Pan-cancer spatially resolved single-cell analysis reveals the crosstalk between cancer-associated fibroblasts and tumor microenvironment.

Cancer-associated fibroblasts (CAFs) are a heterogeneous cell population that plays a crucial role in remodeling the tumor microenvironment (TME). Here, through the integrated analysis of spatial and single-cell transcriptomics data across six common cancer types, we identified four distinct functional subgroups of CAFs and described their spatial distribution characteristics. Additionally, the analysis of single-cell RNA sequencing (scRNA-seq) data from three additional common cancer types and two newly generated scRNA-seq datasets of rare cancer types, namely epithelial-myoepithelial carcinoma (EMC) and mucoepidermoid carcinoma (MEC), expanded our understanding of CAF heterogeneity. Cell-cell interaction analysis conducted within the spatial context highlighted the pivotal roles of matrix CAFs (mCAFs) in tumor angiogenesis and inflammatory CAFs (iCAFs) in shaping the immunosuppressive microenvironment. In patients with breast cancer (BRCA) undergoing anti-PD-1 immunotherapy, iCAFs demonstrated heightened capacity in facilitating cancer cell proliferation, promoting epithelial-mesenchymal transition (EMT), and contributing to the establishment of an immunosuppressive microenvironment. Furthermore, a scoring system based on iCAFs showed a significant correlation with immune therapy response in melanoma patients. Lastly, we provided a web interface ( https://chenxisd.shinyapps.io/pancaf/ ) for the research community to investigate CAFs in the context of pan-cancer.

Low-dose TNF-α promotes angiogenesis of oral squamous cell carcinoma cells via TNFR2/Akt/mTOR axis.

OBJECTIVES: To assess the role of TNF-α/TNFR2 axis on promoting angiogenesis in oral squamous cell carcinoma (OSCC) cells and uncover the underlying mechanisms. MATERIALS AND METHODS: The expression of TNFR2 and CD31 in OSCC tissues was examined; gene expression relationship between TNF-α/TNFR2 and angiogenic markers or signaling molecules was analyzed; the expression of angiogenic markers, signaling molecules, TNFR1, and TNFR2 in TNF-α-stimulated OSCC cells treated with or without TNFR2 neutralizing antibody (TNFR2 Nab) were assessed; the concentration of angiogenic markers in the supernatant of OSCC cells was detected; conditioned mediums of OSCC cells treated with TNF-α or TNF-α + TNFR2 Nab were applied to human umbilical vein endothelial cells (HUVECs), followed by tube formation and cell migration assays. RESULTS: Significantly elevated expression of TNFR2 and CD31 in OSCC tissues was observed. A positive gene expression correlation was identified between TNF-α/TNFR2 and angiogenic markers or signaling molecules. TNFR2 Nab inhibited the effects of TNF-α on enhancing the expression of angiogenic factors and TNFR2, the phosphorylation of the Akt/mTOR signaling pathway, HUVECs migration, and tube formation. CONCLUSIONS: TNFR2 Nab counteracts the effect of TNF-α on OSCC cells through the TNFR2/Akt/mTOR axis, indicating that blocking TNFR2 might be a promising strategy against cancer.

Progranulin inhibits LPS-induced macrophage M1 polarization via NF-кB and MAPK pathways.

BACKGROUND: Macrophage M1 polarization plays a pivotal role in inflammatory diseases. Progranulin (PGRN) has potential anti-inflammation action, however, the effect of PGRN on macrophage M1 polarization has been poorly studied. Our study aimed to investigate the effect of PGRN on lipopolysaccharide (LPS)-induced macrophage M1 polarization and clarify the underlying mechanisms. METHODS: RAW264.7 cells were polarized to M1 macrophage by LPS with or without recombinant PGRN (rPGRN) and tumor necrosis factor alpha antibody (anti-TNF-α). A cell counting kit-8 assay (CCK-8), flow cytometry, Quantitative Real-Time PCR assay (q-PCR), Western blot assay and enzyme-linked immunosorbent assay (ELISA) were used to determine the effect of different treatments on cell proliferation, expression of surface phenotype marker and expressions and secretion of inflammatory cytokines. The activation of NF-κB/mitogen-activated protein kinase (MAPK) pathways and the nuclear translocation of NF-κB p65 were detected by Western blot and immunofluorescence respectively. THP-1 and primary bone marrow-derived monocytes (BMDMs) were also used to demonstrate effect of PGRN on expressions and secretion of inflammatory cytokines induced by LPS. RESULTS: In RAW264.7 cells, rPGRN at concentrations below 80 ng/ml significantly promoted cell proliferation in dose dependent fashion. rPGRN significantly inhibited LPS-induced change of phenotype (CD86/CD206 ratio) and function (tumor necrosis factor (TNF-α) and inducible nitric oxide synthase (iNOS) expressions). LPS-stimulated secretion of TNF-α and activated phosphorylation of IKKα/ß, IкBα, p65, JNK and p38 and the nucleus translocation of NF-кB p65 were also significantly downregulated by rPGRN. In addition, recombinant TNF-α (rTNF-α) significantly boosted TNF-α and iNOS expression vs the control group. Moreover, anti-TNF-α significantly inhibited LPS-induced TNF-α and iNOS expression. In THP-1 and BMDM cells, reversing effect of rPGRN on LPS-enhanced expressions of TNF-α and iNOS and secretion of TNF-α was further demonstrated. CONCLUSIONS: PGRN down-regulates LPS-induced macrophage M1 polarization in phenotype and function via NF-κB/MAPK signaling pathways.

Progranulin promotes osteogenic differentiation of human periodontal ligament stem cells via tumor necrosis factor receptors to inhibit TNF-α sensitized NF-kB and activate ERK/JNK signaling.

OBJECTIVE: To investigate the molecular mechanism of Progranulin (PGRN) in promoting osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in inflammatory environment. BACKGROUND: Progranulin is an antagonist of tumor necrosis factor (TNF) receptors (TNFRs) and is known to promote inflammatory periodontal bone defect regeneration. METHODS: TNFR1- and TNFR2-silenced hPDLSCs designed as hPDLSCs-sh-TNFR1 and hPDLSCs-sh-TNFR2 were cultured with osteoinductive medium containing TNF-α and (or) PGRN. Immunofluorescence, quantitative real-time PCR, and western blot were used to, respectively, detect expressions of TNFR1\TNFR2 and osteogenic differentiation markers as well as phosphorylation level in NF-κB\MAPK-related pathways. RESULTS: Immunofluorescence and real-time PCR showed that TNFR1 and TNFR2 positively expressed in hPDLSCs. TNF-α stimulation could significantly decrease the expressions of ALP and RUNX2 in hPDLSCs, whereas PGRN treatment could significantly enhance their expressions, and reverse TNF-α-mediated expression suppression of ALP and RUNX2 in hPDLSCs. In hPDLSCs-sh-TNFR1, TNF-α mediated osteogenic inhibition decreased, but both TNF-α + PGRN and alone PGRN significantly promoted expression of ALP and RUNX2. PGRN significantly enhanced expression of P-ERK1/2 and P-JNK, while corresponding inhibitors eliminated PGRN-stimulated osteogenic differentiation. In hPDLSCs-sh-TNFR2, no significant difference existed in osteogenic markers and P-JNK expression between the PGRN group and the control group. However, PGRN still activated P-ERK1/2 expression. Besides, PGRN antagonized TNF-α-enhanced NF-κB P65 expression. CONCLUSION: Progranulin promotes osteogenic differentiation of hPDLSCs via TNFR1 to inhibit TNF-α-sensitized NF-κB and via TNFR2 to activate JNK signaling. The mechanism by which PGRN activates ERK signaling remains to be explored.

Curcumin suppresses the proliferation and tumorigenicity of Cal27 by modulating cancer-associated fibroblasts of TSCC.

Cancer-associated fibroblasts (CAFs) are "activated" fibroblasts in the tumor microenvironment (TME) and play a vital role in all steps of cancer development. Increasing evidence focusing on the function of CAFs suggests that CAFs are candidate therapeutic targets and that drugs targeting the modification of CAFs would suppress tumor progression and be beneficial to tumor treatment and prevention. In the present study, we found that curcumin reversed the phenotype of CAFs to that of peri-tumor fibroblast (PTF)-like cells by downregulating the expression of α-SMA (a special marker for CAFs) and inhibiting the secretion of pro-carcinogenic cytokines, including transforming growth factor-ß1 (TGF-ß1), matrix metalloproteinases 2 (MMP2), and stromal cell-derived factor-1 (SDF-1). We further demonstrated that the conditioned medium (CM) derived from CAFs promoted the proliferation of Cal27, and this effect was confirmed by the xenograft model. More importantly, we found that curcumin blocked the CAF-mediated enhancement of Cal27 proliferation in vitro and in vivo. In conclusion, our data suggest that curcumin reverses cell phenotype from CAF to PTF-like cells and suppresses the CAF-mediated proliferation and tumorigenicity of Cal27 by inhibiting TSCC CAFs.

The biological behavior optimization of human periodontal ligament stem cells via preconditioning by the combined application of fibroblast growth factor-2 and A83-01 in in vitro culture expansion.

BACKGROUND: As the optimal source of seed cells in periodontal tissue engineering, periodontal ligament stem cells (PDLSCs) have always been researched to improve cell expansion due to their limited resource and spontaneous differentiation in vitro cultivation. Fibroblast growth factor-2 (FGF-2) has been proven to stimulate bone marrow mesenchymal stem cells (BMMSCs) proliferation and maintain their pluripotency when being added to the culture medium. As a small molecule inhibitor of transforming growth factor-beta receptors (TGF-ßRs), A83-01 can also promote cell proliferation. Therefore, the aim of this study was to verify whether the combined application of FGF-2 and A83-01 could augment cell quantity and quality during in vitro culture. METHODS: PDLSCs were preconditioned with A83-01, FGF-2, or their combination. A cell counting kit-8 (CCK8) assay, cell apoptosis assay, ALP activity assay, Alizarin Red S staining assay, RT-PCR assay, Western blot assay and ELISA were used to determine the sustained effects of different preconditioning strategies on the proliferation, apoptosis, stemness, osteogenic differentiation and paracrine action of PDLSCs. RESULTS: The combined application of FGF-2 and A83-01 significantly augmented cell expansion, reduced cell apoptosis, magnified stemness expression, promoted later osteogenic differentiation and mineralization and increased paracrine action of PDLSCs compared with the control. Moreover, the combination presented significant advantages in enhancing proliferation, stemness expression and paracrine action over FGF-2 alone. CONCLUSIONS: The combined application of A83-01 and FGF-2 may be an improved strategy for PDLSCs biological behavior optimization in culture expansion and advantageous for reinforcing proliferation, stemness expression and cytokine secretion over FGF-2 alone.

C-reactive protein is associated with the development of tongue squamous cell carcinoma.

C-reactive protein (CRP) acts as a biomarker reflecting different degrees of inflammation. Accumulating reports have suggested that there is a close relationship between CRP and various cancers. However, the influence of CRP on the development of tongue squamous cell carcinoma (TSCC) remains unclear. The purpose of this study was to investigate the role of CRP in TSCC. The results of immunohistochemical staining and statistical analyses showed that CRP expression was associated with TSCC tumor size, lymph node metastasis and pathological differentiation. Cell Counting Kit-8 (CCK-8) assay revealed that CRP could enhance TSCC cell proliferation in a dose- and time-dependent manner. Moreover, with CRP stimulation, proliferating cell nuclear antigen (PCNA) expression patterns presented a notable time-dependent up-regulation. In addition, CRP could enhance the invasion and migration of TSCC cells, as revealed by transwell and wound-healing assays, respectively. Annexin V-FITC/PI staining showed that CRP could protect TSCC cells from starvation- and drug-induced apoptosis. With CRP stimulation, the protein expression levels of phosphorylated protein kinase B (pAkt), phosphorylated mammalian target of rapamycin (pmTOR) and phosphorylated S6 ribosomal protein (pS6) were significantly increased, as demonstrated by western blot analysis. Our data suggest that CRP may play an important role in the development of TSCC. Moreover, the biological effects of CRP on TSCC cells might be related to Akt, mTOR, and S6.

BRD4 inhibition suppresses cell growth, migration and invasion of salivary adenoid cystic carcinoma.

BACKGROUND: Bromodomain-containing protein 4 (BRD4) inhibition is a new therapeutic strategy for many malignancies. In this study, we aimed to explore the effect of BRD4 inhibition by JQ1 on in vitro cell growth, migration and invasion of salivary adenoid cystic carcinoma (SACC). METHODS: The human normal epithelial cells and SACC cells (ACC-LM and ACC-83) were treated with JQ1 at concentrations of 0, 0.1, 0.5 or 1 µM. Cell Counting Kit-8 (CCK-8) assay was performed to evaluate cell proliferation. Cell apoptosis and cell cycle distribution was evaluated by Flow cytometry. Immunofluorescence staining was used to examine the expression of BRD4 in SACC cells. The quantitative real-time polymerase chain reaction (qRT-PCR) assay and western blot assay were performed to examine messenger RNA (mRNA) and protein levels in SACC cells. Wound-healing assay and transwell assay were used to evaluate the activities of migration and invasion of SACC cells. RESULTS: JQ1 exhibits no adverse effects on proliferation, cell cycle and cell apoptosis of the normal human epithelial cells, while suppressed proliferation and cell cycle, and induced apoptosis of SACC cells, down-regulated the mRNA and protein levels of BRD4 in SACC cells, meanwhile reduced protein expressions of c-myc and BCL-2, two known target genes of BRD4. Moreover, JQ1 inhibited SACC cell migration and invasion by regulating key epithelial-mesenchymal transition (EMT) characteristics including E-cadherin, Vimentin and Twist. CONCLUSIONS: BRD4 is an important transcription factor in SACC and BRD4 inhibition by JQ1 may be a new strategy for SACC treatment.

Disturbed Expression of EphB4, but Not EphrinB2, Inhibited Bone Regeneration in an In Vivo Inflammatory Microenvironment.

The important role of ephrinB2-EphB4 signaling pathway in bone remodeling has been well established. However, it is still unclear whether this bidirectional signaling also has effects on the regenerative processes of bone defects created in an inflammatory microenvironment. In this study, an experimental animal model of bone defects treated with lentiviruses was prepared and an inflammatory microenvironment was established. Expression levels of bone marker genes were monitored in the newly formed bone tissue using quantitative reverse transcriptase polymerase chain reaction and western blot. Immunohistochemical (IHC) staining and histomorphometric analysis were also performed to evaluate bone healing processes. Compared with the pLenti6.3-ctrl group, the pLenti6.3-ephb4siRNA group exhibited lower expression levels of bone formation marker genes and a higher level of NFATc1 in the new bone tissue. In addition, the newly formed bone was thinner and the number of giant osteoclasts was higher in the pLenti6.3-ephb4siRNA group than that in the pLenti6.3-ctrl group. In contrast, there was no significant difference between the pLenti6.3-efnb2siRNA group and the pLenti6.3-ctrl group. In conclusion, EphB4 plays an irreplaceable role in bone regeneration in an inflammatory microenvironment, whereas the functional loss of ephrinB2 can be effectively compensated, most possibly by other ephrins with similar chemical structures.

Elevated level of serum growth differentiation factor 15 is associated with oral leukoplakia and oral squamous cell carcinoma.

BACKGROUND: Although molecular mechanism of growth differentiation factor 15 (GDF15) in tumorigenesis of oral squamous cell carcinoma (OSCC) is not clear, the diagnostic and prognostic value of serum GDF15 detection has been noticed. However, serum GDF15 levels in patients with oral leukoplakia and GDF15 as a potential predictive biomarker for response to induction chemotherapy in patients with OSCC have not been reported. METHODS: Pretreatment serum GDF15 concentration was detected using an enzyme-linked immunosorbent assay in 30 healthy persons, 24 patients with oral leukoplakia, and 60 patients with OSCC. RESULTS: Serum GDF15 concentration was significantly higher in patients with oral leukoplakia and OSCC, compared with healthy controls (F = 13.701, df = 2, P < 0.001). From a diagnostic standpoint, a cutoff value of 346.9 ng/l of serum GDF15 concentration was calculated using receiver operating characteristic curve, with a sensitivity of 0.750, specificity of 0.867, Youden's Index of 0.617, and area under curve of 0.863. From a prognostic standpoint, patients with serum GDF15 concentration <346.9 ng/l had an improved 3-year disease-free survival rate (64.3% vs 56.5%) compared with those above 346.9 ng/l, but the difference was not statistically significant. A decreased concentration of GDF15 (<346.9 ng/l) showed a predictive trend toward an improved response to induction chemotherapy compared with elevated concentration with clinical response rates of 100% and 71.4%, respectively, but the difference was not significant. CONCLUSION: Elevated GDF15 level may be not only a diagnostic biomarker for oral leukoplakia, but also a prognostic/predictive biomarker associated with decreased survival and diminished response to induction chemotherapy for patients with OSCC.

Progress of research on the surface functionalization of tantalum and porous tantalum in bone tissue engineering.

Tantalum and porous tantalum are ideal materials for making orthopedic implants due to their stable chemical properties and excellent biocompatibility. However, their utilization is still affected by loosening, infection, and peripheral inflammatory reactions, which sometimes ultimately lead to implant removal. An ideal bone implant should have exceptional biological activity, which can improve the surrounding biological microenvironment to enhance bone repair. Recent advances in surface functionalization have produced various strategies for developing compatibility between either of the two materials and their respective microenvironments. This review provides a systematic overview of state-of-the-art strategies for conferring biological functions to tantalum and porous tantalum implants. Furthermore, the review describes methods for preparing active surfaces and different bioactive substances that are used, summarizing their functions. Finally, this review discusses current challenges in the development of optimal bone implant materials.

Single-cell RNA sequencing highlights the immunosuppression of IDO1+ macrophages in the malignant transformation of oral leukoplakia.

Rationale: Immunosuppressive tumor microenvironment (iTME) plays an important role in carcinogenesis, and some macrophage subsets are associated with iTME generation. However, the sub-population characterization of macrophages in oral carcinogenesis remains largely unclear. Here, we investigated the immunosuppressive status with focus on function of a macrophage subset that expressed indoleamine 2,3 dioxygenase 1 (Macro-IDO1) in oral carcinogenesis. Methods: We built a single cell transcriptome atlas from 3 patients simultaneously containing oral squamous cell carcinoma (OSCC), precancerous oral leukoplakia (preca-OLK) and paracancerous tissue (PCA). Through single-cell RNA sequencing and further validation using multicolor immunofluorescence staining and the in vitro/in vivo experiments, the immunosuppressive cell profiles were built and the role of a macrophage subset that expressed indoleamine 2,3 dioxygenase 1 (Macro-IDO1) in the malignant transformation of oral leukoplakia was evaluated. Results: The iTME formed at preca-OLK stage, as evidenced by increased exhausted T cells, Tregs and some special subsets of macrophages and fibroblasts. Macro-IDO1 was predominantly enriched in preca-OLK and OSCC, distributed near exhausted T cells and possessed tumor associated macrophage transformation potentials. Functional analysis revealed the established immunosuppressive role of Macro-IDO1 in preca-OLK and OSCC: enriching the immunosuppression related genes; having an established level of immune checkpoint score; exerting strong immunosuppressive interaction with T cells; positively correlating with the CD8-exhausted. The immunosuppression related gene expression of macrophages also increased in preca-OLK/OSCC compared to PCA. The use of the IDO1 inhibitor reduced 4NQO induced oral carcinogenesis in mice. Mechanistically, IFN-γ-JAK-STAT pathway was associated with IDO1 upregulation in OLK and OSCC. Conclusions: These results highlight that Macro-IDO1-enriched in preca-OLK possesses a strong immunosuppressive role and contributes to oral carcinogenesis, providing a potential target for preventing precancerous legions from transformation into OSCC.

The role of CXCL2-mediated crosstalk between tumor cells and macrophages in Fusobacterium nucleatum-promoted oral squamous cell carcinoma progression.

Dysbiosis of the oral microbiota is related to chronic inflammation and carcinogenesis. Fusobacterium nucleatum (Fn), a significant component of the oral microbiota, can perturb the immune system and form an inflammatory microenvironment for promoting the occurrence and progression of oral squamous cell carcinoma (OSCC). However, the underlying mechanisms remain elusive. Here, we investigated the impacts of Fn on OSCC cells and the crosstalk between OSCC cells and macrophages. 16 s rDNA sequencing and fluorescence in situ hybridization verified that Fn was notably enriched in clinical OSCC tissues compared to paracancerous tissues. The conditioned medium co-culture model validated that Fn and macrophages exhibited tumor-promoting properties by facilitating OSCC cell proliferation, migration, and invasion. Besides, Fn and OSCC cells can recruit macrophages and facilitate their M2 polarization. This crosstalk between OSCC cells and macrophages was further enhanced by Fn, thereby amplifying this positive feedback loop between them. The production of CXCL2 in response to Fn stimulation was a significant mediator. Suppression of CXCL2 in OSCC cells weakened Fn's promoting effects on OSCC cell proliferation, migration, macrophage recruitment, and M2 polarization. Conversely, knocking down CXCL2 in macrophages reversed the Fn-induced feedback effect of macrophages on the highly invasive phenotype of OSCC cells. Mechanistically, Fn activated the NF-κB pathway in both OSCC cells and macrophages, leading to the upregulation of CXCL2 expression. In addition, the SCC7 subcutaneous tumor-bearing model in C3H mice also substantiated Fn's ability to enhance tumor progression by facilitating cell proliferation, activating NF-κB signaling, up-regulating CXCL2 expression, and inducing M2 macrophage infiltration. However, these effects were reversed by the CXCL2-CXCR2 inhibitor SB225002. In summary, this study suggests that Fn contributes to OSCC progression by promoting tumor cell proliferation, macrophage recruitment, and M2 polarization. Simultaneously, the enhanced CXCL2-mediated crosstalk between OSCC cells and macrophages plays a vital role in the pro-cancer effect of Fn.

Low Annexin A1 expression predicts benefit from induction chemotherapy in oral cancer patients with moderate or poor pathologic differentiation grade.

BACKGROUND: The benefit of induction chemotherapy in locally advanced oral squamous cell carcinoma (OSCC) remains to be clearly defined. Induction chemotherapy is likely to be effective for biologically distinct subgroups of patients and biomarker development might lead to identification of the patients whose tumors are to respond to a particular treatment. Annexin A1 may serve as a biomarker for responsiveness to induction chemotherapy. The aim of this study was to investigate Annexin A1 expression in pre-treatment biopsies from a cohort of OSCC patients treated with surgery and post-operative radiotherapy or docetaxel, cisplatin and 5-fluorouracil (TPF) induction chemotherapy followed by surgery and post-operative radiotherapy. Furthermore we sought to assess the utility of Annexin A1 as a prognostic or predictive biomarker. METHODS: Immunohistochemical staining for Annexin A1 was performed in pre-treatment biopsies from 232 of 256 clinical stage III/IVA OSCC patients. Annexin A1 index was estimated as the proportion of tumor cells (low and high, <50% and ≥50% of stained cells, respectively) to Annexin A1 cellular membrane and cytoplasm staining. RESULTS: There was a significant correlation between Annexin A1 expression and pathologic differentiation grade (P=0.015) in OSCC patients. The proportion of patients with low Annexin A1 expression was significantly higher amongst those with moderate/poorly differentiated tumor (78/167) compared to those with well differentiated tumor (18/65). Multivariate Cox model analysis showed clinical stage (P=0.001) and Annexin A1 expression (P=0.038) as independent prognostic risk factors. Furthermore, a low Annexin A1 expression level was predictive of longer disease-free survival (P=0.036, HR=0.620) and locoregional recurrence-free survival (P=0.031, HR=0.607) compared to high Annexin A1 expression. Patients with moderate/poorly differentiated tumor and low Annexin A1 expression benefited from TPF induction chemotherapy as measured by distant metastasis-free survival (P=0.048, HR=0.373) as well as overall survival (P=0.078, HR=0.410). CONCLUSIONS: Annexin A1 can be used as a prognostic biomarker for OSCC. Patients with moderate/poorly differentiated OSCC and low Annexin A1 expression can benefit from the addition of TPF induction chemotherapy to surgery and post-operative radiotherapy. Annexin A1 expression can potentially be used as a predictive biomarker to select OSCC patients with moderate/poorly differentiated tumor who may benefit from TPF induction chemotherapy.

Co-inhibition of adenosine 2b receptor and programmed death-ligand 1 promotes the recruitment and cytotoxicity of natural killer cells in oral squamous cell carcinoma.

Adenosine promotes anti-tumor immune responses by modulating the functions of T-cells and natural killer (NK) cells in the tumor microenvironment; however, the role of adenosine receptors in the progression of oral squamous cell carcinoma (OSCC) and its effects on immune checkpoint therapy remain unclear. In this study, we obtained the tumor tissues from 80 OSCC patients admitted at the Shandong University Qilu Hospital between February 2014 and December 2016. Thereafter, we detected the expression of adenosine 2b receptor (A2BR) and programmed death-ligand 1 (PD-L1) using immunohistochemical staining and analyzed the association between their expression in different regions of the tumor tissues, such as tumor nest, border, and paracancer stroma. To determine the role of A2BR in PD-L1 expression, CAL-27 (an OSCC cell line) was treated with BAY60-6583 (an A2BR agonist), and PD-L1 expression was determined using western blot and flow cytometry. Furthermore, CAL-27 was treated with a nuclear transcription factor-kappa B (NF-κ B) inhibitor, PDTC, to determine whether A2BR regulates PD-L1 expression via the NF-κ B signaling pathway. Additionally, a transwell assay was performed to verify the effect of A2BR and PD-L1 on NK cell recruitment. The results of our study demonstrated that A2BR and PD-L1 are co-expressed in OSCC. Moreover, treatment with BAY60-6583 induced PD-L1 expression in the CAL-27 cells, which was partially reduced in cells pretreated with PDTC, suggesting that A2BR agonists induce PD-L1 expression via the induction of the NF-κ B signaling pathway. Furthermore, high A2BR expression in OSCC was associated with lower infiltration of NK cells. Additionally, our results demonstrated that treatment with MRS-1706 (an A2BR inverse agonist) and/or CD274 (a PD-L1-neutralizing antibody) promoted NK cell recruitment and cytotoxicity against OSCC cells. Altogether, our findings highlight the synergistic effect of co-inhibition of A2BR and PD-L1 in the treatment of OSCC via the modulation of NK cell recruitment and cytotoxicity.

M1 Macrophages Enhance Survival and Invasion of Oral Squamous Cell Carcinoma by Inducing GDF15-Mediated ErbB2 Phosphorylation.

M2 macrophages are generally recognized to have a protumor role, while the effect of M1 macrophages in cancer is controversial. Here, the in vitro and in vivo effects of conditioned medium from M1 macrophages (M1-CM) on oral squamous cell carcinoma (OSCC) cells and a potential mechanism were studied. CCK-8, colony formation, EdU labeling, xenograft growth, and Transwell assays were utilized to observe cell survival/proliferation and migration/invasion, respectively, in OSCC cell lines treated with basic medium (BM) and M1-CM. The ErbB2 phosphorylation inhibitor (CI-1033) and GDF15 knockout cell lines were used to appraise the role of ErbB2 and GDF15 in mediating the effects of M1-CM. Compared with BM, M1-CM significantly enhanced the survival/proliferation of SCC25 cells. The migration/invasion of SCC25 and CAL27 cells also increased. Mechanically, M1-CM promoted GDF15 expression and increased the phosphorylation of ErbB2, AKT, and ErK. CI-1033 significantly declined the M1-CM-induced activation of p-AKT and p-ErK and its protumor effects. M1-CM stimulated enhancement of p-ErbB2 expression was significantly decreased in cells with GDF15 gene knockout vs without. In xenograft, M1-CM pretreatment significantly promoted the carcinogenic potential of OSCC cells. Our results demonstrate that M1 macrophages induce the proliferation, migration, invasion, and xenograft development of OSCC cells. Mechanistically, this protumor effect of M1 macrophages is partly associated with inducing GDF15-mediated ErbB2 phosphorylation.

Characteristics of Microbial Distribution in Different Oral Niches of Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC), one of the most common malignant tumors of the head and neck, is closely associated with the presence of oral microbes. However, the microbiomes of different oral niches in OSCC patients and their association with OSCC have not been adequately characterized. In this study, 305 samples were collected from 65 OSCC patients, including tumor tissue, adjacent normal tissue (paracancerous tissue), cancer surface tissue, anatomically matched contralateral normal mucosa, saliva, and tongue coat. 16S ribosomal DNA (16S rDNA) sequencing was used to compare the microbial composition, distribution, and co-occurrence network of different oral niches. The association between the microbiome and the clinical features of OSCC was also characterized. The oral microbiome of OSCC patients showed a regular ecological distribution. Tumor and paracancerous tissues were more microbially diverse than other oral niches. Cancer surface, contralateral normal mucosa, saliva, and tongue coat showed similar microbial compositions, especially the contralateral normal mucosa and saliva. Periodontitis-associated bacteria of the genera Fusobacterium, Prevotella, Porphyromonas, Campylobacter, and Aggregatibacter, and anaerobic bacteria were enriched in tumor samples. The microbiome was highly correlated with tumor clinicopathological features, with several genera (Lautropia, Asteroleplasma, Parvimonas, Peptostreptococcus, Pyramidobacter, Roseburia, and Propionibacterium) demonstrating a relatively high diagnostic power for OSCC metastasis, potentially providing an indicator for the development of OSCC.

Antifungal drug susceptibility of oral Candida albicans isolates may be associated with apoptotic responses to Amphotericin B.

BACKGROUND: Candida albicans is the important opportunistic fungal pathogens which can cause oral Candidiasis and even more seriously systemic infection. Apoptosis of C. albicans induced by environmental factor such as weak acid and antifungal drugs were studied recently. Illustrating the phenomenon of apoptosis in C. albicans may help us to discover new antifungal therapy by activating the fungal cells to suicide. METHODS: Two oral C. albians clinical isolates which isolated respectively from healthy host [Strain 23C: minimal inhibition concentration (MIC) is 0.125 microg/ml for Amphotericin B (AmB)] and advanced cancer patient (Strain 28A: MIC is 2 microg/ml for AmB), were induced by 1 microg/ml AmB in vitro for 200 min, and then studied the apoptosis markers using terminal deoxynucletidyltransferase-mediated dUTP nick end labeling (TUNEL) (shown by diaminobenzidine and fluorescent isothiocyanate), and the ultrastructure of cell nuclear using transmission electron microscope (TEM), quantitative analysis using flow cytometry for the rapid exposure of phosphatidylserine at the outer membrane and propodium iodide (PI) double staining. C. albicans conference strain YEM30 was used as the control strain. RESULTS: With TUNEL assay and TEM, we detected the typical characteristics of apoptosis. Strain 23C (with low MIC) showed significantly higher percentage of apoptosis (19.92%) compared with Strain 28A (with high MIC) which was isolated from the cancer patient (7.29%) (P < 0.01). In addition, 7.3% of early apoptosis cells of Strain 23C can form colonies on the plates, while 15% for Strain 28A. None of the PI+ cells can form colony. CONCLUSIONS: Apoptosis of oral C. albicans isolates can be induced by AmB. The feature of antifungal drug susceptibility of the oral C. albicans clinical isolates may associate with the response of apoptosis inducing.

EphB4/ TNFR2/ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.

BACKGROUND: Low concentrations of tumor necrosis factor-alpha (TNF-α) and its receptor TNFR2 are both reported to promote osteogenic differentiation of osteoblast precursor cells. Moreover, low concentrations of TNF-α up-regulate the expression of EphB4. However, the molecular mechanisms underlying TNF-α-induced osteogenic differentiation and the roles of TNFR2 and EphB4 have not been fully elucidated. RESULTS: The ALP activity, as well as the mRNA and protein levels of RUNX2, BSP, EphB4 and TNFR2, was significantly elevated in MC3T3-E1 murine osteoblast precursor cells when stimulated with 0.5 ng/ml TNF-α. After TNFR2 was inhibited by gene knockdown with lentivirus-mediated shRNA interference or by a neutralizing antibody against TNFR2, the pro-osteogenic effect of TNF-α was partly reversed, while the up-regulation of EphB4 by TNF-α remained unchanged. With EphB4 forward signaling suppressed by a potent inhibitor of EphB4 auto-phosphorylation, NVP-BHG712, TNF-α-enhanced expressions of TNFR2, BSP and Runx2 were significantly decreased. Further investigation into the signaling pathways revealed that TNF-α significantly increased levels of p-JNK, p-ERK and p-p38. However, only the p-ERK level was significantly inhibited in TNFR2-knockdown cells. In addition, the ERK pathway inhibitor, U0126 (10 µM), significantly reversed the positive effect of TNF-α on the protein levels of RUNX2 and BSP. CONCLUSIONS: The EphB4, TNFR2 and ERK/MAPK signaling pathway comprises a signaling axis to mediate the positive effect of TNF-α on osteogenic differentiation.