The human oral neoplastic cell lines, OSC-19 (cat. no. 0198), OSC-20 cells (cat. no. 0197), SCC4 (cat. no. 9118), SAS (cat. no. 0260), HSC2 (cat. no. 0622), HSC3 (cat. no. 0623), HSC4 (cat. no. 0624), KOSC-2 (cat. no. 0126.1), KON (cat. no. 0194), Ca9-22 (cat. no. 0625), HO-1-u-1 (cat. no. 0828), HO-1-N-1 (cat. no. 0831), SAT (cat. no. 1027) and SKN-3 (cat. no. 1039) were obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank. The aforementioned cell lines were authenticated by JCRB Cell Bank using STR methods. The other human oral neoplastic cell lines, OLC-01 (23) and OSC-70 (24) were gifted from Dr S. Kawashiri (Kanazawa University, Kanazawa, Japan). All cell lines were cultured in high glucose DMEM (Sigma-Aldrich; MilliporeSigma) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO₂.

The assessment of CD44 and SSEA3 was conducted through a process of flow cytometry. This process was facilitated by the use of BD FACSVerse™ Flow Cytometer (cat. no. 651155, version 1.1), which was sourced from BD Biosciences. Before the assessment, the cells were dissociated with Accutase (cat. no. AT104; Invitrogen; Thermo Fisher Scientific, Inc.) and washed with ice cold PBS. A number of the cells was adjusted to a concentration of 1.0×10⁶ cells/ml in ice cold FACS Buffer (PBS, 1% BSA; cat. no. BR-220701651; Biomedical Science, Co., Ltd.). A total of 500 µl cell suspension was stained with a primary antibody that is a rat anti-human SSEA3 monoclonal antibody (MC-631; 1:100; cat. no. MA1-020; Thermo Fisher Scientific, Inc.) and incubated for 30 min at 4°C. After incubation, the cells were washed with ice cold PBS by centrifugation at 200 × g for 5 min and resuspended in 500 µl ice cold FACS Buffer. The cells were stained with the following secondary antibodies: allophycocyanin (APC)-labeled mouse anti-CD44 monoclonal antibody (MEM-85; 1:500; cat. no. ab239294; Abcam) and fluorescein isothiocyanate (FITC)-labeled goat anti-rat IgM polyclonal antibody (1:500; cat. no. 112-095-075; Jackson ImmunoResearch Laboratories, Inc.) and kept at 4°C in a fridge for 30 min. After incubation and washing with PBS, the cells were stained with SYTOX™ Blue Dead Cell Stain (1:500: cat. no. S34857; Invitrogen; Thermo Fisher Scientific, Inc.) to differentiate between viable and nonviable cells. In the flow cytometric analysis, purified rat IgM k isotype control monoclonal antibody (1:100; cat. no. 400801; BioLegend, Inc.) and purified mouse IgG2b k isotype control monoclonal antibody (1:100; cat. no. 202201; BioLegend, Inc.) were used as control primary antibody. These controls were obtained from BD Pharmingen; BD Biosciences. This comprehensive process of flow cytometric assessment allowed for a detailed and accurate appraisal of CD44 and SSEA3 in the cells. Each positive cell population in the entire oral cancer cell population has been analyzed. Values were expressed as percentages.

The isolation of SSEA3 positive (+) and SSEA3 negative (−) cell populations was achieved through a process of cell sorting. This process was facilitated by the use of a Sony SH800S Cell Sorter (Sony Biotechnology Inc.). The sorter was equipped with a 130-µm sorting chip, which was crucial for the segregation of cells. The SCC4 cell lines were prepared for sorting by staining them with a rat anti-human SSEA3 monoclonal antibody (Thermo Fisher Scientific, Inc.) and a goat anti-rat IgM (FITC) antibody (Jackson ImmunoResearch Laboratories, Inc.). This staining process was carried out strictly in accordance with the manufacturer's guidelines provided by Sony Biotechnology. Following the staining, the purity of the partitioned SSEA3(+) and SSEA3(−) cells was assessed. This assessment was carried out through flow cytometry, employing a FACS Verse instrument. The cells were labeled again with the rat anti-human SSEA3 monoclonal antibody and the goat anti-rat IgM FITC antibody for this assessment. Before the sorting process, the stained cells were filtered into a conical tube. A 35-µm nylon filter was used for this filtration process. The cell proliferation, cell cycle, reverse transcription-polymerase chain reaction and chemoresistance assay data reflecting SSEA3 expression were differentiated via cell sorting from SCC4. This comprehensive process ensured the isolation of the specific cell populations for further study.

The study of cellular proliferation was conducted to compare the growth rates between various cellular samples. The procedure began with seeding 1×10⁵ cells in each well of a six-well plate. The cell culture conditions were maintained at 37°C and atmosphere standard for cell growth, specifically within a 5% CO₂ incubator. The growth medium utilized was DMEM, supplemented with 10% FBS to provide necessary nutrients for the cells. These conditions were maintained for a period ranging between 1 to 3 days, allowing for cells to undergo several rounds of proliferation. Post-incubation, the counting of cells was carried out using a Countess Automated Cell Counter (Invitrogen; Thermo Fisher Scientific, Inc.). This device facilitated the accurate and reliable quantification of cells in the sample. To enhance the reliability of the count and to reduce sample bias, a minimum of four different cell images from each well were randomly selected and captured with a phase-contrast microscope for independent verification. This ensured that the cell count was representative of the entire well, providing a more accurate measurement of cellular proliferation. These methods provided a comprehensive and precise examination of the cellular proliferation rates, eliminating any potential for misunderstanding and ensuring the reproducibility of the experiments.

The cell cycle analysis was conducted on SSEA3(+) and negative (−) cells, which were sorted from SCC4 cells. The sorted cells were first resuspended in a solution. This solution was prepared by adding 5 µl of Cell Cycle Assay Solution Blue kit (Dojindo Molecular Technologies, Inc.) to 500 µl of Phosphate-Buffered Saline. This resuspension process was carried out at a controlled temperature of 37°C for a duration of 15 min, strictly adhering to the guidelines provided by the manufacturer of the Cell Cycle Assay Solution Blue. After the resuspension, the cells were stained using the same solution. The stained cells were then assessed using a specific instrument known as BD FACSVerse™ Flow Cytometer. The assessment was followed by a filtration process. In this process, the cells were filtered into a conical tube. A 35-µm nylon filter was used for this purpose to ensure the collection of only the cells of interest. The entire process was carried out under sterile conditions to prevent any contamination. Percentage of cells in the G0/G1, S, and G2/M phases on SSEA3(+) or (−) cells from SCC4 cells has been analyzed.

The mRNA expression levels of brother of the imprinted site regulator (BORIS), sex-determining region Y-box 2 (SOX2), homeobox protein NANOG (Nanog), and octamer-binding transcription factor 4, also known as POU Class 5 Homeobox 1 (POU5F1) were analyzed using a Rotor-Gene Q 2plex System (Qiagen GmbH) with FAM/ZEN/IBFQ probes (Integrated DNA Technologies, Inc.). DNA sequences of pre-designed BORIS primer/probe (ref. 106829301, Hs.PT.58.50443006, NM_001269040, Integrated DNA Technology), pre-designed SOX2 primer/probe (ref. 105825433, Hs.PT.58.237897.g, NM_003106, Integrated DNA Technologies, Inc.), pre-designed Nanong primer/probe (ref. 105825437, Hs.PT.58.21480849, NM_024865, Integrated DNA Technology) and pre-designed POU5F1 primer/probe (ref. 105825441, Hs.PT.58.14648152.g, NM_203289, Integrated DNA Technology) were not available. Assay IDs and RefSeq accession numbers of the tested genes were provided. Total RNA was extracted from SCC4 cells using TRI Reagent (Merck KGaA) and a PureLink RNA Mini kit (Thermo Fisher Scientific, Inc.). cDNA was obtained using a PrimeScript 1st strand cDNA Synthesis kit (Takara Bio, Inc.). All reactions were performed in accordance with the manufacturer's instructions. All PCR reactions were performed on a Qiagen Rotor-Gene Q real-time PCR system with the following thermocycling conditions: Pre-incubation at 94°C for 2 min, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. A total of 10 ng cDNA sample were used for the RT-qPCR reaction following the manufacturer's instructions. The fluorescent signals were analyzed by Rotor-Gene Q software version 2.3.1 (Qiagen). For the 2^−ΔΔCq method for qPCR to be valid, the efficiency of the target amplification and the efficiency of the reference amplification must be ~equal. Thus, Hypoxanthine Phosphoribosyltransferase 1 (Hs.PT.58v.45621572, NM_000194), beta 2-microglobulin (Hs.PT.58v.18759587, NM_004048), TATA-Box Binding Protein (Hs.PT.58v.39858774, NM_003194), Glucuronidase Beta (Hs.PT.58v.27737538, NM_000181), Actin Beta (ACTB) (Hs.PT.39a.22214847, NM_001101), RNA Polymerase II Subunit A (Hs.PT.39a.19639531, NM_000937), Ribosomal Protein Lateral Stalk Subunit P0 (Hs.PT.39a.22214824, NM_001002) and Peptidylprolyl Isomerase A (Hs.PT.58v.38887593.g, NM_021130) were analyzed as potential internal standards. Based on these validation experiments, ACTB was selected as an internal standard using HEX/ZEN/IBFQ probes (Integrated DNA Technologies, Inc.). Relative expression levels were calculated using the 2^−ΔΔCq method for qPCR (25), which presents the data as fold differences in expression relative to a calibrator sample, in this case represented by the mean expression of three experimental measurements.

The chemoresistance assay was conducted using SSEA3(+) or (−) cells that were isolated from SCC4 cells. These cells were seeded at 2.5×10⁴ cells/well and inoculated in 96-well plates and then cultivated with varying concentrations of cisplatin (0, 0.01, 0.1, 1, 5, 10 µM), paclitaxel (0, 0.01, 0.1, 1, 5, 10 nM) and docetaxel (0, 0.01, 0.1, 1, 5, 10 nM) for 72 h. Following this cultivation period, the number of cells and their viability were measured. The percentage of viability was determined. This was achieved by utilizing the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc.), following the manufacturer's protocol. A total of 10 µl Cell Counting Kit-8 solution was added to each well, then incubated for 30 min. The optical density at 490 nm was measured using a microplate reader (iMark™ Microplate Absorbance Reader; Bio-Rad Laboratories, Inc.). The mean IC₅₀ values, which represent the concentration of a drug that gives half-maximal response, were derived from three independent experiments. These values were calculated based on discrete sigmoidal curves that represent the growth inhibition activity in response to cisplatin, paclitaxel, and docetaxel. The cisplatin used in the aforementioned assay was procured from Nichi-Iko Pharmaceutical Co., Ltd., while the paclitaxel and docetaxel were sourced from Nipro Corporation. The data are presented as the mean ± standard deviation.

Data were analyzed by one-way analysis of variance and bilateral Student's t-test using EZR (Saitama Medical Center, Jichi Medical University, version 2.13.0), which is a graphical user interface for R (The R Foundation for statistical computing) (26). P≤0.05 was considered to indicate a statistically significant difference.

To evaluate SSEA3 expression in oral cancer cell lines, flow cytometric analysis was conducted on 16 oral cancer cell lines. This revealed the presence of a small number of SSEA3(+) cells in the several oral cancer cell lines, ranging from 0.02 to 5.88% in OSC-20, Ho-1-u-1, HSC4, SAT, Ho-1-N-1, SAS and SKN-3. SCC4 exhibited the highest expression levels among the tested oral cancer cell lines (Fig. 1A and B).

Flow cytometric analysis revealed a profusion of CD44(+) cells in 16 oral cancer cell lines, ranging from 83.27 to 99.99% (Fig. 2A and B). To elucidate the association between SSEA3 and CD44, SCC4, SKN-3 and SAT were double-stained with CD44 and SSEA3. In the CD44(+) fraction, 52.61% of SCC4 cells, 5.88% of SKN-3 cells, and 0.10% of SAT cells were SSEA3(+) (Fig. 2C). Consequently, further experiments with SCC4 were conducted in order to demonstrate the characteristics of SSEA3 in an oral cancer cell line.

To elucidate the basis for the difference in tumorigenic activity, the cell proliferation capacity of SSEA3(+) or (−) SCC4 cells was investigated. The cell proliferation assay indicated that SSEA3(+) SCC4 cells displayed significantly higher cell proliferation activity compared with SSEA3(−) cells after a 72-h incubation (Fig. 3A and B). SSEA3(+) cells in ES cells and HCT116 cells demonstrated rapid cell cycling (27,28). To determine the cause of the high proliferative capacity of SSEA(+) SCC4 cells, the cell cycle status was examined. Cell cycle analysis revealed that the proportion of SSEA3(+) SCC4 cells in the S and G2/M phases was higher than that of SSEA3 negative (−) cells (Fig. 4A and B).

qPCR was performed to validate the stem cell-related markers in SSEA3(+) or (−) SCC4 cells. The expression levels of BORIS, SOX2, Nanog and POU5F1 in the SSEA3(+) SCC4 cells were 4.18-, 7.09-, 6.33- and 10.92-fold higher than those in the SSEA3(−) SCC4 cells, respectively (Fig. 5).

The chemo-resistant properties of SSEA3(+) or (−) SCC4 cells were assessed by determining their sensitivity to anticancer drugs, including cisplatin, paclitaxel and docetaxel, utilizing the Cell Counting Kit-8 (Fig. 6A). The IC₅₀ of cisplatin in SSEA3(+) SCC4 cells was 8.441±0.528, compared with 8.828±0.520 in SSEA3(−) SCC4 cells (Fig. 6B). SSEA3(+) SCC4 cells exhibited significantly inhibited cell growth with paclitaxel (0.834±0.052) and docetaxel (5.662±0.069) compared with SSEA3(−) SCC4 cells with paclitaxel (3.168±0.142) and docetaxel (9.160±0.160) (Fig. 6B).