Previously untreated primary MDLSCC samples from one female and nine male patients, aged 46–94 years (mean, 64.4 years), were sourced from the Gillies McIndoe Research Institute and used for this study, which was approved by the Central Health and Disabilities Ethics Committee (ref. no. 12/CEN/74).

Hematoxylin and eosin (H&E) staining was performed on 4-μm thick formalin-fixed paraffin-embedded blocks of 10 MDLSCC samples that were subsequently analyzed by an anatomical pathologist (Helen D. Brasch) to confirm the presence of SCC and the histological grading. 3,3-Diaminobenzidine (DAB) IHC staining for NANOG (1:100; cat# ab80892, Abcam, Cambridge, MA, USA), SOX2 (1:200; cat# PA1-094, Thermo Fisher Scientific, Waltham, MA, USA), SALL4 (1:30; cat# CM385M-16, Cell Marque, Rocklin, CA, USA), pSTAT3 (1:100; cat# 9145, Cell Signaling Technology, Danvers, MA, USA), OCT4 (1:1,000; cat# ab109183, Abcam), CD44 (1:1,500; cat# MRQ-13, Cell Marque), and epithelial membrane antigen (EMA, ready-to-use; cat# PA0035, Leica) diluted with Bond™ primary antibody diluent (Leica AR9352) was performed on the tissue sections using the Leica Bond Rx auto-stainer (Leica) as previously described (21, 31).

Immunofluorescent (IF) IHC staining was performed on two representative MDLSCC samples from the original cohort used for DAB IHC staining to investigate protein co-expression. Vectafluor Excel anti-rabbit 594 (ready-to-use; cat# VEDK-1594, Vector Laboratories, Burlingame, CA, USA) and Alexa Fluor anti-mouse 488 (1:500; cat# A21202, Thermo Fisher Scientific) were used to detect combinations that included NANOG, SOX2, or pSTAT3 and VectaFluor Excel anti-mouse (ready-to-use; cat# VEDK2488, Vector Laboratories) and Alexa Fluor anti-rabbit 594 (1:500; cat# A21207, Thermo Fisher Scientific) were used to detect combinations that included OCT4 or SALL4. All IF IHC-stained slides were mounted in Vectashield HardSet Antifade mounting medium with 4′,6-diamidino-2-phenylindole (cat#H-1500, Vector Laboratories).

Positive human control tissues used for the primary antibodies were skin for EMA, tonsil for CD44, pSTAT3 and SOX2, and seminoma for NANOG, SALL4, and OCT4. Negative antibody control was performed on one MDLSCC sample per antibody staining run.

DAB IHC-stained slides were viewed using an Olympus BX53 light microscope (Tokyo, Japan) and the images were captured with the CellSens 2.0 software (Olympus). IF IHC-stained slides were viewed and the images were captured using an Olympus FV1200 biological confocal laser-scanning microscope and processed with the CellSens Dimension 1.11 software using 2D deconvolution algorithm (Olympus).

Total RNA was isolated and quantified as previously described (31) from six snap-frozen MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining. They were subjected to the NanoString nCounter gene expression assay (NanoString Technologies, Seattle, WA, USA) by New Zealand Genomics (Dunedin, New Zealand). Briefly, total RNA was extracted using the MagJET RNA kit (Thermo Fisher Scientific) with the protocol adapted for tissue and run on a KingFisher Duo machine (Thermo Fisher Scientific). RNA samples were then quantitated on a Qubit^® 2.0 fluorometer (Thermo Fisher Scientific) and were subject to RNA integrity analysis via the 2100 Bioanalyzer Instrument (Agilent Technologies). Probes for the genes encoding CD44 (NM_001001392.1), NANOG (NM_024865.2), OCT4 (NM_002701.4), STAT3 (NM_139276.2), and the housekeeping genes glucuronidase beta (GUSB) (NM_000181.1), clathrin heavy chain (CLTC) (NM_4859.2), and hypoxanthine phosphoribosyltransferase 1 (NM_000194.1) were designed and manufactured by NanoString Technologies. Raw data were analyzed using nSolver™ software (NanoString Technologies) using standard settings and normalized against the housekeeping gene.

Total RNA was isolated from six snap-frozen MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining using the RNeasy mini Kit (Qiagen) with a DNase digest and the QIAcube system (Qiagen). Total RNA quantity and quality were assessed using NanoDrop 2000 (Thermo Fisher Scientific). Reverse transcription reactions were performed using the iScript Reverse Transcription Supermix for RT-qPCR (Bio-Rad, Hercules, CA, USA). The expression of stem cell markers was detected using gene-specific TaqMan primers-probe sets (SOX2: Hs01053049_s1; SALL4: Hs00360675_m1) with the Rotor-Gene Multiplex RT-PCR Kit (Qiagen). All measurements were performed in triplicate and relative mRNA expression was determined by the ΔΔCt method. GAPDH was used as the endogenous control and probe-specific gBlocks gene sequences (IDT technologies) were used as calibrators. All samples with a threshold cycle ≥35.0 were considered negative. Graphs were generated with Microsoft Excel and results are shown as relative expression.

Six snap-frozen MDLSCC samples from the original cohort of 10 patients included in DAB IHC staining underwent WB as previously described (32), using primary antibodies for NANOG (1:1,000; cat# ab47102, Abcam), OCT4 (1:2,000; cat# ab109183, Abcam), pSTAT3 (1:1,000; cat# 9145, Cell Signaling Technology), SOX2 (1:1,000; cat# PA1-094, Thermo Fisher Scientific), and β-actin (1:1,000; ab8226, Abcam). Two primary antibodies were used for SALL4 (1:1,000, cat# ab57577 and 1:1,000, cat# ab157172, both from Abcam). Secondary antibodies used were goat anti-rabbit horseradish peroxidase (HRP) conjugate (1:10,000; cat# A16110, Thermo Fisher Scientific) or Alexa Fluor^® 647 rabbit anti-mouse (1:2,000; cat# A21239, Thermo Fisher Scientific) as appropriate. HRP conjugated secondary antibody detection was achieved using Clarity™ Western ECL substrate (Bio-Rad). Membranes were imaged using a ChemiDoc MP imaging system (Bio-Rad).

Hematoxylin and eosin staining confirmed the diagnosis and histological grading of MDLSCC in all 10 samples. Staining patterns for the CSC markers in all 10 samples are shown in Table S1 in Supplementary Material.

DAB IHC staining demonstrated that some cells within the tumor nests (TNs) stained positively with the SCC marker EMA (data not shown), as expected. There was also membranous staining of the CSC marker CD44 (Figure 1A, brown) that was localized to cells within the TNs and the stroma. Staining of NANOG (Figure 1B, red), pSTAT3 (Figure 1C, brown), SALL4 (Figure 1D, brown), and SOX2 (Figure 1E, brown) was localized to cells within the TNs and the stroma. Immunoreactivity for OCT4 (Figure 1F, red) was primarily cytoplasmic and focal in cells within the stroma.

Expected staining patterns for CD44, NANOG, pSTAT3, SALL4, SOX2, and OCT4 (Figure S1 in Supplementary Material) were demonstrated in the respective positive controls. An appropriate negative control by the omission of the primary antibody in MDLSCC samples provided a control for the secondary antibody (Figure S2 in Supplementary Material).

Immunofluorescent IHC staining performed on two representative MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining demonstrated expression of the SCC marker EMA (Figure 2A, green) by cells within the TNs, while SOX2 (Figure 2A, red) was localized to cells within both the TNs and the stroma. Dual staining of SOX2 (Figures 2B,C, red) with CD44 (Figure 2B, green) and SALL4 (Figure 2C, green) showed that SOX2 was localized to cells within the TNs and the stroma that also expressed CD44 and SALL4. Co-staining of NANOG (Figure 2D, red) and pSTAT3 (Figure 2E, red) with CD44 (Figures 2D,E, green) demonstrated that the CD44⁺ cells within the TNs and the stroma also expressed NANOG and pSTAT3. Interestingly, the expression of OCT4 (Figure 2F, green) was confined only to a proportion of SOX2⁺ (Figure 2F, red) cells within the stroma. Images of the individual stains are presented in Figure S3 in Supplementary Material.

NanoString transcriptional profiling of the six MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining normalized against the housekeeping genes GUSB, CLTC, and HPRT1 confirmed the relative abundance of mRNA for CD44 and STAT3 in all the six samples. OCT4 was detected in four out of the six samples, while NANOG was undetectable in all six samples (Figure 3A).

RT-qPCR analysis of six snap-frozen MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining demonstrated abundance of mRNA transcripts for SOX2 and SALL4 in all six samples (Figure 3B).

Western blot analysis of six snap-frozen MDLSCC samples from the original cohort of 10 patients included in DAB IHC staining showed that NANOG was detected in all six samples (Figure 4A). OCT4 was detected in two of the six samples (Figure 4B), pSTAT3 was detected in five of the six samples (Figure 4C), and SOX2 was detected in all six samples (Figure 4D). A band corresponding to the expected size of SALL4 was undetectable by WB in any of the six samples (data not shown).