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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.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.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. ST2IL-33/ST2 signaling enhanced PD-L1-mediated immune escape, ST2

Supplementary Fig. 1. The expression of TLR3 in OSCC patients; Supplementary Fig. 2. The impacts of poly (I:C) on the vitality of OSCC cells; Supplementary Fig. 3. Poly (I:C) could inhibit the expression of drug transporters; Supplementary Fig. 4. The characteristics of combined chemotherapy in vitro; Supplementary Fig. 5. lnc-IL7R is up-regulated by the combined chemotherapy; Supplementary Fig. 6. Lnc-IL7R knockdown shows weak effects on cell apoptosis and cell cycle; Supplementary Fig. 7. Lnc-IL7R knockdown improves the efficiency of combined chemotherapy; Table 1. information of antibodies and reagents; Table 2. Relationships between TLR3 expression on different cell type and clinicalpathologic Characteristics in OSCC patients.

The traditional maximum dose density chemotherapy renders the tumor patients not only the tumor remission but the chemotherapy resistance and more adverse side effects. According to the widely positive expression of Toll-like receptor (TLR)-3 in oral squamous cell carcinoma (OSCC) patients (n = 166), we here provided an alternative strategy involved the orderly treatment of TLR3 agonist polyinosine–polycytidylic acid (PIC) and low-dose cisplatin. The optimal dose of cisplatin, the novel role of PIC and the side effects of the combined chemotherapy were determined in vitro and in distinct human tumor models in vivo. The results in vitro indicated that preculture with PIC downregulated drug transporters (e.g., P-gp and MRP-1) and increased the cytoplasmic residence of cisplatin, and dramatically strengthened the low-dose cisplatin-induced cell death in TLR3- and caspase-3–dependent manner. Meanwhile, the spleen immunocytes were activated but the immunosuppressive cancer-associated fibroblasts (CAF) were dampened. These findings were confirmed in human tumor models in vivo. Pretreatment with PIC promoted the low-dose cisplatin residence for tumor regression with decreased myeloid-suppressive cells (MDSC), tumor-associated macrophages (TAM) and CAFs, and alleviated adverse side effects in the OSCC model, which was further enhanced by the Cetuximab safely. This strategy also repressed the progression of melanoma and lymphoma. Moreover, TLR3 negatively manipulated the inflammation-related long noncoding RNA lnc-IL7R, which was upregulated during this chemotherapy. Knockdown of lnc-IL7R improved the chemotherapy sensitivity. Overall, this study provided preclinically new instructions for the PIC/cisplatin utilization to target tumor microenvironment and strengthen the low-dose cisplatin-based chemotherapy with reduced side effects. Mol Cancer Ther; 16(6); 1068–79. ©2017 AACR.

Therapeutic protocols of tumor models and all other methods were described in Supplementary materials and methodsã€

To determine whether the deletion of p16 can correct tooth and mandible growth retardation caused by Bmi1 deficiency, we compared the tooth and mandible phenotypes of homozygous p16‐deficient (p16−/−) mice, homozygous Bmi1‐deficient (Bmi1−/−) mice, double homozygous Bmi1 and p16‐deficient (Bmi1−/−p16−/−) mice to those of their wild‐type littermates at 4 weeks of age by radiograph, histochemistry and immunohistochemistry. Results showed that compared to Bmi1−/− mice, the dental mineral density, dental volume and dentin sialoprotein immunopositive areas were increased, whereas the ratio of the predentin area to total dentin area and that of biglycan immunopositive area to dentin area were decreased in Bmi1−/−p16−/− mice. These results indicate that the deletion of p16 can improve tooth development in Bmi1 knockout mice. Compared to Bmi1−/− mice, the mandible mineral density, cortical thickness, alveolar bone volume, osteoblast number and activity, alkaline phosphatase positive area were all increased significantly in Bmi1−/−p16−/− mice. These results indicate that the deletion of p16 can improve mandible growth in Bmi1 knockout mice. Furthermore, the protein expression levels of cyclin D, CDK4 and p53 were increased significantly in p16−/− mice compared with those from wild‐type mice; the protein expression levels of cyclin D and CDK4 were decreased significantly, whereas those of p27 and p53 were increased significantly in Bmi1−/− mice; these parameters were partly rescued in Bmi1−/−p16−/− mice compared with those from Bmi1−/− mice. Therefore, our results indicate that Bmi1 plays roles in regulating tooth and mandible development by inhibiting p16 signal pathway which initiated entry into cell cycle.

Objectives: Integrins, the coordinator of extracellular and intracellular signaling, are often found to be aberrant in tumors and can reshape the tumor microenvironment. Although previous studies showed that integrin beta 2 (ITGB2) is important for host defense, its expression profile and role in tumors, especially in cancer associated fibroblasts (CAFs) are still unknown. Methods: Immunofluorescence stain and fluorescence activated cell sorting were used to analyze the ITGB2 expression profile in oral squamous cell carcinoma (OSCC). RT-PCR and western blot were used to compare ITGB2 expression in normal fibroblasts (NFs) and cancer associated fibroblasts (CAFs). Clinical data and function-based experiments were used to investigate the promoting tumor growth ability of ITGB2 expressing CAFs. Enhanced glycolysis activity was identified by using bioinformatics analyses and GC/MS assays. MCT1 knockdown OSCC cell lines were constructed to explore the pro-proliferative mechanisms of ITGB2 expressing CAFs in multiple in vitro and in vivo assays. Results: We found that CAFs exhibited significantly higher ITGB2 expression than the matched NFs. In addition, higher ITGB2 expression in CAFs was correlated with higher TNM stages and more Ki67+ tumor cells, indicating its ability to promote OSCC proliferation. Further, co-culture assay demonstrated that ITGB2-mediated lactate release in CAFs promoted OSCC cell proliferation. Mechanically, ITGB2 regulated PI3K/AKT/mTOR pathways to enhance glycolysis activity in CAFs. Accordingly, lactate derived from ITGB2-expressing CAFs was absorbed and metabolized in OSCC to generate NADH, which was then oxidized in the mitochondrial oxidative phosphorylation system (OXPHOS) to produce ATP. Notably, inhibiting the OXPHOS system with metformin delayed the proliferative capacity of OSCC cells cultured in the ITGB2-expressing CAFs medium. Conclusions: Our study uncovered the ITGB2high pro-tumoral CAFs that activated the PI3K/AKT/mTOR axis to promote tumor proliferation in OSCC by NADH oxidation in the mitochondrial oxidative phosphorylation system.

Saliva is a noninvasive biofluid that can contain metabolite signatures of oral squamous cell carcinoma (OSCC). Conductive polymer spray ionization mass spectrometry (CPSI-MS) is employed to record a wide range of metabolite species within a few seconds, making this technique appealing as a point-of-care method for the early detection of OSCC. Saliva samples from 373 volunteers, 124 who are healthy, 124 who have premalignant lesions, and 125 who are OSCC patients, were collected for discovering and validating dysregulated metabolites and determining altered metabolic pathways. Metabolite markers were reconfirmed at the primary tissue level by desorption electrospray ionization MS imaging (DESI-MSI), demonstrating the reliability of diagnoses based on saliva metabolomics. With the aid of machine learning (ML), OSCC and premalignant lesions can be distinguished from the normal physical condition in real time with an accuracy of 86.7%, on a person by person basis. These results suggest that the combination of CPSI-MS and ML is a feasible tool for accurate, automated diagnosis of OSCC in clinical practice.

Although cancer-associated fibroblasts (CAFs) are crucial stromal cells, characterizing their heterogeneity is far from complete. This study reports a novel subset of CAFs in oral squamous cell carcinoma (OSCC), which positively expressed CD68, the classic marker of macrophages. The spatial and temporal distribution of the CD68+ CAF subset of OSCC (n = 104) was determined by CD68/actin alpha 2, smooth muscle (ACTA2+; α-SMA) immunohistochemistry of serial sections. The CD68+ α-SMA+ CAF subset was elevated from dysplasia to OSCC. Moreover, although both the tumor center and invasive front harbor an abundant CD68+ CAF subset, patients with low-CD68+ CAFs in the tumor center showed more recurrence after operation and shorter survival time, indicating the different function of CD68+ CAFs in tumor initiation and progression. Functional analysis in the OSCC-CAF co-culture system found knockdown of CD68 did not change the phenotype of CAFs, tumor growth, or migration. Unexpectedly, low-CD68+ CAFs were associated with aberrant immune balance. A high proportion of tumor-supportive Tregs was found in patients with low-CD68+ CAFs. Mechanistically, knockdown of CD68 in CAFs contributed to the up-regulation of chemokine CCL17 and CCL22 of tumor cells to enhance Treg recruitment. Thus, up-regulated CD68+ fibroblasts participate in tumor initiation, but the low-CD68+ CAF subset in OSCC is conducive to regulatory T-cell (Treg) recruitment in the tumor microenvironment and contribute to poor prognosis of OSCC patients.

BACKGROUND: Advanced oral squamous cell carcinoma (OSCC) has large lesions and deep infiltration, and the control of safe surgical margins was difficult. If residual tumor remains after incomplete tumor resection, it can lead to local tumor recurrence or even distant metastasis. This study sought to investigate the clinical application of indocyanine green (ICG)-based near-infrared fluorescence (NIF) molecular imaging in the intraoperative detection of surgical margins of advanced OSCC. METHODS: Twenty-nine patients with advanced OSCC treated at the First Ward of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital were divided into the ICG group and non-ICG group. In the ICG group, the tumors were removed with the assistance of ICG fluorescence navigation technology. In the non-ICG group, the tumors were removed with conventional methods, and the cutting-edge tissues of the two groups underwent frozen biopsies. The margin abnormality rates were calculated and compared. RESULTS: Under the excitation of NIF in the ICG group, tumor fluorescence development was observable in all lesions, and the tumor boundary was clear. The abnormal rates of the incisional margin in the ICG group and non-ICG group were 0.78% and 6.25%, respectively (P

As a key histopathological characteristic of tumor invasion, perineural invasion (PNI) assists tumor dissemination, whereas the current definition of PNI by dichotomy is not accurate and the prognostic value of PNI has not reached consensus. To define PNI status in each patient when mixed types of PNI occurred simultaneously, we here further subclassified the traditional PNI in 183 patients with oral squamous cell carcinoma (OSCC). The spatial localization of nerves in OSCC microenvironment was thoroughly evaluated and successfully concluded into four types of PNI: 0, tumor cells away from nerves; 1, tumor cells encircling nerves less than 33%; 2, tumor cells encircling nerves at least 33%; and 3, tumor cells infiltrating into nerve sheathes. Sequentially, patients were stratified by single and mixed types of PNI. Traditionally, types 0 and 1 were defined as PNI−, while types 2 and 3 were PNI+, which predicted shorter survival time. When multiple types of PNI existed within one tumor, patients with higher score of PNI types tended to have a relatively worse prognosis. Therefore, to define the status of PNI more precisely, the new variable worst pattern of PNI (WPNI) was proposed, which was taken as the highest score of PNI types present in each patient no matter how focal. Results showed that patients with WPNI 1 had longest survival time, and WPNI 2 correlated with better overall survival (p= 0.02), local-regional recurrence-free survival (p= 0.03), and distant metastasis-free survival (p= 0.046) than WPNI 3. Multivariate Cox analysis confirmed that only WPNI 3 could independently predict patients’ prognosis, which could be explained by a more damaged immune response in WPNI 3 patients with less CD3+CD8+T cells and CD19+B cells. Conclusively, WPNI by trichotomy provide more meticulous and precise pathological information for tumor-nerve interactions in OSCC patients.

The survival rate of oral squamous cell carcinoma (OSCC) can be greatly improved if intervention could be initiated as early as possible. This poses a technical demand for developing a sensitive screening and specific in vitro diagnosis method for OSCC. Herewith, a large cohort consisting of 241 healthy contrast (HC) and 578 OSCC patients were recruited for conducting the rapid metabolic profiling of trace volume serum using conductive polymer spray ionization mass spectrometry (CPSI‐MS). Statistical analysis picked out 65 metabolite ions as potential characteristic markers for differentiating OSCC from HC. With the aid of a supporting vector machine (SVM), OSCC can be distinguished from HC with an accuracy of 98.0% by cross‐validation in the discovery cohort and 89.6% accuracy in the validation cohort. Furthermore, orthogonal partial least square‐discriminant analysis (OPLS‐DA) also initially showed the potential for OSCC staging, especially between T1/T2 and T3/T4 stages with an accuracy of 90.1%. CPSI‐MS combined with SVM or OPLS‐DA can not only quickly distinguish OSCC from HC but also predict the OSCC progression from T1/2 to T3/4 stages in a few minutes, making it a promising tool for both screening and diagnosing high‐risk population. Key points Sixty‐five characteristic metabolite ions significantly changed in OSCC serum metabolic profile compared to that in the HC group. CPSI‐MS combined with SVM achieved 89.6% accuracy on the validation cohort for OSCC prediction. CPSI‐MS/OPLS‐DA can distinguish T1/T2 from T3/T4 stages with an accuracy of 90.1% by cross‐validation.

Background: Oral squamous cell carcinoma (OSCC) accounts for 90 % of oral cancers. If a necessary intervention before tumorigenesis could be conducted, the current 60% 5-year survival rate would be expected to be majorly improved. This fact motivates the search for developing a highly sensitive and specific in vitro diagnostic method to conduct rapid OSCC screening. Method: Serum samples from 819 volunteers, consisted of 241 healthy contrast (HC) and 578 OSCC patients, were collected, and their metabolic profiles were acquired using conductive polymer spray ionization mass spectrometry (CPSI-MS). Univariate analysis was used to select significantly changed metabolite ions in the OSCC group compared to the HC group. Identities of these metabolite ions were determined by MS/MS experiments and reconfirmed at the tissue level by desorption electrospray ionization mass spectrometry (DESI-MS). The supporting vector machine (SVM) algorithm was employed as the machine learning model to implement the automatic prediction of OSCC. Results: Through statistical analysis, 65 metabolites were selected as potential characteristic marker candidates for serum OSCC screening. In situ validation by DESI-MSI revealed that 8 out of top 10 metabolites showed the same trends of change in tissue and serum. With the aid of machine learning, OSCC can be distinguished from HC with an accuracy of 98.0 % by cross-validation in the discovery cohort and 89.2% accuracy in the validation cohort. Furthermore, orthogonal partial least square-discriminant analysis (OPLS-DA) also showed the potential for recognizing OSCC stages. Conclusion: Using CPSI-MS combined with SVM, it is possible to distinguish OSCC from HC in a few minutes with high specificity and sensitivity, making this rapid diagnostic procedure a promising approach for high-risk population screening.

BackgroundAccurate evaluation of lymph node (LN) status is the key factor to determine the treatment and evaluate prognosis for patients with cancer. However, traditional pathological examination resulted in a 30% false-negative rate of detection of metastases in LNs. This study aimed to utilize Lugol’s iodine (I2-IK)-enhanced micro-CT imaging to reveal the 3-dimensional structure of regional LNs and decrease the false-negative rate in pathological examination.MethodsTo explore the feasibility of I2-IK-enhanced micro-CT imaging in locating metastatic lesion in LNs, nonmetastatic and metastatic LNs from mice were used to mimic the imaging process. Then, the LNs from oral squamous cell carcinoma (OSCC) patients were applied to verify the value of I2-IK-enhanced micro-CT imaging in revealing LN structure and locating metastatic lesions in LNs. The glycogen content in nonmetastatic and metastatic LNs was further detected by the use of a glycogen assay kit and periodic acid–Schiff (PAS) staining to explain the imaging differences between them.ResultsIn nude mice, 0.5% I2-IK staining for 4 h was the best parameter for normal LN. The metastatic foci in metastatic LNs were also clearly outlined in this condition. For nonmetastatic LNs from patients with OSCC, 1% I2-IK staining for 12 h was the best parameter. However, due to the increased volume of metastatic LNs, the image effect of 3% I2-IK staining for 12 h was superior to 1% I2-IK staining [tumor background ratio (TBR), 3% vs. 1%, 1.89 ± 0.10 vs. 1.27 ± 0.07, p < 0.001]. Compared with subsequent pathological sections, we found the CT intensity of metastatic foci in LNs and muscle tissues was significantly higher than in nonmetastatic regions. Meanwhile, the glycogen content of metastatic foci in LNs detected was also significantly higher than in nonmetastatic region.ConclusionsI2-IK-enhanced micro-CT imaging could identify the spatial location of metastatic foci in LNs. This will be an effective method to assist in decreasing the LN false-negative rate for cancer pathology.

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 (CAFs

BackgroundReelin, an extracellular glycoprotein, is expressed on neuronal cells and participates in neuronal migration during brain development. Recently, Reelin also has a vital role in carcinogenesis. However, its role in oral squamous cell carcinoma (OSCC) remains to be explored. The purpose of this study was to explore the roles of Reelin in OSCC.MethodsThe expression of Reelin in cancer-associated fibroblasts (ReelinCAF) and tumor cells (ReelinTC) was analyzed by the Gene Expression Omnibus (GEO) database. Immunohistochemistry (IHC) was used to detect the spatial pattern of Reelin in 75 OSCCs. The diagnostic and prognostic values of Reelin were evaluated and also verified by The Cancer Genome Atlas (TCGA) database. Primary CAFs from 13 OSCC patients were isolated to confirm Reelin expression. Thirty-nine OSCC peripheral blood samples were used to analyze the change of immunocytes based on Reelin levels by flow cytometry. The relationship between Reelin and tumor immune microenvironment in head and neck squamous cell carcinoma (HNSCC) tissues was determined by TISIDB and the Tumor Immune Estimation Resource (TIMER) database.ResultsIn breast cancer, pancreatic cancer and rectal cancer, Reelin in CAFs was significantly upregulated compared with Reelin in TCs. The IHC results in OSCC also showed that Reelin levels were higher in CAFs. Upregulated ReelinTC was related to a decreased pN stage and distant metastasis. Strikingly, patients with enhanced ReelinCAF had a high risk of lymph node metastasis, poor worst pattern of invasion (WPOI), and distant metastasis, but showed comparable Ki-67 level in all OSCC patients, resulting in shorter overall survival (OS) and disease-specific survival (DSS). Unexpectedly, Reelin in tumor-infiltrating lymphocytes (ReelinTIL) was correlated with postoperative relapse. Patients with high ReelinTIL, but not ReelinTC and ReelinCAF, had poor cytotoxicity of CD8+ T cells and higher ratio of CD4/CD8 in peripheral blood. However, Reelin was positively associated with tissue-resident B cells and NK cells in the tumor microenvironment.ConclusionReelin has a versatile function in distinct cell types during the development of OSCC via governing tumor cell and stroma microenvironment.

BackgroundCD38 belongs to the ribosyl cyclase family and is expressed on various hematological cells and involved in immunosuppression and tumor promotion. Although targeting CD38 antibodies has been approved for treatment of multiple myeloma, the function of CD38 in solid tumor, oral squamous cell carcinoma (OSCC)etc., has not been investigated.MethodsThis retrospective study included 92 OSCC samples and analyzed the spatial distribution of CD38 by immunohistochemistry (IHC). The values of diagnosis and prognosis of CD38 were evaluated. Additionally, 53 OSCC preoperative peripheral blood samples were used to be analyzed by flow cytometry. Tumor Immune Estimation Resource (TIMER) and cBioPortal databases were used to study CD38 level in various tumors and its correlation with tumor immune microenvironment in head and neck squamous cell carcinoma (HNSCC).ResultsCD38 ubiquitously presented in tumor cells (TCs), fibroblast-like cells (FLCs), and tumor-infiltrating lymphocytes (TILs). Patients with highly expressed CD38 in TCs (CD38TCs) had higher TNM stage and risk of lymph node metastasis. Upregulation of CD38 in FLCs (CD38FLCs) was significantly associated with poor WPOI. Escalated CD38 in TILs (CD38TILs) led to higher Ki-67 level of tumor cells. Moreover, patients with enhanced CD38TCswere susceptible to postoperative metastasis occurrence, and those with highly expressed CD38TILsindependently predicted shorter overall and disease-free survival. Strikingly, patients with highly expressed CD38TILs, but not CD38TCsand CD38FLCs, had significantly lower CD3+CD4+T cells and higher ratio of CD3−CD16+CD56+NK cells. The imbalance of immune system is attributed to dysregulated immune checkpoint molecules (VISTA, PD-1, LAG-3, CTLA-4, TIGIT, GITR) as well as particular immune cell subsets, which were positively correlated with CD38 expression in HNSCC.ConclusionCD38 is a poor prognostic biomarker for OSCC patients and plays a vital role in governing immune microenvironment and circulating lymphocyte homeostasis. Co-expression between CD38 and immune checkpoint molecules provides new insight into immune checkpoint therapy.

The purpose of this study was to design silver nanoparticles (AgNPs) poloxamer thermoreversible gel (AgNPs-PL) and investigate whether this gel could provide sustained antibacterial activity against Enterococcus faecalis (E. faecalis) in the root canal. The gels fabricated were characterized in terms of gelatin temperature, particle size, in-vitro Ag+ release, and elemental content. Cytotoxicity of AgNPs-PL on primary human periodontal ligament fibroblasts (HPDLFs) was examined by CCK-8 assay. Characterization of AgNPs-PL gel revealed that it contained particles existing as large clumps/fused aggregates of different shapes, with a mean diameter of 21.624 ± 14.689 nm, exhibited sustained release of Ag+ for 9 days, and non-toxic to HPDLFs at a low dose (4–32 μg/mL) through 24, 48, and 72 h exposures. The antibacterial effect of 16 and 32 μg/mL concentrations of AgNPs-PL was compared with blank poloxamer gel (PL) and calcium hydroxide (CH) using three methods: (I) agar counting plate, (II) scanning electron microscope (SEM) observations, and (III) confocal laser scanning microscope (CLSM) analysis. AgNPs-PL at the two doses above was more effective than PL and CH in removing E. faecalis biofilm at 1, 3, 9 days. Thus, AgNPs-PL exhibits strong activity against E. faecalis and is easy to produce, with a continuous release profile of Ag+. AgNPs-PL gel may be a candidate for a new root canal disinfection.

Purpose This was a retrospective analysis of the impact of the expression of p53 in the dys-plastic surgical margins of early oral squamous cell carcinoma (OSCC) (pT1-2, N0). Patients and methods Seventy-two patients with early oral squamous cell carcinoma (OSCC) were recruited. Margin characteristics were abstracted from the pathology report. Expression of p53 in dysplastic surgical margins was examined with the immunohistochemical method and was correlated with clinicopathological parameters and clinical outcomes. Results Patients with moderate/severe dysplasia had poor local relapse-free survival (RFS) compared to those with mild dysplasia. Thirty-two (44.4%) had at least one p53-positive margin, and there was a significant association between the expression of p53 and tumor recurrence (P

Current surgical treatment for oral squamous cell carcinoma (OSCC) must be as precise as possible to fully resect tumors and preserve functional tissues. Thus, it is urgent to develop efficient fluorescent probes to clearly identify tumor delineation, as well as metastatic lymph nodes. Chemo-photothermal therapy combination attracted a growing attention to increase anti-tumor effect in various types of cancer, including OSCC. In the present study, we designed a multimodal NIR-II probe that involves combining photothermal therapy with chemotherapy, imaging OSCC tumors and detecting metastatic lymph nodes. Methods: In this study, we synthesized a novel near infrared (NIR)-II probe named TQTPA [4,4'-((6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo [3,4-g]quinoxaline-4,9-diyl)bis(thiophene-5,2-diyl))bis(N,N-diphenylaniline)] via the Suzuki reaction and prepared multimodal nanoparticles (NPs) loading TQTPA and cis-dichlorodiammine platinum (CDDP) (HT@CDDP) by hyaluronic acid. The characteristics of the NPs, including their photothermal and imaging capabilities were investigated in vitro and in vivo. Their anti-tumor efficacy was evaluated using orthotopic, tongue tumor-bearing, nude mice. Results: The NPs possessed good stability and water solubility and were pH/hyaluronidase sensitive. The good tissue penetration quality and active targeting ability enabled the NPs to draw the outline of orthotopic tongue tumors and metastatic lymph nodes as small as 1 mm in nude mice by IR-808 under NIR exposure. In vitro and in vivo experiments validated the biocompatibility and low systematic toxicity of the NPs. At the same time, the NPs acted as multimodal therapy agents, combining photothermal therapy with chemotherapy. Conclusion: With a good imaging capability and anti-tumor efficacy, our NPs successfully outlined orthotopic tongue tumors and metastatic lymph nodes as well as enabled chemo-photothermal therapy combination. Our study established a solid foundation for the application of new clinical diagnosis and treatment patterns in the future.

Background Although there is consensus that the optimal safe margin is ≥ 5mm, obtaining clear margins (≥5 mm) intraoperatively seems to be the major challenge. We applied a molecular diagnostic method at the lipidomic level to determine the safe surgical resection margin of OSCC by desorption electrospray ionisation mass spectrometry imaging (DESI-MSI). Methods By overlaying mass spectrometry images with hematoxylin-eosin staining (H&E) from 18 recruited OSCC participants, the mass spectra of all pixels across the diagnosed tumour and continuous mucosal margin regions were extracted to serve as the training and validation datasets. A Lasso regression model was used to evaluate the test performance. Findings By leave-one-out validation, the Lasso model achieved 88.6% accuracy in distinguishing between tumour and normal regions. To determine the safe surgical resection distance and margin status of OSCC, a set of 14 lipid ions that gradually decreased from tumour to normal tissue was assigned higher weight coefficients in the Lasso model. The safe surgical resection distance of OSCC was measured using the developed 14 lipid ion molecular diagnostic model for clinical reference. The overall accuracy of predicting tumours, positive margins, and negative margins was 92.6%. Interpretation The spatial segmentation results based on our diagnostic model not only clearly delineated the tumour and normal tissue, but also distinguished the different status of surgical margins. Meanwhile, the safe surgical resection margin of OSCC on frozen sections can also be accurately measured using the developed diagnostic model. Funding This study was supported by Nanjing Municipal Key Medical Laboratory Constructional Project Funding (since 2016) and the Centre of Nanjing Clinical Medicine Tumour (since 2014).

Dysregulated amino acids metabolism reciprocally interplays with evolutionary phenotypic characteristics of cancer cells to enhance metastasis. The high metastasis potential of oral squamous cell carcinoma (OSCC) can manifest with perineural invasion (PNI). We here aimed to determine the role of amino acids metabolism in OSCCs with different PNI statuses. Targeted metabolomics was used to quantify 48 amino acids in 20 fresh OSCC samples and 25 amino acids were successfully detected, within which 9 were significantly up-regulated in PNI positive (PNI+) samples. As its highest area under the curve value (0.9063), l-asparagine was selected as the biomarker to distinguish PNI+ from PNI negative (PNI−). Then, the key enzyme of l-asparagine, asparagine synthetase (ASNS), was investigated using immunohistochemistry with 86 OSCC patients. The results showed that ASNS mainly expressed in tumor epitheliums and positively correlated with lymph node metastasis and PNI. Moreover, subgroup survival analysis revealed that ASNS expression combined with PNI status significantly improved their prognostic value, which was confirmed by the TCGA OSCC cohort (n = 279). To validate whether ASNS promotes PNI, we determined ASNS expression levels in five OSCC cell lines and one normal oral keratinocyte, and HSC3 showed the lowest ASNS level but CAL33 had the highest. Therefore, HSC3 and CAL33 (or PBS as control) were selected and injected separately into sciatic nerves to construct the in vivo PNI mouse models. Although both models eventually developed the hind-limb paralysis, nerve dysfunction in the CAL33 model progressed significantly earlier than HSC3 (Day 9 vs. Day 24). Besides, CAL33 migrated significantly farther than HSC3 in the nerve microenvironment (P = 0.0003), indicating high ASNS expression is indispensable for OSCC progression, especially PNI formation, through l-asparagine metabolism alteration. This study provides novel insights into how amino acids metabolism disorders alter tumor neurotropism which helps cancer metastasis.

Orthodontic force-induced osteogenic differentiation and bone formation at tension side play a pivotal role in orthodontic tooth movement (OTM). Platelet-derived growth factor-BB (PDGF-BB) is a clinically proven growth factor during bone regeneration process with unclear mechanisms. Fibroblasts in periodontal ligament (PDL) are considered to be mechanosensitive under orthodontic force. Thus, we established OTM model to investigate the correlation between PDGF-BB and fibroblasts during bone regeneration at tension side. We confirmed that tensile force stimulated PDL cells to induce osteogenic differentiation via Runx-2, OCN up-regulation, and to accelerate new bone deposition along the periodontium and the alveolar bone interface. Interestingly, PDGF-BB level was remarkably enhanced at tension side during OTM in parallel with up-regulated PDGFRβ+/α-SMA+ fibroblasts in PDL by immunohistochemistry. Moreover, orthodontic force-treated primary fibroblasts from PDL were isolated and, cultured in vitro, which showed similar morphology and phenotype with control fibroblasts without OTM treatment. PDGFRβ expression was confirmed to be increased in orthodontic force-treated fibroblasts by immunofluorescence and flow cytometry. Bioinformatics analysis identified that PDGF-BB/PDGFRβ signals were relevant to the activation of JAK/STAT3 signals. The protein expression of JAK2 and STAT3 was elevated in PDL of tension side. Importantly, in vivo, the treatment of the inhibitors (imatinib and AG490) for PDGFRβ and JAK–STAT signals were capable of attenuating the tooth movement. The osteogenic differentiation and bone regeneration in tension side were down-regulated upon the treatment of inhibitors during OTM. Meanwhile, the expressions of PDGFRβ, JAK2 and STAT3 were inhibited by imatinib and AG490. Thus, we concluded that tensile force-induced PDGF-BB activated JAK2/STAT3 signals in PDGFRβ+ fibroblasts in bone formation during OTM.

A multimodal drug delivery system targeting the tumor microenvironment is an inspiring method for treating cancer tissues, including oral squamous cell carcinomas (OSCC). Such approaches require an efficient and safe drug carrier. Bovine milk derived exosomes are ideal because the source is adequate and have advantages of both synthetic and cell-mediated nano carriers. In the present study, we developed a pH/light sensitive drug system based on milk-exosomes for OSCC therapy. It was called exosome–doxorubicin–anthracene endoperoxide derivative (Exo@Dox–EPT1, NPs). Milk-exosomes were conjugated to doxorubicin (Dox) by a pH-cleavable bond, which can rapture under an acidic microenvironment. Besides, endoperoxides and chlorin e6 (Ce6) were also loaded and the endoperoxides undergo thermal cycloreversion and release singlet oxygen to kill cancer cells. We have also investigated the body distribution, antitumor effects, and biocompatibility of the nanoparticles. The new milk-exosome-based drug delivery system showed controlled drug-release, biocompatibility and, proved to be effective in treating OSCC.

Stromal carcinoma-related fibroblasts (CAFs) are the main type of non-immune cells in the tumor microenvironment (TME). CAFs interact with cancer cells to promote tumor proliferation. Long non-coding RNAs (lncRNAs) are known to regulate cell growth, apoptosis and metastasis of cancer cells, but their role in stromal cells is unclear. Using RNA sequencing, we identified a stromal lncRNA signature during the transformation of CAFs from normal fibroblasts (NFs) in oral squamous cell carcinoma (OSCC). We uncovered an uncharacterized lncRNA, FLJ22447, which was remarkably up-regulated in CAFs, referred to LncRNA-CAF (Lnc-CAF) hereafter. Interleukin-33 (IL-33) was mainly located in the stroma and positively co-expressed with Lnc-CAF to elevate the expression of CAF markers (α-SMA, vimentin and N-cadherin) in fibroblasts. In a co-culture system, IL-33 knockdown impaired Lnc-CAF-mediated stromal fibroblast activation, leading to decreased proliferation of tumor cells. Mechanistically, Lnc-CAF up-regulated IL-33 levels and prevented p62-dependent autophagy-lysosome degradation of IL-33, which was independent of LncRNA-protein scaffold effects. Treatment with the autophagy inducer, rapamycin, impaired the proliferative effect of Lnc-CAF/IL-33 by promoting IL-33 degradation. In turn, tumor cells further increased Lnc-CAF levels in stromal fibroblasts via exosomal Lnc-CAF. In patients with OSCC, high Lnc-CAF/IL-33 expression correlated with high TNM stage (n = 140). Moreover, high Lnc-CAF expression predicted poor prognosis. In vivo, Lnc-CAF knockdown restricted tumor growth and was associated with decreased Ki-67 expression and α-SMA+ CAF in the stroma. In conclusion, we identified a stromal lncRNA signature, which reprograms NFs to CAFs via Lnc-CAF/IL-33 and promotes OSCC development.

The weakened tumour colonization of attenuated Salmonella has severely hampered its clinical development. In this study, we investigated whether an anti-inflammation and antiangiogenesis compound triptolide could improve the efficacy of VNP20009, a highly attenuated Salmonella strain, against mice melanoma. By comparing the effects of conventional VNP20009 monotherapy and a combination therapy that uses both triptolide and VNP20009, we found that triptolide significantly improved the tumour colonization of VNP20009 by reducing the number of infiltrated neutrophils in the melanoma, which led to a larger necrotic area in the melanoma. Moreover, the combination therapy suppressed tumour angiogenesis by reducing the expression of VEGF in a synergistic manner, retarding the growth of the melanoma. Our study revealed that triptolide could significantly enhance the antitumour effect of VNP20009 by modulating tumour angiogenesis and the host immune response, providing a new understanding of the strategy to improve Salmonella-mediated tumour therapy.

The traditional maximum dose density chemotherapy renders the tumor patients not only the tumor remission but the chemotherapy resistance and more adverse side effects. According to the widely positive expression of Toll-like receptor (TLR)-3 in oral squamous cell carcinoma (OSCC) patients (n = 166), we here provided an alternative strategy involved the orderly treatment of TLR3 agonist polyinosine–polycytidylic acid (PIC) and low-dose cisplatin. The optimal dose of cisplatin, the novel role of PIC and the side effects of the combined chemotherapy were determined in vitro and in distinct human tumor models in vivo. The results in vitro indicated that preculture with PIC downregulated drug transporters (e.g., P-gp and MRP-1) and increased the cytoplasmic residence of cisplatin, and dramatically strengthened the low-dose cisplatin-induced cell death in TLR3- and caspase-3–dependent manner. Meanwhile, the spleen immunocytes were activated but the immunosuppressive cancer-associated fibroblasts (CAF) were dampened. These findings were confirmed in human tumor models in vivo. Pretreatment with PIC promoted the low-dose cisplatin residence for tumor regression with decreased myeloid-suppressive cells (MDSC), tumor-associated macrophages (TAM) and CAFs, and alleviated adverse side effects in the OSCC model, which was further enhanced by the Cetuximab safely. This strategy also repressed the progression of melanoma and lymphoma. Moreover, TLR3 negatively manipulated the inflammation-related long noncoding RNA lnc-IL7R, which was upregulated during this chemotherapy. Knockdown of lnc-IL7R improved the chemotherapy sensitivity. Overall, this study provided preclinically new instructions for the PIC/cisplatin utilization to target tumor microenvironment and strengthen the low-dose cisplatin-based chemotherapy with reduced side effects. Mol Cancer Ther; 16(6); 1068–79. ©2017 AACR.