The predictive value of free thyroxine combined with tubular atrophy/interstitial fibrosis for poor prognosis in patients with IgA nephropathy.

Background: IgA nephropathy (IgAN), the most common type of glomerulonephritis, has great individual differences in prognosis. Many studies showed the relationship between thyroid hormones and chronic kidney disease. However, the relationship between free thyroxine (FT4), as a thyroid hormone, and IgAN is still unclear. This study aimed to evaluate the impact of FT4 on IgAN prognosis. Methods: This retrospective study involved 223 patients with biopsy-proven IgAN. The renal composite outcomes were defined as: (1) ESRD, defined as eGFR < 15 ml/(min·1.73 m2) or initiation of renal replacement therapy (hemodialysis, peritoneal dialysis, renal transplantation); (2) serum creatinine doubled from baseline; (3) eGFR decreased by more than 50% from baseline. The predictive value was determined by the area under the curve (AUC). Kaplan-Meier and Cox proportional hazards analyses assessed renal progression and prognosis. Results: After 38 (26-54) months of follow-up, 23 patients (10.3%) experienced renal composite outcomes. Kaplan-Meier survival curve analysis showed that the renal survival rate of the IgAN patients with FT4<15.18pmol/L was lower than that with FT4≥15.18pmol/L (P < 0. 001). Multivariate Cox regression model analysis showed that FT4 was a protective factor for poor prognosis of IgAN patients, whether as a continuous variable or a categorical variable (HR 0.68, 95%CI 0.51-0.90, P =0.007; HR 0.04, 95%CI 0.01-0.20, P <0.001). ROC curve analysis showed that FT4 combined with t score had a high predictive value for poor prognosis of IgAN patients (AUC=0.881, P<0.001). Conclusion: FT4 was a protective factor for IgAN. In addition, FT4 combined with tubular atrophy/interstitial fibrosis had a high predictive value for poor prognosis of IgAN.

Lipid droplets-related Perilipin-3: potential immune checkpoint and oncogene in oral squamous cell carcinoma.

BACKGROUND: Lipid droplets (LDs) as major lipid storage organelles are recently reported to be innate immune hubs. Perilipin-3 (PLIN3) is indispensable for the formation and accumulation of LDs. Since cancer patients show dysregulated lipid metabolism, we aimed to elaborate the role of LDs-related PLIN3 in oral squamous cell carcinoma (OSCC). METHODS: PLIN3 expression patterns (n = 87), its immune-related landscape (n = 74) and association with B7-H2 (n = 51) were assessed by immunohistochemistry and flow cytometry. Real-time PCR, Western blot, Oil Red O assay, immunofluorescence, migration assay, spheroid-forming assay and flow cytometry were performed for function analysis. RESULTS: Spotted LDs-like PLIN3 staining was dominantly enriched in tumor cells than other cell types. PLIN3high tumor showed high proliferation index with metastasis potential, accompanied with less CD3+CD8+ T cells in peripheral blood and in situ tissue, conferring immunosuppressive microenvironment and shorter postoperative survival. Consistently, PLIN3 knockdown in tumor cells not only reduced LD deposits and tumor migration, but benefited for CD8+ T cells activation in co-culture system with decreased B7-H2. An OSCC subpopulation harbored PLIN3highB7-H2high tumor showed more T cells exhaustion, rendering higher risk of cancer-related death (95% CI 1.285-6.851). CONCLUSIONS: LDs marker PLIN3 may be a novel immunotherapeutic target in OSCC.

COMP promotes pancreatic fibrosis by activating pancreatic stellate cells through CD36-ERK/AKT signaling pathways.

BACKGROUND: Pancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP) and pancreatic stellate cells (PSCs) are the key cells of fibrosis. As an extracellular matrix (ECM) glycoprotein, cartilage oligomeric matrix protein (COMP) is critical for collagen assembly and ECM stability and recent studies showed that COMP exert promoting fibrosis effect in the skin, lungs and liver. However, the role of COMP in activation of PSCs and pancreatic fibrosis remain unclear. We aimed to investigate the role and specific mechanisms of COMP in regulating the profibrotic phenotype of PSCs and pancreatic fibrosis. METHODS: ELISA method was used to determine serum COMP in patients with CP. Mice model of CP was established by repeated intraperitoneal injection of cerulein and pancreatic fibrosis was evaluated by Hematoxylin-Eosin staining (H&E) and Sirius red staining. Immunohistochemical staining was used to detect the expression changes of COMP and fibrosis marker such as α-SMA and Fibronectin in pancreatic tissue of mice. Cell Counting Kit-8, Wound Healing and Transwell assessed the proliferation and migration of human pancreatic stellate cells (HPSCs). Western blotting, qRT-PCR and immunofluorescence staining were performed to detect the expression of fibrosis marker, AKT and MAPK family proteins in HPSCs. RNA-seq omics analysis as well as small interfering RNA of COMP, recombinant human COMP (rCOMP), MEK inhibitors and PI3K inhibitors were used to study the effect and mechanism of COMP on activation of HPSCs. RESULTS: ELISA showed that the expression of COMP significantly increased in the serum of CP patients. H&E and Sirius red staining analysis showed that there was a large amount of collagen deposition in the mice in the CP model group and high expression of COMP, α-SMA, Fibronectin and Vimentin were observed in fibrotic tissues. TGF-ß1 stimulates the activation of HPSCs and increases the expression of COMP. Knockdown of COMP inhibited proliferation and migration of HPSCs. Further, RNA-seq omics analysis and validation experiments in vitro showed that rCOMP could significantly promote the proliferation and activation of HPSCs, which may be due to promoting the phosphorylation of ERK and AKT through membrane protein receptor CD36. rCOMP simultaneously increased the expression of α-SMA, Fibronectin and Collagen I in HPSCs. CONCLUSION: In conclusion, this study showed that COMP was up-regulated in CP fibrotic tissues and COMP induced the activation, proliferation and migration of PSCs through the CD36-ERK/AKT signaling pathway. COMP may be a potential therapeutic candidate for the treatment of CP. Interfering with the expression of COMP or the communication between COMP and CD36 on PSCs may be the next direction for therapeutic research.

Immune checkpoint CD161/LLT1-associated immunological landscape and diagnostic value in oral squamous cell carcinoma.

An active host adaptive response is characterized by the existence of programmed cell death protein 1 (PD-1)+ /IFN-γ+ cytotoxic T cells and IFN-γ-induced PD-L1+ tumor cells (TCs), which predicts high response rate to anti-PD-1/L1 therapy. Recently, CD161 and its ligand LLT1 (CLEC2D) have been identified as an emerging checkpoint for immunotherapy. Clarifying its heterogeneous clinical expression pattern and its immune landscape is a prerequisite for maximizing the response rate of CD161 blockade therapy in a specific population of oral squamous cell carcinoma (OSCC) patients. Here, we investigated the expression pattern of CD161/LLT1 and its association with major immunocytes (T cells, B cells, NK cells, and macrophages) by multiplex immunofluorescence, immunohistochemistry, and flow cytometry in 109 OSCC tissues and 102 peripheral blood samples. TCs showed higher LLT1 levels than tumor infiltrating lymphocytes (TILs), whereas CD161 was highly expressed in CD8+ T cells at the tumor front, which was decreased in paracancerous tissue. High expression of TC-derived LLT1 (LLT1TC ) conferred poor clinical outcomes, whereas higher CD161+ and LLT1+ TILs were associated with better prognosis. Meanwhile, patients with high LLT1TC showed a decreased ratio of CD8+ /Foxp3+ T cells in situ, but CD161+ TILs correlated with more peripheral CD3+ T cells. Interestingly, treatment of OSCC patients with nivolumab (anti-PD-1) could restore tumoral CD161/LLT1 signal. Furthermore, an OSCC subgroup characterized by high LLT1+ TCs and low CD161+ CD8+ T cells showed fewer peripheral T cells and a higher risk of lymph node metastasis, leading to a shorter 5-year survival time (29%). More LLT1TC at the invasive front was another risk characteristic of exhausted T cells. In conclusion, in view of this heterogeneity, the LLT1/CD161 distribution pattern should be determined before CD161-based immunotherapy.

IL-36 antagonism blunts the proliferation and migration of oral squamous cell carcinoma cells.

IL-36 is known to mediate inflammation and fibrosis. Nevertheless, IL-36 signalling axis has also been implicated in cancer, although understanding of exact contribution of IL-36 to cancer progression is very limited, partly due to existence of multiple IL-36 ligands with agonistic and antagonistic function. Here we explored the role of IL-36 in oral squamous cell carcinoma (OSCC). Firstly, we analyzed expression of IL-36 ligands and receptor and found that the expression of IL-36γ was significantly higher in head and neck cancer (HNSCC) than that of normal tissues, and that the high expression of IL-36γ predicted poor clinical outcomes. Secondly, we investigated the direct effect of IL-36γ on OSCC cells and found that IL-36γ stimulated proliferation of OSCC cells with high expression of IL-36R expression. Interestingly, IL-36γ also promoted migration of OSCC cells with low to high IL-36R expression. Critically, both proliferation and migration of OSCC cells induced by IL-36γ were abrogated by anti-IL-36R mAb. Fittingly, RNA sequence analysis revealed that IL-36γ regulated genes involved in cell cycle and cell division. In summary, our results showed that IL-36γ can be a tumor-promoting factor, and targeting of IL-36R signalling may be a beneficial targeted therapy for patients with abnormal IL-36 signalling.

ICOSLG-associated immunological landscape and diagnostic value in oral squamous cell carcinoma: a prospective cohort study.

Background: We previously reported that stroma cells regulate constitutive and inductive PD-L1 (B7-H1) expression and immune escape of oral squamous cell carcinoma. ICOSLG (B7-H2), belongs to the B7 protein family, also participates in regulating T cells activation for tissue homeostasis via binding to ICOS and inducing ICOS+ T cell differentiation as well as stimulate B-cell activation, while it appears to be abnormally expressed during carcinogenesis. Clarifying its heterogeneous clinical expression pattern and its immune landscape is a prerequisite for the maximum response rate of ICOSLG-based immunotherapy in a specific population. Methods: This retrospective study included OSCC tissue samples (n = 105) to analyze the spatial distribution of ICOSLG. Preoperative peripheral blood samples (n = 104) and independent tissue samples (n = 10) of OSCC were collected to analyze the changes of immunocytes (T cells, B cells, NK cells and macrophages) according to ICOSLG level in different cellular contents. Results: ICOSLG is ubiquitous in tumor cells (TCs), cancer-associated fibroblasts (CAFs) and tumor infiltrating lymphocytes (TILs). Patients with high ICOSLGTCs or TILs showed high TNM stage and lymph node metastasis, which predicted a decreased overall or metastasis-free survival. This sub-cohort was featured with diminished CD4+ T cells and increased Foxp3+ cells in invasive Frontier in situ, and increased absolute numbers of CD3+CD4+ and CD8+ T cells in peripheral blood. ICOSLG also positively correlated with other immune checkpoint molecules (PD-L1, CSF1R, CTLA4, IDO1, IL10, PD1). Conclusion: Tumor cell-derived ICOSLG could be an efficient marker of OSCC patient stratification for precision immunotherapy.

Prognostic impact of muscle invasion in buccal mucosa squamous cell carcinoma.

OBJECTIVE: The objective of the study was to assess the prognostic value of muscle invasion (MI), a key histopathological feature of tumor aggressiveness, and construct a superior prognostic prediction model combining the current TNM staging system. MATERIALS AND METHODS: MI was analyzed in the whole-slide images from a total of 301 patients with primary buccal mucosa squamous cell carcinoma (BMSCC). Survival times of patients with/without MI were evaluated by Kaplan-Meier analysis. MI was further combined with the TNM staging system to explore its predictive value for prognosis. Moreover, 204 cases of head and neck carcinoma from the TCGA database were included. RESULTS: MI positive rate reached to 76% (229/301) in patients with BMSCC. MI was associated with poor overall survival (p = 0.012) and disease-free survival (p = 0.022). The novel system (TNM staging combined with MI) revealed strong predictive performance, with the largest area under the curve (OS: p < 0.001, DFS: p < 0.004). MI and the established classification system were also had good predictive ability in the TCGA cohort. CONCLUSIONS: MI is an independent predictor of poor prognosis of BMSCC. The inclusion of MI in prediction system can augment the risk stratification of patients with oral squamous cell carcinoma and may assist in the clinical decision-making process.

Development and validation of a prognostic nomogram for predicting cancer-specific survival in advanced endometrial carcinoma after surgery: a retrospective analysis of the SEER Database.

OBJECTIVE: We aimed to construct and validate a prognostic nomogram to predict cancer-specific survival (CSS) after surgery in patients with advanced endometrial carcinoma (EC). DESIGN: Retrospective cohort study. SETTING AND PARTICIPANTS: The Surveillance, Epidemiology, and End Results (SEER) Database contains cancer incidence and survival data from population-based cancer registries in the USA. A total of 5445 patients from the SEER Database diagnosed with advanced EC between 2004 and 2015 were included and randomised 7:3 into a training cohort (n=3812) and a validation cohort (n=1633). OUTCOME MEASURE: CSS. RESULTS: The nomograms for CSS included 10 variables (positive regional nodes, age, tumour size, International Federation of Gynecology and Obstetrics (FIGO) stage, grade, ethnicity, income, radiation, chemotherapy and historical stage) based on the forward stepwise regression results. They revealed discrimination and calibration using the concordance index (C-index) and area under the time-dependent receiver operating characteristic curve, with a C-index value of 0.7324 (95% CI=0.7181 to 0.7468) and 0.7511 (95% CI=0.7301 to 0.7722) for the training and validation cohorts, respectively. Using calibration plots, a high degree of conformance was shown between the predicted and observed results. Additionally, a comparison of the nomogram and FIGO staging based on changes in the C-index, net reclassification index and integrated discrimination improvement demonstrated that the nomogram had better accuracy and efficacy. CONCLUSIONS: We successfully constructed an accurate and effective nomogram to predict CSS in patients with advanced EC, which may help clinicians determine optimal individualised treatment strategies for patients with advanced EC. The predictive performance of the nomogram was evaluated thoroughly, but only internally. Therefore, further validation using different data sources is warranted in future related studies.

Oral tongue squamous cell carcinoma diagnosis from tissue metabolic profiling.

OBJECTIVE: Disease metabolomes have been studied for identifying diagnostic and predictive biomarkers of pathology. Oral tongue squamous cell carcinoma (OTSCC) is one of the most prevalent subtypes of head and neck squamous cell carcinoma, yet the profile and diagnostic value of its tissue metabolite are unclear. SUBJECTS AND METHODS: Tumor tissue samples and matched normal mucosal tissue samples were collected from 40 OTSCC patients. Untargeted metabolic analysis by liquid chromatography-mass spectrometry/mass spectrometry, in positive and negative ion modes, was used to identify dysregulated metabolites in OTSCC. Further, utilizing LASSO regression and receiver operating characteristic analyses, biomarker metabolites were selected and validated, and a diagnostic model was established. RESULTS: One hundred and ninety metabolites were detected. The OTSCC had a total of 89 dysregulated metabolites, of which 73 were elevated. A diagnostic panel of nine metabolites was subsequently created that could accurately identify OTSCC with 100% sensitivity of 100%, 100% specificity and an AUC of 1.00. CONCLUSIONS: This study identified distinct metabolic characteristics of OTSCC and established a diagnostic model. Our research also contributes to the investigation of the pathogenesis of OTSCC.

Multiperspective quantitative tumor-stroma ratio reveals histological areas associated with poor outcomes in oral squamous cell carcinoma.

AIMS: Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological characteristics, and the individual histological characteristics of the tumors are poorly understood. Therefore, calculating the proportion of tumor cells in different regions that allow assessment of the prognostic outcomes for OSCC patients would be of great clinical significance. METHODS AND RESULTS: We established an open-source software-based analytic pipeline that defines the inner tumor and invasive tumor front (ITF) in pancytokeratin-stained whole slide images (WSIs) and quantifies the tumor-stroma ratio (TSR) within the two regions. We applied this method to 114 patients with OSCC and predicted patient prognosis by the TSR. The proportion of tumor area in the inner tumor was generally higher than that in the ITF (p < 0.0001). TSR was an independent prognostic factor for overall survival (OS) (p = 0.016), disease-free survival (DFS) (p = 0.026), and relapse-free survival (RFS) (p = 0.037) in inner tumor, and TSR was an independent prognostic factor for OS (p = 0.00052), DFS (p = 0.035), and metastasis-free survival (MFS) (p = 0.038) in the ITF. Tumor-low status was associated with poorer prognosis. There was a significant correlation between the TSR and perineural invasion (PNI) in the inner tumor (p = 0.009). CONCLUSIONS: The histopathological characteristics of different regions of OSCC may be used to develop the potential prognostic markers. The TSR of the inner tumor is more targeted in predicting prognosis and accurately assesses the risk of PNI+.

Effects of cancer-associated fibroblasts deletion using HSVtk suicide system in OSCC.

OBJECTIVE: To evaluate the biological characteristics of oral cancer cells co-cultured with cancer-associated fibroblasts (CAFs)-HSVtk and to assess the reliability of the CAFs-HSVtk suicide system in a co-culture model. METHODS: CAFs were lentivirus-transfected with PCDH-HSVtk. Ganciclovir (GCV) was added and the survival rates of the CAFs-HSVtk were measured. In parallel with the selective elimination of CAFs, comparison was made of the effects of CAF-HSVtk on tumor cell proliferation/migration in a CAFs-tumor co-cultural system. Cell death of co-cultured oral cancer cells was evaluated by flow cytometry. RESULTS: Q-PCR analysis showed that the expression of HSVtk in the CAFs-HSVtk group was significantly higher than in the control group (p < 0.01). The survival rates of CAFs-HSVtk with GCV were significantly reduced (p < 0.01). Following selective depletion of CAFs-HSVtk, the growth and migration rates of oral cancer cells co-cultured with CAFs-HSVtk were reduced in a mixture ratio of 1:2 (p < 0.01, p < 0.01). CONCLUSIONS: Enhanced proliferation and migration rates of oral cancer cells in co-culture were seriously impaired after deleting CAFs using the HSVtk suicide system, while oral tumor cell death was not affected. Therefore, CAFs-HSVtk can be utilized as a valid model for CAF signature identification.

Tcf21 Alleviates Pancreatic Fibrosis by Regulating the Epithelial-Mesenchymal Transformation of Pancreatic Stellate Cells.

BACKGROUND AND AIMS: The activation of pancreatic stellate cells (PSCs) plays a key role in the occurrence and development of chronic pancreatitis (CP) and pancreatic fibrosis, which is related to the process of epithelial-mesenchymal transition (EMT). This study was designed to investigate the effect and mechanism of Tcf21 (one of tumor suppressor genes) on pancreatic inflammation and fibrosis in vivo and in vitro. METHODS: C57BL/6 male mice were intraperitoneally injected with caerulein for 6 weeks to establish CP animal model. Fixed pancreatic tissue paraffin-embedded sections were used for immunohistochemistry staining of Tcf21, fibrosis-related markers (α-SMA), interstitial markers (Vimentin) and epithelial markers (E-cadherin). Western blotting and qRT-PCR assay were performed to analyze the change of expression of the above markers after stimulation of TGF-ß1 or overexpressed Tcf21 lentivirus transfection in human pancreatic stellate cells (HPSCs). RESULTS: The pancreatic expression of α-SMA and Vimentin of CP mice significantly increased, while the expression of Tcf21 and E-cadherin significantly decreased. TGF-ß1 could promote activation and EMT process of HPSCs, and inhibited the expression of Tcf21. Overexpression of Tcf21 could significantly down-regulate the expression of α-SMA, Fibronectin and Vimentin, and up-regulated the expression of ZO-1 of HPSCs. Cell Counting Kit-8 assay and scratch wound-healing assay results showed that overexpression of Tcf21 could significantly inhibit the cell migration and proliferation of HPSCs. CONCLUSIONS: Overexpression of Tcf21 could significantly alleviate the activation, proliferation, migration of PSCs by regulating the EMT process. Tcf21 had a potential prospect of a new target for CP therapy.

IL-33/ST2 signaling promotes constitutive and inductive PD-L1 expression and immune escape in oral squamous cell carcinoma.

BACKGROUND: Loss-of-function of PD-L1 induces therapy resistance of anti-PD-1/L1 therapy, and the complex regulatory mechanisms are not completely understood. We previously reported that stroma-derived interleukin-33 (IL-33) promoted the progression of oral squamous cell carcinoma (OSCC). We here focused on the immune-regulation role of IL-33 and its receptor ST2 signaling in PD-L1-positive OSCC patients. METHODS: Activated T cells in in situ and peripheral blood were analyzed by IL-33/ST3 expression. Knockdown or overexpression of ST2 combined with IL-33/IFN-γ stimulation were performed to determine PD-L1 expression and PD-L1-dependent immune escape in OSCC/human T cells co-culture system, and OSCC orthotopic model based on humanized mouse with immune reconstitution and C57BL/6 mice models. RESULTS: High IL-33/ST2 correlated with less activated T cells infiltration in situ and peripheral blood. Knockdown of ST2 down-regulated constitutive PD-L1 expression, whereas ST2 also promoted IL-33-induced PD-L1 Mechanistically, IL-33/ST2 activated JAK2/STAT3 pathway to directly promoted PD-L1 expression, and also activated MyD88/NF-κB signaling to up-regulate IFN-γ receptor (IFN-γR), which indirectly strengthen IFN-γ-induced PD-L1. Furthermore, ST2 is required for PD-L1-mediated immune tolerance in vitro and in vivo. ST2high OSCC patients have more PD-L1 and IFN-γR level in situ. CONCLUSIONS: IL-33/ST2 signaling enhanced PD-L1-mediated immune escape, ST2high OSCC patients might benefit from anti-PD-1/L1 therapy.

Halofuginone inhibits tumor migration and invasion by affecting cancer-associated fibroblasts in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor in the oral and maxillofacial regions, with a high rate of metastasis. Cancer-associated fibroblasts (CAFs) play critical roles in tumor growth, metastasis and invasion, making them attractive therapeutic targets for cancer treatment. As an old anti-coccidiosis drug for poultry, Halofuginone (HF) has also been reported to possess anti-fibrosis and anti-cancer activities in the recent decades. However, whether it works by targeting CAFs in OSCC, and the mechanisms involved remain unclear. In the present study, we observed HF dose-dependently inhibits OSCC-derived CAF viability and proliferation. Meanwhile, HF decreased the expressions of α-SMA, FSP-1 and PDGFRß, markers of the malignant phenotype of CAFs, both at mRNA and protein levels. Furthermore, functional studies demonstrated that HF dramatically attenuates the promotion effect of CAFs on OSCC cell migration and invasion. Mechanistically, the inhibition of MMP2 secretion and the upstream TGF-ß/Smad2/3 signaling pathway played an important role in these processes. In the orthotopic transplanted tongue carcinoma in mice model, we confirmed that HF administration inhibited tumor growth and lymph node metastasis (LNM) with reduced CAF population, MMP2 expression and collagen deposition in tumor. Altogether, these results indicate that HF can inhibit the migration and invasion of OSCC by targeting CAFs, which will provide new ideas for the treatment of OSCC.

Identification of Biological Functions and Prognostic Value of NNMT in Oral Squamous Cell Carcinoma.

BACKGROUND: Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) to generate 1-methyl nicotinamide (MNAM). Although previous studies have shown that NNMT is frequently dysregulated to promote the onset and progression of many malignancies, its expression profile, prognostic value and function in oral squamous cell carcinoma (OSCC) are still unknown. METHODS: We used untargeted metabolomics based on mass spectrometry to analyze potential metabolite differences between tumors and matched adjacent normal tissues in 40 OSCC patients. Immunohistochemistry (IHC) was used to analyze the NNMT expression profile in OSCC, and the diagnostic and prognostic values of NNMT were evaluated. Next, qPCR and Western blot were used to compare the expression of NNMT in five OSCC cell lines. Stable transfected cell lines were constructed, and functional experiments were carried out to elucidate the effects of NNMT on the proliferation and migration of OSCC cells. Finally, gene set enrichment analysis (GSEA) was performed using The Cancer Genome Atlas (TCGA) data to investigate the potential functional mechanisms of NNMT in OSCC. RESULTS: We found that the nicotinamide metabolic pathway was abnormally activated in OSCC tumor tissues compared with normal tissues. NNMT was expressed ubiquitously in tumor cells (TCs) and fibroblast-like cells (FLCs) but was absent in tumor-infiltrating lymphocytes (TILs). OSCC patients with highly expressed NNMT in TCs had higher risk of lymph node metastasis and showed a worse pattern of invasion (POI). Moreover, patients with highly expressed NNMT were also susceptible to postoperative recurrence. Highly expressed NNMT can independently predict shorter disease-free survival and recurrence-free survival. Functionally, we demonstrated that the ectopic expression of NNMT promoted OSCC tumor cell proliferation and migration in vitro. Conversely, silencing exerted significantly opposite effects in vitro. In addition, GSEA showed that highly expressed NNMT was mainly enriched in the epithelial-mesenchymal transformation (EMT) pathway, which displayed a significant positive correlation with the six classic EMT markers. CONCLUSIONS: Our study uncovered that NNMT may be a critical regulator of EMT in OSCC and may serve as a prognostic biomarker for OSCC patients. These findings might provide novel insights for future research in NNMT-targeted OSCC metastasis and recurrence therapy.

Impact of preanalytical freezing delay time on the stability of metabolites in oral squamous cell carcinoma tissue samples.

INTRODUCTION: Metabolite stability is critical for tissue metabolomics. However, changes in metabolites in tissues over time from the operating room to the laboratory remain underexplored. OBJECTIVES: In this study, we evaluated the effect of postoperative freezing delay time on the stability of metabolites in normal and oral squamous cell carcinoma (OSCC) tissues. METHODS: Tumor and paired normal tissues from five OSCC patients were collected after surgical resection, and samples was sequentially quenched in liquid nitrogen at 30, 40, 50, 60, 70, 80, 90 and 120 min (80 samples). Untargeted metabolic analysis by liquid chromatography-mass spectrometry/mass spectrometry in positive and negative ion modes was used to identify metabolic changes associated with delayed freezing time. The trends of metabolite changes at 30-120 and 30-60 min of delayed freezing were analyzed. RESULTS: 190 metabolites in 36 chemical classes were detected. After delayed freezing for 120 min, approximately 20% of the metabolites changed significantly in normal and tumor tissues, and differences in the metabolites were found in normal and tumor tissues. After a delay of 60 min, 29 metabolites had changed significantly in normal tissues, and 84 metabolites had changed significantly in tumor tissues. In addition, we constructed three tissue freezing schemes based on the observed variation trends in the metabolites. CONCLUSION: Delayed freezing of tissue samples has a certain impact on the stability of metabolites. For metabolites with significant changes, we suggest that the freezing time of tissues be reasonably selected according to the freezing schemes and the actual clinical situation.

OXTRHigh stroma fibroblasts control the invasion pattern of oral squamous cell carcinoma via ERK5 signaling.

The Pattern Of Invasion (POI) of tumor cells into adjacent normal tissues clinically predicts postoperative tumor metastasis/recurrence of early oral squamous cell carcinoma (OSCC), but the mechanisms underlying the development of these subtypes remain unclear. Focusing on the highest score of POIs (Worst POI, WPOI) present within each tumor, we observe a disease progression-driven shift of WPOI towards the high-risk type 4/5, associated with a mesenchymal phenotype in advanced OSCC. WPOI 4-5-derived cancer-associated fibroblasts (CAFsWPOI4-5), characterized by high oxytocin receptor expression (OXTRHigh), contribute to local-regional metastasis. OXTRHigh CAFs induce a desmoplastic stroma and CCL26 is required for the invasive phenotype of CCR3+ tumors. Mechanistically, OXTR activates nuclear ERK5 transcription signaling via Gαq and CDC37 to maintain high levels of OXTR and CCL26. ERK5 ablation reprograms the pro-invasive phenotype of OXTRHigh CAFs. Therefore, targeting ERK5 signaling in OXTRHigh CAFs is a potential therapeutic strategy for OSCC patients with WPOI 4-5.

Digesting the Role of JAK-STAT and Cytokine Signaling in Oral and Gastric Cancers.

When small proteins such as cytokines bind to their associated receptors on the plasma membrane, they can activate multiple internal signaling cascades allowing information from one cell to affect another. Frequently the signaling cascade leads to a change in gene expression that can affect cell functions such as proliferation, differentiation and homeostasis. The Janus kinase-signal transducer and activator of transcription (JAK-STAT) and the tumor necrosis factor receptor (TNFR) are the pivotal mechanisms employed for such communication. When deregulated, the JAK-STAT and the TNF receptor signaling pathways can induce chronic inflammatory phenotypes by promoting more cytokine production. Furthermore, these signaling pathways can promote replication, survival and metastasis of cancer cells. This review will summarize the essentials of the JAK/STAT and TNF signaling pathways and their regulation and the molecular mechanisms that lead to the dysregulation of the JAK-STAT pathway. The consequences of dysregulation, as ascertained from founding work in haematopoietic malignancies to more recent research in solid oral-gastrointestinal cancers, will also be discussed. Finally, this review will highlight the development and future of therapeutic applications which modulate the JAK-STAT or the TNF signaling pathways in cancers.

Lipid Droplet-Related PLIN2 in CD68+ Tumor-Associated Macrophage of Oral Squamous Cell Carcinoma: Implications for Cancer Prognosis and Immunotherapy.

Background: PLIN2 (adipose differentiation-related protein) belongs to the perilipin family and is a marker of lipid droplets (LDs). Numerous types of tumor exhibit a high PLIN2 level, but its tumorigenic or tumor-suppressive role has been in debate. Recently, LDs serve as innate immune hubs and show antimicrobial capacity. We here aimed to investigate the heterogeneous functions of PLIN2 in the tumor microenvironment and immune regulation. Methods: This retrospective study included 96 oral squamous cell carcinoma (OSCC) samples and analyzed the spatial distribution of PLIN2 by immunohistochemistry (IHC) and LD level by oil red O staining. A total of 21 serial sections were obtained to analyze the relationship between PLIN2 and immune cells by IHC and immunofluorescence (IF). Single-cell sequencing was used to analyze the cell locations of PLIN2. The values of diagnosis and prognosis of PLIN2 were also evaluated. Tumor Immune Estimation Resource (TIMER), cBioPortal databases, and IHC analysis were used to investigate the relationship between PLIN2 and OSCC immune microenvironment. Results: PLIN2 was mainly expressed in tumor-infiltrating immunocytes (TIIs) of OSCC. Patients with high PLIN2 harbored more cytoplastic LDs. CD68+ tumor-associated macrophages (TAMs), instead of T cells and B cells, were found to be the main resource of PLIN2 in OSCC stroma and lung, pancreas, prostate, and testis. However, CD56+ NK cells also showed less extent of PLIN2 staining in OSCC. Moreover, patients with a high PLIN2 level in immune cells had a higher TNM stage and were susceptible to postoperative metastasis, but the escalated PLIN2 level in invasive tumor front independently predicted shorter metastasis-free survival. Furthermore, a high PLIN2 presentation in the microenvironment induced immune suppression which was featured as less infiltration of CD8+ T cells and more CD68+ TAMs and Foxp3+ Tregs, accompanied by more immune checkpoint molecules such as CSF1R, LGALS9, IL-10, CTLA-4, and TIGIT. Conclusion: CD68+ TAM-derived PLIN2 might participate in regulating immune balance of OSCC patients, which provides new insight into immune checkpoint therapy.

Identification of Metabolism-Associated Biomarkers for Early and Precise Diagnosis of Oral Squamous Cell Carcinoma.

The 5-year survival rate for oral squamous cell carcinoma (OSCC), one of the most common head and neck cancers, has not improved in the last 20 years. Poor prognosis of OSCC is the result of failure in early and precise diagnosis. Metabolic reprogramming, including the alteration of the uptake and utilisation of glucose, amino acids and lipids, is an important feature of OSCC and can be used to identify its biomarkers for early and precise diagnosis. In this review, we summarise how recent findings of rewired metabolic networks in OSCC have facilitated early and precise diagnosis of OSCC.