ITGB2-mediated metabolic switch in CAFs promotes OSCC proliferation by oxidation of NADH in mitochondrial oxidative phosphorylation system.

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.

Amino acids signatures of distance-related surgical margins of oral squamous cell carcinoma.

BACKGROUND: Histological assessment of resected margins has some drawbacks. We therefore aimed to identify a panel of metabolic markers for evaluating the surgical margins of oral squamous cell carcinoma during surgery. METHODS: A total of 28 case of OSCC samples were enrolled in the study. Gas chromatography-mass spectrometry based untargeted metabolic analysis was employed to acquire the metabolic perturbation of the distance-related surgical margins in the development group. The acquired MS data were then subjected to univariate and multivariate analysis by MetaboAnalyst. Ultra-high performance liquid chromatography-tandem mass spectrometerbased targeted metabolomics for quantitative analysis of the validation group was performed to verify the results of the development group. Another 60 OSCC patients with dysplastic surgical margins were used to further validate the results of the development group by immunohistochemical examination of key enzyme expression, and correlate them with clinicopathological parameters and clinical outcomes. FINDINGS: We finally identified 4 amino acids as negative margin markers, and 6 amino acids as dysplastic margin markers. IHC analysis showed that asparagine synthetase positive expression in dysplastic surgical margins and its higher expression was correlated with tumor recurrence and local relapse-free survival. INTERPRETATIONS: We developed a panel of metabolic molecular markers to supplement the evaluation of negative and dysplastic margins. FUND: This study was supported by Nanjing Municipal Key Medical Laboratory Constructional Project Funding (Since 2012); Center of Nanjing Clinical Medicine Tumor (Since 2014). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

A near infrared light-triggered human serum albumin drug delivery system with coordination bonding of indocyanine green and cisplatin for targeting photochemistry therapy against oral squamous cell cancer.

To ensure site-specific drug delivery/release in tumor cells and cancer-associated fibroblasts (CAFs) and reduce the systemic toxicity of chemotherapy, a novel drug delivery system called human serum albumin-indocyanine green-cisplatin nanoparticles (HSA-ICG-DDP NPs) was developed in our study. We characterized this system in vitro and in vivo and showed synergistic effects with photodynamic therapy (PDT), photothermal therapy (PTT) and chemotherapy; thereby it can significantly improve therapeutic efficacy compared with cancer monotherapy. High expression of secreted protein acidic and rich in cysteine (SPARC) in oral squamous cell cancer (OSCC) and CAFs was also confirmed in our study. Our study also found that the cellular uptake of HSA-ICG-DDP NPs in tumor cells and CAFs can be enhanced by SPARC-mediated endocytosis. Cisplatin (DDP) release from the NPs in the tumor site can be precisely triggered by the cleavage of the coordination bond of ICG-DDP via a near infrared (NIR)-induced photothermal effect of ICG. Treatment with HSA-ICG-DDP NPs induced generation of reactive oxygen species (ROS) and cytotoxicity in SPARC-highly expressed tumor and CAFs. On in vivo treatment, HSA-ICG-DDP NPs were accumulated within the tumor tissue, where they exhibited stronger antitumor effects, compared to treatment with ICG, HSA-ICG and DDP. Therefore, this novel NIR-triggered drug release system displays potential for the improvement of OSCC treatment through its synergistic effects of PTT/PDT and chemotherapy.

Novel acceptor-donor-acceptor structured small molecule-based nanoparticles for highly efficient photothermal therapy.

Herein, acceptor-donor-acceptor structured small molecule (ITIC) based nanoparticles (NPs) were explored for photothermal therapy application. ITIC NPs show red-shift absorption in the near-infrared region, high photostability, and high photothermal conversion efficiency under laser irradiation, presenting both excellent cancer cell cytotoxicity in vitro and tumor ablation in vivo.

A Novel Multimodal NIR-II Nanoprobe for the Detection of Metastatic Lymph Nodes and Targeting Chemo-Photothermal Therapy in Oral Squamous Cell Carcinoma.

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.

Modulation of Salmonella Tumor-Colonization and Intratumoral Anti-angiogenesis by Triptolide and Its Mechanism.

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 Effect of Superparamagnetic Iron Oxide Nanoparticle Surface Charge on Antigen Cross-Presentation.

Magnetic nanoparticles (NPs) of superparamagnetic iron oxide (SPIO) have been explored for different kinds of applications in biomedicine, mechanics, and information. Here, we explored the synthetic SPIO NPs as an adjuvant on antigen cross-presentation ability by enhancing the intracellular delivery of antigens into antigen presenting cells (APCs). Particles with different chemical modifications and surface charges were used to study the mechanism of action of antigen delivery. Specifically, two types of magnetic NPs, γFe2O3/APTS (3-aminopropyltrimethoxysilane) NPs and γFe2O3/DMSA (meso-2, 3-Dimercaptosuccinic acid) NPs, with the same crystal structure, magnetic properties, and size distribution were prepared. Then, the promotion of T-cell activation via dendritic cells (DCs) was compared among different charged antigen coated NPs. Moreover, the activation of the autophagy, cytosolic delivery of the antigens, and antigen degradation mediated by the proteasome and lysosome were measured. Our results indicated that positive charged γFe2O3/APTS NPs, but not negative charged γFe2O3/DMSA NPs, enhanced the cross-presentation ability of DCs. Increased cross-presentation ability induced by γFe2O3/APTS NPs was associated with increased cytosolic antigen delivery. On the contrary, γFe2O3/DMSA NPs was associated with rapid autophagy. Overall, our results suggest that antigen delivered in cytoplasm induced by positive charged particles is beneficial for antigen cross-presentation and T-cell activation. NPs modified with different chemistries exhibit diverse biological properties and differ greatly in their adjuvant potentials. Thus, it should be carefully considered many different effects of NPs to design effective and safe adjuvants.

Proteomic screening of anaerobically regulated promoters from Salmonella and its antitumor applications.

Solid tumors often contain hypoxic and necrotic areas that can be targeted by attenuated Salmonella typhimurium VNP20009 (VNP). We sought to develop a hypoxia- inducible promoter system based on the tumor-specific delivered strain VNP to confine expression of therapeutic gene specifically or selectively within the tumor microenvironment. A hypoxia-inducible promoter - adhE promoter was screened from the hypoxia-regulated endogenous proteins of Salmonella through two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time-of-flight/time-of-flight MS-based proteomics approaches. The efficiency and specificity of the selected adhE promoter were validated first in both bacteria and animal tumor models. The adhE promoter could specifically drive GFP gene expression under hypoxia, but not under normoxia. Furthermore, luciferase reporter expression controlled by the system was also confined to the tumors. Finally, we investigated the anticancer efficacy of VNP delivering human endostatin controlled by our adhE promoter system in both murine melanoma and Lewis lung carcinoma models. Our results demonstrated that by the dual effects of tumoricidal and anti-angiogenic activities, the recombinant Salmonella strain could generate enhanced antitumor effects compared with those of unarmed VNP treatment or untreated control. The recombinant VNP could retard tumor growth significantly and extend survival of tumor-bearing mice by inducing more apoptosis and more severe necrosis as well as inhibiting blood vessel density within tumors. Therefore, VNP carrying the endostatin gene under our tumor-targeted expression system holds promise for the treatment of solid tumors.

A monoclonal-antibody-based ELISA for the detection of human FADD (Fas-associated death domain).

FADD (Fas-associated death domain) has been widely expressed in various tissues and its expression has been recently demonstrated to correlate with tumour progression and prognosis. Currently, measurement of FADD expression mainly depends on Western-blot or immunohistochemical approaches. To develop a conventional sandwich ELISA avenue for the detection of FADD protein to supplement Western blotting or immunohistochemistry, a series of mAbs (monoclonal antibodies) specific for FADD protein, designated 3A3, 3F9, 3G4, 4B9, 4G1, 7A8, 7B8 and 7F4, were produced by fusing mouse s/p20 myeloma cells with the spleen cells of a mouse immunized with the Escherichia coli-expressed recombinant His(6)-FADD protein. On the basis of the characterization of these mAbs, purified 3F9 was selected as the capture antibody and the biotin-conjugated 3A3 was selected as the detection antibody in sandwich ELISA. The limit of detection for the ELISA was 0.3 ng of purified His(6)-FADD (FADD tagged with hexahistidine), and it could detect both recombinant and native human FADD protein. Furthermore, the positive reaction of the ELISA could be blocked by rabbit anti-FADD sera. All of these results indicated that the ELISA developed in the present paper could be a promising tool for detection of FADD protein.