Analysis Of The Loss Of Caspase 8 In Hnscc Using 3d Organotypic Cultures

Presentation: P005
Topic: Cancer Biology
Type: Poster
Date: Posters
Session: Posters
Authors: Antje Lindemann, PhD, Burak Uzunparmak, Jeffrey N Myers, Curtis R Pickering
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Institution(s): The University of Texas MD Anderson Cancer Center
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The study of cancer genomic alterations should help to improve our understanding of tumor progression and to identify targetable genes and signaling pathways, with the goal of leading to a personalized approach of treatment based on each patient’s individual mutational status. Only a few cancer driver genes are known to be mutated in head and neck squamous cell carcinoma (HNSCC). One of them is caspase 8 (CASP8) which is mutated in 13% of HPV negative HNSCC cases. Caspase 8 functions as an initiator caspase during the extrinsic apoptosis signaling pathway. However, the exact role and function of CASP8 in tumors cells including HNSCC is unknown and needs to be further studied. For that purpose, we generated CASP8 mutant and CASP8 CRISPR knockout clones and characterized these cells using a three dimensional (3D) organotypic culture (OTC). That method allowed us to determine whether caspase 8 plays a role in cell proliferation, cell invasion and differentiation. OTC samples were stained with hematoxylin and eosin and pathway markers and quantified.

In the OTC, 3D epithelial formation was dependent on caspase 8 expression. Control clonal cell lines formed multicellular layers within 14 days of culture, however CASP8 knockout clones, generated by CRISPR-Cas9 technology, showed dramatically reduced numbers of tumor cells and reduced overall thickness of the cell layer. In addition, the knockout clones lost cell-cell contacts and did not invade into the gel substrate. The OTCs were stained with cell death, proliferation, adhesion and differentiation markers to investigate the mechanism of CASP8 regulation of these phenotypes. The expression levels of both wild-type and mutant CASP8 were also modulated in order to further clarify the mechanism of action. In summary, CASP8 is a driver gene in HNSCC that regulates multiple phenotypes in 3D OTC.

 

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