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 Table of Contents  
Year : 2021  |  Volume : 5  |  Issue : 2  |  Page : 170-179

Identification of a subpopulation of chemoresistant cancer cells with adult stem cell properties and embryonic transcription factors in oral squamous cell carcinoma

1 Advanced Research Centre, Narayana Medical College and Hospital, Nellore, Andhra Pradesh, India
2 Department of Biotechnology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India
3 Department of Cardiology, Narayana Medical College and Hospital, Nellore, Andhra Pradesh, India
4 Department of Oral and Maxillofacial Surgery, Narayana Dental College, Nellore, Andhra Pradesh, India

Date of Submission02-Apr-2021
Date of Acceptance01-May-2021
Date of Web Publication16-Jun-2021

Correspondence Address:
Mahaboob Vali Shaik
Advanced Research Centre, Narayana Medical College and Hospital, Nellore, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_55_21

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Background: In South-central Asia, oral cancer ranks among the three most common types of cancer. India alone accounts for 86% of the total oral cancer figures globally. Cancer stem cells (CSCs) are thought to give rise to differentiated tumor cells and to predict tumor recurrence and metastases. This study designed to characterize the CSCs derived from oral squamous cell carcinoma and its identification of correlation with embryonic transcriptional potential. Materials and Methods: Tumor (microscopically ~80% of their areas occupied by tumor cells) and normal counterpart (normal paired noncancerous matched tissue) samples from each histologically confirmed cases of oral squamous cell carcinoma (OSCC) were undertaken in this study. Isolation of stem cells using anti-CD133-positive selection. Expression levels of stem cell surface markers were assessed by flow cytometer. The immunoprofile of these markers was correlated with sex-determining region Y-box 2 (SOX-2), octamer-binding transcription factor 4 (OCT4), and NANOG. The tissue samples of OSCC were studied to identify the localization pattern for CSCs using fluorescence microscopy. Results: Histologically, SOX-2 expression has been identified at all zones exhibiting dysplasia. Isolated CD133+ cells showed differential expression pattern with embryonic transcription factors in tumor cells but not in normal counterpart, which depicts their cancer stemness. Flow cytometry analysis exhibited that SOX-2/OCT4/CD44+with CD133 positive stemness in OSCC malignant tissues was identified to be the best marker for OSCC prediction of the disease. Conclusion: The isolated subpopulation CD133+ cells possess the characteristics of both stem cells and malignant tumors. The findings show that elevated levels of CD133 lead to OSCC invasiveness and metastasis, associated with the upregulation of embryonic and stemness markers. Hence, these tumors may be controlled by restricting the expression of CD133, CD44, OCT4, and SOX2 or by disrupting the molecular pathways that are altered in CSCs.

Keywords: Cancer stem cell, oral squamous cell carcinoma, transcription factor

How to cite this article:
Shaik MV, Shaik M, Subramanyam G, Rajasekhar G. Identification of a subpopulation of chemoresistant cancer cells with adult stem cell properties and embryonic transcription factors in oral squamous cell carcinoma. Biomed Biotechnol Res J 2021;5:170-9

How to cite this URL:
Shaik MV, Shaik M, Subramanyam G, Rajasekhar G. Identification of a subpopulation of chemoresistant cancer cells with adult stem cell properties and embryonic transcription factors in oral squamous cell carcinoma. Biomed Biotechnol Res J [serial online] 2021 [cited 2023 Jun 5];5:170-9. Available from: https://www.bmbtrj.org/text.asp?2021/5/2/170/318432

  Introduction Top

Oral squamous cell carcinoma (OSCC) is the most prevalent subtype of head-and-neck cancers worldwide afflicting 300,000 people annually, with ~150,000 deaths.[1] Histologically, over 95% of oral cancers are squamous cell carcinomas.[2] In India, majority of squamous cell carcinomas arise from preexisting leukoplakias.[3] Betel-quid chewing and smoking are the major risk factors that contribute to the development of oral cancer in South Asia and South-east Asia.[4] The 5-year survival rate for the disease has not improved through the decades, despite advances in treatment modalities that predominantly include surgery and sometimes chemoradiotherapy.[5] The major cause of failure to cure OSCC includes resistance to therapy, recurrence, and metastasis, both local and distant.[6]

Progress in treatment and prognosis for OSCC has been limited, and the molecular mechanisms of OSCC escape from chemo and/or radiation therapies remain mostly unknown. OSCC, like many solid tumors, contains a heterogeneous population of cancer cells. Recent data suggest that a rare subpopulation of cancer cells, termed cancer stem cells (CSCs), is capable of initiation, tumorigenesis, progression, and metastasis.[7] It has been postulated that CSCs within the bulk tumor may escape conventional therapies, thus leading to disease relapse.[8],[9] CSCs undergo self-renewal and differentiation to yield phenotypically diverse nontumorigenic and tumorigenic cancer cells.[10] Therefore, an important goal of therapy could be to identify and kill this CSC population. If CSCs can be identified prospectively and isolated, then we should be able to identify new diagnostic markers and potential therapeutic targets. Defining CSC with specific markers has become difficult due to its frequent phenotypic transitions.[11]

Various markers independently or in combination have been investigated to study CSCs from various tumors. These include specifying molecules based on surface marker expression, drug transporters, enzymatic activity, signaling pathways, and so on.[12],[13]

In the last few years, there has been increasing interest in a broadly distributed family of cell surface glycoproteins such as CD44, CD133, and CD326. CD44 is involved in organ integrity through its ability to contact extracellular matrix (ECM), is signaling active, and serves as a co-receptor for numerous transmembrane proteins such as matrix metalloproteases, receptor tyrosine kinases (ERB) family, and the long known tumor-associated antigens EpCAM (CD326, ESA1).[14] EpCAM plays a morphoregulatory role in normal epithelial and stem/progenitor cells, as well as it may actively drive tumor progression in cancer cells, for example, breast cancer, ovarian cancer, pancreatic, urothelial, gall bladder carcinoma, lung adenocarcinoma tissue, and OSCC.[15],[16],[17],[18],[19],[20] In a study, data suggest that CD133+ cells represent a small subpopulation of CSCs that may contribute to chemoresistance in human OSCC.[21],[22],[23],[24]

Sex-determining region Y-box 2 (SOX2), octamer-binding transcription factor (OCT4) and NANOG which known as embryonic stem cell marker,[25],[26] which are also reported be expressed in various tumor tissues, indicates poor prognosis including prostate cancer, lung adenocarcinoma, gliomas, rectal cancer, gastric carcinoma, and OSCC.[27],[28],[29],[30]

Even though reliable biomarker is needed to develop the individualized treatment strategies for OSCC against CSC, hence, the current study attempted to identify CSC population by a combination of tumor markers with embryonic and adult stem cell marker in OSCC carcinoma versus normal cell counterpart beside the tumor.

  Materials and Methods Top

Sample collection

The protocol is approved by the Narayana Medical College and Hospital Ethics Committee, Nellore, Andhra Pradesh, India Number: IEC/NMC/2015/001-dt.13-06-2015. Tissue samples of oral cancer specimens aseptically collected from the Department of Oral and Maxillofacial Surgery, Narayana Dental College and Hospital, Nellore, India. Diagnosed samples of human OSCC (T) and normal noncancerous lesion (N) from the same individuals were obtained from surgical procedures shifted to the laboratory. Tumor tissues were microscopically screened to have ~ 80% of their areas occupied by tumor cells; the remaining specimen (tumor and normal counterpart lesions) were snap frozen at −80°C for flow cytometry. Immunofluorescence staining of tissue samples of OSCCs to establish the tissular localization pattern for cells expressing various stem cell markers. In addition, quantitatively checked for the same maker using flow cytometry.

Isolation of cancer stem cells

The tissue is processed immediately after collection. The tissue is handled in an aseptic condition and processed in a Biological Safety Cabinet. Tissue was rinsed for a minute in a washing solution containing phosphate-buffered saline (PBS; Invitrogen, CA, USA) and 3X antibiotic (X = 100 units/ml penicillin [Sigma] and 100 μg/ml streptomycin [Sigma]) and shifted to the laboratory in transfer solution containing Dulbecco's Modified Eagle's medium (DMEM) medium and 1X antibiotic and 1X amphotericin B (Gibco 250 μg/ml).

The tissues were then divided into small pieces using surgical scissors and subsequently digested in collagenase type I (3 mg/ml, Invitrogen) and disease (4 mg/ml, invitrogen) for 40 min at 37°C. The pieces are crushed using cell scrappers to disintegrate tissue into cell suspension. The resultant cell suspensions from each sample were then centrifuged with ×1200 g speed for 5 min. Cells are counted using a hemocytometer.

These cells further subjected to magnetically activated cell sorting (MACS) to isolate CSCs using positive selection to CD133 phenotypic markers. CD133-positive cell selection on Midimacs immunomagnetic selection systems (Miltenyi Biotec, Bergisch Gladbach, Germany). According to the manufacturer's instructions, 50 × 107 cells suspended in buffer containing PBS and 0.5% bovine serum albumin were incubated for 15 min at 4°C with a blocking reagent (human immunoglobulin G) and simultaneously with anti-human CD133 antibody (Miltenyi Biotec, Bergisch Gladbach, Germany). The cells were then washed and loaded on the magnetic separation column (LS + column) washed with 3 ml buffer. The unlabeled cells were washed five times through the column using 3 ml buffer. After removing the column from the magnetic separation unit, the labeled (positive) cells are collected by a gentle flush LS + column with 5 ml DMEM. The cell viability was checked by Trypan blue dye exclusion assay and counted by using a hemocytometer in ×10 under a microscope.

Characterization of cancer stem cells

Cells were divided into six fluorescence-activated cell sorting (FACS) round-bottom tubes (Becton Dickinson Falcon, Sunnyvale, CA) at 2 × 105 cells/tube and stained with immunoglobulin G–fluorescein isothiocyanate-conjugated various antibodies, CD326, CD44, CD34, CD90, CD45, CD105, SOX2, Oct4, and NANOG (BD Biosciences). After a 20-min incubation interval at the ambient temperature in the dark, cells were washed twice with 2 mL of FACS wash solution of phosphate-buffered saline containing 0.1% phosphate-buffered solution (PBS) FBS and 0.1% NaN3 and centrifuged for 5 min at 230 g. The supernatant was removed, and cells were fixed with 1% formaldehyde (in phosphate-buffered saline). Respective immunoglobulin G isotype-matched controls (BD Biosciences) were used as negative controls. All of the resultant data was acquired using a FACS Canto II (BD Biosciences, US) and analyzed using FACS Diva software (BD Biosciences).

Immunofluorescence/histochemical staining

All the solutions were prepared with Milli-Q water. Sections of tissue were removed before the collagenase digestion and frozen in −80°C until processing. For SOX-2, OCT4, and NANOG staining in the cytoplasm (Cell Signaling Technology, Inc. USA), sections were fixed by incubating with 2%–4% formaldehyde in PBS for 15 min at the room temperature. Slides were rinsed three times in PBS for 5 min each. Cells were permeabilized with 0.1% Triton X-100/PBS for 10 min. Specimens were blocked in blocking buffer for 60 min. Primary antibody was prepared during blocking by diluting 1:1000 antibody dilution buffer. Blocking solution was aspirated and incubated with diluted primary antibody for overnight at 4°C. Sections were rinsed for three times in PBS for 5 min each. Fluorochrome-conjugated secondary antibody diluted in Antibody Dilution Buffer 1:100 and applied to the specimen and incubated for 1–2 h at the room temperature in dark. Sections were rinsed in PBS and coverslip kept on the slide. Slides were sealed by painting around the edges of coverslips with nail polish.

Isolated total CD133+cells were also subjected to immunocytochemistry as showed above to determine the degree of positivity with stem cell markers: OCT4, SOX2, and NANOG. Negative controls were performed by replacing the first antibodies with the same origin and same isotype antibodies against other nonrelated antigens. Oct4 and Sox2 positive cells and total cells in the same fields were counted. The cells in 10 consecutive fields per section, 5 sections per each fetal liver were counted to assess the percentage of Oct4+ve and SOX2+ve cells. The slides were viewed separately by two pathologists who were blinded to the clinicopathological status. The expression of SOX2, OCT4, and NANOG on the slides was evaluated by the fluorescence immunoreactivity. The proportion of tumor cell staining was evaluated in terms of no staining, weak staining, and strong staining by their intensity levels. In each block, seven random areas were chosen to obtain to identify the percentage of positivity and staining intensity using ×400.

Statistical analysis

Data expressed as a mean and standard deviation. Unpaired t-test used to infer the differences between the groups. Pearson correlation test used to assess correlations between surface markers and intracellular markers of cancerous and normal lesions. For all statistical tests, a P ≤ 0.05 was considered statistically significant. Statistical analysis was performed using IBM Statical Package for the Social Sciences software v16.0 (SPSS Inc.,Chicago, IL, USA).

  Results Top

Characterization of cancer stem cells derived from oral squamous cell carcinoma

Nearly 30 participants suffering from OSSC were selected based on the convenient sample technique after getting clearance from the Institutional Ethical Committee. The investigation has been carried in the Department of Oral and Maxillofacial Surgery, Narayana Dental College and Department of Advanced Research Center, Narayan Medical College, Nellore, India. Tissue samples were collected from the participants with OSCC (group-tumor-T) and as well as adjacent noncancerous region (group-control-N) according to the described procedure. Tissues were processed and cells were separated from both the tissues as described. These separated cells from both tissues were passed through a column of MACS apparatus, which was coated with a monoclonal antibody against CD133. These positive cells were eluted and carried forward for flow cytometry. We assessed the percentage of recovery from the eluted cells. Our results were found to be quite encouraging and the isolated CD133 positive cells were exhibited varied percentage of recovery across the samples in both the groups, as demonstrated in [Table 1]. The maximum recovery of CD133+ cells in T group was found to be 1.4%, whereas in N-control group, it was 0.9%.
Table 1: Stemness of CD133+ cells derived from oral squamous cell carcinoma and their co-expression with other markers at normal and cancerous lesions in ovarian cancer stem cells

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Few studies reveal that these CD133+ cells co-express various cell surface receptors which include CD 34, CD 44, CD 45, CD90, CD105, CD 326, NANOG, Nestin, OCT4, and SOX-2 (reference). However, there is not clear cut evidence on the expression pattern of these receipts in OSCC. Hence, we further screened the isolated CD133-positive cells in both groups by flow cytometer, to analyze and reveal the expression pattern of these receptors and intracellular markers.

The flow cytometric data clearly demonstrated the expression pattern of these receptors, and intracellular markers in OSCC were quite interesting. The observed frequencies of CD133+cells co-expressing CD34 exhibited remarkable difference across the groups. The percentage of co-expression of CD34-positive cells in the cancerous group was 10.03% ±3.06%, higher, when compared to the normal group, 7.41% ±1.43%, with statistical significance (P = 0.0001).

Assessment of co-expression pattern of receptors

The CSC hypothetical theory suggests that only a few subsets of tumor cells have the tendency to initiate into tumors. Therefore, to understand the biology of the tumor, it is essential to identify and characterize such type of tumor cells. Few studies reported few markers for OSCC of CSC using in vitro culture system as a model. However, studies related to the expression of these markers in human clinical samples are still lacking. Hence, based on the literature survey, we decided to investigate to assess the following markers in the CD133-positive population of both groups.

First, we analyzed the co-expression pattern of CD44 cells in the isolated CD133-positive cells of both groups. The obtained results are displayed in [Table 1]. Our results showed the coexpression of CD34-positive cells in the cancerous group was higher with a percentage of 10.03% ± 3.06%, when compared to the normal group which exhibited a percentage of 7.41% ± 1.43%, with statistical significance (P < 0.0001). Next, we studied the pattern of CD 44 coexpression in the CD133 population. Here also, we observed similar results. The average mean percentage of co-expression of CD 44-positive cells was higher in the cancerous group (46.26% ± 11.83%) than that of normal group (11.80% ± 2.01%) with significant (P < 0.0001). Further, we assessed the co-expression pattern of CD 45 cells in the isolated CD133-positive cells. Interestingly, we noticed a declining trend in the expression of CD 45 in the cancerous group (2.46% ± 0.79%) when compared to normal group (8.37% ± 1.54%), which has statistical significance (P < 0.0001).

Further, we studied the co-expression pattern of CD90, CD105, and CD326 in the isolated CD133-positive population. The findings clearly suggest that remarkable variation has been observed which was presented in [Table 1]. The results of frequencies of CD133+cells co-expressing CD90 (cancerous: 32.62% ± 5.23%; normal: 27.1% ± 2.59%; P < 0.0001), CD105 (cancerous: 19.96% ± 3.79%; normal: 17.91% ± 2.83%; P < 0.06) and CD326 (Cancerous: 52.27% ± 10.27%; Normal: 16.88% ± 2.33%; P < 0.0001) were exhibited the varied pattern of expression. An increasing trend in the expression pattern of CD90, CD 105, and CD 326 has been observed in the cancerous group than that of the control group. However, CD105 did not show statistical significance, whereas the others exhibited statistical significance (P < 0.0001) [Figure 1].
Figure 1: Flowcytometry analysis of CD133+cells derived from oral squamous cell carcinoma with CD34, CD90, CD44, CD133, SOX-2, and OCT4 markers in cancerous lesions

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Furthermore, we also screened the co-expression pattern of NANOG, Nestin, OCT4, and SOX-2 in the isolated CD133-positive population. The data of co-expressing intracellular markers such as NANOG (cancerous: 11.06% ±2.99%; normal: 2.01% ± 0.69%; P < 0.0001), nestin (cancerous: 13.77% ±3.71%; normal: 7.68% ± 3.57%; P < 0.0001), OCT4 (cancerous: 50.99% ± 10.08%; normal: 2.86% ± 0.83%; P < 0.0001) and Sox-2 (cancerous: 60.11% ± 12.01%; normal: 2.07% ± 0.82%; P < 0.0001) exhibited elevated levels of expression in the cancerous group when compared to control group, which has statistical significance (P < 0.0001).

Therefore, the co-expression patterns of CD44, SOX-2, and OCT4 markers with CD133+ cells derived from normal and tumor counterpart showed higher variation in patients with OCSC [Figure 2].
Figure 2: Comparative analysis of various stem cell marker expressions in CD133+ sorted cancer cells and unsorted normal cells

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Correlation of markers between cancerous and normal lesions

Next, we tried to investigate the correlation between the markers which include CD34, CD44, CD45, CD90, CD105, CD326, NANOG, Nestin, OCT4, and SOX-2, respectively, in both the groups. The findings are clearly displayed in [Table 1], [Table 2], [Table 3]. Our findings reveal that there is clear cut correlation has been noticed across the groups. The maximum positive correlations observed between the markers such as CD90, CD105, CD44, CD34, CD326, Nestin, SOX-2, and OCT4, respectively, whereas the negative correlation observed in CD326 versus CD45 (r: −0.045), CD44 versus CD45 (r: −0.079), CD44 versus SOX-2 (r: −0.045), CD34 versus CD45 (r: −0.279), CD34 versus NANOG (r: −0.081), CD105 versus CD45 (r: −0.203), CD105 versus NANOG (r: −0.063), Nestin versus CD45 (r: −0.25), Nestin versus NANOG (r: −0.142), SOX-2 versus CD44 (r: −0.045), Sox-2 versus CD45 (r: −0.166), SOX-2 versus NANOG (r: −0.098), OCT4 versus CD44 (r: −0.255), OCT4 versus CD45 (r: −0.013), OCT4 versus NANOG (r: −0.1), NANOG versus CD326 (r: −0.126), NANOG versus CD44 (r: −0.056), NANOG versus CD34 (r: −0.081), NANOG versus CD105 (r: −0.063), NANOG versus CD45 (r: −0.026), NANOG versus Nestin (r: −0.142), NANOG versus SOX-2 (r: −0.098), NANOG versus OCT4 (r: −0.1).
Table 2: Correlations of markers CD34, CD44, CD45, CD90, CD105, CD326, NANOG, Nestin, OCT4, and SOX-2 within cancer group

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Table 3: Correlations of markers CD34, CD44, CD45, CD90, CD105, CD326, NANOG, Nestin, OCT4 and SOX-2 within noncancer group

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There were total negative correlations observed between individually CD45 with CD90, CD44, CD105, CD34, Nestin, Sox-2, OCT4, and Nanog markers.

Sex-determining region Y-box 2 and octamer-binding transcription factor expression in oral squamous cell carcinoma tissue and its normal counterpart

In order to analyze the expression of SOX2 in OSCC tissues, immunohistochemical staining was performed. The results revealed that SOX2 was expressed either in the cytoplasm or the cell membrane of OSCC cells or in both. SOX2 expression in OSCC tissues was positive and included in all tissues collected. By contrast, SOX2 expression in the normal counterpart group was negative. In the tissue section, on immunofluorescence analysis, embryonic markers, i.e. SIX-2, OCT4, and NANOG have been positively stained and given varied interpretations. The signals have been identified at various zonations of the collected cancer samples. The majority of OCT4+ve and SOX2+ve cells were located either within around the small ductal structures or close to them; these were of cytosolic staining, with small numbers of OCT4+ve cells showing nuclear staining, whereas SOX-2 was identified throughout the affected tissue. Similar localizations were observed for NANOG + ve cells [Figure 3].
Figure 3: Immunohisto/cytochemical analysis. (a) SOX-2 ICC; (b) SOX-2 ICC; (c) OCT4 ICC; (d) Sox-2 IHC; (e) OCT4 IHC; (f) SOX-2 IHC (Scale bar: 10–20 μm. Original magnification: a-c: ×100; d: ×20; e: ×40; f: ×20)

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Sex-determining region Y-box 2 and octamer-binding transcription factor expression in CD133+ cells of oral squamous cell carcinoma

The CD133+ from all tissues was much smaller in terms of size (~15 μm). CD133+ cells when subjected to immunofluorescence staining, OCT4 shown positive presence with less affinity in the cytoplasm of small-sized cells (10 μ−20 μ). Whereas, 10 μ–15 μ sized cells in sorted CD133+ cells have been shown OCT4 immunostaining with high positivity within the cytoplasm [Figure 4]. The CD133+ from all tissues was much smaller in terms of size (~15 μm). OCT4 in OSCC was observed to be heterogeneous throughout the field, both in terms of the percentage of positive cells and intensity.
Figure 4: Immunocytochemical analysis of CD133+ cells derived from oral squamous cell carcinoma. (a) OCT4; (b) Nanog; (c) SOX-2 ICC; (d) Sox-2 (Scale bar: 10 μm. Original magnification: ×100)

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On immunofluorescence staining of CD133+ cells with SOX-2 antibody showed a positive signal throughout the field. All Sox-2 positive cells showed 10 μ–20 μ size and expression of SOX-2 observed highly at cytoplasm [Figure 4]. SOX-2 in OSCC was observed to be homogeneous throughout the field, both in terms of the percentage of positive cells and intensity. Both Oct4 and Sox2 expression in sorted CD133+cells were observed with intense cytosolic staining. The frequency of OCT4 and SOX2 expression is varied in CD133+ sorted cells. Sox-2 signals were observed higher than OCT4 signals in the cytoplasm of CD133+ of OSCC, whereas there is less signal observed against Nanog antibody throughout the field.

  Discussion Top

Oral cancer is a devastating disease that ranks as the fifth most common type of cancer affecting humans worldwide.[4] There are approximately 500,000 new oral and pharyngeal cancer cases diagnosed annually, with three quarters being registered in developing countries.[31],[32] Despite increased experience in surgical technology and adjuvant therapies, the overall prognosis of OSCC remains unimproved, resulting in the urgent need for novel treatment strategies. A better understanding of the molecular genetics of OSCC could reveal the mechanisms of the initiation and progression of this malignancy and help to find a new way to develop therapeutic strategies.

Recent studies suggest that CSCs are especially resistant to conventional therapy and are the “drivers” of local recurrence and metastatic potential. Specific markers for this population have been investigated in oral cancers in the hope of developing a deeper understanding of their role in oral cancer pathogenesis, elucidating novel biomarkers for early diagnosis and newer therapeutic strategies.[33] Therefore, the identification and the targeted elimination of these CSCs was considered fundamental for cancer treatment.

Very few studies reveal the cell transition dynamics and characterization of CSCs derived from OSCC. Understanding the cell transition biology of CSCs in OSCC is quite complex. Till date, there is no valid marker in depicting CSCs, especially from OSCC. Therefore, the present study is carried out to understand the biology of cell transition and as well as to identify a potential stem cell marker of OSCC. We selected CD133 to sort to CSC due to its already been identified as a CSC marker derived from neural tissues, prostate, kidney, colorectal, liver, skin, and lung.[5] CD133-positive cells showed relatively high expression of Sox2, Nanog, OCT4, the well-known embryonic stem cell markers. CD326, CD44, and CD90 expression were also consistently high in CD133 cells of OSCC patient samples. CD44 has already been established as a CSC marker in a closely related head-and-neck squamous cell carcinoma (HNSCC).[34] The expression of CD44 is elevated in metastatic cells of breast cancer.[35] Their importance in OSCC is also being recognized along with other cancers.[36],[37] Wright et al. have shown that Brca1 breast tumors contain distinct CD44+/CD24 and CD133+ cells with CSC characteristics.[38] CD44 through its interactions with hyaluronic acid, chondroitin sulfate, and heparin sulfate, CD44 is able to bind growth factors and metalloproteinase MMP9, resulting in inhibition of apoptosis, collagen degradation, invasion, and neovascularization.[39]

In our study, CD326 shown higher expression in the cancerous group among all CD markers. However, the percentage of expression has been consistently changing as reported in another study.[40] EpCAM has been identified as an additional marker for cancer-initiating stem cells.[41],[42] Hwang et al. revealed that EpCAM expression decreased significantly from normal and mild oral epithelial dysplasia (OED) through moderate and severe OED to OSCC.[43] In HNSCC, increased EpCAM expression was observed from hyperplasia to tumor giving clues about its role in oral carcinogenesis.[44] Most studies did not find any association of this marker with clinicopathological parameters but a study specifically on tongue SCC demonstrated a direct relationship between EpCAM expression with larger tumor size, nodal metastasis, and tumor dedifferentiation.[45]

In our study, the expression of OCT4 and SOX-2 markers were high among all intracellular and cell surface markers in isolated CD133+ subpopulation of OSCC patients. The positive correlation of OCT4, SOX-2 with an increased expression status of CD133 depicted a poorer prognosis for oral cancer. OCT4 alone has been shown to be up-regulated in murine Lewis lung carcinoma, human OSCC, bladder cancer, and seminoma cancer.[30] NANOG and SOX2 have been observed to be upregulated in cases of human somatic tumors. Furthermore, it was demonstrated that a triple-positive expression of OCT4, SOX-2, and CD133 in metastatic tissue region of OSCC, indicating their usefulness as invasiveness and predictive marker.[46] OCT4 is reported to maintain the survival of CSCLCs partly by inhibiting apoptosis through the OCT4/TCL1/AKT1 pathway.[47] SOX2 participates in the SOX2/ORAIL/STIM1 pathway. NANOG over expression enhances the expression of many CSC-associated molecules, such as CD133, ABCG2, ALDH1A1, and CD44. Different cancers involve different signaling pathways, but it seems that OCT4, NANOG, and SOX2 are nearly always upregulated to activate or repress the cancer-related pathways.[48],[49] Previously, Nanog has been shown to be a therapeutic target controlling CSC self-renewal in various cancers including HNSCCs.[50] Expression of markers OCT4 and SOX2 in metastatic stage tissue revealed their involvement in the recurrence of cancer cells.

Individually, CD326, CD44, CD34, CD90, CD105, Nestin, Sox-2, OCT4, and Nanog levels positively correlated with all embryonic, mesenchymal stem cell markers with significant difference except CD45. Because CD45 negative will be accepted as tumor cells especially noncirculating tumor cells.[51]

In the present study, Sox2 expression was observed by immunofluorescence staining, showing higher in OSCC tumor region and compared with their counterparts. Increased Sox2 expression is observed in the tumor region. It was noted that the tumors generated by CD133+/CD44+ cells were positive for Sox-2, OCT4 in cytoflourescence studies. In addition, strong positive staining for SOX2/Oct4 was observed not only on the surface of the salivary gland structure but also on the carcinoma cells within the tumor mass. Hence, it was hypothesized that SOX2+/Oct4+ cells may be generated during the in vivo tumor growth from CD133+/CD44+ cell populations. This hypothesis indicates that normal and neoplastic cells can spontaneously convert to a stem-like state. Chaffer et al. showed that CD44+ cells can differentiate into CD44 (low)/CD24+/ESA− and CD44 (low)/CD24+/ESA + progeny, and CD44(low) cells can spontaneously convert to CD44+ cells.[52] Recent studies have also suggested that CSCs in OSCC promote distant metastasis, and this phenotype involves Sox-2 expression and associated pathways.[53],[54] Several transcriptional factors, including zinc-finger proteins snail, slug, and Twist have been identified which negatively regulated E-cadherin expression and were significant in EMT induction and maintenance of cancer stemness. In fact, the positive correlation of SOX-2, OCT4, and CD44 with an increased expression status of CD133 in T-region depicted that these are prognosis markers for oral cancer patients. Over expression of v and Nanog markers, found in CSC-enriched subpopulation derived from HNSCC sphere formation colonies, positively correlated with treatment failure and stage while negatively correlated with differentiation status.[54] Furthermore, it was demonstrated that patients displaying the triple-positive expression of OCT4, Nanog, and CD133 had the worst survival prognosis in OSCC, indicating their usefulness as invasiveness and predictive marker.[46] Cellular heterogeneity in terms of altered pathophysiology and differential expression of various stem cell markers (CD44, CD133, and others) and transcription factors, for example, OCT4, SOX2, and Nanog play a significant role in clonal proliferation. Isolation of CSCs from tumor specimen based on their cell surface markers will be useful in enrichment and establishment of cell lines which may provide in vitro model systems that mimic the functional characteristics of stem cells in the tumor microenvironment and their probable response to therapy.

  Conclusion Top

The current study focused on the cell transition dynamics and characterization of CSCs derived from OSCC. Identification and characterization of CSC from OSCC facilitate the monitoring, therapy, or prevention of OSCC. This data suggest that many potential CSC markers are expressed on different expression levels in OSCC. We have demonstrated that OCSC contain a distinct SOX-2+/OCT4+/CD133+/CD44+ cell subpopulation that possesses CSC-like properties. Hence, these may provide evidence for the existence of CSCs in OSCC. Tumors may be controlled by restricting the expression of CD133, CD44, OCT4, and SOX2 or by disrupting the molecular pathways that are altered in CSCs. The identification of CSCs may provide novel insights into the development of new therapeutic approaches for OCSC.


The authors acknowledge Narayana Medical College and Hospital and Narayana Dental College for providing technical help and material support.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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