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CD44 related stemness maneuvers oral squamous cell carcinoma biology Tandon A, Singh NN, Gulati N



   Abstract  

Introduction: Despite the commendable advancements in oral squamous cell carcinoma (OSCC) diagnostics and therapeutics, it remains a considerable medical challenge. Recent evidence suggests that small populations of stem-like cancer cells are responsible for tumor initiation, progression and metastasis. These cancer stem cells (CSCs) have been identified and characterized in various types of cancers, including OSCCs. CSC hypothesis has been supported by the expression of CD44, CD133, ALDH1 and ABCG2. Amongst them, CD44 (a transmembrane glycoprotein), is the most reported CSC marker in OSCCs. The increasing incidence of OSCC combined with its poor survival rates motivates a need for research into the expression of adhesion molecules and may play a pivotal role in studying tumor biology related to invasion and distant metastasis. Objective: To quantify the expression of CD44 in the different grades of OSCC and to correlate the expression of CD44 with clinicopathological parameters. Method: A total of 20 formalin-fixed paraffin-embedded tissues of OSCC were retrieved from department archives. Immunohistochemical staining was performed using anti-CD44 antibody (Biogenex). The expression was assessed semi-quantitatively in varying histopathological grades of OSCC and were correlated with tumor, node, metastasis (TNM) staging which were obtained from the department records. The results were statistically evaluated. Result: Overexpression of CD44 was detected in 48% of well-differentiated OSCCs followed by a linear decrease in moderately differentiated and poorly differentiated OSCCs and the expression correlated with the tumor size (T) in 23% cases and with lymph node metastases (N) in 42% of cases (P ≤0.05). Conclusion: The results of the present study suggested an altered expression of CD44 in OSCC. This depicts an association of CD44 with tumor aggressiveness and Epithelial Mesenchymal Transition (EMT) related to loss of cell adhesion in a subset of OSCC—clearly stating tumor cell stemness as a key factor in malignant potential of OSCC.

Keywords: CD44, squamous cell carcinoma, stem cells, stemness

How to cite this article:
Tandon A, Singh NN, Gulati N. CD44 related stemness maneuvers oral squamous cell carcinoma biology. Indian J Pathol Microbiol 2022;65:268-73




   Introduction   Top

Head and neck squamous cell carcinoma possess cellular diversity, which is governed by genetic, epigenetic and environmental factors.[1] In view of this, cancer stem cells (CSCs) are cells that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. Such cells are exceedingly tumorigenic compared to the other cancer cells and are believed to be largely responsible for the biological characteristics of cancer, namely, rapid growth, invasion and metastasis.[2],[3] Accounting such features, CSCs are referred as “tumor-initiating cells ” or “tumorigenic cells ”. Surface markers such as CD133, CD24 and CD44 are typically used for isolation and identification of such cells. The CSC model suggests that only certain subpopulations of cancer cells have the ability to drive the progression of cancer. These are specific and more aggressive subtypes of cells, which result in tumor progression and recurrence.[4]

To our interest, CD44 receptor is a type I transmembrane glycoprotein that was initially identified as a leukocyte antigen.[5],[6] The alternative splicing of variable exons of CD44 results in abundant variants, which are denoted CD44v and CD44s (an isoform with no variable exons in the mRNA).[7] The smallest, standard isoform is CD44s, which is generally expressed on vertebrate cells, while CD44v is only expressed on some epithelial cells.[8],[9] Also, CD44 is the major hyaluronan (HA) receptor, and CD44 bound to HA has been proven to participate in controlling tumor biology.[10] Once activated, the cytoplasmic tail of CD44 interacts with the actin cytoskeleton, and CD44 is induced to the leading edge of migrating cells.[10]

The targeted therapies in OSCCs are challenging as conventional therapies may shrink tumors by mainly killing cells with limited proliferative potential. It has been documented that CSCs are less sensitive to targeted therapies and remain viable after therapy and may re-establish the tumor. Truly, if treatment therapies can be targeted specifically against CSCs, then they might more effectively unqualify tumors to maintain themselves or grow. Thus, even if cancer stem cell-directed therapies do not shrink tumors initially, they may eventually lead to cures.[11]

Tumor cells are heterogeneous and only the cancer stem cell subsets have the ability to proliferate extensively and form new tumors. Histologic grade needs to be supplemented with additional information regarding the biologic activity of a tumor. A better understanding of CSC properties is, therefore, crucial for the development of effective alternative strategies, for example, targeting stem cell maintenance, signaling pathways or blocking EMT/Mesenchymal Epithelial Transition (MET) to prevent the switching of CSCs between drug-resistant phenotypes.[12]

Hence, the following study was conducted to analyze the expression of CD44 semi-quantitatively and qualitatively in the different grades of OSCC and to correlate the histomorphological features of OSCC such as tumor budding, perineural invasion, lymphovascular invasion and lymphocytic host response with CD44 immunoexpression.

   Materials and Method   Top

Patients and tissue samples

The study was conducted on archival tissue samples which were submitted for histopathological evaluation. The samples were fixed in 10% neutral buffered formalin and embedded in paraffin wax to obtain 3 μ sections for immune histochemistry procedure. Study samples consisted of 70 cases in total, which included 10 cases of normal oral mucosa (controls) and 60 cases of OSCC which were categorized on the basis of the WHO criteria. Ethical approval from the institutional review board of ITS-Centre for Dental Studies and Research (ITS-CDSR), Muradnagar, Ghaziabad, was obtained for this study (28/6/2019). Clinical data of each case, such as TNM staging, were collected. Archival specimens were grouped based on the WHO (2005) histopathological grading criteria as follows: Group 1 as well-differentiated OSCC (n1 = 20), Group 2 as moderately differentiated OSCC (n2 = 20) and Group 3 as poorly differentiated OSCC (n3 = 20). Recurrent/secondary cases of OSCC and OSCC cases with any therapeutic interventions were excluded from the study sample.

Immunohistochemistry with CD44

Three-micrometer-thick sections from archival formalin-fixed paraffin-embedded tissues were placed on poly-l-lysine-coated slides for immunohistochemistry. CD44 expressions were analyzed by immunohistochemical examination with antibodies. For the antibody, the deparaffinized tissue sections were placed in 10 mmol/L citrate buffer, pH 6.0, and heated to cycles of 95 and 98°C for 13 minutes. Immunohistochemical staining for these proteins was performed by the avidin-biotin complex procedure with a streptavidin biotin complex peroxidase kit. Primary antibody-monoclonal anti-CD44 antibody (Biogenex Ind Pvt Ltd, Fremont, CA, USA, Clone: DF1485, Ready-to-use [Manual]: AM310-5M) along with secondary antibody-poly-Horse Raddish Peroxidase (HRP) secondary detection system (Biogenex Ind Pvt Ltd.) were used. For CD44, tonsil served as the positive control. Positive staining for CD44 was identified as membranous and/or cytoplasmic brown staining.

Assessment of immunoscoring

Immunoscoring of cell positivity and intensity for CD44 was done by the analysis of 1,000 cells from five randomly selected fields using a grid. Qualitative scoring was done as mild, moderate and intense [+, ++ and +++]. For semi-quantitative analysis, immunoreactive score was evaluated by counting the number of positive cells per 1,000 total tumor cells of all grades of OSCC, and further, the percentage of positive cells was calculated.

In the study groups, a few multiple histomorphological parameters were evaluated which included tumor budding (TB),[13] lymphocytic host response (LHR) and[14] vascular invasion (VI).[15] These were correlated to immunoexpression of CD44 alongside the presence of either isolated single cells or small-cell clusters (up to four) scattered in the stroma ahead of the invasive tumor front. Budding represents two malignant features: cellular discohesion and active invasion. The Budding Index (intensity of tumor budding) is scored based on the number of buds/fields; score ‘1’ is specified for <5 buds/field whereas ‘2’ is specified for ≥5 buds/field. The clear presence of tumor cells within a vascular space (lymphatic space or blood vessel) was considered as vascular invasion. The scoring was done based on the presence and absence of the tumor either adhered to the vessel endothelium or attached to the thrombus in the vessel, where ‘0’ was scored for the absence of invasion and ‘1’ was scored for the presence. Lymphocytic infiltrate at the tumor/host interface was quantified as a three-tiered variable. Type 1 as dense complete host-response rimming tumor, type 2 as patches of dense lymphoid infiltrate but the inflammation was discontinuous and Type 3 as limited response that did not form lymphocytic patches, or for no lymphocytic response.

Statistical analysis was performed using IBM SPSS Statistics for Windows, version 20.0. Armonk, NY: IBM Corp. Statistical significance was set at P ≤ 0.05 with 95% confidence interval throughout the study.

   Results   Top

Demographic data

The overall mean age of the study cases was 46.13 years. The cases included in the study had a higher male representation, i.e., 87.5%. The most common site of occurrence was the buccal mucosa (66.3%).

Expression of CD44 in OSCC

As the grade of the tumor advanced, the immunoexpression of CD44 decreased both semi- quantitatively and qualitatively. The qualitative scoring of CD44 immunoexpression decreased in a gradual incremental manner from well to poorly differentiated OSCC with +++ scoring present in 85% cases of well-differentiated OSCC in comparison to + scoring present in 70% cases of poorly differentiated OSCC [Figure 1]. Semi-quantitative scoring of CD44 immunoexpression decreased from well to poorly differentiated OSCC with 55% well-differentiated OSCC showing 25-75% positive cells whereas 75% poorly differentiated OSCC with < 5% positive cells (P ≤ 0.05) [Table 1].

Figure 1: (a and b) Well-differentiated squamous cell carcinoma showing immunopositivity of CD44. All cells in tumor islands showing positive expression of CD44 (IHC, 10x and 40x); (c and d) Moderately differentiated squamous cell carcinoma showing immunopositivity of CD44. Few cells in tumor islands showing positive expression of CD44 (IHC, 10x and 40x); (e and f) Poorly differentiated squamous cell carcinoma showing weak to absent immunopositivity of CD44 (IHC, 10x and 40x)

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Correlation of CD44 immunoexpression with morphological parameters

Tumor budding, lymphovascular invasion and lymphocytic host response were correlated with CD44 immunoexpression. The mean score of CD44 immunoexpression was more where <5 buds were present at the invasive front, whereas perivascular invasion correlated to high immunoexpression of CD44. Increased mean score of CD44 immunoexpression was evident where patchy lymphocytic host response was present [Figure 2].

Figure 2: (a) Graph depicting correlation of mean CD44 immunoscore with tumor budding; (b) Moderately intense CD44 immunoexpression in tumor buds (IHC, 10x) (c); Graph depicting correlation of mean CD44 immunoscore with lymphoplasmacytic host response; (d) Intense CD44 immunoexpression in patchy areas of lymphocytic host response (IHC, 10x); (e) Graph depicting correlation of mean CD44 immunoscore with lymphovascular invasion; (f) Weak CD44 immunoexpression in tumor cells lining the lymphovascular channels (IHC, 10x)

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   Discussion   Top

Apart from the histomorphology, the main focus of the search for prognostic markers targets CSC as these cells, despite comprising only a minority of cancer cells, are able to initiate the formation of new tumors and are responsible for tumor relapse and resistance to therapy. The restricted expression of CD44 in various grades of OSCC was related to the degree of cellular proliferation and differentiation. The decrease in the immunostaining expression of CD44 with an increase in the grade of tumor may suggest a loss of cell–cell adhesion, thereby, contributing to the easy detachment of cells from a rigid configuration.[16]

CD44 deserves considerable attention with respect to its adhesive, locomotive and growth-transducing functions among the cancer cells.[17] CD44 is also a cancer stem cell marker with two distinct phenotypes, such as (i) The epithelial–mesenchymal transition phenotype CSCs that express proteolysis-resistant CD44s isoform and (ii) The CSCs with epithelial phenotype that expresses trypsin-sensitive CD44v isoform. This results in the loss of CD44 expression in epithelial phenotype CSCs and a higher expression in the epithelial–mesenchymal transition phenotype during isolation.[18] The varying patterns of expression in the different grades of OSCC were related to the presence of pleomorphic cells. Highly pleomorphic cells with a weak resemblance to the parent tissue were present in poorly differentiated squamous cell carcinoma, and hence, a weak CD44 immunostaining is associated with poorly differentiated squamous cell carcinoma [Table 1].

We identified the presence of tumor budding activity (TBA) defined as small groups of tumor cells and cell nest size (CNS) as prognostic parameters indicating an unfavorable outcome in OSCC, providing information additional to the Tumor Node Metastasis/ Union for International Cancer Control (TNM/UICC) systems. TBA has been assumed to represent a feature of epithelial–mesenchymal-transition (EMT). Recent evidence suggests that cells that undergo EMT gain stem-cell-like abilities and can be referred to as CSCs. Also, at the invasive front, tumor budding was assessed where the mean score of CD44 immunoexpression was more where <5 buds were present at the invasive front. Our findings were similar to the study conducted by Wang et al.,[13] who proposed a novel prognostic model for early stage oral tongue cancer and stated that tumor budding is actually a sign of epithelial to mesenchymal transition of carcinoma cells, reflecting the powerful invasive growth of tongue carcinomas. Tumor budding resides ahead of the invasive front and characterizes poor prognosis. It is a marker of discohesion and active invasion of tumor cells.

We found that CD44 overexpression is significantly correlated with high TBA, small CNS and poor histopathological tumor differentiation. As CD44 functions as a CSC marker, the expression of CD44 in tumor buds and single cells—both features that are linked to EMT—might reflect the presence of CSC within these specific carcinoma cells. CD44-positive CSCs might, therefore, be present in increased numbers in poorly differentiated OSCC with features of EMT. In addition, the significant correlation High CD44 with Lymphatic Invasion possibly reveals CSCs in OSCC as immunogenic cells—a finding that might be interesting for potential immunological treatment approaches. Besides, the CD44+ cells in OSCC might as well be therapeutic targets for anti-CSC-based therapies as specific medications against the isoform CD44v6 have been developed and are under investigation in head and neck squamous cell carcinoma.[19],[20]

The Histological Risk Model, and more specifically, the lymphocyte host response significantly correlates with the disease progression and survival for patients. We, therefore, evaluated the host response and observed increased mean score of CD44 immunoexpression where patchy lymphocytic host response was present. Tumor microenvironment encompasses cellular interactions between cancer cells, immune effectors and inflammatory cells as well as cells of the tumor vasculature and the stroma. Inflammatory cells play a decisive role in different stages of tumor development, including initiation, promotion, progression, invasion and metastasis.[14]

On the other hand, perivascular invasion is classified according to the presence or absence of neoplastic cells located in the wall or lumen of blood or lymphatic vessels and has been found to be correlated with low rates of survival and high risk of recurrence. In our study, perivascular invasion correlated to high immunoexpression of CD44, suggesting that in relation to oral carcinomas vascular invasion not only does have a strong correlation with disease prognosis but also to the presence of stem-cell-like property of tumor cells. To the best of our knowledge, ours is the first study to correlate CD44 expression with lymphovascular invasion and lymphocytic host response.[15]

CD44 also signals for the orientation of epithelial cells that differentiate and migrate upward. These events are switched off as the cells reach their end differentiation and are disclosed from their intercellular junctions. CD44 down regulation might reflect an early cellular change from normal cell–cell and cell–matrix interactions to a bizarre heterotypic cell surface adhesion property that predisposes the cells to attain invasiveness.[16] Our findings were in accordance with many others such as Mende et al.,[21] Kuo et al.,[22] Satoa et al.,[23] Carinci et al.,[24] González-Moles et al.,[25] Mostaana et al.,[26] Krump et al.[27] and Kaza et al.[16]

Critical aspect in the growth of the tumor is its deep invasion into the nodes in which the vascular invasion and metastatic spread are likely to occur. On evaluation of the OSCC cases, apart from the membranous staining, cytoplasmic labeling was also evident. This could be due to the interaction of the cytoplasmic domain of CD44 with cytoskeletal linker proteins such as ezrin and ankyrin through which it can mediate cell migration on hyaluronan.[28],[29] With the evidence of accumulated data, we have formulated a working pathway to understand the positive feedback loop of CD44 in OSCC [Figure 3] alongside summarizing how CD44 is at the forefront of transcending the disease toward an unfavorable outcome.

   Conclusion   Top

CSCs are functionally defined by their extensive selfrenewal capacity and challenge successful treatment of Squamous Cell carcinoma (SCCs). Identification of regulators that control the tumorigenic potential of CSCs and their correlation with histomorphologic and clinicopathologic features might provide new insight into therapeutic strategies against CSCs to improve treatment approaches in OSCCs. Aforementioned tumor characteristics may be clubbed to derive a model to decide appropriate treatment of primary and/or recurrent OSCCs since CSC confers increased radio and chemo resistance in tumors resulting in tumor recurrence and metastatic spread.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

   References   Top


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Correspondence Address:
Ankita Tandon
Department of Oral Pathology, Microbiology and Forensic Odontology, Dental Institute, RIMS, Ranchi, Jharkhand
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_1139_20

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[Figure 1], [Figure 2], [Figure 3]
 
 
 
[Table 1]

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