Cytomorphology and Gene Expression Signatures of Anchorage-independent Aggregations of Oral Cancer Cells

Abstract

Background/Aim: Cancer cells with high anchorage independence can survive and proliferate in the absence of adhesion to the extracellular matrix. Under anchorage-independent conditions, cancer cells adhere to each other and form aggregates to overcome various stresses. In this study, we investigated the cytomorphology and gene expression signatures of oral cancer cell aggregates. Materials and Methods: Two oral cancerderived cell lines, SAS and HSC-3 cells, were cultured in a low-attachment plate and their cytomorphologies were observed. The transcriptome between attached and detached SAS cells was examined using gene expression microarrays. Subsequently, gene enrichment analysis and Ingenuity Pathway Analysis were performed. Gene expression changes under attached, detached, and re-attached conditions were measured via RT-qPCR. Results: While SAS cells formed multiple round-shaped aggregates, HSC-3 cells, which had lower anchorage independence, did not form aggregates efficiently. Each SAS cell in the aggregate was linked by desmosomes and tight junctions. Comparative transcriptomic analysis revealed 1,698 differentially expressed genes (DEGs) between attached and detached SAS cells. The DEGs were associated with various functions and processes, including cell adhesion. Moreover, under the detached condition, the expression of some epithelial genes (DSC3, DSP, CLDN1 and OCLN) were up-regulated. The changes in both cytomorphology and epithelial gene expression under the detached condition overall returned to their original ones when cells re-attached. Conclusion: The results suggest specific cytomorphological and gene expression changes in oral cancer cell aggregates. Our findings provide insights into the mechanisms underlying anchorage-independent oral cancer cell aggregation and reveal previously unknown potential diagnostic and therapeutic molecules.Oral cancer is the most common type of malignancy in the head and neck region and the most cases are histologically squamous cell carcinoma (1). The 5-year survival rate in the early stages of oral cancer is more than 80%. However, the survival rate is much lower in advanced cases (2). Oral 64