Identification of different gene expressions between diffuse- and intestinal-type spheroid-forming gastric cancer cells
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Three-dimensional in vitro spheroid models are unique because they are considered for enrichment of specific cell populations with self-renewal ability. In this study, we explored the different mechanisms of gastric cancer spheroid-forming cells according to the Lauren classification.
We isolated and enriched cells with self-renewal ability using spheroid-forming methods from gastric cancer cell lines. The expression of candidate target genes was investigated using western blot and qRT-PCR analysis. Lentiviral shRNA knockdown of target gene expression was performed and the effects on spheroid, colony forming, and tumorigenic ability were analyzed.
The SNU-638, SNU-484, MKN-28, and NCI-N87 successfully formed spheroid from single cell and enriched for self-renewal ability from 11 gastric cancer cell lines, including diffuse and intestinal types. The expression of SOX2 and E-cadherin increased in spheroid-forming cells in a diffuse-type cell line (SNU-638 and SNU-484), but not in the intestinal type (MKN-28 and NCI-N87). In contrast, ERBB3 expression was only increased in intestinal-type spheroid cells. The depletion of each candidate target gene expression suppressed self-renewal ability to grow as spheroids and colonies in a soft agar assay. In particular, down-regulated ERBB3 in the intestinal-type cell lines inhibited tumor growth in a mouse xenograft model. We found that high ERBB3 gene expression correlates with decreased survival in the intestinal type of gastric cancer.
Our results suggest that diffuse- and intestinal-type spheroid-forming cells express genes differently. Our data suggest that these candidate genes from spheroid-forming cells can be used in applications in targeted therapy.
KeywordsGastric cancer Cellular spheroid Lauren classification Diffuse type Intestinal type
This study was supported by a Grant from the National Research Foundation of Korea (NRF), which is funded by the Korean government (MEST) (No. 2010-0020986).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
All institutional and national guidelines for the care and use of laboratory animals were followed.
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