IDENTIFICATION OF CANCER STEM CELLS IN ORAL SQUAMOUS CELL CARCINOMA USING CRIPTO-1.

Yasmine Alaa El-Din 1 , Amina Fouad Farag 1 and Nadia Fathy Hassabou 2 . 1. Assistant Lecturer, Oral Pathology Department, Faculty of Dentistry, October 6 University, Egypt. 2. Lecturer, Oral Pathology Department, Faculty of Dentistry, October 6 University, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 6(1), 411-419 412 Such capabilities can be attributed to the presence of a small undifferentiated subpopulation of cells with stem-like, self-renewing properties capable of differentiating within the tumor termed cancer stem cells (CSCs) (Ye et al., 2015).
CSCs are highly tumorigenic with a greater capacity for migration, invasion, and proliferation compared to the other cancer cells (Chen, 2009 andOkamoto et al., 2009) and are believed to be largely responsible for the biological characteristics of cancer namely, rapid growth, invasion, metastasis  and even recurrence after conventional therapy due to their increased resistance against radio and chemo-therapeutic modalities (Oshimori et al., 2015).
Over the past years, a substantial number of studies on many solid tumors including glioblastoma (Tysnes et al., 2013), breast cancer (Al-Hajj et al., 2003), prostate cancer (Collins et al, 2005), pancreatic cancer (Hermann et al., 2007), colorectal cancer  and OSCC ) support the role of CSCs where their targeted elimination can present a new conceptual framework for oral cancer risk assessment, early detection, prognosis, prevention and treatment and becomes increasingly crucial for clinical and basic tumor research (Tirino et al., 2008, Prince et al., 2007and Satpute et al., 2013. However, most of these stem cell markers including CD133, CD44,...etc. used in the above studies were empirical and derived from normal stem cells which are known to be largely tissue specific which inturn had raised an important question regarding the specificity and reliability of such CSC markers (Baillie et al., 2017 andLiu et al., 2017).
This finding is further complicated by the presence of multiple CSC subtypes within the same tumor and among different tumors, making the investigation more reliant on the use of multiple markers and rise the urge to identify a CSC marker specific for the OSCC in order to improve the diagnostic capabilities and treatment efficacy (Baillie et al., 2017).
Cripto-1 (CR-1), also referred to as teratocarcinoma-derived growth factor-1 (TDGF-1), has showed a promising potential as a functional marker for the identification and isolation of CSC from OSCC based on the finding that the CR-1 high OSCC cells express high level of stemness-related genes and possess high potential of self-renewal and high ability of tumorigenesis .
CR-1 is a member of the epidermal growth factor-Cripto/FRL1/Cryptic as a cysteine-rich region (EGF-CFC) gene family that is involved in embryonic development, embryonic stem cell self-renewal, pluripotency capabilities‫و‬ tumor progression and maintenance. Remarkably, it is expressed in CSCs compartments, modulating and boosting cancer cell proliferation, migration, invasion, epithelial mesenchymal transition (EMT) and angiogenesis (Klauzinska et al., 2014).
It also participates in multiple cell signaling pathways including Wnt, Notch, and TGF-β which are directly involved in embryogenesis, cell differentiation, proliferation, migration, organogenesis, epithelial-to-mesenchymal transition (EMT) and apoptosis (Bianco et  Overexpression of CR-1 is found to be correlated between CR-1 with high grade, moderately-and welldifferentiated breast and cervical carcinomas, gastric adenocarcinomas, OSCC and bladder cancer (Wei et al., 2015, Gong et al., 2007, although no correlation was reported between CR-1 expression and the tumor grade in pancreatic neoplasms (Wu et al. 2009). Such results had presented it as an effective marker to predict cancer initiation and progression and even as a potential therapeutic target for a number of human cancers.
Although the important role of this CSCs marker in different cell signaling pathways and its possible association with tumorgenesis in various human malignancies, Only few studies has investigated its role in OSCC .
Therefore, this study aimed to estimate the potentiality of CR-1 as a specific CSC marker for OSCC and to investigate its expression among the different grades of OSCC and its association with tumor progression.

Material and Methods:-Tissue Specimens and Histopathological Study:-
Forty specimens were collected as 20 specimens of well differentiated and another 20 poorly differentiated OSCC obtained from the archives of the Oral and Maxillofacial Pathology Department, Faculty of Dentistry, Cairo University and the Department of Surgical Pathology, National Cancer Institute, Cairo University were used in the present study. In addition to 6 specimens of normal appearing oral mucosa were taken from free tumor margins to be employed as control group Two serial 4 µm thick sections were cut from each formalin-fixed, paraffin-embedded tissue block, one section was mounted on ordinary glass slide, for routine stain with H&E to be examined under ordinary light microscope in order to confirm previously made diagnosis of the selected specimens. The other section was mounted on positively charged slides for immunostained with anti-CR-1 antibody.

Immunofluorescence Study:-
The cells were 4% formaldehyde fixed (10 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block nonspecific protein-protein interactions. The cells were then incubated with the primary anti-Cripto1 antibody, rabbit polyclonal antibody of IgG type (ab19917, 5μg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM. All markers were supplied by Abcam, UK.

Identification and Counting of CR-1 Positive Cells:-
Fluorescent images were visualized using a fluorescence microscope equipped with digital camera.
Manual counting was used to determine the number of CR-1 cells and the data were expressed as number of counted CR-1+ cells where: + is few (CR-1+ cell count is <1000), ++ is medium (CR-1+ cell count is between 1000-5000) and +++ is many (CR-1+ cell count is > 5000), while mean percentage represents the mean total counts / slides number in each group (31).
Statistical Analysis:-Data were statistically analyzed using Statistical Package for Social Scientists (SPSS) 10.00 for windows. All values were expressed at mean percentages and standard deviation (mean±S.D).
At confidence interval of 95%, p-value was considered significant when it is below 0.05 level.

Discussion:-
OSCC is still considered the most common malignancy of the oral cavity and the most lethal cancer due to the late detection of this cancer in its advanced stages despite the easy accessibility of the oral cavity for regular examinations (Perez et al., 2013). Several markers were discovered to correlate the relation between this cancer and CSCs and the role of these cells in tumor aggressiveness, such as CD44 and CD109 (Hema et al., 2014).
CSCs have shared a new era in the field of pathogenesis of OSCC in oral cavity. They are the most promising technology to conduit between the bench and beside. This importance comes from their ability for perpetual selfrenewal and proliferation, producing downstream progenitor cells and cancer cells that drive tumor growth. Studies on many cancer types including OSCC identified CSCs using specific markers, but it is still unclear as to where in the stem cell hierarchy these markers fall (Baillie et al., 2017).
Considering the promising results of CR-1 as a member of the EGF-CFC protein family and its involvement in the activation of several different signaling pathways during embryonic development, we have chosen it in this work. Although the CR-1 protein was over expressed in several human cancers including breast, colon, cervix, gastric, and pancreatic cancer (Bianco et al., 2010 andFrancescangeli et al., 2015), no one shed the light on its importance as a novel marker for CSC and to the best of our knowledge, only one study evaluated CR-1 expression in OSCC . Therefore, the current work investigated the CR-1 expression in clinical samples of OSCC using immunoflurescence, to analyze the correlation between CR-1 expression as a CSC marker and the different grades in OSCC.
In the current work, the immunoflurescent results showed high expression in well differentiated samples comparing to low expression in poorly differentiated ones. These results were supported by the immunohistochemical findings by (Yoon et al., 2011). Also, was in the same line of our previous study (Hassabou et al, 2011) in which we used CD44v6 as a CSC marker and also, was in agreement with ( Yu et al. (2015) found no prognostic significance of CD44v6 expression in OSCC. The significant decrease in CSCs count with increasing the grade of OSCC detected in the current work could be explained on the basis of the earlier observations which implicated CSCs in initiation of different solid tumors including HNSCC (Burkert et al., 2006). Moreover Choi et al. (2008) and Tsai et al. (2014) were in accordance with our results using Oct-4 as CSC marker. They clarified that Oct-4 plays a role in tumor transformation, tumorigenicity, invasion and metastasis within OSCC and proposed that Oct-4 promotes tumor initiation by playing a role in the regulation of EMT.
In agreement with our results were the PCR results of Mahmoudian et al. (2017). There was a significant correlation observed between CR-1 expression and tumor differentiation grade and progression stage of the studied cases of esophageal squamous cell carcinoma (ESCC). This was clarified that over expression of CR-1 is involved in the development of ESCC. A small subpopulation of either adult or progenitor stem cells can contribute to CSCs, which support the growth and maintenance of tumor cells in most types of cancers including colon, breast, prostate and ESCC (Forghanifard et al., 2014). Therefore, investigation of stem cell regulatory genes in cancers may help to identify potential biological markers for early diagnosis. Therefore, this indicated that CR-1 might represent an example of an embryonic gene that is re-expressed, possibly in stem cells in adult tissues, in an inappropriate fashion and thereby may contribute to the pathogenesis of cancer. Furthermore, over expression of CR-1 in human tumors may confirm the linkage between stem cell biology and tumor development (Schier et al., 2003) and increase the demand of identifying effective markers to predict cancer initiation and progression (Wei et al., 2015).
CR-1 has a key regulatory role in early embryogenesis, oncogenic proliferation, and transformation via its activity in multiple signal transduction pathways, which include TGF-β, Notch, and Wnt signaling cascades. CR-1 over expression can induce cell proliferation, EMT and tumour angiogenesis . CR-1 induces invasion, tumor cell proliferation, cellular migration, and cell motility in many different cancers . The expression profiles of a number of protein markers have been studied as putative CSC markers within OSCC samples and cell lines. Finally, no single marker has been shown to unequivocally identify CSCs and it is likely that CSCs exist in an overlapping hierarchy of cell population subsets. Consequently, the majority of CSC characterization research relies on using combinations of these markers (Liu et al., 2017).

Conclusion:-
From the current study it can be concluded that there is a small population of cells with stem cell properties referred to CSCs which play an important role in tumor initiation.