The squamous cell carcinoma cell line OM-1 retains both p75-dependent stratified epithelial progenitor potential and cancer stem cell properties

Abstract

The low-affinity nerve growth factor receptor p75 is a stratified epithelial stem/progenitor marker of human epithelia. We found OM-1, a human squamous cell carcinoma (SCC) cell line, showed distinct cells with p75 cluster, especially located at the center of a growing colony in a monolayer culture. A cell with p75 cluster was surrounded by cytokeratin 14- and cytokeratin 13-expressing cells that settled at the outer margin of the colony. OM-1 cells were also capable of forming tumor spheres in a cell suspension culture, an ability which was attenuated by the inhibition of p75-signaling. Intriguingly, we also found a p75-negative cell population from a growing culture of OM-1 that re-committed to become p75-clustering cells. These results indicated the possibility that SCC with epithelial multi-layering capacity can exploit the p75-dependent stratified epithelial progenitor property for the cancer stemness.

Keywords: Cancer stem cell (CSC); Squamous cell carcinoma (SCC); p75.

Fig. 1

RT-7 cells expressed the possible epithelial stem/progenitor marker protein p75. (A) mRNA expression profile for p75, CK13, CK14, and G3PDH in gingival tissue and RT-7 cells. CK13 was undetectable in proliferating RT-7 cells grown in serum-free keratinocyte growth media while gingival tissue displayed CK14 and CK13 as lineage markers. (B) Immunostaining with anti-p75 antibody in growing RT-7 cells (green: p75, blue: DAPI). Distinct RT-7 cells expressed p75 with a characteristic clusters. (C) Double immunostaining with anti-CK14 antibody and anti-CK13 antibody in RT-7 cells (green: CK13, red: CK14). All cells expressed CK14 only; CK13 was undetectable. (D) RT-7 cells gained CK13 mRNA upon growth in DMEM with 10% FBS. (E) Histological analysis via HE staining of air-exposed 3D cultures of RT-7 grown in DMEM with 10% FBS. RT-7 cells failed to form a multi-layered stratified structure. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

OM-1 cells maintained stratified epithelial progenitor and CSC properties. (A) The mRNA expression profiles of p75, CK13, CK14, andG3PDH in various squamous cell carcinoma cell lines are shown. A431 cells were used as the positive control for CK13 and CK14 expression [23]. (B) Immunocytochemistry using anti-p75 antibody in growing OM-1 cell cultures (green: p75, blue: DAPI). Some cells expressed p75 with the characteristic clusters. (C) Immunofluorescence of CK14 and CK13 in growing OM-1 cultures (green: CK13, red: CK14). CK14 and CK13 expression were exclusive in a given cell. (D, E) Immunofluorescence of a colony of OM-1 cells derived from a single cell. The cells with the characteristic p75 cluster were located at the center. CK14-negative cells can be found at the outer layer of the colony in D (green: p75, red: CK14, blue: DAPI). Outer cells displayed CK13 expression in E (green: CK13, red: CK14, blue: DAPI). (F) Distinct slow-growing small colonies with p75 clustering. (G) Histological analysis with HE staining of air-exposed 3D cultures of OM-1 cells on a dermis-mimic collagen gel. OM-1 cells built a multi-layered stratified epithelium. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Suppression of epithelial stem/progenitor and CSC properties of OM-1 by an intracellular p75 signal inhibitor TAT-Pep5. (A) OM-1 formed tumor spheres. (B) The average number of tumor spheres obtained from 200 cells with or without the p75 signal inhibitor TAT-Pep5. Double asterisks indicate a statistical difference (p < 0.01). (C) Immunofluorescences (green: CK13, red: CK14, blue: DAPI) of single cell-derived colonies formed under treatment with TAT-Pep5. Outer cells lacked CK14 failed to express CK13. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Re-emergence of p75-positive cells from p75-negative cells. (A) p75-negative cells were isolated by a Becton Dickenson FACSAria II flow cytometer as indicated. The sorted cells were recovered as a monolayer culture. (B) Flow cytometry confirmed that the aliquoted cells were p75-negative. (C) Immunofluorescences of a representative colony formed by p75-negative OM-1 cells (green: CK13, red: CK14, blue: DAPI). (D) Flow cytometry of cells after expansion for two weeks showed the re-emergence of a p75-positive population. (E), (F) Immunofluorescences of re-emerged p-75 positive cells in colony. Cells with and without clusters were in E (green: p75, blue: DAPI). In F, CK13-expressing cells in a colony were recovered (green: CK13, red: CK14, blue: DAPI), but lacking the layered distributions observed in Fig. 2 (G) The number of tumor spheres obtained from 400 cells of the indicated cell types. The initial p75-negative population in A turned to be comparable to the parental OM-1 cells. Double asterisks indicate statistical differences (p < 0.01). . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)