A Preliminary Study of the Role of Endothelial-Mesenchymal Transitory Factor SOX 2 and CD147 in the Microvascularization of Oral Squamous Cell Carcinoma.

INTRODUCTION: The aim of this study was to detect the possible endothelial expression of embryonic-type cancer stem cells (CSC) marker SOX2 and the stemness-type CSC marker CD147 in oral potential malignant disorders (OPMDs), oral leukoplakia (OL) in particular, and oral squamous cell carcinoma (OSCC). METHODS: This study focuses on the immunohistochemical pattern of expression of CSC protein-biomarkers SOX2 and CD147 in paraffin-embedded samples of 21 OSCCs of different grades of differentiation and 30 cases of OLs with different grades of dysplasia, compared to normal oral mucosa. RESULTS: The protein biomarker SOX2 was expressed in the endothelial cells, but without establishing any statistically significant correlation among OSCC, OL, and normal tissue specimens. However, SOX endothelial staining was noticed in 7/30 (23.3%) cases of OL (one non-dysplastic, one mildly dysplastic, one moderately dysplastic, and four severely dysplastic cases) and 5/21 (23.8%) cases of OSCC (two well-differentiated, one moderately differentiated, and two poorly differentiated cases). Although CD147 is expressed in normal oral epithelium, OL, and OSCC neoplastic cells, its vascular-endothelial expression was noticed in only 2/5 (40%) cases of normal oral epithelium, 1/30 (3.3%) cases of OL (one severely dysplastic case), and 4/21 (19%) cases of OSCC (two well-differentiated, one moderately differentiated, and one poorly differentiated case). Therefore, no statistically significant correlation among OSCC, OL, and normal tissue specimens was established. CONCLUSION: The endothelial presence of SOX2 both in oral potentially malignant and malignant lesions suggests that SOX2 may be implicated in the microvascularization process and associated with the degree of dysplasia in OL. The expression of CD147 may be attributed both to local inflammation and tumorigenesis. The implementation of CD147 in larger groups of tissue samples will shed some light on its role in cancer and inflammation. The evidence so far supports the need for more studies, which may support the clinical significance of these novel cancer stem cell biomarkers.

Expression of the Embryonic Cancer Stem Cells' Biomarkers SOX2 and OCT3/4 in Oral Leukoplakias and Squamous Cell Carcinomas: A Preliminary Study.

INTRODUCTION: Cancer stem cells (CSCs) are incriminated for initiating the process of carcinogenesis either de novo or through the transformation of oral potentially malignant disorders (OPMDs) to oral squamous cell carcinoma (OSCC). The aim of this study was to detect the expression of embryonic-type CSC markers OCT3/4 and SOX2 in OSCCs and oral leukoplakias (OLs), the most common of OPMDs. MATERIALS AND METHODS: The study type is experimental, and the study design is characterized as semiquantitative research, which belongs to the branch of experimental research. The experiment was conducted in the Department of Oral Medicine/Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece. This study focuses on the semiquantitative immunohistochemical (IHC) pattern of expression of CSCs protein-biomarkers SOX2 and OCT3/4, in paraffin embedded samples of 21 OSCCs of different grades of differentiation and 30 cases of OLs with different grades of dysplasia, compared to five cases of normal oral mucosa in both terms of cells' stain positivity and intensity. Statistical analysis was performed through SPSS 2017 Pearson Chi-square and the significance level was set at 0.05 (p=0.05). The expression of the respective genes of SOX2 and OCT3/4 was studied through quantitative polymerase chain reaction (qPCR), in paraffin-embedded samples of 12 cases of OLs with mild/non dysplasia and 19 cases moderately/poorly differentiated OSCCs(n=19) and five normal mucosa using the Independent Paired T-test. RESULTS: The genes SOX2 and Oct3/4 were expressed in all examined cases although no statistically significant correlations among normal, OL and OSCC, were established. A nuclear/membrane staining of OCT3/4 was noticed only in three out of 21 OSCCs but in none of OLs or normal cases (without statistical significance). A characteristic nuclear staining of SOX2 was noticed in the majority of the samples, mostly in the basal and parabasal layers of the epithelium. SOX2 was significantly detected in the OSCCs group (strong positivity in 17/21) than in the OL group (30 cases, mostly mildly stained) (p-value=0.007), and the normal oral epithelium (mild stained, p=0.065). Furthermore, SOX2 was overexpressed in well differentiated OSCCs group (5/OSCCs, strongly stained) rather than in mildly dysplastic and non-dysplastic OLs samples (14/OLs, mildly stained) (p-value =0.035). CONCLUSION: The characteristic expression of SOX2 but not of OCT3/4 in OLs' and OSCCs' lesions suggests the presence of neoplastic cells with certain CSC characteristics whose implication in the early stages of oral tumorigenesis could be further evaluated. The clinical use of SOX2, as prognostic factor, requires further experimental evaluation in larger number of samples.

Aldehyde Dehydrogenase: An Off-Label Marker of Endothelial Activation in Oral Squamous Cell Carcinoma.

INTRODUCTION: The vascular endothelial (VE) expression of aldehyde dehydrogenase (ALDH) 1/2 family in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) cases has not been studied so far. The aim of this study was to illustrate the "off-label" endothelial expression of cancer stem cell (CSC) biomarker, ALDH1/2, adjacent to oral potentially malignant and malignant lesions in order to shed some light on the mechanisms leading to oral carcinogenesis.  Materials and methods: The expression of CSC protein-biomarker ALDH1/2 was detected through immunohistochemistry (IHC) in 30 paraffin-embedded samples of OL and 21 samples of OSCC compared to five samples of normal oral mucosa. Statistical analysis was done using SPSS, Pearson Chi-square, and Fischer's exact test. The significance level was set at 0.05 (p≤ 0.05).  Results: In oral mucosal vessels, ALDH1/2 was not expressed. It was expressed significantly more in the vessels of OSCCs compared to the OLs (Fisher's exact test, p-value= 0,001). However, when endothelial expression of ALDH1/2 in the vasculature of OLs was compared with that of the normal oral mucosa, no significant change was noticed (Fisher's exact test, p-value=1.000).  Discussion: The IHC VE expression of ALDH1/2 in OSCC vasculature but not in OL indicates a possible significantly stronger activation of endothelial cells during carcinogenesis, which could be an indicator of the role of inflammation in the development of field cancerization and of prognostic value for (vascular/lymphatic) metastasis.

Fabrication of 3D Printed Hollow Microneedles by Digital Light Processing for the Buccal Delivery of Actives.

In the present study, two different microneedle devices were produced using digital light processing (DLP). These devices hold promise as drug delivery systems to the buccal tissue as they increase the permeability of actives with molecular weights between 600 and 4000 Da. The attached reservoirs were designed and printed along with the arrays as a whole device. Light microscopy was used to quality control the printability of the designs, confirming that the actual dimensions are in agreement with the digital design. Non-destructive volume imaging by means of microfocus computed tomography was employed for dimensional and defect characterization of the DLP-printed devices, demonstrating the actual volumes of the reservoirs and the malformations that occurred during printing. The penetration test and finite element analysis showed that the maximum stress experienced by the needles during the insertion process (10 N) was below their ultimate compressive strength (240-310 N). Permeation studies showed the increased permeability of three model drugs when delivered with the MN devices. Size-exclusion chromatography validated the stability of all the actives throughout the permeability tests. The safety of these printed devices for buccal administration was confirmed by histological evaluation and cell viability studies using the TR146 cell line, which indicated no toxic effects.

Preliminary Study of the Cancer Stem Cells' Biomarker CD147 in Leukoplakia: Dysplasia and Squamous Cell Carcinoma of Oral Epithelial Origin.

Objectives Cancer stem cells (CSCs) are responsible for initiating the process of carcinogenesis de novo, as well as through the transformation of oral potential malignant disorders (OPMDs) to oral squamous cell carcinoma (OSCC). The aim of our study was to detect the expression of stemness-type CSC marker CD147 in oral leukoplakias (OLs), the most common OPMD, and OSCCs as well. Materials and methods This study focuses on the semiquantitative immunohistochemical pattern of the expression of the CSC protein biomarker CD147 in paraffin-embedded samples of 20 OSCCs of different grades of differentiation and 30 cases of OLs without or with different grades of dysplasia, compared to the normal oral epithelium in terms of cells' stain positivity. Statistical analysis was performed through Statistical Package for Social Sciences (SPSS) version 25.0 (IBM SPSS Statistics, Armonk, NY) with Pearson chi-square test, and the significance level was set at 0.05 (p=0.05). In addition, the study clarified the expression of the respective gene of CD147 through quantitative polymerase chain (qPCR), in paraffin-embedded samples of the two extreme graduations: OLs of mildly dysplastic or non-dysplastic cases (n=10 cases) and OSCCs of moderately/poorly differentiated cases (n=17). Statistical analysis was then performed through SPSS version 25.0 with an independent paired t-test, and the significance level was set at 0.05 (p=0.05). Results The gene CD147 was expressed in all cases, although no statistically significant correlations were established. Regarding its protein products, the characteristic membranous staining of CD147 was noticed in the majority of the samples, mostly in the basal and parabasal layers of the epithelium. CD147 was upregulated significantly in the moderately and severely dysplastic OLs than in the mildly dysplastic and non-dysplastic OLs (p=0.008). Also, CD147 was upregulated significantly in the mildly dysplastic and non-dysplastic OLs than in the normal oral epithelium (p=0.012). Discussion The characteristic expression of CD147 in OLs and OSCCs' lesions suggests the presence of stemlike cancer cells, illustrating an underlying effect on the early stages of oral dysplasia, in the OL stage. The clinical application of CD147 as prognostic factor requires the experimental evaluation in larger number of samples. Conclusion Stem cells play an important role in the process of carcinogenesis. A major goal in cancer research is the identification of specific biomarkers for the detection of cancer stem cells. CD147 is considered as an innovative stem cell marker. Our findings in oral mucosal potentially malignant disorders showed that CD147 is expressed more intensely in parallel with the progression of the grade of dysplasia in OL. On the other hand, in oral squamous cell carcinoma, CD147 expression remains stable regardless of the degree of differentiation.

Histological study of eyelid hidrocystoma: A clinical case.

Hidrocystomas are benign lesions with frequent localization on the eyelids and common appearance as isolated translucent cystic lesions. The multiple and bilateral localization has been associated with systemic diseases or epidermodysplasia. The histological and ultrastructural study contributes to the distinction in eccrine or apocrine. In this clinical case, multiple bilateral skin lesions of the eyelids were removed surgically from a 55-year-old male patient with free medical history. The lesions were fully removed surgically without rupture of the cysts. The obtained tissues were fixed and prepared for an anatomical pathology study as well as observation under the electron microscope. The histological study revealed cystic lesions with low cuboid epithelium and presence of cells and collagen fibrils, while the ultrastructural study also revealed the presence of apoptotic and secretory cells as well as vacuolization of the cytoplasm. No relapse of the lesions was obtained. In conclusion, the histological and ultrastructural study of multiple hidrocystomas can highlight special features that help to better classify and diagnose eccrine or apocrine hidrocystomas, while surgical removal without rupture of the cyst can prevent reappearance.

Immunohistochemical Evaluation of CD3, CD4, CD8, and CD20 in Decidual and Trophoblastic Tissue Specimens of Patients with Recurrent Pregnancy Loss.

Recurrent miscarriages affect up to 5% of couples. CD3+ (T-lymphocytes), CD4+ (helper T-lymphocytes), CD8+ (cytotoxic T-lymphocytes), and CD20+ (B-lymphocytes) cells may participate in the pathophysiology of recurrent pregnancy loss (RPL). The aim of this study was to investigate the complicity of these molecules in RPL. The experimental specimens were obtained from 20 females who underwent miscarriages in the first gestational trimester, while the control group's specimens consisted of 20 females who proceeded with voluntary pregnancy termination during the same period. Tissue samples were taken from the decidua basalis, decidua parietalis, and trophoblast (placental chorionic villi) and were studied using immunohistochemical methods. Monoclonal antibodies were used against CD3, CD4, CD8, and CD20 cells. The lymphocyte levels in the decidua parietalis displayed profound disparities among the two groups. The decidua basalis and trophoblast exhibited almost the same disparities regarding positive CD cells. The comparison of CD4+ and CD8+ cells in the endometrial tissue revealed a significant difference between the two groups of study. The analysis uncovered a strong relationship between RPL and the presence of CD3+, CD4+, CD8+, and CD20+ cells in the decidua parietalis tissue. The number of positive T cells was decreased in the decidual basalis and chorionic villi, proving that their absence significantly disrupts the balance of the immunological environment.

Synergistic Antitumor Potency of a Self-Assembling Peptide Hydrogel for the Local Co-delivery of Doxorubicin and Curcumin in the Treatment of Head and Neck Cancer.

Combination therapy has been conferred with manifold assets leveraging the synergy of different agents to achieve a sufficient therapeutic outcome with lower administered drug doses and reduced side effects. The therapeutic potency of a self-assembling peptide hydrogel for the co-delivery of doxorubicin and curcumin was assessed against head and neck cancer cells. The dual loaded peptide hydrogel enabled control over the rate of drug release based on drug's aqueous solubility. A significantly enhanced cell growth inhibitory effect was observed after treatment with the combination drug-loaded hydrogel formulations compared to the respective combination drug solution. The synergistic pharmacological effect of selected hydrogel formulations was further confirmed with enhanced apoptotic cell response, interference in cell cycle progression, and significantly altered apoptotic/anti-apoptotic gene expression profiles obtained in dose levels well below the half-maximal inhibitory concentrations of both drugs. The in vivo antitumor efficacy of the drug-loaded peptide hydrogel formulation was confirmed in HSC-3 cell-xenografted severe combined immunodeficient mice and visualized with µCT imaging. Histological and terminal deoxynucleotidyl transferase dUTP nick end labeling assay analyses of major organs were implemented to assess the safety of the topically administered hydrogel formulation. Overall, results demonstrated the therapeutic utility of the dual drug-loaded peptide hydrogel as a pertinent approach for the local treatment of head and neck cancer.