub-type of head and neck cancers. Multiple components of these pathways are amplified, such as Inhibitor of Apoptosis (IAP) proteins or mutate (CASP8), allowing cancer cells to evade apoptosis, and contribute to treatment resistance. Smac-mimetics are a new drug class and target the IAPs (e.g cIAP1/cIAP2) and have been shown to restore treatment sensitivity, possibly through the promotion of apoptosis but also work as an adjuvant to immunotherapy checkpoint inhibitors. Adjuvant Smac-mimetics therapy may enhance treatment response and open avenues to improve patient survival and quality of life by altering survival/death signalling and thus OSCC immunobiology. We therefore aimed to first establish models to test adjuvant Smac-mimetics response in 2-D cell models and 3D tumouroids.
Human OSCC cell lines with known CASP8 allele status were tested by proliferation assay comparing response to traditional chemotherapy, Smac-mimetics (birinapant and compound A) and in combination. Using the genome editing tool CRISPR/CAS9, CASP8 was removed for testing in a parallel manner. A 4-NQO oral carcinogenesis model was established in wild-type and Casp8 genetically diverse C57BL/6 mice. Fresh tumour tissue was harvested and matched to confirm OSCC histopathology, for the development of tumour organoid. Human OSCC cell lines with functional CASP8 were significantly more sensitive to Smac-mimetic therapies (birinapant and compound A) in combination with chemotherapy. A workflow for murine organoid harvesting, culture and expansion was tested and optimised. Both murine wild-type and modified Casp8 sourced organoids were successfully expanded.
We have also established methods to grow and establish OSCC tumour organiods from both wt and Casp8- modified 4-NQO treated mice. Human cell lines with modified CASP8 are anticipated to respond further to adjuvant Smac-mimetics. Step follow include mutation-associated response to high throughput screening methods using a low-viscosity matrix suspension method to test sensitivity to chemotherapy, Smac-mimeticsor irradiation in single, double and triple combinations.