Drift of 4T2 cancer cell line induced byPrss14/epithin shedding and medium component

초록

4T1 cell line has been useful for a mouse metastatic breast cancer model, however, is well known for unstable behavior in culture. During culturing our 4T1 cell obtained from ATCC for several months, we noticed morphological drifts including loosening cell contacts, cell sizes, actin fibers forms with three different media, RPMI (4T1-R), IMDM (4T1-I), DMEM (4T1-D). The levels and forms of PRSS14/epithin membrane serine protease, an important human triple negative metastatic breast cancer marker, were also drifted in the three media. In IMDM, shedding of PRSS14/epithin protein was greatly enhanced, and even in the case of media shift from DMEM for one day. N-terminal fragment of PRSS14/epithin protein translocating to nucleus from membrane upon shedding through regulated intramembrane proteolysis was found differentially located among 4T1-varients. 4T1-I cell migration toward serum gradient was the highest among 4T1-varients and PRSS14/epithin knockdown severely affected cellular migration in all media. This cell migration defect is also affected by the absence of shedding form of protein. When cellular heterogeneity 4T1 cellular population were examined at the single cell level by flow cytometry, the results showed that Sca-I+ population are variable percentages among 4T1 variants and this population were greatly reduced when PRSS14/epithin is knocked down in all media. Instead, Syndecan1, a molecule involved in cell adhesion was upregulated in different levels. RNAseq analyses of 4T1 variant populations showed gene expression variations in EMT marker genes, suggesting components of the media as well as PRSS14/epithin are contributes to 4T1 cell drifts at the gene expression level. For the analysis of cells responsible for in vivo heterogeneity, intra-tumor immune cells are to be investigated in orthotopic Balb/c animal model.