Cancer-associated fibroblasts contribute to the immunosuppressive landscape and influence the efficacy of the combination therapy of PD-1 inhibitors and antiangiogenic agents in hepatocellular carcinoma.

BACKGROUND: Chronic inflammation is considered the most critical predisposing factor of hepatocellular carcinoma (HCC), with inflammatory cell heterogeneity, hepatic fibrosis accumulation, and abnormal vascular proliferation as prominent features of the HCC tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) play a key role in HCC TME remodeling. Therefore, the level of abundance of CAFs may significantly affect the prognosis and outcome in HCC patients. METHODS: Unsupervised clustering was performed based on 39 genes related to CAFs in HCC identified by single-cell RNA sequencing data. Patients of bulk RNA were grouped into CAF low abundance cluster and high abundance clusters. Subsequently, prognosis, immune infiltration landscape, metabolism, and treatment response between the two clusters were investigated and validated by immunohistochemistry. RESULTS: Patients in the CAF high cluster had a higher level of inflammatory cell infiltration, a more significant immunosuppressive microenvironment, and a significantly worse prognosis than those in the low cluster. At the metabolic level, the CAF high cluster had lower levels of aerobic oxidation and higher angiogenic scores. Drug treatment response prediction indicated that the CAF high cluster could have a better response to PD-1 inhibitors and conventional chemotherapeutic agents for HCC such as anti-angiogenic drugs, whereas CAF low cluster may be more sensitive to transarterial chemoembolization treatment. CONCLUSIONS: This study not only revealed the TME characteristics of HCC with the difference in CAF abundance but also further confirmed that the combination therapy of PD-1 inhibitors and anti-angiogenic drugs may be more valuable for patients with high CAF abundance.

Efficient Editing of an Adenoviral Vector Genome with CRISPR/Cas9.

Immunotherapy based on genetic modification of T cells has played an important role in the treatment of tumors and viral infections. Moreover, adenoviral vectors engineered with improved safety due to their inability to integrate into the host genome have been key in the clinical application of T cell therapy. However, the commonly used adenoviral vector Ad5 exhibits low efficiency of infection of human T cells and the details of the intracellular trafficking pathway of adenoviral vectors in human primary T cells remains unclear. Resolution of these issues will depend on successful modification of the adenoviral vector. To this end, here we describe the successful establishment of a simple and efficient method for editing adenoviral vectors in vitro using the CRISPR-Cas9 gene editing system to target the adenoviral fiber gene.

Overexpression of Malic Enzyme 2 Indicates Pathological and Clinical Significance in Oral Squamous Cell Carcinoma.

Our study investigated the expression of malic enzyme 2 (ME2) in human oral squamous cell carcinoma (OSCC) and associated pathological and clinical pattern. We demonstrated that human OSCC tissues expressed a high level of ME2, and the overexpression of ME2 is closely connected to a high pathological grade, lymphatic metastasis, large tumor size and human papillomavirus (HPV) (P < 0.001). Similarly, high levels of ME2 expression in OSCC tissue were shown to be correlated with poor prognosis (P < 0.05). The expression of ME2 was correlated with Slug, SOX2, and aldehyde dehydrogenase-1 (ALDH1) immunoreactivity.ME2 was shown to be overexpressed in OSCC tissue and indicated a poor prognosis for OSCC. ME2 may be correlated with several immune markers.

Increased salivary microvesicles are associated with the prognosis of patients with oral squamous cell carcinoma.

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.

Inhibition of SRC family kinases facilitates anti-CTLA4 immunotherapy in head and neck squamous cell carcinoma.

The immune system plays a critical role in the establishment, development, and progression of head and neck squamous cell carcinoma (HNSCC). As treatment with single-immune checkpoint agent results in a lower response rate in patients, it is important to investigate new strategies to maintain favorable anti-tumor immune response. Herein, the combination immunotherapeutic value of CTLA4 blockade and SFKs inhibition was assessed in transgenic HNSCC mouse model. Our present work showed that tumor growth was not entirely controlled when HNSCC model mice were administered anti-CTLA4 chemotherapeutic treatment. Moreover, it was observed that Src family kinases (SFKs) were hyper-activated and lack of anti-tumor immune responses following anti-CTLA4 chemotherapeutic treatment. We hypothesized that activation of SFKs is a mechanism of anti-CTLA4 immunotherapy resistance. We, therefore, carried out combined drug therapy using anti-CTLA4 mAbs and an SFKs' inhibitor, dasatinib. As expected, dasatinib and anti-CTLA4 synergistically inhibited tumor growth in Tgfbr1/Pten 2cKO mice. Furthermore, dasatinib and anti-CTLA4 combined to reduce the number of myeloid-derived suppressor cells and Tregs, increasing the CD8+ T cell-to-Tregs ratio. We also found that combining dasatinib with anti-CTLA4 therapy significantly attenuated the expression of p-STAT3Y705 and Ki67 in tumoral environment. These results suggest that combination therapy with SFKs inhibitors may be a useful therapeutic approach to increase the efficacy of anti-CTLA4 immunotherapy in HNSCC.

Blockage of the NLRP3 inflammasome by MCC950 improves anti-tumor immune responses in head and neck squamous cell carcinoma.

The NLRP3 inflammasome is a critical innate immune pathway responsible for producing active interleukin (IL)-1ß, which is associated with tumor development and immunity. However, the mechanisms regulating the inflammatory microenvironment, tumorigenesis and tumor immunity are unclear. Herein, we show that the NLRP3 inflammasome was over-expressed in human HNSCC tissues and that the IL-1ß concentration was increased in the peripheral blood of HNSCC patients. Additionally, elevated NLRP3 inflammasome levels were detected in tumor tissues of Tgfbr1/Pten 2cKO HNSCC mice, and elevated IL-1ß levels were detected in the peripheral blood serum, spleen, draining lymph nodes and tumor tissues. Blocking NLRP3 inflammasome activation using MCC950 remarkably reduced IL-1ß production in an HNSCC mouse model and reduced the numbers of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and tumor-associated macrophages (TAMs). Moreover, inhibiting NLRP3 inflammasome activation increased the numbers of CD4+ and CD8+ T cells in HNSCC mice. Notably, the numbers of exhausted PD-1+ and Tim3+ T cells were significantly reduced. A human HNSCC tissue microarray showed that NLRP3 inflammasome expression was correlated with the expression of CD8 and CD4, the Treg marker Foxp3, the MDSC markers CD11b and CD33, and the TAM markers CD68 and CD163, PD-1 and Tim3. Overall, our results demonstrate that the NLRP3 inflammasome/IL-1ß pathway promotes tumorigenesis in HNSCC and inactivation of this pathway delays tumor growth, accompanied by decreased immunosuppressive cell accumulation and an increased number of effector T cells. Thus, inhibition of the tumor microenvironment through the NLRP3 inflammasome/IL-1ß pathway may provide a novel approach for HNSCC therapy.

TRAF6 regulates tumour metastasis through EMT and CSC phenotypes in head and neck squamous cell carcinoma.

Epithelial-mesenchymal transition (EMT) is associated with metastasis formation, generation and maintenance of cancer stem cells (CSCs). However, the regulatory mechanisms of CSCs have not been clarified. This study aims to investigate the role of TNF receptor-associated factor 6 (TRAF6) on EMT and CSC regulation in squamous cell carcinoma of head and neck (SCCHN). We found TRAF6 was overexpressed in human SCCHN tissues, and high TRAF6 expression was associated with lymphatic metastasis and resulted in poor prognosis in patients with SCCHN. In addition, elevated TRAF6 expression was observed in several HNSCC cell lines, and wound healing and transwell assay results showed that TRAF6 knockdown inhibited the migration and invasion ability of the SCCHN cells. Moreover, the expression of Vimentin, Slug and N-cadherin was down-regulated and that of E-cadherin was elevated after TRAF6 knockdown but decreased by transforming growth factor beta 1 (TGF-ß1) and CAL27 similar to mesenchymal cells formed after TGF-ß1 induction. In addition, the expression levels of CD44, ALDH1, KLF4 and SOX2 were inhibited after TRAF6 knockdown, and the anchor-dependent colony formation number and sphere number were remarkably reduced. Flow cytometry showed TRAF6 knockdown reduced ALDH1-positive cancer stem cells. We also demonstrated that TRAF6 is closely associated with EMT process and cancer stem cells using a Tgfbr1/Pten 2cKO mice SCCHN model and human SCCHN tissue microarray. Our findings indicate that TRAF6 plays a role in EMT phenotypes, the generation and maintenance of CSCs in SCCHN, suggesting that TRAF6 is a potential therapeutic target for SCCHN.

Specific blockade CD73 alters the "exhausted" phenotype of T cells in head and neck squamous cell carcinoma.

The adenosine-induced immunosuppression hampers the immune response toward tumor cells and facilitates the tumor cells to evade immunosurveillance. CD73, an ecto-5-nucleotidase, is the ectoenzyme dephosphorylating extracellular AMP to adenosine. Here, using immunocompetent transgenic head and neck squamous cell carcinoma (HNSCC) mouse model, immune profiling showed high expression of CD73 on CD4+ and CD8+ T cells was associated with an "exhausted" phenotype. Further, treatment with anti-CD73 monoclonal antibody (mAb) significantly blunted the tumor growth in the mouse model, and the blockade of CD73 reversed the "exhausted" phenotype of CD4+ and CD8+ T cells through downregulation of total expression of PD-1 and CTLA-4 on T cells. Whereas the population of CD4+ CD73hi /CD8+ CD73hi T cells expressed higher CTLA-4 and PD-1 as compared to untreated controls. In addition, the human tissue microarrays showed the expression of CD73 is upregulated on tumor infiltrating immune cells in patients with primary HNSCC. Moreover, CD73 expression is an independent prognostic factor for poor outcome in our cohort of HNSCC patients. Altogether, these findings highlight the immunoregulatory role of CD73 in the development of HNSCC and we propose that CD73 may prove to be a promising immunotherapeutic target for the treatment of HNSCC.

γ-Secretase inhibitor reduces immunosuppressive cells and enhances tumour immunity in head and neck squamous cell carcinoma.

Immune evasion is a hallmark feature of cancer, and it plays an important role in tumour initiation and progression. In addition, tumour immune evasion severely hampers the desired antitumour effect in multiple cancers. In this study, we aimed to investigate the role of the Notch pathway in immune evasion in the head and neck squamous cell carcinoma (HNSCC) microenvironment. We first demonstrated that Notch1 signaling was activated in a Tgfbr1/Pten-knockout HNSCC mouse model. Notch signaling inhibition using a γ-secretase inhibitor (GSI-IX, DAPT) decreased tumour burden in the mouse model after prophylactic treatment. In addition, flow cytometry analysis indicated that Notch signaling inhibition reduced the sub-population of myeloid-derived suppressor cells (MDSCs), tumour-associated macrophages (TAMs) and regulatory T cells (Tregs), as well as immune checkpoint molecules (PD1, CTLA4, TIM3 and LAG3), in the circulation and in the tumour. Immunohistochemistry (IHC) of human HNSCC tissues demonstrated that elevation of the Notch1 downstream target HES1 was correlated with MDSC, TAM and Treg markers and with immune checkpoint molecules. These results suggest that modulating the Notch signaling pathway may decrease MDSCs, TAMs, Tregs and immune checkpoint molecules in HNSCC.

Inhibition of JAK2/STAT3 reduces tumor-induced angiogenesis and myeloid-derived suppressor cells in head and neck cancer.

Angiogenesis is an essential event in tumor growth and metastasis, and immune system also contributes to the tumor evasion. Emerging evidences have suggested the bidirectional link between angiogenesis and immunosuppression. Myeloid-derived suppressor cell (MDSC) is a kind of immunosuppressive cells and plays an important role in this process. However, the actual regulatory mechanisms of angiogenesis and MDSCs in head and neck squamous cell carcinoma (HNSCC) were unclear. In this study, through analyzing the immunohistochemistry staining of human HNSCC tissue microarray, we found that the microvascular density (MVD) was significantly increased in HNSCC patients. We also characterized angiogenic factors p-STAT3, VEGFA, CK2, and MDSCs marker CD11b in HNSCC tissue array, and found the close expression correlation among these markers. To determine the role of JAK2/STAT3 pathway in tumor microenvironment of HNSCC, we utilized AG490 (an inhibitor of JAK2/STAT3) for further research. Results showed that inhibition of JAK2/STAT3 suppressed angiogenesis by decreasing VEGFA and HIF1-α both in vitro and vivo. Moreover, in HNSCC transgenic mouse model, inhibiting JAK2/STAT3 not only suppressed angiogenesis but also reduced MDSCs in the tumor microenvironment through suppressing VEGFA and CK2. Our findings demonstrated the close relationship between angiogenesis and MDSCs in HNSCC, and inhibition of JAK2/STAT3 could reduce tumor-induced angiogenesis and decrease MDSCs.

Overexpression of p21-activated kinase 2 is correlated with high-grade oral squamous cell carcinomas.

AIM: p21-activated kinase 2 (PAK2) is overexpressed in several tumors but the expression of PAK2 in oral squamous cell carcinomas (OSCCs) remains unclear. MATERIALS & METHODS: Immunohistochemistry was performed on human tissue microarrays containing 165 primary OSCC, 48 oral epithelial dysplasia and 43 normal oral mucosa. RESULTS: PAK2 expression was increased in primary OSCC compared with normal mucosa and significantly increased in primary OSCC grade III compared with grade I, but independent of overall survival rate. Moreover, the expression of PAK2 was statistically correlated with Lck/Yes novel tyrosine kinase (LYN), zinc finger transcription factor Slug, tumor-associated macrophage marker CD163 and LAG3. CONCLUSION: Overexpression of PAK2 in OSCC may be associated with an advanced pathology grade.

Platelet-Facilitated Photothermal Therapy of Head and Neck Squamous Cell Carcinoma.

Here, we present a platelet-facilitated photothermal tumor therapy (PLT-PTT) strategy, in which PLTs act as carriers for targeted delivery of photothermal agents to tumor tissues and enhance the PTT effect. Gold nanorods (AuNRs) were first loaded into PLTs by electroporation and the resulting AuNR-loaded PLTs (PLT-AuNRs) inherited long blood circulation and cancer targeting characteristics from PLTs and good photothermal property from AuNRs. Using a gene-knockout mouse model, we demonstrate that the administration of PLT-AuNRs and localizing laser irradiation could effectively inhibit the growth of head and neck squamous cell carcinoma (HNSCC). In addition, we found that the PTT treatment augmented PLT-AuNRs targeting to the tumor sites and in turn, improved the PTT effects in a feedback manner, demonstrating the unique self-reinforcing characteristic of PLT-PTT in cancer therapy.

Selective blockade of B7-H3 enhances antitumour immune activity by reducing immature myeloid cells in head and neck squamous cell carcinoma.

Immature myeloid cells including myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (TAMs) promote tumour growth and metastasis by facilitating tumour transformation and angiogenesis, as well as by suppressing antitumour effector immune responses. Therefore, strategies designed to reduce MDSCs and TAMs accumulation and their activities are potentially valuable therapeutic goals. In this study, we show that negative immune checkpoint molecule B7-H3 is significantly overexpressed in human head and neck squamous cell carcinoma (HNSCC) specimen as compared with normal oral mucosa. Using immunocompetent transgenic HNSCC models, we observed that targeting inhibition of B7-H3 reduced tumour size. Flow cytometry analysis revealed that targeting inhibition of B7-H3 increases antitumour immune response by decreasing immunosuppressive cells and promoting cytotoxic T cell activation in both tumour microenvironment and macroenvironment. Our study provides direct in vivo evidence for a rationale for B7-H3 blockade as a future therapeutic strategy to treat patients with HNSCC.

Blockade of adenosine A2A receptor enhances CD8+ T cells response and decreases regulatory T cells in head and neck squamous cell carcinoma.

BACKGROUND: Cancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC. METHODS: The expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model. RESULTS: Immunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4+Foxp3+ regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4+ Foxp3+ Tregs and enhanced the anti-tumor response of CD8+ T cells. CONCLUSIONS: These results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy.

Inhibition of SRC family kinases reduces myeloid-derived suppressor cells in head and neck cancer.

SRC family kinases (SFKs), a group of nonreceptor tyrosine kinases, modulate multiple cellular functions, such as cell proliferation, differentiation and metabolism. SFKs display aberrant activity in progressive stages of human cancers. However, the precise role of SFKs in the head and neck squamous cell carcinoma (HNSCC) signaling network is far from clear. In this study, we found that the inhibition of SFKs activity by dasatinib effectively reduced the tumor size and population of MDSCs in the HNSCC mouse model. Molecular analysis indicates that phosphorylation of LYN, rather than SRC, was inhibited by dasatinib treatment. Next, we analyzed LYN expression by immunostaining and found that it was overexpressed in the human HNSCC specimens. Moreover, LYN expression in stromal cells positively correlated with myeloid-derived suppressor cells (MDSCs) makers CD11b and CD33 in human HNSCC. The dual positive expression of LYN in epithelial and stromal cells (EPI+ SRT+ ) was associated with unfavorable overall survival of HNSCC patients. These findings indicate that SFKs may be a potential target for an effective immunotherapy of HNSCC by decreasing MDSCs and moreover, LYN will have an impact on such therapeutic strategy.

Expression of VISTA correlated with immunosuppression and synergized with CD8 to predict survival in human oral squamous cell carcinoma.

V-domain Ig suppressor of T cell activation (VISTA), a novel immune checkpoint regulatory molecule, suppresses T cell mediated immune responses. The aim of the present study was to profile the immunological expression, clinical significance and correlation of VISTA in human oral squamous cell carcinoma (OSCC). Human tissue microarrays, containing 165 primary OSCCs, 48 oral epithelial dysplasias and 43 normal oral mucosae, were applied to investigate the expression levels of VISTA, CD8, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), programmed death ligand 1 (PD-L1), PI3Kα p110, IL13Rα2, phospho-STAT3 at tyrosine 705 (p-STAT3) and myeloid-derived suppressor cell (MDSC) markers (CD11b and CD33) by immunohistochemistry and digital pathology analysis. The results demonstrated that the protein level of VISTA was significantly higher in human OSCC specimens, and that VISTA expression in primary OSCCs was correlated with lymph node status. VISTA expression did not serve as an independent predictor for poor prognosis, while patient subgroup with VISTA high and CD8 low expression (22/165) had significantly poorer overall survival compared with other subgroups based on the multivariate and Cox hazard analyses among the primary OSCC patients in the present cohort. Additionally, the expression of VISTA was significantly correlated with PD-L1, CTLA-4, IL13Rα2, PI3K, p-STAT3, CD11b and CD33 according to Pearson's correlation coefficient test. Taken together, the results indicated that the VISTA high and CD8 low group, as an immunosuppressive subgroup, might be associated with a poor prognosis in primary OSCC. These findings indicated that VISTA might be a potential immunotherapeutic target in OSCC treatment.

Expression and associations of TRAF1, BMI-1, ALDH1, and Lin28B in oral squamous cell carcinoma.

Tumor necrosis factor receptor-associated factor 1, an adaptor protein of tumor necrosis factor 2, is involved in classical nuclear factor (NF)-κB activation and lymphocyte recruitment. However, less is known about the expression and association of tumor necrosis factor receptor-associated factor 1 with cancer stem cell markers in oral squamous cell carcinoma. This study aimed to investigate the expression of tumor necrosis factor receptor-associated factor 1 and stem cell characteristic markers (lin28 homolog B, B cell-specific Moloney murine leukemia virus integration site 1, and aldehyde dehydrogenase 1) in oral squamous cell carcinoma and analyze their relations. Paraffin-embedded tissues of 78 oral squamous cell carcinomas, 39 normal oral mucosa, and 12 oral dysplasia tissues were employed in tissue microarrays, and the expression of tumor necrosis factor receptor-associated factor 1, B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B was measured by immunohistostaining and digital pathological analysis. The expression of tumor necrosis factor receptor-associated factor 1 was higher in the oral squamous cell carcinoma group as compared with the expression in the oral mucosa (p < 0.01) and oral dysplasia (p < 0.001) groups. In addition, the expression of tumor necrosis factor receptor-associated factor 1 was associated with those of B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B (p = 0.032, r2 = 0.109; p < 0.0001, r2 = 0.64; and p < 0.001, r2 = 0.16) in oral squamous cell carcinoma. The patient survival rate was lower in the highly expressed tumor necrosis factor receptor-associated factor 1 group, although the difference was not significant. The clustering analysis showed that tumor necrosis factor receptor-associated factor 1 was most related to aldehyde dehydrogenase 1. These findings suggest that tumor necrosis factor receptor-associated factor 1 has potential direct/indirect regulations with the cancer stem cell markers in oral squamous cell carcinoma, which may help in further analysis of the cancer stem cell characteristics.

[Effect of miRNA from Glycyrrhiza uralensis decoction on gene expression of human immune cells].

MicroRNAs(miRNA) are small non-coding RNAs that regulate the expression of protein coding genes by repressing translation of protein coding mRNA or enhancing mRNA degradation. Its functions have attracted more and more attention from the public. In recent years, the cross-border regulation of miRNA has become a new research direction, and provides a new perspective for people to comprehensively understand the functions of miRNA. Plant miRNA is usually methylated and not easy to degrade. According to our previous researches, there were abundant small RNAs in the decoction of dried liquorice, which provides a new way to study the mechanism of action of licorice. In this study, small RNAs extracted from Glycyrrhiza uralensis decoction and synthesized miRNA mimics were used to treat peripheral blood mononuclear cells(PBMC) isolated from healthy volunteers. The gene expression of toll-like receptors(TLRs), some transcription factors, signal molecules and cytokines were analyzed by RT-PCR. The results showed that glycyrrhiza miRNA could significantly regulate PBMC by inhibiting the expression of genes involved in T cell differentiation, inflammation and apoptosis. The study brought new ideas to us in comprehensively studying the mechanism of licorice and developing the traditional Chinese medicine.

B7-H4 expression indicates poor prognosis of oral squamous cell carcinoma.

Checkpoint blockade therapy utilizing monoclonal antibodies to reactivate T cells and recover their antitumor activity makes an epoch in cancer immunotherapy. The role of B7-H4, a novel negative immune checkpoint, in oral squamous cell carcinoma (OSCC) has still not been elucidated. In this study, tissue samples from human OSCC, which contains 165 primary OSCC, 48 oral epithelial dysplasia and 43 normal oral mucosa specimens, and Tgfbr1/Pten 2cKO mice OSCC model were stained with B7-H4 antibody to analyze the correlations between B7-H4 expression and clinicopathological characteristics. Kaplan-Meier analysis was used to compare the survival of patients with high B7-H4 expression and patients with low B7-H4 expression. We found B7-H4 is highly expressed in human OSCC tissue, and the B7-H4 expression level was associated with the clinicopathological parameters containing pathological grade and lymph node status. Moreover, we confirmed that B7-H4 was overexpressed in Tgfbr1/Pten 2cKO mice OSCC model. Our data also indicated that patients with high B7-H4 expression had poor overall survival compared with those with low B7-H4 expression. Furthermore, this study demonstrated that B7-H4 was positively associated with PD-L1, CD11b, CD33, PI3Kα p110, and p-S6 (S235/236). Taken together, these findings suggest B7-H4 is a potential target in the treatment of OSCC.

Syngnathia Between the Palate and Mouth Floor, Cleft Palate, and Funnel Chest.

Syngnathia is a rare malformation involving soft tissue and/or bony adhesions between the maxilla and mandible. Less than 40 patients have been reported in the literature. Here the authors report a 6-month-old infant diagnosed as syngnathia of the palate and mouth floor combined with cleft palate and funnel chest in the Department of Oral and Maxillofacial Surgery at Henan Provincial People's Hospital in January 2015. The authors discussed and evaluated the diagnostic and treatment difficulties on surgical and anesthetic procedure. There is no standard treatment protocol, but early treatment is necessary to improve airway functions and infant feeding, and to support proper nutrition for the growth of maxillofacial region.