Background: Tumor-initiating cells (TICs) play a pivotal role in the unfavorable outcomes of laryngeal tumor proliferation, recurrence, and resistance to chemoradiotherapy. This study aims to explore the expression of CD271 (p75 neurotrophin receptor (p75NTR) in human laryngocarcinoma Hep2 cells and unravel its potential biological functions as a marker of laryngeal TICs.
Materials and methods: Immunomagnetic cell sorting was utilized to separate subsets of Hep-2 cells based on high and low expression levels of CD271. Various aspects such as proliferation activity, colony formation ability, cell cycle distribution, and the expression of cancer-related proteins in each subpopulation were evaluated using immunofluorescence, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, soft agar gel assay, flow cytometry, and western blot assay. Furthermore, the tumor-forming potential of the subsets displaying high and low CD271 expression was examined through an in vivo experiment involving nude mice. The proteins associated with the phosphorylated signal transducer and activator of transcription 3 (p-STAT3)/Octamer-binding transcription factor 4 (OCT4) pathway were detected via western blot assay.
Results: The expression of CD133 was the highest in the CD271 high-expression group, and the expression of CD133 was the lowest in the CD271 low-expression group. Hep2 cells with high CD271 expression exhibited enhanced proliferation capacity, in contrast to those with low CD271 expression which showed reduced proliferation (p < 0.05). The CD271 high-expression group of Hep2 cells demonstrated superior clonogenic ability, a higher proportion in the S and G2/M phases of the cell cycle, and an increased sphere-forming capacity. Moreover, Hep2 cells with high CD271 expression displayed enhanced tumor formation capability in nude mice (p < 0.001). Western blot analysis indicated significantly elevated levels of specific proteins such as OCT4, Nanog Homeobox (NANOG) and p-STAT3/STAT3 in the CD271 high-expression group were significantly higher than those in the control group (p < 0.01), and the protein levels of low-expression group were significantly lower than those in the control group (p < 0.01).
Conclusions: CD271 serves as a marker for TICs in Hep-2 cells, presenting a novel target for further investigation.
Keywords: CD133; CD271 (p75NTR); Hep-2; laryngocarcinoma.