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Bettina Muller, Bernadette Brockmann-Ahmed, Henning Thomasen, Marina Schock, Klaus-Peter Steuhl, Daniel Meller; Performance of comparative studies between healthy conjunctiva, SCC tissue and a conjunctival SCC cell line. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3041.
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© ARVO (1962-2015); The Authors (2016-present)
Our aim was to understand the squamous cell carcinoma (SCC) from the human bulbar conjunctiva in more detail. Therefore, we analyzed healthy conjunctival tissue in comparison to the SCC tissue and the cell line PeCa-UKHb-01 derived out of SCC of the bulbar conjunctiva. We set the focus to genes involved in tumor development, stem cell characteristics and in tissue specific ones.
For the comparison of conjunctival tissue, tumor tissue and PeCa-UkHb-01 a qRT-PCR based on the TaqMan® Assays as duplex reactions was performed. Beforehand, the endogenous control genes were tested and GAPDH as well as PPIA were suited the best ones. Tissue specific genes of interest were EpCAM, K3, K15, K13, K19 and CD90. The cancer and stem cell genes of interest were ABCG2, EGFR, KIT, Myc, Nestin, Notch1, Oct4, and SOX2, also NANOG a gene known for stem cell characteristics. Furthermore, Pax6 a gene in eye development was analyzed. Additional information was obtained by immunofluorescent staining.
The expression pattern of EGFR, KIT, Myc, Nestin, NANOG and Oct4 did not show any conspicuousness. Notch1 expression was lower in PeCa-UkHb-01. Only the conjunctival tissues had higher Pax6 expression. ABCG2 expression was higher in the cell line. The expression pattern of Sox2 was different, in the conjunctival tissue low, in the malignant tissue low and high and PeCa-UkHb-01 in the middle. The expression of EpCAM was in the cell line higher than in the tissue samples. K3 served as a control for the purity of the samples. K13 and K15 expression was observed in all samples. The conjunctival samples as well as PeCa-UkHb-01 showed expression of K19, none was seen in the tumor tissue samples. The other way around was it with CD90.
Through this study we could show that there was a variance between conjunctival tissue and tumor tissue in the genes CD90 and K19. Sox2 needs further analyses to prove that there could be a difference between distinguished tumor samples. A variation between cell line and tissue samples was witnessed for ABCG2, EpCAM and Notch1. Pax6 expression was equal for the malignant tissue and the cell line. It is a gene for eye development and therefore it was expected that both of these samples do not show any expression. Further genes need to be analyzed to find one which shows similarity between the cell line and tumor but not to the healthy conjunctival tissue.
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