An Increase in the Chemokine Mediators (CCL28 and CCR10) Associated with the Progression of Oral Squamous Cell Carcinoma: A Cross-Sectional Investigation

Abstract

Oral cancer ranks as the 16th most prevalent form of cancer worldwide and is a significant concern in Southeast Asia, primarily due to the extensive use of tobacco. Chemokines contribute to a wide range of illness situations. This study aimed to investigate the immunohistochemistry expression of CCL28 and its receptor CCR10 in oral squamous cell carcinoma and examine their relationship with tumor progression. This study retrospectively examined tissues of oral squamous cell carcinoma that were preserved by formalin fixation and paraffin embedding. The study includes two groups: Group I included 50 cases of OSCC with varying histological grades, whereas Group II comprised 50 samples of normal oral mucosa (NOM) obtained from healthy individuals who did not engage in any tobacco use. The cytoplasmic placement of the study groups allowed us to determine the expression patterns of CCL28 and CCR10. The study found a strong link between CCL28 and CCR10 immunohistochemical expression in oral squamous cell carcinoma. There were big differences in the amount of CCR10 staining in the 96 cases that showed positive immunoexpression. This shows that these two molecules have a big impact on how OSCC acts biologically. Various stages of oral squamous cell carcinoma and healthy oral mucosa exhibited simultaneous expression of CCL28 and CCR10. In summary, we can utilize CCR10 and CCL28 co-expression, along with other biomarkers, to aid in the prognosis of oral squamous cell carcinoma (OSCC).

Introduction

In India, non-communicable diseases account for 63% of all deaths, and cancer is one of the leading causes (9%) [1]. Cancer is a multistep process with a multifactorial etiology that involves the tumor's initiation, promotion, and progression. Cancer is the second-most common cause of morbidity and mortality globally, next to cardiovascular diseases [2]. The Global Cancer Observatory (GLOBOCAN) 2022 reports that approximately 3,89,846 new cancer cases and 188,438 million cancer deaths occur worldwide, with India accounting for 1.3 million new cancer cases and 851,678 cancer deaths [3].

Oral cancer ranks as the sixteenth most common cancer in the world; in India, oral cancer and breast cancer rank first in males and females, respectively; and it is a major concern in Southeast Asia due to prevalent oral habits [4, 5]. Gaining knowledge about the genetic mechanisms and evolutionary development of cancer has the potential to improve the results of medical treatment. Our comprehension of oncogenesis, as well as our ability to detect oral malignancies using molecular approaches and new biomarkers, has greatly improved in the past ten years [6]. The hallmarks of cancer are considered to play an essential role in the growth and progression of tumors [7]. Various signaling pathways mainly regulate the initiation and maintenance of the invasion process of cancer cells [8]. Epithelial-mesenchymal transition (EMT) is a biological process in which epithelial cells undergo downregulation, enabling cancer cells to invade nearby tissue and spread to other parts of the body (metastasize). The tumor microenvironment (TM) consists of soluble proteins, signaling molecules, and chemokines, which play a role in the invasion of tumors [9]. Chemokines are essential in various pathophysiological processes like diseases, inflammation, and tumor growth. They indirectly control tumor growth by preventing apoptosis and inducing cancer cell proliferation. They also release growth and angiogenic factors from tumor stromal cells, facilitating metastasis [9, 10].

CCL28 (Mucosae-associated epithelial chemokine; MEC) is a CC chemokine ligand for CCR10 and CCR3. It drives the mucosal homing of T and B lymphocytes that express CCR10 and the migration of eosinophils expressing CCR3 [11, 12]. It seems to have different functions in different types of human cancer. CCL28 protein expression in colon and salivary gland tumors is lower than normal, promoting tumor progression [13]. However, it is upregulated in lung adenocarcinoma and breast carcinoma, promoting cell proliferation and growth [14, 15].

CCR10 is a member of the chemokine receptor family that is typically expressed by melanocytes, plasma cells, and skin-homing T cells and can be activated by CCL27 or CCL28 [16]. CCR10 and its ligands are expressed on various epithelia-homing or cancer cells and play an essential role in their specific tissue location, survival, and metastasis. CCR10 is overexpressed in malignant melanoma, and the CCL27-CCR10 interaction can enhance the growth and migration of melanoma cancer cells [17].

In oral squamous cell carcinoma (OSCC), the upregulation of the CCL28-CCR10 axis inhibited invasion and epithelial-mesenchymal transition by inducing retinoic acid receptor-β and preventing RANKL expression in OSCC. CCL28, CCR10, and RARβ are useful markers for predicting and treating OSCC bone invasion [18]. In this study, we evaluated the intensity of expression of CCL28 and CCR10 and correlated the expression of CCL28 and CCR10 with the biological behavior of OSCC.

Material and Methods

Study Design

This cross-sectional study was done on the formalin-fixed paraffin-embedded tissue specimens of oral squamous cell carcinoma and normal oral mucosa. The study was conducted in the Department of Oral Maxillofacial Pathology and Oral Microbiology, FODS, KGMU, Lucknow, India, between April 2020 and March 2021. Before the commencement of the study, ethical approval was obtained from King George's Medical University (KGMU), (101st ECM II B Thesis/P65), Lucknow. The sample size is calculated based on a 35.7% proportion of high expression of CCL28 and CCR10 in different grades of OSCC cases, and the 100% high expression is assumed to be in control under the null hypothesis using the formula. The alpha value is 0.05 with 90% power of the study and keeping the data loss factor at 10%. The calculated minimum sample size for the study was 50 in each group. Group I: 50 cases of different histological grades of OSCC, and Group II: 50 normal oral mucosae (NOM) from healthy controls without any associated adverse habits. We included patients with well-, moderately-, and poorly differentiated squamous cell carcinoma. Clinical information for all cases, including age, sex, tumor location, and associated risk factors, is gathered from the requisition forms.

Immunohistochemical Analysis

Before the IHC staining procedure commenced, gelatin solution, citrate buffer, and phosphate buffer saline were prepared according to the standard process. From the clinically and histopathologically confirmed formalin-fixed paraffin-embedded tissue blocks, 3.5-micron-thick sections were stained with routine hematoxylin and eosin (H & E) staining. The histological grading of malignancy was analyzed under light microscopy according to Broder’s grading system (1920). Cases that met the histologic criteria of well-, moderate-, and poorly differentiated squamous cell carcinoma were selected for the immunohistochemical analysis. Later, these slides were subjected to the IHC procedure as per standard protocol. To determine CCL28 and CCR10 expression, the tissue sections were deparaffinized in xylene, followed by increasing grades of alcohol for rehydration. The tissues were subjected to antigen retrieval. After the retrieval, the sections were incubated with the primary antibodies CCR10 (1:100) and CCL28 (1:50) and kept in a humidified chamber for 2 h at 370 °C, followed by 40 °C overnight. We then washed the slides with PBS, incubated them with HRP (polymer) for 30 min, and then washed them in PBS. These slides were subsequently incubated with fresh DAB chromogen for 20 min. DAB chromogen was prepared by adding DAB to the stable buffer at a ratio of 1:20. The slides were washed in distilled water to stop the chromogen reaction and to remove excess DAB, and finally, the tissues were counterstained with Mayer’s hematoxylin for 2 min. The slides were dehydrated with graded isopropyl alcohol, cleared using xylene, and mounted with DPX.

Evaluation of CCL28 and CCR10 Immunoexpression

The slides were observed and assessed under a Carl Zeiss Axioscope A1 Penta head microscope with an attached Zeiss Camera Axio Cam ICc5. Immunohistochemical staining was evaluated by the intensity of CCL28 and CCR10 expression in the epithelium and connective tissue. CCL28 and CCR10 immunopositivity were evaluated by the brown color of the immunostaining of the cytoplasm (Fig. 1) .

Fig. 1.

Immunohistochemical expression of OSCC and Normal mucosa. CCL28 (A–C) A Well differentiated (strong expression), B Moderately differentiated (weak expression), C Normal oral mucosa (moderate expression), and CCR10 (D–F) D Well differentiated (strong expression), E Moderately differentiated (weak expression), F Normal oral mucosa (no expression)

Observation Criteria of CCL28 and CCR10 Immunoexpression

The CCL28 and CCR10 expression patterns in study groups were recorded based on their localization as cytoplasmic expression (Fig. 1).

The intensity of staining was estimated as follows:

• Score 1: negative staining

• Score 2: weak positive staining

• Score 3: moderate positive staining

• Score 4: strong positive staining

The proportion of stained cells.

• 1 = No stained cell.

• 2 = Less than 25% stained cells.

• 3 = 26–50% stained cells.

• 4 = More than 50% stained cells.

An ordinal scoring method [19] was performed to determine each sample's immunostaining intensity distribution (IID) index, specifically for the cytoplasmic expression of CCL28 and CCR10. The IID index is calculated by multiplying the overall staining intensity for each layer by the proportion of stained cells in that layer.

Two expert oral pathologists manually evaluated the specimens independently and in a blinded manner. The average number of five fields in a representative segment from each specimen was calculated. The observers were blinded by the grades of OSCC. Interexaminer variability was calculated using the rank correlation coefficient.

Statistical Analysis

Data were analyzed using Statistical Package for Social Sciences (SPSS) version 21, IBM Inc. Descriptive statistics such as frequency and percentages of all categorical variables were calculated, and summarized data were presented using tables and graphs. The Shapiro–Wilk test was used to check which variables followed a normal distribution, while the Chi-square test was applied to compare categorical data among groups. The level of statistical significance was set at a p-value less than 0.05.

Results

The present study was carried out in the Department of Oral Maxillofacial Pathology and Oral Microbiology, KGMU Lucknow, to study the expression of CCL28 and CCR10 in oral squamous cell carcinoma. We conducted a thorough examination and analysis of various factors in our study. These factors include the distribution of subjects among different age groups in the study groups, the distribution of gender among the study population, the distribution of subjects with oral squamous cell carcinoma (OSCC) across different sites, the history of habits among the study population, the distribution of study cases based on histopathological diagnosis, the distribution of CCL28 immunohistochemical expression intensity among the study groups, the distribution of CCR10 immunohistochemical expression intensity among the study groups, and the correlation between CCL28 and the biological behavior of oral squamous cell carcinoma.

The site-wise distribution of the majority of OSCC cases was found on the buccal mucosa (46%), followed by the mandible (26%), tongue (8%), maxilla (6%), lip (6%), palate (4%), and combined lesion (4%) (Fig. 2). Habit history was present in 42 cases of OSCC samples out of 50, and none among the control group exhibited a significant difference in the study population when compared using the Chi-square test (p-value 0.00). The majority of the subjects with OSCC had a history of tobacco consumption. The distribution of study cases according to histopathological diagnosis showed significant differences in the histopathological diagnosis among the subjects (p-value 0.00) (Table 2).

Fig. 2.

Site-wise distribution of subjects having OSCC

Table 2.

Correlation of CCL28 with the biological behavior of oral squamous Cell carcinoma

Intensity of staining (CCL28)	No expression	Weak expression	Moderate expression	Strong expression
Well-Differentiated	0	7 (35%)	12 (60)	1 (5%)	0.007
Moderately differentiated	0	13 (65%)	6 (30%)	1 (5%)
Poorly differentiated	4 (40%)	4 (40%)	1 (10%)	1 (10%)
Intensity of staining (CCR10)
Well-differentiated	0	8 (40%)	11 (55%)	1 (5%)	0.0001
Moderately differentiated	0	14 (70%)	6 (30%)	0
Poorly differentiated	6 (12%)	2 (20%)	1 (10%)	1 (10%)

The distribution according to the intensity of immunohistochemical expression of CCL28 among study groups is as follows: a strong positive cytoplasmic staining was observed in the section with a normal human colon, which was selected as a positive control for CCL28 IHC procedures. 4 cases (4 OSSC and 0 NOM) did not show any expression of CCL28, along with 96 cases showing positive immunoexpression. Among these, 4 OSCCs showed negative staining, 24 OSCCs showed weak staining, 19 cases showed moderate staining and 3 cases showed strong staining. Whereas none of the cases with NOM showed negative staining, 16 showed weak staining, 20 showed moderate staining, and 14 showed strong staining. We also observed a significant difference in the distribution of cases according to the intensity of CCL28 immunostaining (p-value 0.00).

The distribution according to the intensity of immunohistochemical expression of CCR10 among study groups is as follows: a strong positive cytoplasmic staining was observed in the section with a normal human colon, selected as a positive control for CCR10 IHC procedures. We have observed positivity for CCR10 among 44 cases and negativity among 56 cases. Among OSCC cases, 6 have shown negative results, 24 showed weak results, 18 showed moderate results, and 2 showed strong immunostaining. In the case of normal mucosal tissues, none of the cases showed positive expression under immunohistochemistry. A significant difference was seen in the intensity of CCR10 staining (p-value 0.00) (Table 1, Figs. 3 and 4). On examining the correlation of CCL28 with the biological behavior of oral squamous cell carcinoma, a significant correlation was found when compared using the Chi-square test with a p-value of 0.00. On the correlation of CCR10 with the biological behavior of oral squamous.

Table 1.

Distribution of age, gender, histopathological grade, and immunohistochemical expression of CCL28 and CCR10

	OSCC	Control	p-value
Age group
21-40 yrs	12 (24%)	23 (46%)	0.046
41-60 yrs	28 (56%)	17 (34%)
61–80	10 (20%)	10 (20%)
Gender
Male	44 (88%)	41 (82%)	0.288
Female	6 (12%)	9 (18%)
Histopathological Diagnosis
Well-differentiated	20 (40%)		0.0001
Moderately differentiated	20 (40%)
Poorly differentiated	10 (20%)
Normal oral mucosa		50 (100%)
Immunohistochemical expression of CCL28
No expression	4 (8%)	0	0.007
Weak expression	24 (48%)	16 (32%)
Moderate expression	38 (20%)	20 (40%)
Strong expression	3 (6%)	14 (28%)
Immunohistochemical expression of CCR10
No expression	6 (12%)	50 (100%)	0.0001
Weak expression	24 (48%)
Moderate expression	18 (36%)
Strong expression	2 (4%)

Fig. 3.

Correlation of CCL28 with the biological behavior of oral Squamous cell carcinoma.

Fig. 4.

Correlation of CCR10 with the biological behavior of oral Squamous cell carcinoma.

A cell carcinoma difference was seen in the intensity of CCR10 staining (p-value 0.00). We have found a statistically significant correlation between CCR10 immunopositivity and the biological behavior of OSCC (p-value 0.00) (Table 2).

Discussion

Oral cancer is the sixteenth most common cancer in the world, and it is a significant concern in Southeast Asian regions due to prevalent deleterious oral habits [4]. In India, oral cancer ranks fourth and accounts for 50–70% of total cancer-related mortality [5]. India has become a high-risk region for OSCC due to increased consumption of tobacco products. Besides tobacco and alcohol, dietary factors, socioeconomic status, human papillomavirus (HPV) infection, genetic factors, and oral hygiene are also risk factors [20].

The tissue microenvironment plays a major role in the development and progression of a developing tumor comprising proliferating tumor cells, tumor stroma, feeder blood vessels, infiltrating inflammatory cells, and a variety of associated tissue cells [21]. Different subsets of the immune cell are recruited into the tumor microenvironment via interactions between chemokines and chemokine receptors. These populations have distinct effects on tumor progression and therapeutic outcomes [22, 23].

Chemokines are small, secreted proteins well-known for their roles in mediating immune cell trafficking, immune surveillance, and lymphoid tissue development [22]. They also play a critical role in a variety of pathophysiological processes such as infectious and autoimmune diseases, acute and chronic inflammation, hypersensitivity reactions, modulation of angiogenesis, proliferation of hematopoietic progenitor cells, tumor immune escape, and even tumor development [10]. Chemokines are essential coordinators of cellular migration and cell–cell interactions and greatly influence tumor initiation and growth. In the tumor, tumor-associated host cells and cancer cells release different chemokines, resulting in the recruitment and activation of different cell types that mediate the balance between antitumor and pro-tumor responses. In addition to their primary role as chemoattractants, chemokines are also involved in other tumor-related processes, such as angiogenesis and metastasis [9].

In OSCCs, upregulation of the CCL28–CCR10 axis has involved inhibiting epithelial-mesenchymal transition and invasion by inducing retinoic acid receptor-β and preventing RANKL expression on osteoblasts, respectively. The CCL28, CCR10, and RARβ are being studied to be used as markers for the prediction of prognosis and treatment planning of OSCCs [18].

Thus, in the present study, we have evaluated the immunohistochemical expression of CCL28 and its receptor CCR10 to demonstrate its role in oral squamous cell carcinoma progression. Among the 100 cases studied in the present study, 50 were from normal oral mucosa that was employed as controls, 20 were well-differentiated, 20 were moderately differentiated, and 10 were poorly differentiated OSCCs. The IHC reactivity of both CCL28 and CCR10 expression was evaluated based on variable cytoplasmic staining intensities in all the cases, including the control group.

CCL28 is a chemokine that binds to CCR10 and CCR3 and is expressed in various body parts. It drives the mucosal homing of T and B lymphocytes expressing CCR10 and the migration of eosinophils expressing CCR3, respectively [11, 12]. Recent studies on colon and salivary gland tumors have suggested that low CCL28 protein expression could promote tumor progression [13]. On the other hand, it was upregulated in lung adenocarcinomas under hypoxic conditions and has been reported to promote angiogenesis via CCR3 [14]. In breast carcinomas, CCL28 overexpression through the mitogen-activated protein kinase (MAPK) signal promoted cancer cell proliferation and tumor growth by activating Bcl-2 and increased invasiveness and metastatic ability by suppressing β-catenin expression [15].

The study found that among 20 cases of WDSCC, 7 had weak staining (35%), 12 had moderate staining (60%), and 1 had strong staining (5%). In MDSCC, 13 had weak staining (65%), 6 had moderate staining (30%), and 1 had strong staining (5%). The staining intensity was statistically significant across OSCC grades, and a correlation was found between weak CCL28 expression and poorer OSCC differentiation. Our results support the inference obtained in the studies conducted on breast carcinomas, pleomorphic adenomas, and adenolymphomas of salivary glands and OSCCs, where they found a poor prognosis with a decreased level of CCL28 [13, 15, 18]. It has been hypothesized that the expression of CCL28 is solely dependent on the degree of cellular differentiation and maturation, with only expression on terminally differentiated cells. Whereas, in higher grades of OSCCs, the tumor cells retain their pluripotency and show very poor histological differentiation, describing the decreased staining of CCL28 in PDSCCs.

CCL28 staining was found in normal oral mucosal tissues, with varying intensities in basal, parabasal, and spinous layers. Out of 50 tissue samples, 32% showed mild staining, 40% moderate, and 28% strong staining. This aligns with a study by Park et al., suggesting that normal epithelial cells secrete CCL28 to regulate host mucosal defense mechanisms, thereby confirming its positive expression in the study subjects [18].

CCR10 is a member of the chemokine receptor family that is normally expressed by melanocytes, plasma cells, and skin-homing T cells and can be activated by CCL27 or CCL28 [16]. CCR10 and its ligands are expressed on various epithelial and/or cancer cells, which play an important role in their specific tissue location, survival, and metastasis. CCR10 overexpression has been reported in malignant melanoma, and the CCL27-CCR10 interaction could enhance the growth and migration of melanotic cancer cells [17].

Among 20 cases of WDSCC, 8 cases showed weak staining (40%), 11 cases showed moderate staining (55%), and 1 case showed strong staining (5%). 14 cases showed weak staining (70%) in moderately differentiated carcinoma, and 6 cases showed moderate staining (30%). Among 10 cases of PDSCC, 2 cases showed mild staining (20%), 1 case showed moderate staining (10%), 1 case showed strong staining (10%), and in 6 (60%) cases, negative staining was observed. The staining intensity of CCR10 was statistically significant between the three histopathological grades of OSCC (p-value 0.001). Our results are backed up by the study conducted on melanoma by Simonetti et al., who have observed an increased ability of neoplastic cells to grow, invade tissue, disseminate to lymph nodes, and escape the host immune response with increased levels of CCR10 [17].

In the present study, none of the normal oral mucosa tissues have expressed CCR10. Our findings are similar to a study conducted by Kai et al., who also did not find any expression of CCR10 IHC in normal epidermis or Bowen’s disease [24]. They have not mentioned any specific reason for this phenomenon and concluded their study by highlighting the diagnostic and prognostic role of SCC [24]. Homey et al. pointed out that CCR10-specific mRNA is significant for the formation and mobilization of CCR10 over the cell [25]. They have reported that the epidermal keratinocytes lack those mRNAs and do not express any CCR10 on their cellular membrane, which explains the possible reason for the negative expression of CCR10 among CCL28-positive cases in our study. Contrary to our findings, a study done by Park et al. reported the expression of CCR10 in normal oral mucosal tissues and suggested that the expression of CCL28 strongly influences the level of CCR10, and both of them are interrelated [18].

Park et al. have stated that the invasion of OSCC cells can be blocked via CCR10 by an elevation of CCL28 levels in the tumor microenvironment. It was observed that CCL28 functions as a negative regulator of OSCC bone invasion. CCL28 inhibited invasion and epithelial-mesenchymal transition (EMT), and its inhibition of EMT was characterized by induced E-cadherin expression and reduced nuclear localization of β-catenin in OSCC cells with detectable RUNX3 expression levels. CCL28 signaling via CCR10 increased retinoic acid receptor-β (RARβ) expression by reducing the interaction between RARα and HDAC1. It was suggested that higher expression of CCL28, CCR10, or RARβ had significantly better overall survival [18]. Hence, these findings indicate that decreased CCL28 and CCR10 expression was associated with advanced clinical staging of the disease, consistent with our findings.

The study found significant differences in CCL28 and CCR10 expression in different OSCC histological grades, with weak immunostaining intensity in PDSCCs, higher in MDSCCs, and highest in WDSCCs. Low expression correlates with higher malignancy grade, progression, and aggressiveness. A statistically significant association was found between CCL28 and CCR10 expression and OSCC biological behavior, supporting Park et al.'s findings on their positive correlation with OSCC.

However, there are a few shortcomings, like the fact that the fact that different grades of OSCC were not separately analyzed owing to a smaller sample size. Due to the predominance of male participants in our retrospective investigation, it was challenging to find gender-matched controls. The personal histories of the individuals in all the cases analyzed could not be evaluated. Consequently, important risk factors for oral squamous cell carcinoma (OSCC), such as smoking, drinking, and betel quid chewing, were not accounted for or assessed in this study. Along with a lack of molecular investigations, we found a consistent expression of CCL28 and CCR10 in all the study cases, which was at its maximum level in well-differentiated malignancies and decreased with decreasing grades of cytological differentiation and maturation of OSCCs. It is recommended to conduct future studies with larger sample sizes and incorporate clinical follow-up to validate and expand upon our findings. Molecular detection techniques could improve the present study's results and may help evaluate the prognostic significance of the CCL28 and CCR10 axes in OSCCs.

Conclusion

We have observed the expression of CCR10 and CCL28 in normal oral mucosa and differing histological grades of OSCCs. The integrated expression of both IHC markers was absent to less in the unaffected oral mucosa. In contrast, it was abundant in the initial stages of OSCC but decreased with poor differentiation. From the results obtained through the present study, we hereby conclude that the co-expression of CCR10 and CCL28 could be used as an adjunctive in the prognostication of OSCCs. Further studies are warranted shortly to validate our results, and we also suggest that including both of these markers in the routine prognostication of OSCCs might improve the survival of the affected individuals by detecting the malignancy at its earliest. Moreover, combined immunostaining of the aforementioned antibodies would aid in a better understanding of the rationale behind the malignant transformation of potentially malignant oral disorders, which might pave new ways for scrutinizing contemporary early diagnostic techniques.

Authors' Contributions

All the authors contributed significantly to this manuscript.

Funding

None.

Availability of Data and Materials

Not Applicable.

Code Availability

Not Applicable.

Declarations

Conflicts of interest

The authors declare that they have no known conflict of interest.

Ethical Approval

Obtained from King George's Medical University (KGMU), (101st ECM II B Thesis/P65) Lucknow.

Consent to Participate

Not Applicable.

Consent for Publication

Not Applicable.