Moderately differentiated lip squamous cell carcinoma samples from one female and nine male patients aged 46–94 (mean, 64.4) years sourced from the Gillies McIndoe Research Institute Tissue Bank, were used for this study, which was approved by the Central Regional Health and Disability Ethics Committee (ref. no. 12/CEN/74).

Hematoxylin and eosin (H&E) staining was performed on 4-μm thick formalin-fixed paraffin-embedded sections of MDLSCC samples by an anatomical pathologist (HDB) to confirm the appropriate histological grade and to identify areas of SCC within the tissue sections. 3,3-Diaminobenzidine (DAB) IHC staining was performed on the sections using primary antibodies for PRR (1:100; cat # HPA003156, Sigma, St. Louis, MO, USA), ACE (1:100; cat# MCA2054, AbD Serotec, Raleigh, NC, USA), ATIIR1 (1:30; cat# ab9391, Abcam), ATIIR2 (1:2,000; cat# NBP1-77368, Novus Biologicals, Littleton, CO, USA), and ETS-related gene (ERG; 1:200; cat# EP111, Cell Marque, Rocklin, CA, USA). All slides were mounted in Surgipath Micromount (cat# 3801732, Leica, Richmond, IL, USA).

To determine co-expression of two proteins, immunofluorescent (IF) IHC staining was performed on two MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining. This was done utilizing a combination of VectaFluor Excel anti-mouse 488 (ready-to-use; cat# VEDK2488, Vector Laboratories, Burlingame, CA, USA) and Alexa Fluor anti-rabbit 594 (1:500; cat# A21207, Life Technologies, Carlsbad, CA, USA) to detect combinations that included OCT4 (1:1,000; cat# EPR2054, ab109183, Abcam), SALL4 (1:30; cat# CM385M-16, Cell Marque, Rocklin, CA, USA), SOX2 (1:200; cat# PA1-094, Thermo Fisher Scientific, Waltham, MA, USA), and ERG (1:200; cat# EP111, Cell Marque) with PRR (1:2,000; cat#, ab40790, Abcam), ACE (1:100; cat# MCA2054, AbD Serotec), ATIIR1 (1:30; cat# ab9391, Abcam), and ATIIR2 (1:2,000; cat# NBPI 1-12677368, Novus Biologicals). All IF IHC-stained slides were mounted in Vectashield HardSet mounting medium with 4′,6-diamidino-2-phenylindole (Vector Laboratories).

Positive human control tissues used for primary antibodies for IHC staining were placenta for PRR and ERG; liver for ATIIR1, kidney for ACE and ATIIR2, skin for SOX2, and seminoma for OCT4 and SALL4. A negative control by omitting the primary antibody on a MDLSCC sample was also prepared (data not shown).

All antibodies were diluted with Bond primary antibody diluent (cat# AR9352, 143 Leica) and all DAB and IF IHC staining was carried out on the Leica Bond Rx autostainer as previously described (26).

Total protein was extracted from six MDLSCC specimens, from the original cohort of 10 patients used for DAB IHC staining, by homogenization in ice-cold RIPA buffer (Sigma-Aldrich, St. Louis, MA, USA) containing 10 mM dithiothreitol (Sigma-Aldrich) and 1× HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Western blotting (WB) analysis was performed as described previously (27), with changes to the antibodies used as follows: PRR (1:500; cat# HPA003156, Sigma-Aldrich), ACE (1:200; cat# sc-12184, Santa Cruz Biotechnology, Dallas, TX, USA), ATIIR1 (1:200; cat# sc-1173, Santa Cruz Biotechnology), ATIIR2 (1:5,000; cat# ab92445, Abcam), and β-actin (1:2,000; cat# ab8226, Abcam). Appropriate secondary antibodies used were goat anti-rabbit horseradish peroxidase (HRP) conjugate (1:10,000; cat# A16110, Life Technologies), donkey anti-goat HRP conjugate (1:5,000; cat# ab97120, Abcam) or Alexa Fluor^® 647 rabbit anti-mouse (1:2,000; cat# A21239, Life Technologies). All primary and secondary antibodies were diluted in TBS containing 0.1% Tween-20. Detection of the HRP-conjugated secondary antibodies was achieved using Clarity™ Western ECL substrate (Bio-Rad, Hercules, CA, USA). Membranes were imaged using the ChemiDoc MP imaging system (Bio-Rad).

Six snap-frozen MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining were utilized for isolation of total RNA for NanoString nCounter™ Gene Expression Assay (NanoString Technologies, Seattle, WA, USA). RNA was extracted from tissues using a RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantitated by Qubit^® 2.0 Fluorometer (Life Technologies). This RNA was subjected to NanoString nCounter™ gene expression assay completed by New Zealand Genomics Ltd. (Dunedin, NZ), according to the manufacturer’s protocol. Probes for PRR (ATP6AP2, NM_005765.2), ACE (NM_000789.2), ATIIR1 (AGTR1, NM_000685.3), ATIIR2 (AGTR2, NM_000686.3), and the housekeeping gene GUSB (NM_000181.1) were designed and synthesized by NanoString Technologies. Data analysis was done using nSolver™ software (NanoString Technologies) with standard settings. Results were normalized against the housekeeping gene and graphed using Excel (Microsoft Office 2013).

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

H&E staining confirmed the histological grade and the presence of MDLSCC for all 10 samples. DAB IHC staining showed weak to moderate cytoplasmic expression of PRR localized predominantly to cells within the TNs and occasional cells within the stroma (Figure 1A, brown). ACE was present within the endothelium of the microvessels within the stroma between the TNs (Figure 1B, brown) but not the cells within the TNs. Weak to moderate cytoplasmic and nuclear staining of ATIIR1 was observed in cells within the TNs and cells within the stroma (Figure 1C, brown). There was strong perinuclear and cytoplasmic staining of ATIIR2 in cells within the TNs, the stroma and skeletal muscle (Figure 1D, brown). A summary of staining patterns across all 10 samples is given in Table S1 in Supplementary Material.

Positive human control tissues demonstrated the expected staining pattern in placenta for PRR (Figure S1A in Supplementary Material) and ERG (data not shown), liver for ACE (Figure S1B in Supplementary Material) and ATIIR1 (Figure S1C in Supplementary Material, brown) and kidney for ATIIR2 (Figure S1D in Supplementary Material, brown). The omission of the primary antibody in a MDLSCC sample provided a control for the secondary antibody (data not shown).

Double IF IHC staining showed that PRR (Figure 2A, red) was expressed by the SALL4⁺ cells (Figure 2A, green) within the TNs and the stroma. PRR (Figure 2B, red) was also detected in the OCT4⁺ CSC subpopulation (Figure 2B, green) and a separate OCT⁻ CSC subpopulation (Figure 2B, red) within the stroma. Expression of ACE (Figure 2C, green) was observed in endothelium of the microvessels that also expressed SOX2 (Figure 2C, red). Expression of ACE was localized exclusively to the endothelium of the microvessels that expressed the endothelial marker ERG (Figure 2D, red). ATIIR1 (Figure 2E, green) was expressed by cells within the TNs as well as the microvessels that also expressed SOX2 (Figure 2E, red). Perinuclear expression of ATIIR2 (Figures 2F,G, red) was demonstrated in cells within the TNs that expressed SALL4 (Figure 2F, green) as well as cells within the stroma, regardless of whether they were OCT4⁺ (Figure 2G, green) or OCT4⁻ (Figure 2G, red). Separated IF IHC images of Figure 2 are shown in Figure S2 in Supplementary Material.

Western blotting confirmed the presence of PRR at an expected molecular weight of 35 kDa (Figure 3A) within the extracts of all six MDLSCC samples examined. ACE (Figure 3B) was detected in five of the six MDLSCC samples analyzed, at the expected molecular weight of 195 kDa. ATIIRI was detected at 41 kDa in five samples, however, its proximity to the control ruled out significance (data not shown). ATIIR2 was detected in four of the six MDLSCC samples at approximately 50 kDa (Figure 3C), which is larger than the native form of the protein and may be due to glycosylation. β-actin confirmed approximately equal total protein loading in all samples (Figures 3A–C).

NanoString gene analysis for PRR, ACE, ATIIR1, and ATIIR2 performed on six MDLSCC samples from the original cohort of 10 patients used for DAB IHC staining, confirmed the presence of PRR and ACE mRNA in all six samples, and ATIIR1 mRNA in one sample. ATIIR2 mRNA was below detectable levels in all six samples (Figure 4).