Twelve cell groups from three cell lines were used in the present study. hTERT⁺-OME is an immortalized cell line created by hTERT gene transduction into primary cultured oral mucosal epithelial (OME) cells (13). Human tongue squamous cell carcinoma cell line (Cal27) was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Gca1551 is a cell line established by primary cultured cells from a 64-year-old man with gingival squamous cell carcinoma with lymph node metastasis (T2N2M0). hTERT⁺-O⁺-OME, hTERT⁺-S⁺-OME, hTERT⁺-OS⁺-OME, Cal27-O^low, Cal27-S^low, Cal27-O^lowS^low, Gca1551-O^low, Gca1551-S^low and Gca1551-O^lowS^low cells were derived by our group (see below). Ethical approval was obtained from the Ethics Committee of Zhengzhou University (reference no., 20130523-10-2).

Human gingival carcinoma primary tumor samples were obtained within 1 h after surgery. The tissues were minced with blades into small pieces. These pieces were enzymatically digested using 0.25% dispase II (Sigma, St. Louis, MO, USA) at 4°C overnight. After digestion with 0.25% trypsin (Sigma) for 10 min at 37°C, the tissue was triturated with a pipette and passed through a 200-mm cell strainer. Then, the cells were centrifuged at 300 × g for 5 min, re-suspended in Dulbecco's modified Eagle's medium:nutrient mixture (DMEM/F12) with 10% fetal bovine serum (FBS), and plated in 6-well plates. Once the cell clones emerged, they were removed by 0.25% trypsin digestion and cultured in plates. The cells that were not attached after 20 min were collected to purify floating cancer cells from the more rapidly adhering fibroblasts. The collected cells were centrifuged and plated in the new flasks at a density of 1,000 cells/cm². The process was repeated several times. The purified cancer cells were acquired and this cell line was named as Gca1551.

All the cell lines were cultured in a basic medium that was comprised of DMEM/F12 supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (all from Sigma). This basic medium was named as 'A' in the present study and the culturing details of all cell lines are described in Table I.

In the process of cell reprogramming, hTERT⁺-OME, hTERT⁺-O⁺-OME, hTERT⁺-S⁺-OME and hTERT⁺-OS⁺-OME were cultured in TeSR™-E7 and TeSR™-E8 media (StemCell Technologies, Vancouver, BC, Canada) according to the manufacturer's protocol.

The sphere medium and culture method were the same as previously described (13). Briefly, the medium contained no FBS and was replaced by B27 (serum-free supplement from Invitrogen, Carlsbad, CA, USA). In addition, 20 ng/ml of epidermal growth factor (Invitrogen) and basic fibroblast growth factor (BD Biosciences, San Jose, CA, USA) were added.

hTERT⁺-OME cells were transduced with lentiviral vectors encoding nuclear reprogramming factors. Briefly, the lentiviral vector plasmids pMXs-hOCT4-GFP (GenePharma, Shanghai, China) and mCMV-hSOX2-mCherry (BioWit Technologies, Shenzhen, China) were introduced into 293T cells using RNAi-Mate transfection reagent. After 48 h, the virus-containing supernatants were passed through a 0.45-mm filter and supplemented with 5 mg/ml Polybrene. hTERT⁺-OME cells were seeded at 6×10⁵ cells/100-mm dish 24 h before incubation in the virus/Polybrene-containing supernatants for 24 h. The cells were then washed and returned to fresh DMEM/F12 medium. Subsequently, the GFP and mCherry-positive clones were acquired by adding 2.5 µg/ml puromycin and 200 µg/ml hygromycin, respectively. After 7 days, the cells were cultured in ultra-low attachment dishes and the medium was replaced with TeSR™-E7 medium. After 1 week, the cells were attached on Matrigel (BD Biosciences)-coated plates with TeSR™-E7 medium for 7 days. Then, the culture medium was replaced with TeSR™-E8 medium for an additional 7 days. After transduction and reprogramming, cell colonies were plated in an attachment dish with DMEM/F12 medium supplemented with 10% FBS. The cells were maintained at 37°C in a 5% CO₂ incubator for these days. Thereafter, three cell lines (hTERT^+-O⁺-OME, hTERT⁺-S⁺-OME and hTERT⁺-OS⁺-OME) were obtained.

Two different shRNA sequences were inserted into plasmid vector pVSV-G. 48–72 h after transient transfection. The transfection efficiency was examined using fluorescence microscopy (Axio Observer A1; Carl Zeiss, Jena, Germany) to choose the cells with the most effective transfection. The sequence was then cloned into the pGLV3/H1 and pGLV10/U6 vectors for lentiviral-mediated knockdown of Oct4 and Sox2, respectively. The chosen short hairpin sequence specific for Oct4 was as follows: (5′-CCTTCGCA1551AGCCCTCATTT-3′) and Sox2 (5′-GCA1551GACTTCACATGTCCCAGC-3′). These complete vectors were named as LV3 (H1/GFP&Puro)-Oct4 and LV10 (U6/RFP&Puro)-Sox2. The Cal27 and Gca1551 cells were stably transfected with LV3 (H1/GFP&Puro)-Oct4 and LV10 (U6/RFP&Puro)-Sox2, respectively, according to the manufacturer's protocol (GenePharma). Subsequently, the single GFP- or RFP-positive clones were acquired by adding 2.5 µg/ml puromycin. The double GFP- and RFP-positive clones were selected by sorting using a flow cytometer (Aria II; BD Biosciences). After that, 6 fresh cell lines (Cal27-O^low, Cal27-S^low, Cal27-O^lowS^low, Gca1551-O^low, Gca1551-S^low and Gca1551-O^lowS^low) were formed. All cells were tested for Oct4 and Sox2 protein expression by western blotting.

Eight cell lines (Cal27, Cal27-O^low, Cal27-S^low, Cal27-O^lowS^low, Gca1551, Gca1551-O^low, Gca1551-S^low and Gca1551-O^lowS^low) were analyzed by western blotting. The total protein was extracted from the cultured cells using lysis buffer (Bio-Rad, Hercules, CA, USA). Proteins (40 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Bio-Rad). The membranes were then incubated with primary antibodies overnight at 4°C, washed twice and incubated with horseradish peroxidase-conjugated secondary antibody (Bio-Rad) for 2 h at room temperature. Subsequently, the protein bands were detected by enhanced chemiluminescence, visualized using a VersaDoc-MP Imaging system (Bio-Rad). The antibodies used were goat anti-rabbit-Oct4, goat anti-rabbit-Sox2 (both from Stemgent, San Diego, CA, USA) and goat anti-mouse-GAPDH (Boster, Wuhan, China). The concentrations used were 1:500 (Oct4), 1:500 (Sox2) and 1:1,000 (GAPDH), respectively.

One hundred and twenty 5-week-old non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice (16–18 g, all males) (Vital River Laboratories, Beijing, China) were divided into 12 groups, with 10 mice in each group. Twelve groups of cells were washed twice with antibiotic- and serum-free cell culture medium and finally resuspended in 0.1 ml of serum-free culture medium. The cell suspension was then mixed with an equal volume of Matrigel (BD Biosciences) and injected subcutaneously into the mice. The tumors that formed were surgically removed after 21 days. All the xenografts of oral squamous cell carcinoma were weighed. Representative tumor tissues were fixed in 3% formalin, embedded in paraffin wax and sectioned at a thickness of 10 µm. The sections were stained with hematoxylin and eosin (H&E) for pathological examination, or processed for immunohistochemical analysis. The experiments were carried out in accordance with the guidelines issued by the ethics Committee of Zhengzhou University.

For immunohistochemistry, paraffin-embedded tissue sections were removed of paraffin in xylene, and dehydrated in alcohol. Afterwards, the sections were subjected to antigen retrieval with a steam pressure cooker (120°C, 5 min) in citrate buffer (pH 6.0), washed with phosphate-buffered saline (PBS) and then blocked with 5% goat serum. Primary antibodies were diluted in 0.1% bovine serum albumin (BSA) and incubated with the sections overnight at 4°C. After incubation with the secondary antibody at room temperature for 60 min, the sections were stained with SP link detection kits (ZSGB-BIO, Beijing, China) according to the manufacturer's instructions. The primary antibodies used in the present study were as follows: goat anti-rabbit-Oct4 (1:200), goat anti-rabbit-Sox2 (1:200) (both from Stemgent), goat anti-rabbit-CK5 (1:100), goat anti-rabbit-CK19 (1:100), goat anti-rabbit-vimentin (1:100), goat anti-rabbit-Ki-67 (1:100), goat anti rabbit-CD31 (1:100) and goat anti rabbit-calponin (1:100) (all from Bioss, Beijing, China).

All values for tumors weight are presented as mean ± standard deviation. Comparisons between four groups of two cell lines were performed by one-way ANOVA. The significance level was assigned at P<0.05. The statistical tests were performed with the program, Statistical Package for Social Sciences (IBM SPSS 22.0; IBM SPSS, Armonk, NY, USA).

The lentiviral-mediated introduction of Oct4 and Sox2 (both or individual) into hTERT⁺-OME cells, followed by a series of reprogramming steps (Fig. 1), gave rise to the expression of Oct4 and Sox2 proteins in the nuclei of the cells after 28 days. The entire process was recorded by fluorescence microscopy (Fig. 2).

The ability of hTERT⁺-OS⁺-OME cells to form tumors in the xenograft model was assessed by injection of hTERT⁺-OME, hTERT⁺-O⁺-OME, hTERT⁺-S⁺-OME and hTERT⁺-OS⁺-OME cells subcutaneously into NOD/SCID mice, respectively and then the tumors were monitored for 21 days. As a result, only the hTERT⁺-OS⁺-OME group of cells was able to initiate tumor formation in the mice (Fig. 3A). Histological examinations of the tumors revealed that hTERT⁺-OS⁺-OME cells produced tumors with local invasion (Fig. 3B). Immunohistochemical studies demonstrated that the tumors had differentiation from epithelial cells (CK5- and CK19-positive) with mesenchymal properties (vimentin-positive). In addition, we detected that Oct4 and Sox2 transduced into the hTERT⁺-OS⁺-OME cells were positively expressed in the xenografts (Fig. 3B).

Western blot analysis showed that the expression levels of Oct4 and Sox2 were effectively altered by shRNA transfection in the Cal27 and Gca1551 cells. Downregulation of Sox2 by shRNA led to the slight downregulation or no change of Oct4 expression, while shRNA-mediated repression of Oct4 induced the upregulation of Sox2 expression (Fig. 4). In the xenograft model, Sox2^low and Oct4^lowSox2^low cells decreased the size of tumors in the immunodeficient mice equally. In contrast, the single knockdown Oct4^low cancer cells had increased tumor size when compared to the untreated cells (Fig. 5). The weight of each group of tumors was measured and the results are detailed in graphical representation (Fig. 6A). Moreover, the histological examinations demonstrated that Sox2^low or Oct4^lowSox2^low cells caused low histological grade carcinoma in the xenografts, while the Oct4^low cancer cells formed more aggressive xenografts comprised of high grade carcinoma (Fig. 6B).