Edited by: Vincent Vander Poorten, KU Leuven, Belgium
Reviewed by: A. B. Zulkiflee, University Malaya Medical Centre, Malaysia; Tommaso Lombardi, Hôpitaux Universitaires de Genève, Switzerland
†Equal senior authors.
Specialty section: This article was submitted to Otorhinolaryngology - Head and Neck Surgery, a section of the journal Frontiers in Surgery
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To identify and characterize cancer stem cells (CSCs) in moderately differentiated lip squamous cell carcinoma (MDLSCC).
MDLSCC samples underwent 3,3-diaminobenzidine (DAB) immunohistochemical (IHC) staining for squamous cell carcinoma marker EMA, CSC marker CD44 and embryonic stem cell markers NANOG, octamer-binding transcription factor 4 (OCT4), spalt-like transcription factor 4 (SALL4), sex-determining region Y-box 2 (SOX2), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3). Immunofluorescent IHC staining was performed on two MDLSCC samples. Western blotting (WB) was used to confirm the expression of the aforementioned proteins and their transcription activation was investigated using NanoString and RT-qPCR.
IHC staining demonstrated the presence of (1) an EMA+/CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4− CSC subpopulation within the tumor nests (TNs); (2) a CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4− CSC subpopulation; and (3) a CD44+/SALL4+/NANOG+/pSTAT3+/SOX2+/OCT4+ CSC subpopulation within the stroma, between the TNs. NanoString and RT-qPCR confirmed the presence of mRNA for CD44, SALL4, STAT3, SOX2, and OCT4, and WB confirmed the presence of NANOG, pSTAT3, SOX2, and OCT4.
This study demonstrates three putative CSC subpopulations within MDLSCC.
Lip cancer constitutes a subsite of oral cavity cancer with more than 80% being squamous cell carcinoma (SCC) (
The etiological factors for oral cavity SCC (OCSCC) including lip SCC include allelic imbalance involving tumor suppressor genes (
There is growing evidence supporting the hierarchical model of carcinogenesis, which proposes that the development, growth, and spread of cancer are driven by a small population of cancer stem cells (CSCs) (
CD44 is a transmembrane glycoprotein, a receptor for the glycosaminoglycan hyaluronan (
We have recently identified and characterized CSC subpopulations within oral tongue (
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 (
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 (
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 (
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
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
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
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
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
This study adds to the growing body of evidence for the presence of CSCs in OCSCC. Our study demonstrates the presence of three distinct CSC subpopulations within MDLSCC (
The novel identification of more than one subpopulation of CSCs within MDLSCC parallels our recent findings in oral tongue (
NANOG and SOX2 were expressed on cells within the TNs and the stroma. Overexpression of SOX2 in tumors has been correlated with increased tumor thickness and invasion, metastasis in esophageal cancer, drug resistance, and decreased survival in tumors such as breast cancer and lung adenocarcinoma (
Interestingly, OCT4 is expressed exclusively by the CSC subpopulation within the stroma. It is intriguing that the expression of OCT4 demonstrated by IHC staining in the stroma of all 10 MDLSCC samples was supported by NanoString and WB analyses in only two out of six samples. This may be due to sampling bias and/or antibody specificity. Investigating the expression of OCT4 in head and neck SCC samples of 119 patients, Koo et al. (
Wu et al. (
This report demonstrates three putative subpopulations of CSCs within MDLSCC. It adds to the increasing support of the hierarchical concept of cancer. Further study may provide insights into the role CSCs may play in the biology of MDLSCC. It is exciting to speculate that CSCs may be a potential novel therapeutic target.
This study included a relatively small sample size. A larger study is needed to confirm the observed expression pattern.
Further work is needed on well and poorly differentiated lip SCC lesions to compare the expression patterns observed in this study.
Functional cell culture work is needed to demonstrate the ability for the three putative CSC subpopulations to form an orthoptic model for this cancer.
TI and ST formulated the study hypothesis and designed the study. RR, HB, TI, and ST interpreted the DAB and IF IHC data. JD performed WB analysis. JD, TI, PD, and ST interpreted the WB data. TI and ST interpreted the NanoString data. RR, TI, and ST drafted the manuscript. All authors commented on and approved the manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. TI, PD, and ST are inventors of the PCT patent application (No. PCT/NZ2015/050108) cancer diagnosis and therapy.
We thank Ms. Liz Jones and Ms. Alice M. Chibnall of the Gillies McIndoe Research Institute for their assistance in IHC staining and performing tissue processing for NanoString and analysis of the data, respectively. TI was supported, in part, by the Pacific Emerging Researcher First Grant, Health Research Council of New Zealand (grant no. HRC 16/434). Aspects of this work were presented at the Australia and New Zealand Head & Neck Cancer Society Annual Scientific Meeting and the International Federation of Head and Neck Oncologic Societies 2016 World Tour, 25-27 October 2016, Auckland, New Zealand.
The Supplementary Material for this article can be found online at
DAB, 3,3-diaminobenzidine; CSC, cancer stem cell; ESC, embryonic stem cell; H&E, hematoxylin and eosin; IF, immunofluorescent; IHC, immunohistochemical; MDLSCC, moderately differentiated lip squamous cell carcinoma; OCSCC, oral cavity squamous cell carcinoma; SCC, squamous cell carcinoma; TN, tumor nest; WB, Western blotting.