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Objectives: Various types of cells comprising a complex and diverse cell population are required for the biological activities of odontogenic keratocyst (OKC). Immune and non‐immune cells collaborate via cytokine‐ or chemokine‐mediated communication and direct cell–cell interactions. This study aimed to characterize the immune ecosystem and understand the potential chemotactic role of OKC fibroblasts in immune cell migration. Materials and Methods: Mass cytometry of 41 markers was employed for the classification of OKC cells from six OKC samples. Immunofluorescence staining and single‐cell RNA sequencing (GSE176351) were used for the detection of fibroblast subpopulations. Enzyme‐linked immunosorbent assay and immunofluorescence staining were employed for chemokine detection in hypoxia‐ and/or HIF‐1α inhibitor‐treated OKC fibroblasts and tissues. Chemotaxis assay was employed to determine the chemotactic effect of fibroblasts via co‐culture with peripheral blood mononuclear cells. A cell communication network was constructed based on the single‐cell RNA sequencing data. Results: The characterization of the immune cell types of OKC evidenced the enrichment of macrophages, neutrophils and B cells. The majority (41.5%) of fibroblast subsets consisted of chemokine ligand‐enriched myofibroblasts. The activation of the HIF‐1α signaling pathway in fibroblasts was associated with chemokine release. The chemokines released by OKC fibroblasts remarkably promoted the migration of peripheral blood mononuclear cells in the co‐culture system. Close interactions between myofibroblasts and immune cells were validated by cell–cell interaction analysis. Increased RANKL expression was detected in OKC fibroblasts in the co‐culture system with peripheral blood mononuclear cells. Conclusions: Our results provided deep insights into the immune ecosystem and highlighted the potential chemotactic effects of chemokine‐enriched myofibroblasts within OKCs. The close interaction between immune cells and fibroblasts demonstrated in this study may be responsible for the osteoclastogenic effects of OKC fibroblasts. [ABSTRACT FROM AUTHOR]

Background: Salivary gland pleomorphic adenoma (SPA) is a common neoplasm of salivary glands that displays remarkable histological diversity. Previous studies have demonstrated the involvement of gene rearrangements and cytoskeleton‐remodeling‐related myoepithelial cells in SPA tumorigenesis. Cytoskeleton remodeling is necessary for epithelial‐mesenchymal transition (EMT), a key process in tumor progression. However, the heterogeneity of tumor cells and cytoskeleton remodeling in SPA has not been extensively investigated. Methods: An analysis of single‐cell RNA sequencing (scRNA‐seq) was performed on 27 810 cells from two donors with SPA. Bioinformatic tools were used to assess differentially expressed genes, cell trajectories, and intercellular communications. Immunohistochemistry and double immunofluorescence staining were used to demonstrate FOXC1 and MYLK expression in SPA tissues. Results: Our analysis revealed five distinct cell subtypes within the tumor cells of SPA, indicating a high level of intra‐lesional heterogeneity. Cytoskeleton‐remodeling‐related genes were highly enriched in subtype 3 of the tumor cells, which showed a close interaction with mesenchymal cells. We found that tumoral FOXC1 expression was closely related to MYLK expression in the tumor cells of SPA. Conclusion: Tumor cells enriched with cytoskeleton‐remodeling‐related genes play a crucial role in SPA development, and FOXC1 may partially regulate this process. [ABSTRACT FROM AUTHOR]

The article presents the discussion on exploring more valuable diagnostic and prognostic methods for improving the long-term survival of OSCC patients. Topics include OSCC treatment modalities increasing patients' survival mainly include surgery, chemotherapy, radiation therapy, and adjuvant treatment; and CD8+ T cell-derived extracellular vesicles significantly destroying the tumor stroma.

Surgery with the assistance of conventional radiotherapy, chemotherapy and immunotherapy is the basis for head and neck squamous cell carcinoma (HNSCC) treatment. However, with these treatment modalities, the recurrence and metastasis of tumors remain at a high level. Increasingly, the evidence indicates an excellent anti-tumor effect of chimeric antigen receptor T (CAR-T) cells in hematological malignancy treatment, and this novel immunotherapy has attracted researchers' attention in HNSCC treatment. Although several clinical trials have been conducted, the weak anti-tumor effect and the side effects of CAR-T cell therapy against HNSCC are barriers to clinical translation. The limited choices of targeting proteins, the barriers of CAR-T cell infiltration into targeted tumors and short survival time in vivo should be solved. In this review, we introduce barriers of CAR-T cell therapy in HNSCC. The limitations and current promising strategies to overcome barriers in solid tumors, as well as the applications for HNSCC treatment, are covered. The perspectives of CAR-T cell therapy in future HNSCC treatment are also discussed. [ABSTRACT FROM AUTHOR]

Tumour cell‐secreted microvesicles (MVs) contribute immensely to tumour progression. However, the role of tumoral salivary MVs in oral squamous cell carcinoma (OSCC) remains unclear. Herein, we elucidated the role of non‐apoptotic salivary tumoral MVs in OSCC development, especially relating to the migration ability. We purified and compared non‐apoptotic salivary tumoral MVs from 63 OSCC patients and orthotopic OSCC mice model. Next, we compared the protein difference between apoptotic and non‐apoptotic MVs by Western blot, proteomics and flow cytometry from saliva and CAL27 cells. Finally, we collected the non‐apoptotic MVs and co‐cultured with normal oral epithelial cells, the migration ability was examined by wound healing assay and Western blot assay. Our results indicated that the levels of non‐apoptotic tumoral S‐MVs were significantly higher in OSCC patients with T3 to T4 stages than in patients with T1 to T2 stages or healthy donors. In OSCC mice model, we found elevations of non‐apoptotic tumoral MVs associated with tumoral volume. EGFR overexpression increased the generation of non‐apoptotic tumoral MVs which could significantly promote normal epithelial cell migration. In conclusion, elevated levels of non‐apoptotic tumoral S‐MVs are associated with clinicopathologic features of OSCC patients, implying that non‐apoptotic tumoral S‐MVs are a potential progressive marker of OSCC. [ABSTRACT FROM AUTHOR]

Background. Interleukin 8 (IL-8) is a chemotactic cytokine released by various cells including leukocytes, endothelial cells, and epithelial cells. IL-8 has multiple functions in inflammation, tumour invasion, or angiogenesis. Human odontogenic cystic lesions are chronic and frequently inflamed. Tissue-derived extracellular vesicles (Ti-EVs) are widely present in various tissues and could more accurately reflect the characteristics of the primary tissue. However, the involvement of IL-8 in Ti-EVs of human odontogenic lesions is still unclear. This study aimed to explore the expression of IL-8 in Ti-EVs of human odontogenic lesions and the potential roles of Ti-EVs that carried IL-8. Methods. Fresh tissue samples of dentigerous cyst (DC, n = 5) and odontogenic keratocyst (OKC, n = 5) were collected for Ti-EVs isolation. Ti-EVs were characterised by transmission electron microscopy and nano-flow cytometry analysis. The cytokine profile of Ti-EVs was explored by cytokine antibody array. The IL-8 expression was examined by immunochemical staining in tissue of odontogenic lesions (DC, n =12; OKC, n =28). Antioxidants (N-acetyl-L-cysteine and diphenyleneiodonium) were employed to treat HaCaT cells, and the expression of IL-8 was detected by enzyme-linked immunosorbent assay. The gene expression of MMP9 was explored by quantitative real-time polymerase chain reaction in co-culture system of fibroblasts of OKC with Ti-EVs. Results. Compared with DC, the expression of IL-8 in Ti-EVs and fixed tissue specimens of OKC was markedly upregulated. The antioxidants decreased the expression level of IL-8 protein in the supernatant of HaCaT cells. The Ti-EVs treatment (10 μg/ml) of fibroblasts significantly induced the MMP9 mRNA expressions in OKC fibroblasts. Conclusions. IL-8 was upregulated in Ti-EVs of OKC and might be involved in the tissue destruction of OKC. [ABSTRACT FROM AUTHOR]

Signal transducer and activator of transcription 3 (STAT3), a member of the STAT family, discovered in the cytoplasm of almost all types of mammalian cells, plays a significant role in biological functions. The duration of STAT3 activation in normal tissues is a transient event and is strictly regulated. However, in cancer tissues, STAT3 is activated in an aberrant manner and is induced by certain cytokines. The continuous activation of STAT3 regulates the expression of downstream proteins associated with the formation, progression, and metastasis of cancers. Thus, elucidating the mechanisms of STAT3 regulation and designing inhibitors targeting the STAT3 pathway are considered promising strategies for cancer treatment. This review aims to introduce the history, research advances, and prospects concerning the STAT3 pathway in cancer. We review the mechanisms of STAT3 pathway regulation and the consequent cancer hallmarks associated with tumor biology that are induced by the STAT3 pathway. Moreover, we summarize the emerging development of inhibitors that target the STAT3 pathway and novel drug delivery systems for delivering these inhibitors. The barriers against targeting the STAT3 pathway, the focus of future research on promising targets in the STAT3 pathway, and our perspective on the overall utility of STAT3 pathway inhibitors in cancer treatment are also discussed. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are lipid‐bilayer membrane structures secreted by most cell types. EVs act as messengers via the horizontal transfer of lipids, proteins, and nucleic acids, and influence various pathophysiological processes in both parent and recipient cells. Compared to EVs obtained from body fluids or cell culture supernatants, EVs isolated directly from tissues possess a number of advantages, including tissue specificity, accurate reflection of tissue microenvironment, etc., thus, attention should be paid to tissue‐derived EVs (Ti‐EVs). Ti‐EVs are present in the interstitium of tissues and play pivotal roles in intercellular communication. Moreover, Ti‐EVs provide an excellent snapshot of interactions among various cell types with a common histological background. Thus, Ti‐EVs may be used to gain insights into the development and progression of diseases. To date, extensive investigations have focused on the role of body fluid‐derived EVs or cell culture‐derived EVs; however, the number of studies on Ti‐EVs remains insufficient. Herein, we summarize the latest advances in Ti‐EVs for cancers and non‐cancer diseases. We propose the future application of Ti‐EVs in basic research and clinical practice. Workflows for Ti‐EV isolation and characterization between cancers and non‐cancer diseases are reviewed and compared. Moreover, we discuss current issues associated with Ti‐EVs and provide potential directions. [ABSTRACT FROM AUTHOR]

Purpose: We aimed to define cell subpopulations of odontogenic keratocyst (OKC), particularly relating to angiogenesis and explored the potential regulation mechanism for angiogenesis. Materials and Methods: Single-cell RNA sequencing (scRNA-seq) analysis was investigated on 14,072 cells from 3 donors with OKC. The differential expressed genes, cell trajectory and intercellular communications were evaluated by bioinformatic analysis. Hydrostatic pressure (80 mmHg, 6h) was applied to the primary fibroblasts of OKC and the supernatant was collected for cytokines detection by cytokine antibody array. The chemokine (C-X-C motif) ligand 12 (CXCL12) and CD31 expressions were explored by immunohistochemistry in tissue microarray of OKC. Results: Five different cell types were identified in the epithelium of OKC and 3 different cell types in the OKC fibroblasts were characterized, indicating high intra-lesional heterogeneity. CXCLs were highly enriched in the subset of fibroblasts and showed close interactions with endothelial cells. Hydrostatic pressure (80mmHg) significantly increased CXCL12 secretions in OKC fibroblasts. Stromal CXCL12 expressions were closely related to CD31 expressions of tissue microarray of OKC. Conclusion: CXCLs enriched fibroblasts are crucial for angiogenesis of OKCs which could be partially regulated by hydrostatic pressure. [ABSTRACT FROM AUTHOR]

Microvesicles (MVs), which are cell‐derived membrane vesicles present in body fluids, are closely associated with the development of malignant tumours. Saliva, one of the most versatile body fluids, is an important source of MVs. However, the association between salivary MVs (SMVs) and oral squamous cell carcinoma (OSCC), which is directly immersed in the salivary milieu, remains unclear. SMVs from 65 patients with OSCC, 21 patients with oral ulcer (OU), and 42 healthy donors were purified, quantified and analysed for their correlations with the clinicopathologic features and prognosis of OSCC patients. The results showed that the level of SMVs was significantly elevated in patients with OSCC compared to healthy donors and OU patients. Meanwhile, the level of SMVs showed close correlations with the lymph node status, and the clinical stage of OSCC patients. Additionally, the ratio of apoptotic to non‐apoptotic SMVs was significantly decreased in OSCC patients with higher pathological grade. Consistently, poorer overall survival was observed in patients with lower ratio of apoptotic to non‐apoptotic SMVs. In conclusion, the elevated level of SMVs is associated with clinicopathologic features and decreased survival in patients with OSCC, suggesting that SMVs are a potential biomarker and/or regulator of the malignant progression of OSCC. [ABSTRACT FROM AUTHOR]

Aims: The purpose of this study was to explore the potential involvement of Fra‐1, c‐Jun and c‐Fos, three vital members of the AP‐1 complex, in the pathogenesis of odontogenic keratocysts (OKCs). Methods and results: Tissue samples, containing 10 normal oral mucosa (OM), 10 dentigerous cysts (DC) and 32 OKC specimens, were applied to investigate the expression levels of Fra‐1, c‐Jun and c‐Fos by immunohistochemistry and real‐time–quantitative polymerase chain reaction (RT–qPCR). The association between Fra‐1, c‐Jun and c‐Fos expression levels and markers of proliferation [Ki‐67, proliferating cell nuclear antigen (PCNA)], anti‐apoptosis (Bcl‐2) was then investigated in the OKC serial tissue sections. The results showed that Fra‐1, c‐Jun and c‐Fos expression levels were increased significantly in OKCs compared to these in OM and DC tissue samples. Meanwhile, the expression levels of Fra‐1, c‐Jun and c‐Fos were associated positively with the expression levels of Ki‐67, PCNA and Bcl‐2, as confirmed further by double‐labelling immunofluorescence analysis and hierarchical analysis. Conclusions: This study revealed for the first time that Fra‐1, c‐Jun and c‐Fos were overexpressed in OKCs and had a close correlation with proliferation and anti‐apoptosis potential of OKCs. [ABSTRACT FROM AUTHOR]

Bone resorption in the jaws is one of the most severe complications of keratocystic odontogenic tumors (KCOTs), and can be treated by either enucleation or marsupialization. However, the effects of marsupialization on bone regeneration adjacent to KCOTs, and the mechanisms involved, are still unclear. In this study, samples of 27 KCOTs were collected (20 before marsupialization and 7 after marsupialization) to detect the expression of bone regeneration-related molecules adjacent to KCOTs by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). The results showed that bone formation was significantly enhanced in the KCOT capsule wall adjacent to bone after marsupialization, as demonstrated by alkaline phosphatase activity assay and immunostaining for bone morphogenetic protein. Moreover, immunohistochemistry revealed that osteoprotegerin (OPG) was up-regulated in the KCOT capsule wall adjacent to bone after marsupialization, while receptor activator of nuclear factor-κB ligand (RANKL) was down-regulated. Real-time qPCR also demonstrated increased expression of OPG after marsupialization, accompanied by a decrease in the expression of osteoclastogenesis-related molecules such as cathepsin K, matrix metalloproteinase-9, NFATc1, RANK and RANKL. This study provides further evidence that marsupialization may promote bone regeneration adjacent to KCOTs partially through regulation of the OPG/RANK/RANKL signaling pathway. [ABSTRACT FROM AUTHOR]

The aim of this study is to clarify whether YAP/TAZ is involved in the pathogenesis and proliferative growth of keratocystic odontogenic tumor (KCOT). The expression levels of YAP/TAZ and downstream proteins and genes in normal oral mucosa (OM) and KCOT were determined and compared by immunohistochemistry and real-time quantitative PCR. The results showed that the expression of YAP/TAZ and downstream proteins (Cyr61, CTGF) was significantly upregulated in KCOT with upregulation of Ki-67 compared to OM. Importantly, the mRNA levels of transcription factors (TEAD1, TEAD4, and RUNX2) and cell cycle related genes (CDK2, PCNA), which interact with the transcriptional coactivators YAP/TAZ, are also upregulated in the KCOT. In addition, the results from Spearman rank correlation test revealed the close relationship between YAP/TAZ and Ki-67, which was further evidenced by double-labelling immunofluorescence that revealed a synchronous distribution for YAP/TAZ with Ki-67 in KCOT samples. All the data suggested YAP/TAZ might be involved in the proliferative behavior of KCOT. [ABSTRACT FROM AUTHOR]

Objectives Methods Results Conclusions This study aimed to investigate the expression and biological significance of Semenogelin 1 (SEMG1), a member of the cancer‐testis antigen family, in oral squamous cell carcinoma (OSCC). Further, we explored its potential association with metabolism‐related molecules.SEMG1 expression levels in OSCC were determined through immunohistochemistry, flow cytometry, and Western blot analyses. To decipher the biological implications of SEMG1 in OSCC, the CAL27 OSCC cell line was either stably overexpressed with SEMG1 or subjected to SEMG1‐shRNA knockdown. The relationship between clinicopathological parameters and SEMG1 expression in OSCC patients was also assessed.SEMG1 was found to be overexpressed in OSCC, though its expression was not influenced by the pathological grade. The fluorescent dihydroethidium assay indicated that SEMG1 augmented reactive oxygen species production. The mitochondrial membrane potential assay suggested a significant upregulation of mitochondrial membrane potential by SEMG1. Cell cycle assessments highlighted that SEMG1 overexpression led to a notable rise in cells entering the S‐phase. Additionally, a strong correlation between SEMG1 expression and both ENO1 and PKM2 expression in OSCC was observed.The findings underscore the elevated expression of SEMG1 in OSCC and its contributory role in the tumorigenesis of OSCC patients. [ABSTRACT FROM AUTHOR]

Objectives Materials and Methods Results Conclusions Ameloblastoma (AM), a locally aggressive tumor with extensive growth capacity, causes significant damage to the jaw and affects facial appearance. Although the high prevalence of BRAF V600E mutation in AM is known, its specific impacts on patients with AM remain unclear. Thus, the present study investigated the role of BRAF V600E mutation, thereby focusing on its impact on AM invasion and growth.Immunohistochemical analysis was used to compare BRAF V600E, MMP2, MMP9, and Ki‐67 expressions in AM (n = 49), normal oral mucosa (NOM) (n = 10), and odontogenic keratocyst (OKC) (n = 15) tissues. AM was further classified according to the presence or absence of BRAF V600E. The relationship between BRAF V600E and invasion as well as growth was evaluated. In addition, correlation analysis was performed using immunohistochemistry and confirmed via double‐labeling immunofluorescence. Finally, comparative analyses using mass spectrometry, immunohistochemistry, and immunofluorescence were performed to explore and identify underlying mechanisms.AM exhibited a higher incidence of BRAF V600E mutation than NOM and OKC. BRAF V600E expression was positively correlated with the invasion‐associated proteins MMP2 and MMP9 and the growth‐related protein Ki‐67. Proteomic data revealed that BRAF V600E primarily activates the MAPK signaling pathway in AM, particularly driving the phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2).In summary, the findings suggested that the BRAF V600E mutation enhances the invasion and growth abilities of AM via the MAPK/ERK signaling pathway. Thus, targeting BRAF V600E or the MAPK/ERK pathway may be a potential AM therapy. [ABSTRACT FROM AUTHOR]

Hypoxia plays a critical role in head and neck squamous cell carcinoma (HNSCC) prognosis. However, till now, robust and reliable hypoxia-related prognostic signatures have not been established for an accurate prognostic evaluation in HNSCC patients. This article focused on establishing a risk score model to evaluate the prognosis and guide treatment for HNSCC patients. RNA-seq data and clinical information of 502 HNSCC patients and 44 normal samples were downloaded from The Cancer Genome Atlas (TCGA) database. 433 samples from three Gene Expression Omnibus (GEO) datasets were incorporated as an external validation cohort. In the training cohort, prognostic-related genes were screened and LASSO regression analyses were performed for signature establishment. A scoring system based on SRPX, PGK1, STG1, HS3ST1, CDKN1B, and HK1 showed an excellent prediction capacity for an overall prognosis for HNSCC patients. Patients were divided into high- and low-risk groups, and the survival status of the two groups exhibited a statistically significant difference. Subsequently, gene set enrichment analysis (GSEA) was carried out to explore the underlying mechanisms for the prognosis differences between the high- and low-risk groups. The tumor immune microenvironment was evaluated by CIBERSORT, ESTIMATE, TIDE, and xCell algorithm, etc. Then, we explored the relationships between this prognostic model and the levels of immune checkpoint-related genes. Cox regression analysis and nomogram plot indicated the scoring system was an independent predictor for HNSCC. Moreover, a comparison of predictive capability has been made between the present signature and existing prognostic signatures for HNSCC patients. Finally, we detected the expression levels of proteins encoded by six-HRGs via immunohistochemical analysis in tissue microarray. Collectively, a novel integrated signature considering both HRGs and clinicopathological parameters will serve as a prospective candidate for the prognostic evaluation of HNSCC patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Man and Liu.)

Bone resorption in the jaws is one of the most severe complications of keratocystic odontogenic tumors (KCOTs), and can be treated by either enucleation or marsupialization. However, the effects of marsupialization on bone regeneration adjacent to KCOTs, and the mechanisms involved, are still unclear. In this study, samples of 27 KCOTs were collected (20 before marsupialization and 7 after marsupialization) to detect the expression of bone regeneration-related molecules adjacent to KCOTs by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). The results showed that bone formation was significantly enhanced in the KCOT capsule wall adjacent to bone after marsupialization, as demonstrated by alkaline phosphatase activity assay and immunostaining for bone morphogenetic protein. Moreover, immunohistochemistry revealed that osteoprotegerin (OPG) was up-regulated in the KCOT capsule wall adjacent to bone after marsupialization, while receptor activator of nuclear factor-κB ligand (RANKL) was down-regulated. Real-time qPCR also demonstrated increased expression of OPG after marsupialization, accompanied by a decrease in the expression of osteoclastogenesis-related molecules such as cathepsin K, matrix metalloproteinase-9, NFATc1, RANK and RANKL. This study provides further evidence that marsupialization may promote bone regeneration adjacent to KCOTs partially through regulation of the OPG/RANK/RANKL signaling pathway.

Osteoradionecrosis (ORN) is one of the most serious complications of oral and nasopharyngeal malignant neoplasms after radiotherapy. The incidence of mandibular ORN is significantly higher than that of maxilla. A radical surgical intervention such as mandibulectomy for advanced ORN is appropriate and effective to relieve pain and control infection. However, recovery of functionality and aesthetics is usually not the primary purpose of the one-stage radical surgery. Some patients often need a 2-stage operation owing to the bone defect, scar contracture, or recurrence. Although free flap has been proven as a reliable way to repair the complex maxillofacial defects, it is still a great challenge for surgeons to reconstruct the secondary complex mandibular ORN defects. Here, we report a case of secondary composite mandibular ORN reconstruction using the preoperative 3-dimensional biomodel planning and flowthrough radial forearm flap for free fibula osteocutaneous flap.