Oral squamous cell carcinoma (OSCC), which accounts for approximately 90% of oral cancers, has a high rate of local recurrence and a poor prognosis despite improvements in treatment. Exosomes released from OSCC cells promote cell proliferation and metastasis. Although it is clear that the biogenesis of exosomes is mediated by the endosomal sorting complex required for transport (ESCRT) machinery, the gene expression pattern of ESCRT, depending on the cell type, remains elusive. The exosomal release from the human OSCC cell lines, HSC-3 and HSC-4, and their corresponding gefitinib-resistant sub-cell lines, HSC-3/GR and HSC-4/GR, was assessed by western blot and flow cytometry. The levels of ESCRT machinery proteins, including Hrs, Tsg101, and Alix, and whole-cell ubiquitination were evaluated by western blot. We observed that the basal level of exosomal release was higher in HSC-3/GR and HSC-4/GR cells than in HSC-3 and HSC-4 cells, respectively. Long-term gefitinib exposure of each cell line and its corresponding gefitinib-resistant sub-cell line differentially induced the expression of the ESCRT machinery. Furthermore, whole-cell ubiquitination and autophagic flux were shown to be increased in gefitinib-treated HSC-3 and HSC-4 cells. Our data indicate that the expression patterns of the ESCRT machinery genes are differentially regulated by the characteristics of cells, such as intracellular energy metabolism. Therefore, the expression patterns of the ESCRT machinery should be considered as a key factor to improve the treatment strategy for OSCC.

Introduction
Materials and Methods
    1. 세포 배양 및 시약
    2. 엑소좀 분리
    3. Western blot analysis
    4. Flow cytometry
    5. Ubiquitination assay
    6. Statistical analysis
Results
    1. Gefitinib-내성 획득으로 인한 구강편평세포암 세포 유래엑소좀 생성 증가
    2. 구강편평세포암 세포주별 gefitinib-내성 획득에 따른ESCRTs machinery 단백 발현 변화
    3. 구강편평세포암 세포주별 gefitinib-내성 획득에 따른 세포내 유비퀴틴화 및 자가포식 정도(autophagic flux)의 변화
Discussion
Funding
Acknowledgements
Conflicts of Interest
References

• Mi Seong Kim(Department of Oral Physiology, Institute of Biomaterial-Implant, School of Dentistry, Wonkwang University, Wonkwang Dental Research Institute, School of Dentistry, Wonkwang University)
• Min Seuk Kim(Department of Oral Physiology, Institute of Biomaterial-Implant, School of Dentistry, Wonkwang University) Corresponding author