In-vitro study of stemness characteristics of cells originating from oral squamous cell carcinoma

Abstract

CSC) are accountable for tumour initiation, progression and metastasis. Until now, studies were focused exclusively on the characterization of these cell populations within the tumour itself, while tumour margins were neglected, although it is known that the histological and molecular status of tumour margins may play a significant role in the course of the disease. In the present study tumor and margin cell cultures obtained from patients with oral squamous cell carcinoma were used to determine the expression patterns in the course of time, of CSC-related markers (CD44, CD133, Oct-4, Sox2, Nanog), epithelial to mesenchymal transition (EMT)-related markers (Vimentin, αSMA, SLUG and SNAIL), and features, i.e. the clonal, proliferative and migratory potential of the two types of cells. The aims of the study were to isolate cells from oral squamous cell carcinomas and their respective margins, to characterize these cells using CSC/EMT markers, to assess their self-renewal, proliferation and migration potential and determine their chemoresistance. Cell cultures were obtained from 12 tissue specimens (6 tumors and 6 margins). Total RNA was extracted and gene expression analysis was done by real-time PCR. Flow cytometry, immunocytometry, immunohistochemistry, Raman micro-spectroscopy, sphere formation, cell proliferation, colony forming, scratch wound healing and MTT assays were conducted to fully characterize the two cell types. With minor differences, cells originating from both tumors and tumor margins showed the presence of stem cell markers CD133, Nanog, Sox2, CD44, and Oct-4, had the capacity to form spheroids and showed chemoresistance/sensitivity. All the studied EMT markers were expressed in both tumor and margin cells, without statistically significant difference (p>0.05). With few exceptions, for both EMT and CSC markers, the expression was higher in the 5th passage compared to the 1st, probably as the consequence of culture enrichment with CSC in the course of time...

oviji podaci ukazuju na postojanje male subpopulacije kancerskih matičnih ćelija (KMĆ) koje su odgovorne za inicijaciju, progresiju i metastaziranje tumora. Do sada, su studije bile fokusirane isključivo na karakterizaciju ovih ćelijskih populacija unutar samog tumora, dok su margine tumora bile zanemarene, iako je poznato da histološki i molekularni status margina tumora može imati značajnu ulogu u toku bolesti. U ovoj studiji, primarne kulture ćelija tumora i margina dobijenih od pacijenata sa oralnim planocelularnim karcinomom su korišćene za ispitivanje ekspresije markera povezanih sa KMĆ (CD44, CD133, Oct-4, Sox2, Nanog), markera epitelno mezenhimske tranzicije (EMT) (E-kadherin, N-kadherin, Vimentin, ɑSMA, SLUG i SNAIL), a ispitivan je i klonalni, proliferativni i migracijski potencijal ova dva tipa ćelija. Stoga su ciljevi ove studije bili da se izoluju ćelije oralnog planocelularnog karcinoma i njegovih margina, uspostave primarne ćelijske kulture i ispitaju populacije ćelija sa karakteristikama kancerske matičnosti. Ćelijske kulture su dobijene iz 12 uzoraka tkiva (6 tumora i 6 margina). RNK je ekstrahovana i analiza ekspresije gena je urađena pomoću lančane reakcije polimeraze u realnom vremenu. Korišćene su i protočna citometrija, imunocitohemija, imunohistohemija, Raman mikro- spektroskopija, testovi formiranja sfera, ćelijske proliferacije, formiranja kolonija, migracije i MTT test citotoksičnosti, kako bi se u potpunosti okarakterisala ova dva tipa ćelija. Sa manjim odstupanjima, ćelije koje potiču od tumora i ćelije poreklom od margine pokazale su prisustvo markera matičnih ćelija CD133, Nanog, Sox2, CD44 i Oct-4, imale su sposobnost da formiraju sferoide i pokazale su hemorezistenciju. Takođe, svi ispitivani EMT markeri kao dodatni dokaz kancerske matičnosti, bili su eksprimirani u tumorskim i ćelijama margine, bez statistički značajne razlike (p> 0,05). Uz nekoliko izuzetaka, ekspresija EMT i KMĆ markera je bila viša u petoj pasaži u poređenju sa prvom, što bi moglo da se tumači obogaćenjem ćelijskih kultura subpopulacijom KMĆ tokom vremena...