Tumor progression is closely related to tumor tissue metabolism and reshaping of the microenvironment. Oral squamous cell carcinoma (OSCC), a representative hypoxic tumor, has a heterogeneous internal metabolic environment. To clarify the relationship between different metabolic regions and the tumor immune microenvironment (TME) in OSCC, Single cell (SC) and spatial transcriptomics (ST) sequencing of OSCC tissues were performed. The proportion of TME in the ST data was obtained through SPOTlight deconvolution using SC and GSE103322 data. The metabolic activity of each spot was calculated using scMetabolism, and k-means clustering was used to classify all spots into hyper-, normal-, or hypometabolic regions. CD4T cell infiltration and TGF-β expression is higher in the hypermetabolic regions than in the others. Through CellPhoneDB and NicheNet cell-cell communication analysis, it was found that in the hypermetabolic region, fibroblasts can utilize the lactate produced by glycolysis of epithelial cells to transform into inflammatory cancer-associated fibroblasts (iCAFs), and the increased expression of HIF1A in iCAFs promotes the transcriptional expression of CXCL12. The secretion of CXCL12 recruits regulatory T cells (Tregs), leading to Treg infiltration and increased TGF-β secretion in the microenvironment and promotes the formation of a tumor immunosuppressive microenvironment. This study delineates the coordinate work axis of epithelial cells-iCAFs-Tregs in OSCC using SC, ST and TCGA bulk data, and highlights potential targets for therapy.
Humans ; Carcinoma, Squamous Cell/metabolism* ; Squamous Cell Carcinoma of Head and Neck ; Mouth Neoplasms/metabolism* ; Immunosuppression Therapy ; Transforming Growth Factor beta ; Head and Neck Neoplasms ; Gene Expression Profiling ; Tumor Microenvironment

OBJECTIVES: Tongue squamous cell carcinoma (TSCC) is a common cancer in the oral and maxillofacial region, which seriously endangers people's life and health.Heterogeneous nuclear ribonucleoprotein A2/B1(hnRNP A2/B1) is an RNA-binding protein that regulates the expression of a variety of genes and participates in the occurrence and development of a variety of cancers. This study aims to investigate the role of hnRNP A2/B1 in TSCC progression. METHODS: The differential expression of hnRNP A2/B1 in oral squamous cell carcinoma (OSCC) and normal oral mucosa cells and tissues was analyzed based on the gene expression profiles of GSE146483 and GSE85195 in the Gene Expression Omnibus (GEO) database. The correlation between hnRNP A2/B1 expression and disease-free survival of TSCC patients was analyzed based on TSCC related chip of GSE4676. TSCC cancer and paracancerous tissue samples of 30 patients were collected in Hunan Cancer Hospital from July to December 2021. Real-time RT-PCR and Western blotting were used to verify the mRNA and protein expression of hnRNP A2/B1 in TSCC patients'samples, respectively. Human TSCC Tca-8113 cells were transfected with hnRNP A2/B1 empty vector (a sh-NC group), knockdown plasmid (a sh-hnRNP A2/B1 group), empty vector overexpression plasmid (an OE-NC group) and overexpression plasmid (an OE-hnRNP A2/B1 group), respectively. The knockdown or overexpression efficiency of hnRNP A2/B1 was detected by Western blotting. The proliferation activity of Tca-8113 cells was detected by cell counting kit-8 (CCK-8), and the apoptosis rate of Tca-8113 cells was detected by flow cytometry. RESULTS: Based on the analysis of OSCC-related chips of GSE146483 and GSE85195 in the GEO database, it was found that hnRNP A2/B1 was differentially expressed in the OSCC and normal oral mucosa cells and tissues (all P<0.01). Meanwhile, the analysis of TSCC related chip GSE4676 confirmed that the expression of hnRNP A2/B1 was negatively correlated with the disease-free survival of TSCC patients (P=0.006). The results of real-time RT-PCR and Western blotting showed that the relative expression levels of hnRNP A2/B1 mRNA and protein in TSCC tissues were significantly up-regulated compared with those in adjacent tissues (all P<0.01). The results of Western blotting showed that the expression level of hnRNP A2/B1 in Tca-8113 cells was significantly inhibited or promoted after knockdown or overexpression of hnRNP A2/B1 (all P<0.01). The results of CCK-8 and flow cytometry showed that inhibition of hnRNP A2/B1 expression in Tca-8113 cells reduced cell proliferation activity (P<0.05) and increased cell apoptic rate (P<0.01). Overexpression of hnRNP A2/B1 in Tca-8113 cells significantly increased cell proliferation (P<0.05) and decreased cell apoptosis (P<0.01). CONCLUSIONS HnRNP A2/B1 is a key factor regulating the proliferation and apoptosis of TSCC cells. Inhibition of hnRNP A2/B1 expression can reduce the proliferation activity of TSCC cells and promote the apoptosis of TSCC cells.
Humans ; Carcinoma, Squamous Cell/genetics* ; Sincalide/metabolism* ; Tongue Neoplasms/genetics* ; Mouth Neoplasms ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism* ; RNA, Messenger ; Tongue/metabolism* ; Cell Line, Tumor

Tumor-associated macrophages (TAMs) play crucial roles in tumor progression and immune responses. However, mechanisms of driving TAMs to antitumor function remain unknown. Here, transcriptome profiling analysis of human oral cancer tissues indicated that regulator of G protein signaling 12 (RGS12) regulates pathologic processes and immune-related pathways. Mice with RGS12 knockout in macrophages displayed decreased M1 TAMs in oral cancer tissues, and extensive proliferation and invasion of oral cancer cells. RGS12 increased the M1 macrophages with features of increased ciliated cell number and cilia length. Mechanistically, RGS12 associates with and activates MYC binding protein 2 (MYCBP2) to degrade the cilia protein kinesin family member 2A (KIF2A) in TAMs. Our results demonstrate that RGS12 is an essential oral cancer biomarker and regulator for immunosuppressive TAMs activation.
Mice ; Humans ; Animals ; Tumor-Associated Macrophages/metabolism* ; Carcinoma, Squamous Cell ; Squamous Cell Carcinoma of Head and Neck ; Mouth Neoplasms ; GTP-Binding Proteins/metabolism* ; Head and Neck Neoplasms ; Ubiquitin-Protein Ligases/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism* ; RGS Proteins/metabolism* ; Kinesins/metabolism* ; Repressor Proteins/metabolism*

Oral squamous cell carcinoma (OSCC) escape from the immune system is mediated through several immunosuppressive phenotypes that are critical to the initiation and progression of tumors. As a hallmark of cancer, DNA damage repair is closely related to changes in the immunophenotypes of tumor cells. Although flap endonuclease-1 (FEN1), a pivotal DNA-related enzyme is involved in DNA base excision repair to maintain the stability of the cell genome, the correlation between FEN1 and tumor immunity has been unexplored. In the current study, by analyzing the clinicopathological characteristics of FEN1, we demonstrated that FEN1 overexpressed and that an inhibitory immune microenvironment was established in OSCC. In addition, we found that downregulating FEN1 inhibited the growth of OSCC tumors. In vitro studies provided evidence that FEN1 knockdown inhibited the biological behaviors of OSCC and caused DNA damage. Performing multiplex immunohistochemistry (mIHC), we directly observed that the acquisition of critical immunosuppressive phenotypes was correlated with the expression of FEN1. More importantly, FEN1 directly or indirectly regulated two typical immunosuppressive phenotype-related proteins human leukocyte antigen (HLA-DR) and programmed death receptor ligand 1 (PD-L1), through the interferon-gamma (IFN-γ)/janus kinase (JAK)/signal transducer and activator transcription 1 (STAT1) pathway. Our study highlights a new perspective on FEN1 action for the first time, providing theoretical evidence that it may be a potential immunotherapy target for OSCC.
Humans ; Carcinoma, Squamous Cell/pathology* ; DNA ; Down-Regulation ; Flap Endonucleases/metabolism* ; Head and Neck Neoplasms ; Interferon-gamma/metabolism* ; Mouth Neoplasms/pathology* ; Phenotype ; Squamous Cell Carcinoma of Head and Neck ; Tumor Microenvironment ; Janus Kinases/metabolism*

Diosgenin, a steroidal sapogenin, obtained from Trigonella foenum-graecum, Dioscorea, and Rhizoma polgonati, has shown high potential and interest in the treatment of various cancers such as oral squamous cell carcinoma, laryngeal cancer, esophageal cancer, liver cancer, gastric cancer, lung cancer, cervical cancer, prostate cancer, glioma, and leukemia. This article aims to provide an overview of the in vivo, in vitro, and clinical studies reporting the diosgenin's anticancer effects. Preclinical studies have shown promising effects of diosgenin on inhibiting tumor cell proliferation and growth, promoting apoptosis, inducing differentiation and autophagy, inhibiting tumor cell metastasis and invasion, blocking cell cycle, regulating immunity and improving gut microbiome. Clinical investigations have revealed clinical dosage and safety property of diosgenin. Furthermore, in order to improve the biological activity and bioavailability of diosgenin, this review focuses on the development of diosgenin nano drug carriers, combined drugs and the diosgenin derivatives. However, further designed trials are needed to unravel the diosgenin's deficiencies in clinical application.
Male ; Humans ; Carcinoma, Squamous Cell/drug therapy* ; Diosgenin/metabolism* ; Mouth Neoplasms/drug therapy* ; Apoptosis ; Prostatic Neoplasms/drug therapy*

Oral squamous cell carcinoma (OSCC) is a prevalent malignant tumor affecting the head and neck region (Leemans et al., 2018). It is often diagnosed at a later stage, leading to a poor prognosis (Muzaffar et al., 2021; Li et al., 2023). Despite advances in OSCC treatment, the overall 5-year survival rate of OSCC patients remains alarmingly low, falling below 50% (Jehn et al., 2019; Johnson et al., 2020). According to statistics, only 50% of patients with oral cancer can be treated with surgery. Once discovered, it is more frequently at an advanced stage. In addition, owing to the aggressively invasive and metastatic characteristics of OSCC, most patients die within one year of diagnosis. Hence, the pursuit of novel therapeutic drugs and treatments to improve the response of oral cancer to medication, along with a deeper understanding of their effects, remains crucial objectives in oral cancer research (Johnson et al., 2020; Bhat et al., 2021; Chen et al., 2023; Ruffin et al., 2023).
Humans ; Mouth Neoplasms/pathology* ; Carcinoma, Squamous Cell/metabolism* ; Luteolin/therapeutic use* ; Squamous Cell Carcinoma of Head and Neck/drug therapy* ; Head and Neck Neoplasms/drug therapy* ; Cell Line, Tumor

Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2β1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2β1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.
Humans ; Paxillin/metabolism* ; Protein-Lysine 6-Oxidase/metabolism* ; Carcinoma, Squamous Cell/pathology* ; Epithelial-Mesenchymal Transition ; Integrin alpha2beta1/metabolism* ; Mouth Neoplasms/pathology* ; Collagen/metabolism* ; Fibroblasts ; Extracellular Vesicles/metabolism* ; Cell Line, Tumor ; Tumor Microenvironment

Oral potentially malignant disorders (OPMDs) are precursors of oral squamous cell carcinoma (OSCC). Deregulated cellular energy metabolism is a critical hallmark of cancer cells. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC1α) plays vital role in mitochondrial energy metabolism. However, the molecular mechanism of PGC1α on OPMDs progression is less unclear. Therefore, we investigated the effects of knockdown PGC1α on human dysplastic oral keratinocytes (DOKs) comprehensively, including cell proliferation, cell cycle, apoptosis, xenograft tumor, mitochondrial DNA (mtDNA), mitochondrial electron transport chain complexes (ETC), reactive oxygen species (ROS), oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and glucose uptake. We found that knockdown PGC1α significantly inhibited the proliferation of DOKs in vitro and tumor growth in vivo, induced S-phase arrest, and suppressed PI3K/Akt signaling pathway without affecting cell apoptosis. Mechanistically, downregulated of PGC1α decreased mtDNA, ETC, and OCR, while enhancing ROS, glucose uptake, ECAR, and glycolysis by regulating lactate dehydrogenase A (LDHA). Moreover, SR18292 (an inhibitor of PGC1α) induced oxidative phosphorylation dysfunction of DOKs and declined DOK xenograft tumor progression. Thus, our work suggests that PGC1α plays a crucial role in cell proliferation by reprograming energy metabolism and interfering with energy metabolism, acting as a potential therapeutic target for OPMDs.
Humans ; Carcinoma, Squamous Cell/metabolism* ; Cell Proliferation ; DNA, Mitochondrial ; Energy Metabolism ; Glucose ; Mouth Neoplasms/metabolism* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism* ; Phosphatidylinositol 3-Kinases ; Reactive Oxygen Species

Interferon-γ (IFN-γ), a key effector molecule in anti-tumor immune response, has been well documented to correlate with the intratumoral infiltration of immune cells. Of interest, however, a high level of IFN-γ has been reported in salivary adenoid cystic carcinoma (SACC), which is actually a type of immunologically cold cancer with few infiltrated immune cells. Investigating the functional significance of IFN-γ in SACC would help to explain such a paradoxical phenomenon. In the present study, we revealed that, compared to oral squamous cell carcinoma cells (a type of immunologically hot cancer), SACC cells were less sensitive to the growth-inhibition effect of IFN-γ. Moreover, the migration and invasion abilities of SACC cells were obviously enhanced upon IFN-γ treatment. In addition, our results revealed that exposure to IFN-γ significantly up-regulated the level of programmed death ligand 1 (PD-L1) on SACC cell-derived small extracellular vesicles (sEVs), which subsequently induced the apoptosis of CD8⁺ T cells through antagonizing PD-1. Importantly, it was also found that SACC patients with higher levels of plasma IFN-γ also had higher levels of circulating sEVs that carried PD-L1 on their surface. Our study unveils a mechanism that IFN-γ induces immunosuppression in SACC via sEV PD-L1, which would account for the scarce immune cell infiltration and insensitivity to immunotherapy.
B7-H1 Antigen/metabolism* ; CD8-Positive T-Lymphocytes/pathology* ; Carcinoma, Adenoid Cystic/pathology* ; Carcinoma, Squamous Cell/pathology* ; Cell Line, Tumor ; Humans ; Immunosuppression Therapy ; Interferon-gamma/pharmacology* ; Mouth Neoplasms/metabolism* ; Programmed Cell Death 1 Receptor/metabolism* ; Salivary Gland Neoplasms/pathology*

Objective: To investigate the difference of programmed death-ligand 1 (PD-L1) expression between primary lesions and lymph node metastatic lesions in oral squamous cell carcinoma. Methods: Eighty-two patients diagnosed with oral squamous cell carcinoma from December 2020 to July 2021 in Department of Oral Pathology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine & College of Stomatology, Shanghai Jiao Tong University, were enrolled in this study. All the patients underwent neck dissection concurrently and had lymph node metastasis. Among them, there were 28 females and 54 males. The age range was 24-79 years old [(58.6±11.7) years old]. The expression of PD-L1 protein in primary tumors and lymph node metastatic lesions was detected by immunohistochemistry. Combined positive score (CPS) was used to evaluate the PD-L1 expression. And the difference of PD-L1 expression between primary tumors and metastatic lesions was analyzed. Results: Among 82 primary tumors, 9 cases (11%) had PD-L1 CPS<1, 31 cases (38%)≥ 1 and <20, and 42 cases (51%)≥20. Cases with perineural invasion had lower CPS (χ²=6.35, P=0.042). Among 82 matched lymph node metastatic lesions, 9 cases (11%) had CPS<1, 38 cases (46%)≥1 and<20, and 35 cases (43%)≥20. The CPS of 27 pairs (33%) of primary and metastatic lesions were discordant. The statistical results showed that the Kappa value of consistency evaluation was 0.446, indicating that the consistency of PD-L1 CPS in primary and metastatic lesions of OSCC was medium. Conclusions: There are differences in PD-L1 expression between the primary lesion of OSCC and cervical lymph node metastatic lesions, and the consistency is medium. In the routine practice, lymph node metastatic lesions should be carefully used to replace the primary lesion for PD-L1 CPS evaluating.
Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; Young Adult ; B7-H1 Antigen/metabolism* ; Biomarkers, Tumor ; Carcinoma, Squamous Cell/pathology* ; China ; Head and Neck Neoplasms ; Lymph Nodes/pathology* ; Mouth Neoplasms/pathology* ; Squamous Cell Carcinoma of Head and Neck