Schwann cell derived pleiotrophin stimulates fibroblast for proliferation and excessive collagen deposition in plexiform neurofibroma.

Neurofibromatosis type 1 associated plexiform neurofibroma (pNF) is characterized by abundant fibroblasts and dense collagen, yet the intricate interactions between tumor-origin cells (Schwann cells) and neurofibroma-associated fibroblasts (NFAFs) remain elusive. Employing single-cell RNA sequencing on human pNF samples, we generated a comprehensive transcriptomics dataset and conducted cell-cell communication analysis to unravel the molecular dynamics between Schwann cells and NFAFs. Our focus centered on the pleiotrophin (PTN)/nucleolin (NCL) axis as a pivotal ligand-receptor pair orchestrating this interaction. Validation of PTN involvement was affirmed through coculture models and recombinant protein experiments. Functional and mechanistic investigations, employing assays such as CCK8, EdU, Western Blot, ELISA, Hydroxyproline Assay, and Human phospho-kinase array, provided critical insights. We employed siRNA or inhibitors to intercept the PTN/NCL/proline-rich Akt substrate of 40 kDa (PRAS40) axis, validating the associated molecular mechanism. Our analysis highlighted a subset of Schwann cells closely linked to collagen deposition, underscoring their significance in pNF development. The PTN/NCL axis emerged as a key mediator of the Schwann cell-NFAF interaction. Furthermore, our study demonstrated that elevated PTN levels enhanced NFAF proliferation and collagen synthesis, either independently or synergistically with TGF-ß1 in vitro. Activation of the downstream molecule PRAS40 was noted in NFAFs upon PTN treatment. Crucially, by targeting NCL and PRAS40, we successfully reversed collagen synthesis within NFAFs. In conclusion, our findings unveil the pivotal role of the PTN/NCL/PRAS40 axis in driving pNF development by promoting NFAFs proliferation and function. Targeting this pathway emerges as a potential therapeutic strategy for pNF. This study contributes novel insights into the molecular mechanisms governing pNF pathogenesis.

Identification of Exosome-Related Genes Associated with Prognosis and Immune Infiltration Features in Head-Neck Squamous Cell Carcinoma.

The highly immunosuppressive nature of head-neck squamous cell cancer (HNSCC) is not fully understood. Exosomes play crucial roles in the communication between cancer and non-cancer cells, but the clinical significance of the expression of exosome-related genes (ERGs) remains unclear in HNSCC. This study aimed to establish an HNSCC-ERGs model by using mass spectrometry (MS)-based label-free quantitative proteomics in combination with the TCGA primary HNSCC dataset. The study managed to classify the HNSCC patients into two subtypes based on the expression level of prognostic ERGs, which showed significant differences in prognosis and immune infiltration. LASSO regression algorithm was used to establish a risk prediction model based on seven risky genes (PYGL, ACTN2, TSPAN15, EXT2, PLAU, ITGA5), and the high-risk group was associated with poor survival prognosis and suppressive immune status. HPRT1 and PYGL were found to be independent prognostic factors through univariate and multivariate Cox regression analyses. Immune and ssGSEA analysis revealed that HPRT1 and PYGL were significantly related to immunosuppression, immune response, and critical signaling transduction pathways in HNSCC. Immunohistochemistry results further validated the expression level, clinical value, and immunosuppressive function of HPRT1 and PYGL in HNSCC patients. In conclusion, this study established molecular subtypes and a prediction risk model based on the ERGs. Furthermore, the findings suggested that HPRT1 and PYGL might play critical roles in reshaping the tumor microenvironment.

An integrated bioinformatics analysis of the S100 in head and neck squamous cell carcinoma.

Background: This study aims to evaluate the expression profile and clinical value of the S100 family in head and neck squamous cell carcinoma (HNSCC). Methods: The expression patterns, clinicopathological features, prognostic significance and underlying correlations of S100 family genes in HNSCC were determined by bioinformatics analysis with the application of several databases, including the The Cancer Genome Atlas (TCGA) and Oncomine for differential expression gene (DEG) analysis, and a series of analysis tools, including Database for Annotation, Visualization and Integrated Discovery (DAVID), cBioPortal, Kaplan-Meier Plotter, Tumor Immune Estimation Resource (TIMER) and R software packages. Results: The results from the study demonstrated that S100A4, S100A10, and S100A13 may act as prognostic markers through overall survival (OS), disease-free survival (DFS) and tumor infiltrating immune cell enrichment and a prognostic S100 family gene model comprising S100A1-A4, S100A8, S100A10, S100A12, and S100A13 was identified. The messenger RNA (mRNA) expression of S100A1, S100A9, S100A14, and S100A7A was significantly different in HNSCC patients, together with a high mutation rate of the S100 family was found. Evaluation of clinicopathological value demonstrated the heterogeneity of S100 family functions. S100A1, S100A7, S100A8, S100A9, S100A13, S100A14, and S100A16 were observed to significantly correlate with multiple biological processes (BPs) of HNSCC, including initiation, lymph node metastasis, and lymphovascular invasion. In addition, the S100 family were significantly associated with epithelial-mesenchymal transition (EMT)-related genes. Conclusions: This present study demonstrated that S100 family members are implicated in the initiation, progression, metastasis and survival of HNSCC.

Inhibition of YAP Sensitizes the Selumetinib Treatment for Neurofibromatosis Type 1 Related Plexiform Neurofibroma.

Background: Targeted therapy of Neurofibromatosis type 1 (NF1) related plexiform neurofibroma (pNF) aiming at MEK molecule has not demonstrated a convincing result for complete disease inhibition, probably due to other signal pathways crosstalk. Our previous study revealed an increased nuclear translocation of YAP molecule in NF1 related pNF. Herein, we decided to further investigate the therapeutic relations of YAP interference during the MEK treatment against NF1 related pNF. Methods: By means of selumetinib (MEK-inhibitor), RNA-sequencing was firstly performed to identify the changes of signal pathways in pNF Schwann cells, which was probably related to YAP regulation. Nuclear-cytoplasmic fractionation and western blotting were performed to show the intracellular YAP changes under selumetinib treatment. Thirdly, a series of in vitro assays were performed including flow cytometry, CCK-8, and colony/sphere formation under dual treatment of selumetinib and verteporfin (YAP-inhibitor). In addition, Chou-Talalay method was adopted to evaluate the synergistic inhibiting effects of such drug combination. Xenograft study was also used to detect the combining effects in vivo. Results: RNA-sequencing revealed that selumetinib treatment might be associated with the undesirable activation of Hippo pathway in NF1 related pNF tumor cells, which might reduce its pharmaceutic effects. Next, nuclear-cytoplasmic fractionation and further studies demonstrated that selumetinib could promote the nuclear translocation and transcriptional activation of YAP in vitro, which might cause the aforementioned resistance to selumetinib treatment. Additionally, when combined treatments were performed based on verteporfin and selumetinib, synergistic effects were observed on cytotoxicity of NF1 related pNF tumor cells in vitro and in vivo xenograft models. Conclusion: YAP inhibition can effectively sensitize NF1 related pNF tumor cells to selumetinib. Dual targeting of YAP and MEK might be a promising therapeutic strategy for treating NF1 related pNF.

Single-Cell Transcriptome Analysis Reveals Changes of Tumor Immune Microenvironment in Oral Squamous Cell Carcinoma After Chemotherapy.

Induction chemotherapy in oral squamous cell carcinoma is a controversial issue in clinical practice. To investigate the evolution of cancer cells and tumor microenvironment (TME) response to chemotherapy in oral squamous cell carcinoma, single-cell transcriptome analysis was performed in a post-chemotherapy squamous cell carcinoma located in oral cavity. The main cell types were identified based on gene expression patterns determined using dimensionality reduction and unsupervised cell clustering. Non-negative matrix factorization clustering of the gene expression of Cancer-associated fibroblasts (CAFs) and macrophages was performed. Kyoto Encyclopedia of Genes and Genomes pathway analyses and gene set enrichment analysis were performed to explore significant functional pathways. CellPhoneDB and NicheNet were used to detect the intercellular communication between cell types. CAFs were divided into "inflammatory CAFs," "antigen-presenting CAFs" and "myofibroblastic CAFs." Three classic subgroups of tumor-associated macrophages (TAMs) were detected, namely C1Q (+), FCN1 (+) and SPP1(+) TAMs. The inflammatory cytokine expression is elevated, and several molecular pathways, such as PI3K/Akt/mTORC1, TNF-α via NFκB, TGF-ß, IL-6/JAK2/STAT3 and CXCL12/CXCR4 axis associated with epithelial-mesenchymal transition were enriched in TME. Also, CD74-MIF/COPA/APP interactions were expressed in TME of oral squamous cell carcinoma after chemotherapy. The results revealed the characteristics of TME in post-chemotherapy oral squamous cell carcinoma at single-cell transcriptome level, providing new insights and clues for further investigation.

M1-like tumor-associated macrophages cascade a mesenchymal/stem-like phenotype of oral squamous cell carcinoma via the IL6/Stat3/THBS1 feedback loop.

BACKGROUND: Tumor-associated macrophages (TAMs) have a leading position in the tumor microenvironment. Previously, we have demonstrated that M1-like TAMs activated by exosome-transferred THBS1 promote malignant migration in oral squamous cell carcinoma (OSCC). However, the functional roles and associated molecular mechanisms of the activated M1-like TAMs need to be further clarified in OSCC. METHODS: Conditioned Media (CM) were harvested from the exosome activated M1-like TAMs. We measured the malignant behaviors of OSCC under the treatment of CM from M1-like TAMs by performing colony forming assays, invasion assays, wound-healing assays, spheroid forming assays and in vivo xenograft experiments. The underlying mechanisms were investigated by RNA-seq, cytokines analysis, intracellular signaling pathway analysis, ChIP assays, bioinformatics analysis and validation. RESULTS: M1-like TAMs significantly promoted the epithelial-mesenchymal transition (EMT) process, and induced the cancer-stem like cells (CSCs) by upregulating the expression of MME and MMP14 in OSCC cells. Cytokine analysis revealed a shark increase of IL6 secretion from M1-like TAMs. Blocking IL6 in the CM from M1-like TAMs could significantly weaken its effects on the colony forming, invasion, migration, microsphere forming and xenograft forming abilities of OSCC cells. Cellular signaling assays indicated the activation of Jak/Stat3 pathway in the OSCC cells treated by the CM from M1-like TAMs. Blocking the activation of the Jak/Stat3 pathway could significantly weaken the effects of M1-like TAMs on the colony forming, invasion, migration, microsphere forming and xenograft forming abilities of OSCC cells. Further RNA-seq analysis and bioinformatics analysis revealed an increased expression of THBS1 in the OSCC cells treated by M1-like TAMs. Bioinformatics prediction and ChIP assays revealed the activation of Stat3 by CM from M1-like TAMs could directly promote the transcription of THBS1 in OSCC cells. CONCLUSIONS: We proposed that M1-like TAMs could cascade a mesenchymal/stem-like phenotype of OSCC via the IL6/Stat3/THBS1 feedback loop. A better understanding on the functional roles and associated molecular mechanisms of M1-like TAMs might facilitate the development of novel therapies for supplementing the current treatment strategies for OSCC patients.

Primary undifferentiated pleomorphic sarcoma in oral-maxillary area: retrospective study and molecular analysis.

Undifferentiated pleomorphic sarcoma (UPS) in oral-maxillary area is rarely reported. Herein, we aimed to investigate the clinical characteristics, treatment strategies, prognosis, and molecular features of the oral-maxillary UPS. In total, 10 cases with primary oral-maxillary UPS were included. The rapidly progressive UPS can easily develop to an advanced and life-threatening stage, especially concerning the complex anatomical structures and spaces in the oral-maxillary area. The final diagnosis for UPS greatly depended on histological findings and immunohistochemistry staining after the exclusion of all possible differential diagnoses. Retrospectively, the treatment strategies for the included cases still referred to those of oral squamous cell carcinoma (OSCC). Statistically, the median overall survival (OS) for all the included cases was 7.75 months (range: 5-17 months). Comparatively, 3 cases had improved OS (median survival: 17 months, range: 17-18 months) and experienced PR/SD with neoadjuvant chemotherapy (anlotinib). The molecular features were demonstrated by using whole exonic sequencing for 1 included case. Cancer driver gene detection revealed GBP4 as a candidate driver gene for the primary oral-maxillary UPS. Additionally, a missense mutation in gene PIK3CA (p.E545K) was also identified. Our findings could greatly expand the knowledge about primary oral-maxillary UPS, and provide molecular evidences to improve the therapeutic options for primary oral-maxillary UPS.

Increased nuclear translation of YAP might act as a potential therapeutic target for NF1-related plexiform neurofibroma.

Plexiform neurofibroma (pNF) in the head and neck is a characteristic feature in patients with neurofibromatosis type 1 (NF1) and is associated with significant disfigurement and psychological distress. Yes-associated protein (YAP), the key molecule involved in the Hippo pathway, is a vital transductor that regulates the proliferation and remyelinating of Schwann cells. The functional status of YAP and its feasibility as a potential target are still unknown in pNF. A total of 17 pNF tumor tissue specimens from the head and neck were collected at the Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. Histologically, diagnosis of the Schwann cell region in pNF was achieved with hematoxylin-eosin staining, positive reactions for S100, SOX10, ERK and p-ERK, and low identification of Ki67 and SMA. Compared with normal nerve tissue, obviously increased nuclear YAP was detected in the Schwann cell region of pNF, with a mean nuclear staining rate of 67.11%. Based on the shNF1 Schwann cell model (the RSC96 cell line), with upregulated expression of RAS, ERK and p-ERK, p-YAP (Ser127) and p-YAP (Ser397) were significantly decreased and total YAP and nuclear YAP were increased. According to a confocal assay, the interference of shNF1 substantially promoted YAP nuclear translocation. Compared with control Schwann cells, the YAP inhibitor CA3 might have a more sensitive effect (IC50: NC=0.96±0.04, shNF1=0.71±0.02, P<0.05) on the shNF1 Schwann cell model than the classic MEK1/2 inhibitor selumetinib (IC50: NC=14.36±0.95, shNF1=24.83±0.98, P>0.05). For in vivo inhibition, the CA3 group and the selumetinib group displayed a similar inhibition effect with no significant difference. Increased nuclear translation and the functional state of YAP implies that the YAP-Hippo pathway might play an important role in the formation and remyelination of pNF. Compared with selumetinib, the YAP inhibitor can exhibit a similar but more sensitive effect on NF1-/- Schwann cells. These observations imply that YAP as a novel or adjuvant therapy target in the treatment of pNF.

Bioinformatics analysis for the biomarkers of the tumor-infiltrating lymphocytes in head and neck squamous cell carcinoma.

BACKGROUND: Immunotherapy strategies are successful in only a subset of patients with advanced head and neck squamous cell carcinoma (HNSCC). The roles of tumor-infiltrating lymphocytes (TILs) in HNSCC are less clear. Herein, we present a preliminary study to identify the underlying heterogeneity and correlations among TILs in HNSCC by bioinformatics analysis of TIL-related biomarkers. METHODS: The expression patterns, clinicopathological values and underlying correlations for the TILs related genes were analyzed based on the TCGA primary HNSCC cohort. The prognostic significance for the involved genes in the HNSCC patients was evaluated by using an online tool, Kaplan-Meier Plotter. Bioinformatics prediction for the co-expression, physical interactions, pathway, and genetic interactions of the involved genes was performed by using GeneMANIA. RESULTS: The expression of programmed death-ligand 1 (PD-L1) was significantly correlated with the expression of CD4 (P<0.01), CD8 (P<0.01), and CD20 (P=0.011), but not CD56 (P=0.065) based on the TCGA primary HNSCC cohort. For the expression of programmed cell death 1 (PD-1), a significant correlation was also observed with the expression of CD4 (P<0.01), CD8 (P<0.01), and CD20 (P<0.01), but not CD56 (P=0.861). For the clinically significant evaluation, variable roles for the biomarkers were compared and demonstrated. Increased expression of CD8 (P=0.029), CD20 (P<0.01), or PD-1 (P=0.0084) significantly correlated with improved overall survival (OS), and increased CD56 expression indicated obviously decreased OS for HNSCC patients (P=0.0033). Significantly positive correlations between human papilloma virus (HPV) status and the expression of CD8 (P<0.01), CD20 (P<0.01) and PD-1 (P<0.01) were demonstrated and a significantly negative correlation between HPV status and CD56 expression was also demonstrated (P<0.01). In addition, IL2RB was determined to be a hub factor that regulates the infiltration of TILs and the expression of PD-1/PD-L1 in HNSCC. CONCLUSIONS: A systematic evaluation for the TILs status can be informative to predict prognosis and direct the immunotherapy for HNSCC patients, but further investigations are still needed.

Genetic landscape of common venous malformations in the head and neck.

OBJECTIVE: Common venous malformations (VMs) are a frequent sporadic subtype of vascular malformations. Given the TEK and PIK3CA mutations identified, this study aims to investigate the genetic landscape of VMs in the head and neck. METHODS: Patients from published sequencing studies related to common VMs were reviewed. Detailed data regarding clinical characteristics, sequencing strategies, and mutation frequency were synthesized. Lesion distribution of common VMs in the head and neck were further retrospectively analyzed by the pathologic database of the Department of Oral and Maxillofacial-Head and Neck Oncology, Shanghai Ninth People's Hospital. For the frequently affected sites in the head and neck, patients were selected for targeted sequencing with a designed vascular malformation-related gene panel or whole exome sequencing. Detected variants were analyzed by classical bioinformatic algorithms (SIFT23, PolyPhen-2 HDIV, LRT, MutationTaster, Mutation Assessor, and GERP++). To confirm the expression pattern of particular candidate gene, specimens were examined histochemically. Gene ontology enrichment analysis and a protein-protein interaction network were also constructed. RESULTS: Three hundred patients from eight sequencing studies related to common VMs were reviewed. The total prevalence rates of TEK and PIK3CA mutations were 41.3% and 26.7%, respectively. The most frequent TEK/PIK3CA mutations were TEK-L914F/PIK3CA-H1047R. TEK/PIK3CA mutations existed in 70.3% and 2.7% of VMs in the head and neck. In retrospective data from 649 patients carrying cervicofacial VMs at Shanghai Ninth Hospital, the most frequent sites were the maxillofacial region (lips, cheek, parotid-masseteric region, submandibular region) and the oral and oropharyngeal region (buccal mucosa, tongue). Targeted sequencing for 14 frequent lesions detected TEK variants in three patients (21.4%), but no PIK3CA mutations. On whole exome sequencing of two patients without TEK/PIK3CA mutations, CDH11 was the only shared deleteriously mutated gene. Bioinformatic analyses of CDH11 implied that genes involved in cellular adhesion and junctions formed a significant portion. CONCLUSIONS: Common VMs of the head and neck have a unique genetic landscape. Novel CDH11 and TEK variants imply that pathogenesis is mediated by the regulatory relationship between endothelial cells and extracellular components.

Biological responses of human bone mesenchymal stem cells to Ti and TiZr implant materials.

BACKGROUND: Titanium-zirconium alloy (TiZr1317) is a new material used for biological implants. There are several studies on the effects of TiZr implants on the biological characteristics of human bone mesenchymal stem cells (hBMSCs). PURPOSE: The purpose of this study was to investigate the biological responses of hBMSCs to implant holes affected by the physicochemical properties of oral implants (TiSLA , TiSLActive , TiZrSLA , and TiZrSLActive ). MATERIALS AND METHODS: Grade 4 Ti and TiZr (13-17% Zr) substrates were modified by sand-blasted large-grit acid-etched (SLA) or hydrophilic sand-blasted large-grit acid-etched (SLActive), resulting in four types of surface with complex microstructures corresponding to the commercially-available implants SLA, RoxolidSLA, SLActive, and RoxolidSLActive (Institute Straumann AG, Basel, Switzerland). Physicochemical properties were detected and the biological responses of hBMSCs were observed. RESULTS: Surface morphology characterization by scanning electron microscopy and atomic force microscopy revealed differences between the four groups. SLActive had higher surface energy/wettability than SLA, indicating that increased surface energy/wettability can promote the absorption of osteogenic proteins and enhance osseointegration. hBMSCs seeded on SLActive substrates exhibited better performance in terms of cell attachment, proliferation and osteoblastic differentiation than cells seeded on SLA. CONCLUSION: Because of their more suitable physicochemical properties, TiSLActive and TiZrSLActive materials demonstrated more pronounced effects on the biological responses of hBMSCs compared with TiSLA and TiZrSLA .