Ultrafast and broad spectral bandwidth photodetectors are desirable attributable to their unique bandstructures. Photodetectors based on graphene have great potential due to graphene’s outstanding optical and electrical properties. However, the highest reported values of the photoresponsivity of pure graphene are less than 10 mA/W at room temperature, which significantly limits its potential applications. Here, we report a photoresponsivity of 32 A/W in pure monolayer graphene photodetectors, an improvement of over one order of magnitude for functional graphene nanostructures (<3 A/W). The high photocurrent generation in our devices can be attributed to the high sensitivity of graphene’s resistivity to a local change of the electric field induced by photo-excited carriers generated in the light-doping substrate. This dramatically increases the feasibility of using graphene for the next generation of photodetectors. As a novel two-dimensional (2D) material, graphene possesses a unique conic band structure –3 and at2) substrate. We demonstrate a higher photoresponsivity at room temperature at visible range. The photoresponsivity value reaches a maximum of 32 AW−1 at room temperature measured from biased source-drain and back-gate voltage modulation, one order of magnitude higher than the values reported in recently published works. Our work potentially reveals the importance of the effects of the doping substrate for graphene photodetectors and offers insight on how to optimize pure graphene-based photodetectors and transistors for optoelectronic and electronic practical applications.To date, work on creating graphene photodetectors with a high response speed and a broad spectral range at room temperature is still ongoing. More recent efforts concentrate on graphene functionalization techniques by creating a bandgap to enhance the photoresponsivity. However, not much work has been done on understanding and controlling the substrate effect on graphene photodetectors. Several known mechanisms contribute to photocurrent generation in biased graphene with a heavily doped silicon substrate , 24, 26.2 dielectric and confirmed with Raman spectroscopy. In order to detect the excessive photo field-effect, we used a lightly doped (p-type 10–20 ohm-cm) silicon dioxide on silicon substrate. In situ sample cleaning by the electric field were performed to clean the surface, enhance carrier mobility, and reduce intrinsic defects. After this treatment, the Dirac point of the devices were near 25 V. The photocurrent measurements were performed with a helium–neon laser with a wavelength of λ ≈ 632 nm at room temperature. Incident laser power values were fixed at 5 mW, and the frequency of the laser pulse could be adjusted from 5–5000 Hz. The laser spot diameter on the sample could be adjusted from d ≈ 1 μm to 1 mm using a microscope objective lens. The photoresponsivity data was collected with a semiconductor device analyzer . Considering the electric field enhancement attributable to the gate stack, we calculated that the absorption of the incident light in the graphene device was 2.5%. The photocurrent amplitudes shown are peak-to-background values throughout. The photoresponsivity measurements were carried out on multiple samples.Graphene photodetectors with an FET configuration were fabricated by mechanical exfoliation from bulk highly oriented pyrolytic graphite . Photolithography and e-beam evaporation of 5 and 80 nm of chromium and gold, respectively, were used to create the source and drain electrodes. The sample of monolayer graphene was identified by optical contrast on the 300 nm SiOI–V) characteristics of the graphene photodetector with back-gate bias (VG) varying from −10 to 10 V. Note that the current–voltage (I–V) with respect to Vs-d = 0 is asymmetric; this asymmetric behavior indicates that an intrinsic internal electric field  exists in the device. This plays an important role in the overall photocurrent response of graphene photodetectors, possibly as a result of both fabrication-induced defects and potential barriers between the edges of the graphene sheets. The inset in Fig. pD2/pG > 1 confirms that the sample is monolayer graphene. In addition, as the D peak is not seen in the Raman spectrum, the graphene sample must be relatively free of defects , P is the incident laser power [W], SG and SL are the area of the sample and laser spot, respectively, and λincident is the laser wavelength. The laser-induced charge carrier generation and subsequent separation at the graphene–metal interface results in photocurrent generation. When light is incident on the interface between the graphene and the electrodes, the photo-excited electron–hole pairs are generated and accelerated in opposite directions by the internal electric field, thus generating an observable photocurrent Iph = VOC/Rg .We investigated the photoresponsivity of the pure graphene photodetector device (refer to Sample A in the latter part of the paper) at source-drain voltage see Fig. . In thesVG = − 1V. As shown in Fig. Iph and photoresponsivity are strongly dependent on the size of the laser spot. The photocurrent Iph increases linearly with the laser spot size and saturates at spot size d = 1.0 mm. Such behavior indicates that the photocurrent originates from several different mechanisms [2 interface under illumination without a source-drain bias, leading to a photogating effect in the graphene FET [Iph reduces to 10 nA. This value agrees with the previously reported results of a low photoresponsivity of pure graphene of about 10 mAW−1 [To clarify the origin of the photocurrent, we investigated the photocurrent dependence on the laser spot size. This allowed us to clearly identify the photocurrent contributions from both the silicon substrate and the graphene–electrode interfaces, where internal electric fields are produced and separate the photo-generated carriers . In realchanisms , 25, 26.hene FET , 34. TheOwing to the work function mismatch between silica and silicon, the valence and conduction bands in silicon bend at the interface . In our VG stays fixed at 0 V) of sample A are shown in Fig. I is the sum of the photocurrent and dark current: I = Iph + Id. Interestingly, the photocurrent Iph increases with increasing source-drain bias voltage , but decreases for 0 > Vs − d > − 55mV and changes photocurrent sign at Vs − d < − 55mV  and gain (G) rise linearly and saturate at Vs − d = 2V. A maximum photoresponsivity of S = 0.17 AW− 1 is achieved at room temperature at a source-drain bias of Vs-d = 2000 mV, representing a 17-fold improvement over our previously reported value for our graphene photodetector at room temperature. The enhanced photoresponsivity with the addition of the substrate indicates that the lightly doped substrate significantly improves the functionality of the graphene photodetector. This result holds promise for prospective applications in a new era of high-performance optoelectronic devices.Figure V ≤ Vg ≤ 12 V) applied to the silicon back-gate at Vs − d = 5mV. With a positively biased back-gate voltage, the photocurrent Iph decreases with increasing back-gate bias voltage. In contrast, the photocurrent Iph increases with the negative back-gate biases. Figure S = 1.38 AW− 1 is obtained at a back-gate bias of VG = − 12V. These observed values are larger than those reported in literature based on pure monolayer graphene-based photodetectors at room temperature with bias voltage. The increasing photocurrent with the decreasing back-gate voltage can be attributed to the hole impact ionization effect with respect to the external electrical field. As the gate bias decreases, more holes are involved in the impact ionization process, generating more electron–hole pairs. The observed results indicate that the back-gate voltage induced doping of the graphene channel and shifted the position of the Fermi level. Interestingly, the photo-induced current and the bias back-gate voltage are linearly related, indicating that higher photoresponsivity can be readily obtained by applying a larger negative bias back-gate voltage. Furthermore, the photoresponsivity and gain can be gate-modulated, providing a convenient on-off switching control for the graphene photodetector.Moreover, the external photoresponsivity and gain can also be enhanced by applying a back-gate bias within the photocurrent generation path and by augmenting the interaction between graphene and the incident light. Figure VG) and source-drain (Vs-d) voltages. Moreover, the proposed configuration is superior to the previously reported structure, and a larger scale of the proposed device can be easily fabricated. Further device performance improvement can also be obtained through plasmonic nanostructures and waveguide-integrated configurations, providing that a method of growing high-quality graphene with high carrier mobility that is compatible with modern semiconductor technologies can be developed.In conclusion, we have demonstrated a highly effective method of increasing the sensitivity of a pure monolayer graphene photodetector by using lightly p-doped silicon dioxide on silicon as the substrate. Our pure graphene photodetector exhibits an increased photoresponsivity, one order of magnitude higher than that of similar graphene photodetectors in previous reports at room temperature. The observed phenomena in our experiments point to a clear pathway toward practical applications of pure graphene in the design of photodetectors that can be manipulated by back-gate (


PIN+]. Although overexpression of Sis1 altered the TDP-43 aggregation pattern, we did not detect physical association of Sis1 with TDP-43, suggesting the possibility of indirect effects on TDP-43 aggregation. Furthermore, overexpression of the mammalian Sis1 homologue, DNAJB1, relieves TDP-43 mediated toxicity in primary rodent cortical neurons, suggesting that Sis1 and its homologues may have neuroprotective effects in ALS.Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective loss of motor neurons with inclusions frequently containing the RNA/DNA binding protein TDP-43. Using a yeast model of ALS exhibiting TDP-43 dependent toxicity, we now show that TDP-43 overexpression dramatically alters cell shape and reduces ubiquitin dependent proteolysis of a reporter construct. Furthermore, we show that an excess of the Hsp40 chaperone, Sis1, reduced TDP-43’s effect on toxicity, cell shape and proteolysis. The strength of these effects was influenced by the presence of the endogenous yeast prion, [ Many neurodegenerative diseases are associated with aggregation of specific proteins. Thus we are interested in factors that influence the aggregation and how the aggregated proteins are associated with pathology. Here, we study a protein called TDP-43 that is frequently aggregated in the neurons of patients with amyotrophic lateral sclerosis (ALS). TDP-43 aggregates and is toxic when expressed in yeast, providing a useful model for ALS. Remarkably, a protein that modified TDP-43 toxicity in yeast successfully predicted a new ALS susceptibility gene in humans. We now report a new modifier of TDP-43 toxicity, Sis1. We show that expression of TDP-43 in yeast inhibits degradation of damaged protein, while overexpression of Sis1 restores degradation. Thus suggests a link between protein degradation and TDP-43 toxicity. Furthermore we show that a mammalian protein similar to Sis1 reduces TDP-43 toxicity in primary rodent neurons. This identifies the mammalian Sis1-like gene as a new ALS therapeutic target and possible susceptibility gene. In aPIN+] dependent polyQ aggregation and toxicity  dependent toxicity of an overexpressed prion-like protein Pin4C. When overexpressed in the presence of [PIN+], Pin4C formed large hyperphosphorylated aggregates that co-localized with Sis1 and reduced degradation of a ubiquitin proteasome system (UPS) reporter protein. Aggregation, hyperphosphorylation and toxicity were suppressed by overexpression of Sis1  dependent toxicity caused by overexpression of the [PIN+] prion protein, Rnq1. Here, overexpressed Sis1 appears to enhance aggregation of toxic detergent soluble Rnq1 oligomers into apparently non-toxic larger detergent resistant aggregates in the nucleus  cells [PIN+] and [pin-] yeast [PIN+] and [pin-] cells  ,49–51 wepin-] and [PIN+] cells in three strain backgrounds. The rescue appeared to be weaker in 74D-694 [PIN+] cells where TDP-43 is more toxic PIN+], bePIN+] cells reduced degradation of a UPS reporter, CG*  and [PIN+] 74D-694 cells but was more dramatic in [PIN+] cells where TDP-43 is more toxic. Furthermore, simultaneous overexpression of Sis1 and TDP-43 resulted in rapid degradation of CG*. This is consistent with the hypothesis that TDP-43 toxicity in part results from interference with the UPS system.Previous results showed that overexpression of polyQ or Pin4C in  than [pin-] 74D-694 cells and likewise has a greater effect on UPS in [PIN+] vs. [pin-] cells. By analogy, the presence vs. absence of heterologous aggregates in mammalian cells may also influence the proteostasis of disease-causing aggregates.Aggregation of TDP-43 is frequently associated with ALS and other neurological diseases –79 and oPIN+] effects toxicity and UPS inhibition is unknown. While [PIN+] dramatically enhances aggregation and toxicity of polyQ and Pin4C [PIN+] vs. [pin-] cells are indistinguishable. Nonetheless, the fact that [PIN+] prion aggregates sequester some essential chaperones including Sis1 [PIN+] cells more vulnerable to TDP-43 overexpression, e.g. the lower level of Sis1 available for the UPS in [PIN+] cells could make cells more sensitive to the inhibitory effect of TDP-43 on the UPS. Indeed, we found that overexpression of Sis1 reduces the deleterious effects of TDP-43 on cell growth, cell shape and UPS inhibition. Several genetic screens in yeast have identified modifiers of TDP-43 toxicity; however, Sis1 was not among them  and other unknown differences between strains alter the toxicity of TDP-43, repeating screens for toxicity modifiers in additional yeast strain backgrounds may reveal new modifiers.Why and how  cells  cells . In that+] cells ,38,66,71Thus, the mechanism by which Sis1 rescues cells from TDP-43 toxicity appears to be distinct from its rescue of polyQ, Pin4C or Rnq1 toxicity. Previous studies implicated a direct effect of a different HSP40 chaperone, DNAJB6 (a member of the DNAJB7 and 8 family) on TDP-43 via interactions with the TDP-43 C-terminal domain ,83. In cDBR1 knockdown)  versions of strains were obtained on YPD plates with 5 mM GuHCl and were confirmed to be [pin-] by checking for lack of aggregates after transforming with an Rnq1-GFP plasmid  and [pin-] versions of 74D-694, W303 and BY4741 were doubly transformed with GAL1 controlled TDP-43-DsRed, GAL1 controlled modifier or control empty vector plasmids listed below. Transformants were selected on SD-Leu-Ura plates. Normalized suspensions of cells taken from SD-Leu-Ura were 10X serially diluted in water and 15 μl were spotted on SD-Leu-Ura (dextrose), and 2% Gal-Leu-Ura  plates, which were photographed after 3 (dextrose) or 5  days of incubation at 30°C. We show hUpf1 because it had a bigger effect than the yeast homologue Ecm32 found in the initial screen  and [pin-] versions of 74D-694 (A), BY4741 (B) and W303 (C) carrying pGAL1-TDP-43-DsRed (p2173) or pAG415 GAL1-ccdB-DsRed (p2302) were grown on 2% galactose plates for 4 days and then imaged at the same magnification. Shown are representative fields of many fields photographed and examined.[(PDF)Click here for additional data file.S3 Figpin-] 74D-694 with pTETR-TDP-43-YFP (p2223) were selected on SD-Trp supplemented with doxycycline (10 μg/ml). Transformants were then grown in liquid SGal-Trp media with the indicated amount of doxycycline for 24 h and were examined and photographed at same magnification. The levels of TDP-43-YFP were determined by immunoblotting SDS-PAGE gels of normalized cell lysates probed with anti-TDP-43 antibodies, and anti-Pgk1 antibodies as an internal loading control.Transformants of [(PDF)Click here for additional data file.S4 FigPIN+] and [pin-] 74D-694 doubly transformed with pGAL1-TDP-43-DsRed (p2173) and pGAL-Sis1 (p1759) were grown in plasmid selective synthetic liquid media containing 2% galactose and 2% raffinose for 2 days. Cells were then crossed to [pin-] 64D-694 MATα (L2642) bearing plasmid p1185 (pCUP1-RNQ1-GFP). Diploids were selected on SD-Leu-Ura-Trp, expressed on SD-Leu-Ura-Trp supplemented with Cu++ (50 μg/ml), and examined for Rnq1-GFP dots or diffuse fluorescence, which was diagnostic for the presence or absence, respectively, of the [PIN+] prion.[(PDF)Click here for additional data file.S5 FigHSP104 does not prevent Sis1 overexpression from reducing TDP-43 toxicity. Isogenic [psi-] [pin-] versions of 74D-694 with (hsp104Δ) and without (WT) a deletion of HSP104 doubly transformed with p2042 (pGAL1-TDP-43-YFP) and p1767 (pGAL1-SIS1) or vector control p1752 (pGAL1-YFP) and p1768 (pGAL1) were selected on SD-Ura-Trp plates. Normalized suspensions of cells taken from SD-Ura-Trp were 10X serially diluted in water and 15 μl were spotted on SD-Ura-Trp (dextrose), and 2% SGal-Ura-Trp  plates which were photographed after 3 (dextrose) or 7  days of incubation at 30°C. B. Reduction of Ssa1 activity does not prevent Sis1 overexpression from reducing TDP-43 toxicity. Strain 1014 bearing a deletion of SSA2 and expressing a dominant negative allele of SSA1 (SSA1-21 ssa2Δ) and its isogenic parent strain, L3504 (WT) were doubly transformed with pGAL1-TDP-43-DsRed (p2173), pGAL-SIS1 (p1759), or vector controls (p2302 or p484). Normalized suspensions of cells taken from plasmid selective SD-Leu-Ura medium were 10X serially diluted in water and 15 μl were spotted on SD-Leu-Ura (dextrose), and 2% Gal-Leu-Ura  plates, which were photographed after 3 (dextrose) or 5  days of incubation at 30°C.A. Deletion of (PDF)Click here for additional data file.S6 Fig-5; # HR 3.18, p < 2x10-16, Cox proportional hazards analysis. Results were pooled from two independent experiments.*Rodent primary cortical neurons were dissected and transfected with plasmids encoding EGFP and TDP-43(WT)-mApple or mApple. In each case, neurons were also transfected with scrambled siRNA or siRNA targeting DNAJB1. (A) Knockdown was validated by immunocytochemistry using antibodies against DNAJB1. Scale bar, 50 μm. (B) Transfection with siRNA against DNAJB1 resulted in a 60% reduction in anti-DNAJB1 antibody reactivity  In longitudinal assays of neuronal survival, DNAJB1 knockdown enhanced the risk of death by 20% in control neurons expressing EGFP alone and in neurons overexpressing TDP43. * HR 1.20, p 0.004; ** HR 1.21, p 2.3x10(PDF)Click here for additional data file.


Patients (N = 165) with AGC treated at Peking University Cancer Hospital from October 2008 to February 2013 were retrospectively studied. Tissue samples prior to chemotherapy were assessed for PD-L1 expression using RNA in situ hybridization (an RNAscope assay) and immunohistochemistry (IHC). The correlations of PD-L1 expression to patient characteristics and clinical outcomes were statistically analyzed. PD-L1 mRNA signals were located in tumor compartments or the mesenchyme in a brown dotted or clustered pattern, and PD-L1 mRNA expression in gastric cancer was heterogeneous. PD-L1-positive expressions were observed in 33.9% (56/165) and 35.1% (46/131) patients in mRNA level and protein level, respectively. A positive relationship was found between PD-L1 mRNA and PD-L1 protein, and compared to IHC, RNAscope assay could provide an intuitional and quantitative data with potential clinical application. No statistically significant differences occurred between PD-L1 expression and clinical response to chemotherapy, or survival. However, we found that PD-L1 expression was higher in intestinal type than in diffuse type. These findings suggested that the RNAscope assay may be a promising method for patient assessment in gastric cancer clinical trials, which would be illustrated in further study.PD-L1 expression may be a predictive marker for anti-PD-1 therapeutic efficacy. No standard detection method of PD-L1 expression was available for advanced gastric cancer (AGC), which would be investigated in this study using RNA  Gastric cancer is one of the most lethal cancers, partially due to few effective therapies. Despite substantial efforts to develop better treatments, gastric cancer prognoses, especially that which is advanced is poor . To allehttp://www.clinicaltrials.gov/).Activation of the PD-1/PD-L1 signaling pathway may cause an immunosuppressive tumor microenvironment that protects tumor cells from immune surveillance and killing , 5. ThusAs a main target of anti-PD-1/PD-L1 therapy, PD-L1 expression by tumor cells may be a controversial marker for therapeutic efficacy , 9. Almoin situ hybridization in tumor FFPE tissues using an RNAscope assay is favored for its specificity and interpretative objectivity [Recently, a novel antibody-independent assay for RNA ectivity , 13. In in situ hybridization. Of these, 131 patients had samples evaluable for PD-L1 IHC. The screening diagram of eligible patients is depicted in Figure Overall, 165 patients were eligible for the study and had samples evaluable for PD-L1 RNA PD-L1 mRNA signals were located in tumor compartment or mesenchyme by brown dotted or clustered patterns, and PD-L1 mRNA expression in gastric cancer was heterogeneous Figure .No standard scoring criteria for PD-L1 expression in gastric cancer was determined, so we adopted criteria from the literature. First, PD-L1 mRNA expression occurred in 33.9% patients based on positive signals. Second, the PD-L1 mRNA positivity threshold was defined as PD-L1 expression in the mesenchyme or ≥ 1% tumor cells according to criteria from the KEYNOTE-012 trial . So 33.9P = 0.122, McNemar's test; Among all eligible patients, 131 patients had samples evaluable for PD-L1 IHC, and 46 (35.1%) patients presented PD-L1-positive expression. A positive relationship was found between PD-L1 mRNA and PD-L1 protein (P > 0.05). Positive PD-L1 mRNA expression in patients with gastroesophageal junction exceeded that of patients with non-gastroesophageal junction, but this was not statistically significant .Among all subjects, 93.3% had clinical response evaluations. Patients with partial response (PR), stable disease (SD), and progressive disease (PD) who positively expressed PD-L1 mRNA were 33.9%, 53.6%, and 7.1%, respectively, and these were not significantly different from those  who were negative for PD-L1 mRNA expression. Objective response and disease control rates of both groups were 33.9%, 87.5% and 40.4%, 77.1%, respectively  and 11.9 months (95% CI = 9.5–14.3), respectively , fluorescent in situ hybridization (FISH), and IHC for HER2 amplification and expression in breast cancer [in situ hybridization using RNAscope assay may hold promise for future research due to its specificity, reproducible, and interpretative objectivity.Alba's group compared RNA t cancer . Data sht cancer , 13. In P > 0.05). PD-L1 mRNA expression in patients with gastroesophageal junction exceeded that of patients with non-gastroesophageal junction (43.3% vs. 28.6%), although the difference was not statistically significant (P = 0.054). Also, PD-L1 expression was higher in intestinal type than in diffuse type (P = 0.010), which suggested the chronic inflammation caused by H. pylori infection or EB virus typically related to intestinal type might affect PD-L1 expression. Furthermore, PD-L1 mRNA expression in AGC as not associated with clinical response to first-line chemotherapy, PFS, or OS based on our results.PD-L1 expression has been associated with clinicopathological characteristics and prognosis in different cancers –24. We fIn conclusion, our findings suggested that the RNAscope assay may be a promising method to detect PD-L1 expression in gastric cancer patients, and might play an important role in clinical trials.A total of 165 patients with advanced gastric cancer treated in the Department of Gastroenterology of Peking University Cancer Hospital from October 2008 to February 2013 were retrospectively studied. All patients received first-line fluorouracil-based chemotherapy and had FFPE tumor sections containing at least 100 tumor cells prior to chemotherapy. Subjects provided written informed consent for their samples to be used in the research. Clinical data containing clinicopathological characteristics, clinical response and patient survival retrospectively obtained from medical records, and this study was approved by the ethics committees of Peking University Cancer Hospital.PD-L1 mRNAs in FFPE tumor samples were measured with RNAscope assay  following the manufacturer's instructions. In brief, 5 μm sections were deparaffinized, incubated with pretreatment reagents 1, 2, and 3 and room temperature for 10 min, boiled for 15 min, 40°C for 30 min, respectively. FFPE samples were hybridized with Hs-CD274-probes (ACD) at 40°C for 2 h. Hybridization signals were amplified and visualized with RNAscope 2.0 HD detection kit (Brown). Images were captured with a fluorescent microscope (Nikon ECLIPSE 80i) and the data were analyzed either by scoring or using Spot Studio software.RNAscope results were examined under a standard bright field microscope at 200–400× magnification. Positive signals showed as brown punctuate dots. PPIB and DapB were positive and negative probes, respectively, to control tissue RNA conditions and non-specific hybridization. Figure PD-L1 protein expression was determined by immunohistochemistry (IHC) assay using anti-PD-L1 antibody  according to previous procedure . BrieflyP < 0.05 with two-sided test was considered statistical significant.SPSS 16.0 software was used for statistical analysis. The association between PD-L1 expressions in mRNA level and protein level was analyzed by Kappa and McNemar's tests. The associations between PD-L1 expression and clinical characteristics were analyzed using a Chi-square test or Fisher's exact test. Kaplan-Meier survival curves and a log-rank test were used to assess patient survival.


A stable modulation depth up to −23.26 dB can be achieved. Due to the cooperative effect of the “bright-bright” elements, the amount of the gold strips in each unit cell determines the number of the stop-bands, providing a simple and flexible approach to develop multispectral devices. Further investigations illustrate that the location of the stop bands not only can be adjusted by varying the length of gold strips, but also can be dynamically controlled by tuning the Fermi energy level of graphene, and deep modulation is acquired through designing the carrier mobility. With the sensitivity as high as 2393 nm/RIU of the resonances to the varieties of surrounding medium, the structure is also enabled to be an index based sensor. The results will benefit the on plane or integrated micro-structure research with simple structure and flexible tunability, and can be applied in multi-band stop filters, sensors and other graphene-based multispectral devices.Dynamically tunable band stop filter based on metal-graphene metamaterials is proposed and numerically investigated at mid-infrared frequencies. The proposed filter is constructed by unit cells with simple gold strips on the stack of monolayer graphene and the substrate of BaF By controlling the geometry of microelements in the metamaterial structures, the electric permittivity and magnetic permeability can be arbitrarily designed. Recently, the combination of metamaterials and plasmonics reveals an analogous physical phenomena of EM induced transparency (EIT), which is termed as plasmon induced transparency5 (PIT). The unit cell of PIT based metamaterials usually consists of bright mode and dark mode resonators10. The bright mode or super-radiant mode can be excited directly by an external incident field, and has a large scattering cross section5. In contrast, the dark mode or sub-radiant mode cannot be directly excited, but it can be excited by the local field of the bright mode via near field coupling and exhibits a larger quality factor due to the weak radiation coupling11.Electromagnetic (EM) metamaterials, a kind of artificially structured EM materials, are usually composed of periodically arranged subwavelength microstructures or ‘meta-molecules’, and have attracted great attention for their unique EM properties that the natural materials cannot realize. Some of the most typical meta-molecules are the metal split ring resonators14 and the bright to dark mode coupling16, but usually come with an unsatisfactory modulation depth. Moreover, the optical properties of metamaterials based on metallic structures cannot be changed once the structure is fabricated. To improve above situation, some actively controlled elements, such as liquid crystals19, liquid metals21 and semiconductors14, are applied in these metamaterials devices. In recent years, graphene, a flat monolayer of carbon atoms packed into a dense 2D honeycomb crystal lattice, has become a very promising material for its unique electrical properties24, such as high electron mobility, flexible tunability, relatively low loss and tight field confinement. The most attractive one among these properties is that the conductivity of graphene can be dynamically tuned by changing the Fermi energy through chemical doping26 or electrostatic gating27. Therefore, the combination of graphene and metamaterials can provide an effective approach to improve the dynamically tunable resonant properties of the multispectral devices31.Metamaterials with multiple PIT windows have many promising applications in optical information processing systems, effective switchers and ultrasensitive sensors. Traditional PIT based multispectral devices are usually designed by two approaches: the bright to bright mode couplingIn this work, by introducing a monolayer of graphene into a simple metamaterial structure, dynamically tunable dual-band and multi-band stop filters are proposed and researched. With two gold strips acting as typical bright mode resonators, a dual-band stop filter with an ideal modulation depth as high as −23.26 dB is achieved. To reveal the cooperative effect of the bright mode resonators, the electric field and surface current distributions are further investigated. The number of the stop-band can be easily increased by adding the gold strips in each unit cell, hence a filter with multi-band stop is obtained. The dynamically tunable properties of the proposed filters are realized by adjusting the graphene Fermi energy, and the modulation depth can be designed through varying the carrier mobility. Furthermore, the proposed structure can also be used as an index based sensor for its ultra-high sensitivity to the surrounding medium. All these results demonstrated that the presented metal-graphene based metamaterials provide promising applications in multi-band stop filters, sensors and other graphene-based multispectral devices.2 substrate assuming the refractive index n = 1.46 are separated by a monolayer of graphene and a thin layer of SU-8 photoresist. As shown in Fig. w = 0.5 μm are symmetrically attached on the center of the unit cell and separated from each other by a small gap d = 0.2 μm. The lengths of the short and long gold strips are set as l1 and l2, respectively. The periods of the unit cell are 4.2 μm both on x and y directions. The top metallic pad serves as the electrodes along with the square metallic ring at the back of the dielectric layer to tune the Fermi energy level of the graphene by applying a gate voltage. The thickness of the gold strips and the SU-8 photoresist layer are 75 nm and 300 nm, respectively.The proposed dual-band stop filter is schematically depicted in Fig. 33:w, kB, e is the incident angular frequency, Boltzmann constant, reduced Planck constant and electron charge, respectively. T is the temperature in kelvin, which is fixed to 300 K in this study. The value of chemical potential μc is equal to that of the Fermi energy level Ef when kB < μc, which can be written as n is the carrier concentration and the Fermi velocity is 6 m/s. Hence the Fermi energy is determined by the carrier concentration, which is proportional to the gate voltage Vg on the graphene as n = εdε0Vg/eh, where εd and h is the permittivity and thickness of the insulated substrate material. The relaxation time μ is the carrier mobility. Recent literatures showed that the graphene carrier mobility could reach higher than 105 cm2V−1s−1 in the experiment34, and is set as 2050 cm2V−1s−1 corresponding to the relaxation time Ef = 0.1 eV.In the mid-infrared region, the complex conductivity of monolayer graphene, consisting of interband and intraband contributions, can be given by Kubo formulax-direction (p-polarized) is used as the normal incident light. Figure 5. As depicted by the dash curves in Fig. l = 2.8 μm (black) and 2.0 μm (red) show strong resonance dips at 34.6 THz and 43.3 THz, respectively, which are exactly corresponding to the two resonances of the proposed filter. So, different from the traditional PIT effect of near-field coupling between bright elements and black elements, both the short and long gold strips in the dual-band stop filter are act as bright elements, and the two deep transmission dips are actually resulted from the simple cooperative effect of the two bright elements.A plane wave polarized parallel to the x-component electric field Ex, y-component electric field Ey and the amplitude of electric field |E| with surface current density are calculated and depicted in Fig. In order to further demonstrate the cooperative effect of the gold strips in the proposed structure, distributions of l leads to a significant red shift of the resonance frequency, suggesting that the required stop band can be reached by designing the special length of the gold strips. Figure l2 increases from 2.4 μm to 2.8 μm, while the higher one remains unchanged for the fixed length of short gold strip. Similarly, the varieties of the short gold strip length l1 decreasing from 2.2 μm to 1.8 μm corresponds to a blue shift of the high-order stop band, while the location of the low-order stop band keeps as a constant because of the fixed length l2. The shift of the resonance frequency results a broader transmission band. All of the results are in good agreement with those in Fig. As we noted above, the length of gold strip plays a key role in the location of the transmission dip. To explore this relationship, the resonance frequencies of the single gold strip structure versus its length are displayed in Fig. A(f) = 1 − R(f) − T(f), where T(f) and R(f) is the transmission and reflection coefficient. As shown in Fig. Ef = 0.1 eV and 0.55 eV at the corresponding center frequencies of the low-order stop bands. As expected, stronger confined electric fields are observed around the long gold strip at the Fermi energy of 0.55 eV.As discussed above, the graphene conductivity is a function of the Fermi energy, which can be flexibly tuned by the applied gate voltage between two metallic pads. Figure τ ranging from 0.01 ps to 0.1 ps, corresponding to the values of the graphene carrier mobility μ changing from 1000 cm2V−1s−1 to 10000 cm2V−1s−1. It is evident that the increasing of graphene carrier mobility has no effect on the location of the resonance frequencies, but raises the peak of the transmission band contributing to the increment of the modulation depth, due to the reduction of the graphene absorption. Thus, by introducing the monolayer graphene into the filter, its stop bands can realize dynamical tunability through controlling the graphene Fermi energy, and then a relatively good modulation depth can be achieved by designing the carrier mobility of graphene.Though the structure couldn’t be dynamically adjusted by graphene carrier mobility in the experiment, the modulation depth can be further improved by choosing the monolayer graphene with a high value. When the Fermi energy of graphene is fixed as 0.1 eV, Fig. Note that the resonance frequencies of the proposed structure are sensitive to the refractive index of the surrounding medium. Figure l1 = 1.8 μm, l2 = 2.2 μm and l3 = 2.6 μm. Similarly, the four-band stop structure can be obtained simply by increase the number of gold strips to four as depicted in Fig. Finally, multispectral metamaterials are realized with the Fermi energy and relaxation time are fixed as 0.1 eV and 0.0629 ps, respectively. The diagram of the structure with triple-band stop are shown in Fig. In conclusion, dynamically tunable band stop filters based on metal-graphene metamaterials are investigated at mid-infrared frequencies. The calculated transmission spectrums exhibit broad stop bands, and the number of the stop-band can be increased simply by adding the gold strips in each unit cell. The center frequency of the stop band or the location of resonance not only can be adjusted by varying the length of gold strips, but also can be dynamically controlled through the graphene Fermi energy. The increment of graphene carrier mobility will enhance the amplitude of the transmission peak to realize low loss transmission in pass band. Furthermore, the proposed structure can also be used as an index based sensor for its high sensitivity to the surrounding medium. The results will benefit the on plane or integrated micro-structure research with simple structure and flexible tunability, can be applied in multi-band stop filters, sensors and other graphene-based multispectral devices.x and y directions is performed with the periodic boundary condition. A p-polarized plane wave is used as the incident light. The conductivity of the gold is described by the Drude model with the plasma frequency wp = 1.36 × 1016 rad/s and the scattering rate Γ = 3.33 × 1013 rad/s in this work. The monolayer graphene is modeled as a surface current in the boundary conditions, which is defined as the product of graphene conductivity and the electric field in the frequency domain. The surrounding medium are assume to be uniform with the refractive index nr = 1. The transmission coefficient T(f) and reflection coefficient R(f) are calculated from S-parameters in the simulated results, and given as T(f) = |S21|2, R(f) = |S11|2 respectively.The proposed structures are numerically calculated using finite element method (FEM). In the three-dimensional simulations, Floquet ports with perfectly matched layer (PML) absorbing boundary conditions are used in the z-direction, and the unit cell both in


Hydrophilic graphene sheets were synthesized from a mixture of magnesium and gallic acid (GA) modified graphene oxide (GO) in a self-propagating high-temperature synthesis (SHS) process, and hydrophilic graphene sheets displayed the higher C/O ratio (16.36), outstanding conductivity (~88900 S/m) and excellent water-solubility. GO sheets were connected together by GA, and GA was captured to darn GO structure defects through the formation of hydrogen bonds and ester bonds. In SHS process, the most oxygen ions of GO reacted with magnesium to prevent the escape of carbon dioxide and carbon monoxide to from the structure defects associated with vacancies, and GA could take place the high-temperature carbonization, during which a large-area graphene sheets formed with a part of the structure defects being repaired. When only GO was reduced by magnesium in SHS process, and the reduced GO (rGO) exhibited the smaller sheets, the lower C/O ratio (15.26), the weaker conductivity (4200 S/m) and the poor water-solubility because rGO inevitably left behind carbon vacancies and topological defects. Therefore, the larger sheet, less edge defects and free structure defects associated with vacancies play a key role for graphene sheets good dispersion in water. When N2H4 was used as reducing agent, N can be doped in the as-prepared graphene. Although NaBH4 showed excellent ability in GO reduction, the hydrolysis property made it difficult to get a stable NaBH4 aqueous solution2 elimination from the carbon lattice to form the atomic vacancies or graphene fragment, which cause the structural deterioration of as-prepared grapheneKnown as GO rich in oxygen containing groups such as hydroxyl, carbonyl and epoxy group, and the chemical reduction of GO is the most efficient method for low-cost and large-scale production of graphene121314–4~10−3 m/s), generation of high temperatures , and rapid heating (103~104 K/s). The SHS technique is also characterized by process simplicity, short reaction time, easy-to-build equipment, low energy requirements, and the possibility of obtaining complex or metastable phases. GA has three hydroxyl and one carboxyl groups in its benzene ring which has the same oxygen-containing groups with GO. The network holes of GO can be darn with GA by hydrogen bonds and ester bonds, and GO sheets can also be connected together by gallic acid. In this paper, GO was modified by GA to form GOGA, and GOGA was further reduced by magnesium in SHS process under air atmosphere condition 4 (8.6)4 (12)GA and rGO, indicating that the delocalized π-conjugated structure was well restored in rGOGA and rGOGA was higher than that of rGO, but rGOGA displayed much better hydrophilic than rGO. Those results are far beyond our expectations. A hypothesis was proposed that graphene composition is not the main factor to influence the hydrophilic or hydrophobic property, but its structure characteristics must play a key role on its hydrophilic or hydrophobic property.In order to investigate the reasons for excellent water-solubility of rGOvey scan , and no GA, rGOGA and rGO. A series of absorption bands ranging from 1000 to 1750 cm−1 exhibited the presence of C=O bonds (at 1720 cm−1), O–H bending (at 1621 cm−1), O-H deformation (at 1406 cm−1), C-O groups in epoxy (at 1221 cm−1) and C-O groups in alkoxy (at 1057 cm−1)29−1, due to the presence of moisture intercalated within hydrophilic GO sheetsGA), the characteristic bands for oxygen containing groups did not change remarkably, but some new FT-IR absorptions of GOGA appeared in the range of 1490–1190 cm−1, which indicated that gallic acid molecules were adsorbed or darned on GOGA is shifted by 44 cm−1 from 1720 cm−1 to 1676 cm−1 compared to GO, corresponding to the formation of O=C-O-C groups, which demonstrates that a portion of gallic acid are combined with GO by esterification. After samples undergo magnesium self-propagating high temperature synthesis process, the characteristic bands for oxygen-containing groups of rGO and rGOGA almost completely disappeared, but the bands of aromatic carbon  at 1575 cm−1 and (Larger π-conjugated C=C structure) at 1560 cm−1 appeared. These observations suggested that GO and GOGA were well reduced, and the larger-area π-conjugated structure was established in rGOGA.GA, rGOGA and rGO were further characterized by Raman spectroscopy. As shown in −1) and G band (~1585 cm−1), which are E2g vibration mode in-plane and A1g breathing modeGA was shifted by 7 cm−1 from 1589 to 1582 cm−1, displaying a high reduction degree2 cluster regions is the inverse of the ratio of the D and the G band integrated intensities (ID/IG)3234D/IG ratio was 0.89, and the ID/IG ratio of GOGA increased to 0.91. However, ID/IG ratio of rGO was 0.97, rGOGA ID/IG ratio declined to 0.89. From those results, the following conclusions can be drawn: (1) the most oxygen ions of GO reacted with magnesium to form MgO, reducing the number of carbon atoms vacancies of the carbon lattice; (2) the average size of the sp2 domains of rGOGA was larger than rGO slightly; (3) gallic acid can repair the structure defects of graphene during magnesium thermal reaction. Apart from D band and G band, there are two Raman bands with weaker intensity called 2D and D + G located at 2680–3000 cm−1 as shown in insets of et al.GA by comparing these two bands. Compared with rGO (or GO), the enhancement of I2D/ID+G ratio of rGOGA can be clearly observed, which suggests that the full recovery and the larger-area π-conjugated structure for rGOGA.GO, GOGA and rGO. GA shows an obvious shift to higher 2θ angles , suggesting that rGOGA was well ordered with two-dimensional sheets and well removal of surface functional groupsGA, a weak shoulder peak at 26.5° of rGO was found, which revealed a small portion of the reduced graphene sheets agglomerated together or formed graphite structureXRD was used to characterize graphite, GO, rGOGA and rGO red shifted to 264 nm owing to the restoration of the conjugated structure, which indicated the removal of oxygen-containing functional groups and the disappearance of n→π* transition band41GA formed with a part of the structure defects being repaired during a SHS process.Samples were also characterized by UV-vis absorption, as shown in 373839GA. By comparison, GA was adsorbed on the surface and edge of GO by the interaction between GA molecule and defects of GO  as precursor. The merit of this method is that the process carried out in the air atmosphere without external heating source, and gallic acid can repair the structure defects of graphene during SHS process. The obtained graphene sheets could be stably dispersed in water for more than one month. The wrinkles, scrolls, irregular structure defect associated with vacancies and grain boundaries (rather than C/O atomic ratio) determine the hydrophilic property. While the less edge defects and free structure defects associated with vacancies play a key role for graphene sheets good dispersion in H2O. Moreover, the synthetic process can be utilized to produce large-scale graphene by an environmentally friendly way. Obviously, these advantages will facilitate its industrial applications.In conclusion, a novel and facile approach to the synthesize graphene sheets based on magnesium SHS process is reported using GO modified by gallic acid (GO3 (99.8%), KMnO4 (99.5%), H2SO4 (98%), HCl (35%), magnesium powder and magnesium ribbon et al. were purchased from Tianjin Kermel Chemical Reagent Co., Ltd.Natural graphite powder (80 mesh) was supplied by Tianjin Chemical Co., Ltd and used as received. NaNO−1. Raman spectra were obtained by using a micro-Raman system on  with an excitation energy of 2.41 eV (l = 514.5 nm). XRD patterns were measured on a Bruker XPS (Germany) using Cu Kα radiation. UV-vis detection was carried out by UV/Vis Spectrometer . TEM and HRTEM images were obtained from a JEM-2100 transmission electron microscope . Electrical conductivity of samples was measured on a ST2722 semiconductor powder resistivity tester . X-ray photoelectron spectroscopy (XPS) analysis was carried by .FT-IR was recorded from a TESOR 37  and operated by attenuated total reflectance (ATR) in the wavenumber range of 4000–500 cm3, and 125 ml H2SO4 were loaded in a 1000 ml round-bottom flask with magnetic stirring. After being cooled with ice-water bath at 0 °C for 1 h, 17.5 g KMnO4 was added into above reaction system. After reaction was kept at 0–5 °C for 1 h and then heated at 35 °C for 2 h, the mixture was diluted with 250 ml deionized water and reacted at 98 °C for 45 min. 200 ml deionized water and 50 ml H2O2 (30%) were added into above suspension under the condition of magnetic stirring. After centrifugation and dry, GO was obtained.GO was prepared by modified Hummers’ methodGA was prepared in three steps: (1) 1.5 g gallic acid was loaded into 500 ml GO (5 mg/ml) solution, and the mixture was dispersed by ultrasound for 40 min. (2) the mixed solution was allowed to stand at room temperature for 3 h. (3) The mixed solution was dried at 90 °C under air atmosphere for 24 h to obtain the mixture of gallic acid-modified GO (GOGA). As a control, different addition amount of gallic acid (1.0 g and 2.0 g) in equal mass of GO (were labeled as GOGA1 and GOGA2) were prepared with the same method.Exfoliation of GO was obtained by ultrasonication in water for 2 h. GOGA powder were mixed evenly. The mixture was put in a crucible, and mixture was covered with a thin layer of magnesium powder on the surface of mixture. Mixture was ignited by magnesium ribbon under air atmosphere condition. After the reaction is over, the mixed products were transferred to a beaker containing 250 ml of 3 M HCl. Products were transferred into the acid solution to remove MgO and residual Mg. The resulting rGOGA was collected with filtration, washed with deionized water until the filtrate turned out to be of neutral. Obtained rGOGA was dried at 60 °C under high vacuum conditions for 10 h. As a control, rGOGA1, rGOGA2 and rGO were prepared from GOGA1, GOGA2 and GO reduction by magnesium with the same methods.3.0 g Magnesium powder and 1.5 g GOThe contact angle of the samples was measured on DSA 100 (KRUSS) contact angle system at ambient temperature. Before the measurement, 20 mg power samples were dispersed in 500 ul ethanol with ultrasonic treatment 1 h. In the following, the suspension was dropped onto a glass slide to form a uniform film.Electrical conductivity of samples was measured on a ST2722 semiconductor powder resistivity tester . The powder sample was filled into a test slot, then applying a pressure about 18 MPa. The electrical resistivity tester would reflect the electrical resistivity of the sample directly. The electrical conductivity of samples was calculated through the following equations:How to cite this article: Cao, L. et al. Hydrophilic Graphene Preparation from Gallic Acid Modified Graphene Oxide in Magnesium Self-Propagating High Temperature Synthesis Process. Sci. Rep.6, 35184; doi: 10.1038/srep35184 (2016).


The combination treatment also produced synergistic increase in P-ERK1/2 level in spleen. Immunohistochemistry shown that SEP induced the PD-L1 expression in melanoma tissue possibly by promoting IFN-γ excretion, which led to the synergistic anti-tumor effects of aPD-L1 and SEP. Furthermore, in the purified T lymphocyte from the naive mice, the combination of SEP and αPD-L1 had more potent than SEP or αPD-L1 in promoting T lymphocyte proliferation and cytokines secretion including IL-2 and IFN-γ, at least partially by activating MEK/ERK pathway. Our study provides the scientific basis for a clinical trial that would involve combination of anti-PD-L1 mAb and SEP for sustained melanoma control.The increased PD-L1 induces poorer prognosis in melanoma. The treatment with PD-1/PD-L1 antibodies have a low response rate. The combination immunotherapies are the encouraging drug development strategy to receive maximal therapeutic benefit. In this study, we investigated the enhanced antitumor and immunomodulatory activity of combined SEP and αPD-L1 in B16-F10 melanoma-bearing mice. The results shown that combined SEP and αPD-L1 presented significant synergistic antitumor effects, increased the frequency of CD8 The average survival time was 6–12 months and a 5-year survival rate under 10% with traditional therapies2. Recently, due to the profound understanding of immunobiology for melanoma development, immunotherapies have become the standard treatment regimens for the patients with advanced melanoma2.Melanoma comprises only 5% of all skin cancers, but approximately 80% of all skin cancer-related deaths are caused by melanoma3. However, the coinhibitory receptors such as anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed cell death 1 (PD-1) are able to down-regulate the immune system by preventing T cell over-activation, promoting self-tolerance and avoiding autoimmunity4. After expressed approximately 48 h after T cell activation, CTLA-4 binds to B7-1 in limiting T cell activation2. When the activated T cells enter tumor microenvironment, they become “tolerated”  by engagement of PD-1/B7-H1 (PD-L1) signaling pathway5. Therefore, these negative regulation mechanisms decrease T cell anti-tumor activity in cancer immunotherapy. So far, the immunotherapeutic drugs such as CTLA-4 antibody (ipilimumab) and PD-1 antibodies (pembrolizumab and nivolumab), have been approved for the treatment of the advanced melanoma by the US Food and Drug Administration (FDA)2. In addition, the PD-1 receptor ligand (PD-L1) antibodies (BMS-936559 and atezolizumab) have demonstrated promising activity for treating melanoma in preclinical mouse models and clinical trials6. Furthermore, concurrent CTLA-4 and PD-1/PD-L1 inhibition and combination with other immunotherapeutic strategies, have been a promising approach for the melanoma patients with the increased benefits7. In a randomized phase III trial in treating patients with metastatic melanoma, an increased response rate and improved progression-free survival were observed with the ipilimumab-nivolumab combination when compared with ipilimumab alone8.T lymphocytes play a critical role in cell-mediated immunity and cancer immunotherapy. The classic two-signal activation model includes both TCR signaling pathways and CD28/B7 costimulatory pathway9. Several immunoceuticals composed of polysaccharides have been used for treating cancers such as lentinan, schizophyllan and krestin9. These polysaccharides are be characterized by low toxicity and limited side effects10.Many polysaccharides, isolated from mushrooms, fungi, yeasts and plants, have attracted more attention recently due to their immunomodulatory and anti-cancer effects9. Strongylocentrotus nudus egg polysaccharide (SEP), a D-glucan containing an a-1,4-linked backbone and a-1,6-linked branches, was isolated and purified from Strongylocentrotus nudus eggs11. In our previous studies, SEP prevented the growth of both S180 and H22 hepatocellular carcinomas in vivo by enhancing splenocyte proliferation, CD4+ and CD8+ T cell numbers as well as cytotoxic T lymphocyte (CTL) activity, and increasing IL-2 and TNF-α secretion levels in the serum12. The studies also found that SEP regulates intracellular signaling pathway associated with splenocytes proliferation and cytokine expression12.Sea urchins belong to the echinoderm phylum. Sea urchin eggs are a kind of favorite seafood for their good taste and high nutrition in ChinaAccording to the above studies, SEP can activate T cells possibly by mediating signaling pathway in lymphatic tissues, while the PD-L1 antibodies can upragulate T-cell effector function by blocking PD-1/B7-H1 (PD-L1) signaling pathway in peripheral tissues including the tumor microenvironment. Therefore, we hypothesize that the combined treatment with SEP and anti-PD-L1 mAb produce an additive and even synergistic antitumor effect via immuneregulation in melanoma.in vivo tumor growth, T lymphocytes counts, CTL cytotoxicity and cytokines expression. We also investigated the potential effects of combination treatment on MEK/ERK signaling pathway in spleen. In addition, by using the purified T lymphocyte from the untreated mice, we studied the effects of MEK/ERK signaling pathway on the T lymphocyte proliferation and cytokines secretion induced by the combined SEP and αPD-L1 in vitro.In the present study, we investigated the enhanced antitumor and immunomodulatory activity of combined SEP and anti-PD-L1 mAb in B16-F10 melanoma-bearing mice via analyzing in vitro. As expected, neither SEP or αPD-L1 mAb alone, nor combination could inhibit B16-F10 cells growth compared with the control group   and tumor volumes  compared with the control group  and tumor volumes . Furthermore, we noticed that combination of SEP with αPD-L1 showed more potent than SEP or αPD-L1 on reducing tumor weights  and tumor volumes .We examined the oup Fig. . αPD-L1 P < 0.05 at every ratio, n = 3). Furthermore, combination of SEP and αPD-L1 resulted in the additive upregulation of cytotoxicity activity of splenocytes in B16-F10-bearing mice compared with SEP  or αPD-L1 .CTL plays an important role in inhibiting tumor cell. Therefore, we measured the cytotoxic activity of splenocytes against B16-F10 cells. The results showed that the cytotoxic activity of splenocytes at E/T ratios of 10:1, 20:1 and 40:1 were increased in three treatment groups in B16-F10-bearing mice compared with the control group . Furthermore, the combination of SEP and αPD-L1 led to significantly additive increase of CD3+, CD4+ or CD8+ T cells in spleen compared with SEP  or αPD-L1 .To investigate the effect of each treatment on the cellular immunity, the cell counts for CD3+, CD4+ or CD8+ T cells was also observed in three treatment groups compared with the control group   or αPD-L1 .Similarly, in tumor, the increased percentage of CD3P < 0.05 for each cytokine, respectively, n = 6). Furthermore, combination of SEP and αPD-L1 promoted significantly additive increase of IFN-γ and IL-2 levels compared with SEP  or αPD-L1  in spleen . Furthermore, combination of SEP and αPD-L1 significantly increase IFN-γ and IL-2 levels compared with SEP  or αPD-L1  in tumor . Furthermore, combination of SEP and αPD-L1 had more potent than SEP  or αPD-L1 . The results suggests that these facilitated the T cell proliferation and cytokines in B16-F10-bearing mice partially via the MEK/ERK signaling pathway.One of the key pathways in the cytokine expression involves the MEK/ERK signaling pathway in spleen. Therefore, we investigated the effect of the combination of SEP and αPD-L1 on the ERK1/2 phosphorylation. As shown in Fig. We used ICH methods to examine PD-L1 expression in melanoma cancer tissue. All immunostained tumor sections were examined by light microscopy, and images were photographed with a microscope camera  at a magnification of × 100. We found that the PD-L1 expression was significantly induced by SEP in melanoma cancer tissue compared with that in the control group Fig. . The αPDP < 0.05, respectively, n = 3). Furthermore, combination of SEP and αPD-L1 had more potent than SEP  or αPD-L1 . However, after the pretreatment with MEK inhibitor PD98059 for 30 min, the the increased ratios induced by SEP or/and αPD-L1 was partially decreased .To assess the role of MEK/ERK signaling pathway in T lymphocytes immunomodulation during SEP or/and αPD-L1 treatment, we used western blot analysis to detect the expression of ERK1/2 and p-ERK1/2 in T lymphocytes after 48 h exposure. Our results in Fig. P < 0.05, respectively, n = 6). Furthermore, combination of SEP and αPD-L1 had more potent than SEP  or αPD-L1 . However, after the pretreatment with MEK inhibitor PD98059 for 30 min, the T cell proliferation induced by SEP or/and αPD-L1 was partially inhibited .To investigate the effects of MEK/ERK signaling in T lymphocyte proliferation, CCK-8 kit was used. As shown in Fig. P < 0.05, respectively, n = 6). Furthermore, combination of SEP and αPD-L1 had more potent than SEP  or αPD-L1 . However, after the pretreatment with MEK inhibitor PD98059, the increased IL-2 and IFN-γ secretion in SEP or/and αPD-L1 groups was partially inhibited  secrete a series of cytokines, such as IL-2, IFN-γ and TNF-α, to enhance the production of CD8+ cytotoxic T cells9. The IL-2 can stimulate T cell proliferation and differentiation into mature CD4+ and CD8+ T cells to enhance the adaptive immunity16. The activated CD8+ T cells can concentrate to the tumor microenvironment and secrete some cytokines, including IFN-γ. The IFN-γ expression levels by CD8+ T cells are closely related with the specific cytotoxic activity for antitumor effects17. In our study, the ELISA results showed that combined SEP and αPD-L1 induced T cells to produce the additive IL-2 and IFN-γ increase in splenocytes and tumor from B16-F10 melanoma-bearing mice when compared with single treatment, which either promote proliferation and differentiation of immune cells to inhibit tumor growth or lead to cytotoxic effects to tumor cells18.CD419. Previous studies have demonstrated that many drugs can induce PD-L1 expression on tumor cells by increasing the IFN-γ excretion20. In our study, SEP can induce IFN-γ excretion in melanoma tumor tissue, which could inderectly stimulate the PD-L1 expression, which provide the rationale for combined cancer treatment of SEP and αPD-L1.We used ICH methods to examine PD-L1 expression in B16-F10 melanoma tumor tissue. The results shown that SEP significantly induced the PD-L1 expression in melanoma cancer tissue, which may partially responsible for the synergistic anti-tumor effects of aPD-L1 and SEP as tumor PD-L1 expression disabling the antitumor function of preexisting tumor antigen-specific T cells has been thought to be prerequisite for αPD-1/αPD-L1 mAb therapy to work21. However, blocking the PD-1/PD-L1 immune checkpoint pathway by anti-PD-1 or anti-PD-L1 antibodies, causes activation of T cells partially by increasing MEK/ERK pathway, promoting differentiation of effector and memory T cells, which suppresses cancer cell survival and enhances the antitumor responses of T cells21. Our previous study shown that SEP can activate MEK/ERK signaling pathway associated with T cells proliferation and cytokines excretion12. In the present study, the treatment with αPD-L1 or SEP in B16-F10 melanoma-bearing mice enhanced the ERK1/2 phosphorylation level in the spleen compared with the control group. However, the combination of SEP and αPD-L1 had synergistic effects in increasing ERK1/2 phosphorylation level in spleen, which allowed more effective T cells activation, proliferation and cytokines secretion such as IL-2 and IFN-γ, and further increase of antitumor effect.The actived PD-1/PD-L1 pathway inhibits the signaling pathways that lead to activation and expansion of T cells that recognize tumor antigens with the impaired generation of T effector and memory cells, which promote the survival of cancer cells and tumor tolerancein vitro from naive mice with/without MEK inhibitor PD98059 for 30 min, followed by 48 h of the coculture with the aPD-L1 and/or SEP with ConA. We found that the combination of SEP and αPD-L1 had more potent than SEP or αPD-L1 in the increase of P-ERK1/2 level. However, the pretreatment with PD98059 significantly reduced the increased P-ERK1/2 level induced by SEP or/and αPD-L1. Furthermore, SEP and αPD-L1 also significantly had more potent than SEP or αPD-L1 in promoting T cells proliferation and cytokines secretion including IFN-γ and IL-2. Similarly, the pretreatment with PD98059 significantly reduced T cells proliferation and cytokines secretion induced by SEP or/and αPD-L1. All these data indicated that the MEK/ERK signaling was at least partially responsible for T lymphocyte proliferation and cytokines secretion induced by SEP or/and αPD-L1.In order to further investigate the effects of MEK/ERK signaling in T lymphocyte proliferation and cytokines secretion, we pretreated splenocytes in vivo and in vitro in combination with αPD-L1 partially by activating the MEK/ERK signaling. Our study provides the scientific basis for a clinical trial that would involve combination of αPD-L1 and SEP for sustained tumor control in cancer patients.The antibodies targeting PD-1/PD-L1 pathways such as pembrolizumab and nivolumab has already been approved by FDA for treatment of melanoma. The αPD-L1 has demonstrated promising activity for treating melanoma in preclinical mouse models and clinical trials. It will be critical to increase the frequency of effector T cells and cytokines excretion in order to receive maximal therapeutic benefit. Our work suggests that administration of SEP may harness the therapeutic potential of effector T cells Strongylocentrotus nudus eggs as described previously11. Briefly, the crude, water-soluble polysaccharide from the eggs of sea urchins was separated and sequentially purified via Cellulose DE-52 and Sephacryl S-400 to yield the SEP for subsequent analysis. High performance liquid chromatography (HPLC) system with a TSK gel 4000 PWXL column and a Waters 2414 refractive index detector  was applied for analyzing the purity of SEP . The profile showed a single symmetrical and sharp peak with the purity of 98.0%. To remove endotoxin contamination, 1 ml of Affi-Prep Polymyxin Matrix was packed in a Bio-spin column; centrifuged at 200 × g for 2 min; and then 0.5 ml of either SEP (100 µg/ml), LPS (2 µg/ml) or a mixture of SEP (100 µg/ml) and LPS (2 µg/ml) was added. After an overnight incubation at 4 °C, the effluent was collected and centrifuged under the same condition. The endotoxin level in SEP was determined by using the E-TOXATE kit22.SEP was obtained from FITC-conjugated anti-CD3 mAb(#555274), FITC-conjugated anti-CD4 mAb (#553729) and PE-conjugated anti-CD8 mAb (#553033) were provided by BD Biosciences . Mice interferon gamma (IFN-γ) and interleukin-2 (IL-2) ELISA kits were purchased from R&D Systems, USA. The αPD-L1 antibody (clone 10 F.9G2) was bought form Bio-XCell (USA). ERK1/2 (SC-135900), P-ERK1/2 (Thr202) (SC-101760) and lipopolysaccharide (LPS) were from Santa Cruz Biotechnology . The anti-PD-L1 antibody  was from Cell Signaling Technology (USA) for immunohistochemistry (ICH). PD98059 (MEK inhibitor) was purchased from Calbiochem .® Number: CRL-6475™) was obtained from Cell Culture Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. The cells were maintained in DMEM supplemented with 10% (v/v) heat-inactivated fetal bovine serum in a humidified 5% CO2 atmosphere at 37 °C.Mouse melanoma cell line B16-F10  were used for 3 cells/well. After 24 h incubation, the cells were exposed to SEP and/or aPD-L1 at the designated concentrations and cultured at 37 °C in a humidified atmosphere for 48 h, 72 h and 96 h. 20 μL of MTT (5 mg/ml) was added to each well and incubated for additional 4 h at 37 °C. The medium was subsequently discarded, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a Molecular Devices Spectra Max M5 .To determine the cell viability of SEP and/or aPD-L1 on melanoma cell lines, cell survival was measured by MTT assay. Cells were plated in 96-well plate with a density of 5 × 105 cells mixed with matrigel into the dorsal area of male mice. On 2rd day, the mice bearing tumors were randomly divided into four groups (12/each group). Administration doses of SEP and aPD-L1 were designed according to the references14. Mice were intraperitoneally administrated with SEP at 16 mg/kg or aPD-L1 at 9 mg/kg with a volume of 0.1 ml/10 g for each drug. The mice in combination drug group were administrated with SEP at 16 mg/kg and aPD-L1 at 9 mg/kg. Control mice were given the same volume of saline. Figure 3) = 0.5 × length (mm) × width (mm) × width (mm). On day 16, all the mice were decapitated between 9:00 a.m. and 11:00 a.m. The tumors were obtained. And the inhibition rate (IR) of tumor growth was calculated by the following formula: IR (%) = [(A − B)/A] × 100, where A and B were the mean tumor weight in the control and treatment groups, respectively.B16-F10 tumors were established by injecting 1 × 1023. Briefly, B16-F10 cells were applied as target cells (T) and seeded in 96-well U-bottom microtiter plate at 1 × 104 cells/well in DMEM complete medium. The spleens were collected from treated mice in each group (n = 3) at the end of the experiment (day 16), and gently homogenized and passed through a 200-mesh sieve to obtain single-cell suspensions. After treatment with erythrocyte lysis buffer, the splenocytes were washed with phosphate-buffered saline (PBS) three times and resuspended in DMEM complete medium, which were used as effector cells (E). The ratios of effector cells to target cells were 10:1, 20:1, and 40:1. The plates were then continuously incubated for 20 h at 37 °C. 20 µl of MTT solution (5 mg/ml) was added to each well and the plates were incubated for another 4 h. To determine the percentage of the killed target cells, the following equation was used: CTL activity (%) = (ODT − (ODS − ODE))/ODT × 100%, where ODT, optical density value of target cells control, ODE, optical density value of effector cells control, and ODS, optical density value of samples.The cytotoxic activity of CTL was analyzed by MTT assay as previously described6 cells were used for antibody staining by using FITC-conjugated anti-CD3 mAb, FITC-conjugated anti-CD4 mAb, or PE-conjugated anti-CD8 mAb. The cell counts for the CD3+, CD4+ and CD8+ T lymphocytes were assessed via flow cytometry  and expressed as a percentage of the total number of lymphocytes.For flow cytometric analysis, spleen and tumor tissues were harvested at the end of the experiment (day 16). Single cell suspensions were prepared and a Ficoll-Hypaque purification step was carried out for the tumor-derived cell suspension. After the cells were washed twice with PBS and resuspended in 1% paraformaldehyde (PFA), about 2 × 10Mouse splenocytes in each group were obtained as described above. After incubation with LPS (4 μg/ml) for 48 h, the supernatants were collected for detecting IFN-γ and IL-2 levels using murine ELISA kits according to the manufacturer’s instructions. And IFN-γ and IL-2 levels in the tumor supernatants were measured by ELISA kits.The protein content of the spleen homogenates from the melanoma-bearing mice were measured by BCA method. The protein samples (20–30 μg per lane) were separated by SDS polyacrylamide gel electrophoresis, and transferred onto a nitrocellulose membrane. These membranes were incubated with 1% bovine serum albumin in Tween-Tris-buffered saline and reacted overnight at 4 °C with the mouse or rabbits antibody (1:1000) against P-ERK1/2 and ERK1/2. After repeated washings, the immunoreactive bands were reacted with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody, then visualized by FluorChem™ Q chemiluminescent System . The O.D. value of reactive bands visible was determined densitometrically.2O for 10 min, and then counterstained with hematoxylin. After dehydration by using an ethanol series and xylene, each stained section was examined by light microscopy .The collected melanoma tumors were fixed in 4% paraformaldehyde for 24 h and then embedded in paraffin. Serial sections (4-μm) were prepared for ICH. After deparaffinization and antigen retrieval, the sections were preincubated in blocking serum, and then incubated overnight at 4 °C with the anti-PD-L1 antibody for ICH. Following incubation, the sections were washed three times in PBS and then incubated with biotin-conjugated secondary antibody for 2 h. Next, the sections were washed 3 times in PBS. Diaminobenzidine substrate was placed on each tissue section. After color development, the slides were immediately washed with H24. The T cells were cultured at 5 × 106–6 × 106 cells/ml in RPMI-1640 medium supplemented with 10% fetal calf serum. After the cells were pretreated with/without MEK inhibitor PD98059 (40 μM) for 30 min25, they were cultured with the aPD-L1 (10 nM) and/or SEP (50 μg/ml) with concanavalin A  for additional 48 h. Then the cell survival was determined by CCK-8 method, or the cell supernatants were collected, and IFN-γ and IL-2 levels were mesured by murine ELISA kits.T cells were purified from the spleens from naive C57BL/6NCrl mice, by using magnetic negative selection . The purity of T cells was more than 95% by as determined by flow cytometric analysisFor the effects on MEK/ERK signaling pathways, after the treatment with/wihtout PD98059 (40 μM) followed by aPD-L1 and/or SEP with ConA according to the above method, the T cells were collected and lysed in lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors . Western blot analysis was conducted for the detection of P-ERK1/2 and ERK1/2 expression accoriding to the above method.P < 0.05 was considered statistically significant.The statistical analyses were performed using one-way ANOVA, followed by LSD post hoc test in SPSS software (V 16.0).


P<0.001) but not with gender, primary site, or lymph node status. The PD-L1 positivity rate and signal intensity are directly correlated (P<0.001) with a grade increase from G1 to G3. In particular in G3 cases, we observed a dichotomy between the morphology (WD- and PD-NENs) and Ki67. Moreover, our study demonstrated a significant association with the grade and PD-L1 expression levels in immune-infiltrating cells (P<0.001). In particular, G3 tumors are characterized by strong PD-L1 expression in both the tumor and infiltrating immune cells (P<0.001), reflecting an unfavorable environment for T-cell-mediated tumor aggression. These findings suggest that NENs might acquire resistance to immune surveillance by upregulating PD-L1 and inhibiting peritumoral and intratumoral infiltrating lymphocytes. Here we demonstrate that PD-L1 is currently the best-known biomarker for G3 NENs, becoming the new gold standard for G3 NEN discrimination. Furthermore, pharmacological approaches using anti-PD-1 antibodies may become the logical choice for the treatment of G3 cases with a poor prognosis.Neuroendocrine neoplasms (NENs) are rare, heterogeneous and ubiquitous tumors commonly localized in the gastrointestinal tract, lung, and pancreas. The clinical behavior of NEN is highly unpredictable; in fact, low-grade cases can unexpectedly be associated with metastases. Currently, the 2010 WHO NEN classification employs histological differentiation and the proliferation index for grading tumors but fails to provide reliable prognostic and therapeutic indications. Therefore, there is an urgent need for a better characterization of G2/G3 NENs. Similar to several other tumors, NENs possess immune-escape mechanisms, but very little has yet been done to characterize this crucial aspect. There are no available data describing PD-L1 expression in these tumors. Here we provide, for the first time, evidence of PD-L1 tissue expression in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). PD-L1 expression was significantly associated with a high-grade WHO classification (G3) ( In 2000 and 2010, the WHO provided two different classifications of NENs based on the histopathological pattern, tumor grade, and stage intended to improve the therapeutic and prognostic evaluation.6 Most gastrointestinal NENs are well differentiated (WD) and characterized by a slow growth rate (hence the old term carcinoid). Gastrointestinal neuroendocrine carcinomas are highly aggressive malignancies and are mainly treated with a platinum-based chemotherapy.7 However, at certain locations, such as the esophagus or colon, poorly differentiated NENs are more frequent than their WD counterparts.8 The tumor morphology and Ki67 immunostaining have been defined as prognostic factors that can subdivide gastrointestinal NENs into two or even three subgroups with significantly different survival rates.9 However, the two current prognostic evaluation systems fail to guide the most efficient therapeutic strategy on the basis of the histological differentiation of the tumoral cell and their proliferative index. In particular, the 2010 WHO NEN classification identified three groups: NENs G1, NENs G2, and G3, also defined as neuroendocrine carcinoma (NEC).11 In general, the WD NENs (G1/G2) are much more common (70%) than the poorly differentiated NENs.12 However, some WD cases exhibit a high level of proliferation and, on the contrary, some poorly differentiated cases show a low mitotic activity. Besides, the defined range  appears very broad, including many heterogeneous clinical, morphological and prognostic conditions.13 A few recent papers14 indicated that despite the assessment of the proliferation fraction, some tumors, identified as NECs, do not show the expected poorly differentiated morphology. These reports indicate that these patients do not respond to cisplatin-based chemotherapy, the gold standard therapeutic approach to high-grade PD-NECs,14 and have a better prognosis. In contrast, WD-NENs, despite mostly featuring a low/intermediate differentiation grade, may contain regions with an increased proliferation rate that places them, in accordance with the WHO, in the WHO G3 category.15 Specifically, G3 NENs with a WD morphology are commonly Ki67 positive  and respond poorly to cisplatin-based treatment. In contrast, patients with a poorly differentiated NEC, but Ki67 expression of >60%, respond to cisplatin-based chemotherapy.18 In addition, PD-NEC may also include a combined component of a conventional carcinoma (MANEC),19 such as a squamous cell carcinoma or adenocarcinoma, but they do not typically contain a lower-grade WD-NEN.21 Therefore, the availability of biomarkers that could better characterize G3 GEP NENs could have crucial therapeutic implications, indicating the most efficient pharmacological strategy. Immunotherapy, which allows immune cells to attack cancer cells, results in long-term survival in patients with several different tumors, such as melanoma, non-small cell lung cancer, and renal cell carcinoma.22 Cancer immunotherapy has undergone major breakthroughs during the past decades, impressive results being reported by several studies.24 One key target of cancer immunotherapies has been the PD-1–PD-L1 pathway.26 Programmed death ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274), is expressed on many cancer cells. PD-L1 binding to PD-1 results in a pattern of inhibitory signals, which inhibits CD8+ T cells’ proliferation, thus blocking the antitumor immune response and hence favoring tumor growth and metastasis.27 PD-1 has been detected in tumor-infiltrating lymphocytes (TILs) present in the tumor microenvironment, where PD-L1 aberrant expression is associated with a poor prognosis in several human tumors.29 Taube et al.30 demonstrated that the expression of PD-L1 in tumor cells and tumor-infiltrating immune cells was highly correlated with PD-1 expression in infiltrating lymphocytes. They also demonstrated that PD-L1-positive tumors are those that respond better to Nivolumab administration. Nivolumab is a monoclonal anti-PD-1 antibody that can block the aforementioned inhibitory signals that prevent activated T cells from attacking the cancer. Anti-PD-1 administration may improve the prognosis of patients characterized by both tumor cells’ PD-L1 expression and TILs, as shown by multiple studies.32 PD-L1 expression on tumor cells and immune-infiltrating cells, such as tumor-infiltrating macrophages, appears to be a crucial predictor of the antitumor T cells’ response. Importantly, in most clinical trials, the response rates of various tumor types to PD-L1/PD1 targeting therapies were correlated with the immune-histochemical expression of PD-L1, suggesting that PD-L1 expression could be a predictive biomarker of the tumor sensitivity to immunotherapy.33 Immunological approaches against NENs have not yet been explored, but in the present manuscript, we provide data that may better support the possible benefit deriving from the revolution in immunotherapy as regards the clinical evaluation of NENs and the identification of the most effective pharmacological strategy. PD-L1 tissue expression has not been addressed in gastroenteropancreatic NENs (GEP-NENs). Here we provide evidence of PD-L1 tissue expression in different NENs. PD-L1 expression, together with Ki67, offers a new and efficient benchmark for a broad range of grading of GEP-NENs. Therefore, results of the present study indicate that PD-L1 tissue expression may be the best GEP/NEN morphological characterization and grading marker. Furthermore, Nivolumab administration may become the pharmacological choice for Ki67>60% PD-NENs, identified in this study as the tumors with the highest PD-L1 expression.Neuroendocrine neoplasms (NENs) are commonly localized in the gastrointestinal tract, lung, and pancreas.The patients characteristics of the 57 cases of NETs are summarized in P=0.345; P<0.001; P<0.001) from G1 to G3 (P<0.001). Furthermore, we highlighted significant differences between the PD-L1 score and the Ki67 index and, in particular, observed a remarkable difference between individual Ki67 mean levels and the PD-L1 score  while 16 cases were positive (28%). The relationship between PD-L1 expression and gender was not significant P=0.345; . ImportaG1 to G3 . In addiG1 to G3  from 0 tG1 to G3 . PD-L1 tP<0.001; .P<0.001; PD-L1 expression was also detected in peritumoral and intratumoral immune-infiltrating cells. Immune-infiltrating cells were most frequently located at the interface between tumor cells and stroma, with similar morphological features to the lymphoepithelial lesions frequently present in MALT lymphomas. In our study, we highlighted a variable PD-L1 expression by grade in immune-infiltrating cells, as shown in 37 The PD-L1-positive rate was ∼30% in melanoma cases and 25–36% in non-small cell lung cancer; in NENs, we found 28% cases.39 Some studies explored, by immunohistochemistry (IHC), PD-L1 expression in different tumors and demonstrated that PD-L1 membranous expression on tumor cells and/or infiltrating immune cells is correlated to a better chance of response to anti PD-1 drugs.41 However, the prognostic role of PD-L1 remains controversial, mainly due to tumor heterogeneity, variability in the assays, location of intratumoral expression, and cutoff values for positive versus negative expression. Therefore, in the present study we investigated the impact of PD-L1 tissue expression on the grading of 57 GEP/NEN patients, associating PD-L1 expression with the morphological characterization. The characteristics of indolent or aggressive GEP-NEN were determined by tumor grade, differentiation, and proliferation status.42 The current WHO 2010 grading system was designed to provide clinicians with an effective tool for patients stratification and clinical management. Years later, this WHO classification fails to provide an efficient cutoff between G1, G2, and G3 likely because existing biomarkers discriminate only partially between different grades. Currently, the Ki67 expression level is associated with different prognoses and can only partially indicate the most efficient pharmacological strategy. Similarly, high-grade G3 NECs are defined by morphology and Ki67 expression .15 The broad interval indicated by Ki67 expression for G3 disease (21–100%) may include a variety of different neoplasms, with potentially different responses to therapies.43 Overall, the morphology-based classification of different categories is still challenging, especially for G3 cases. Our results indicate, for the first time, that PD-L1 tissue expression is present in 100% of G3 NENs, those cases in which the Ki67 expression is an unreliable benchmark for treatment choice and intratumoral grade heterogeneity assessment. The terminology of ‘neuroendocrine carcinoma’ for G3 neoplasms implies that they are histologically poorly differentiated, but some morphologically WD-NETs also have proliferation rates  that meet the threshold for G3 NECs. A recent landmark study showed that G3 NECs with a Ki67 index of <55% do not respond to cisplatinum-based chemotherapy, unlike G3 NECs with a Ki67 index >55%.45 This supports the concept that the current WHO G3 category is heterogeneous and that tumors at the lower end of the G3 range are, in fact, WD-NETs with an elevated proliferation rate . Membranous PD-L1 expression by tumor cells and immune infiltrates varied significantly by grade and was significantly associated with the high-grade WHO classification (G3) (P=0.001) but not with gender, primary site, or number of metastatic sites. Our findings highlight that the expression of PD-L1 is present in the proliferative, aggressive G3 type of GEP-NET and, only in G3, shows a moderate-to-strong signal. In particular, poorly differentiated NENs (NEN G3 and NEC) have a constant positive PD-L1 membrane expression that increases with the tumor aggressiveness. Consistently with previous studies based on different tumors,47 PD-L1 expression is correlated with a more aggressive subset of NENs identified using morphological data and routine staining. In accordance with this possibility, PD-L1 expression has been reported as the most important predictor of the responsiveness of various cancers to PD-L1 or PD-1 blocking antibodies.48 Moreover, our study demonstrated a significant association with grading and PD-L1 expression levels in infiltrating immune cells (P=0.001). PD-L1 expression on tumor cells and tumor-associated immune cells was also positively correlated with the progression of human malignant tumors.49 PD-1 is expressed by activated T cells50 and its engagement with PD-L1 inhibits TCR-mediated activation of IL-2 production and T-cell proliferation. These findings suggest that NENs might acquire resistance to immune surveillance by upregulating PD-L1 and inhibiting peritumoral and intratumoral infiltrating lymphocytes. Thus this hypothesis provides a strong rationale for PD-1/PD-L-targeted immunotherapy for NENs. Recent studies52 have demonstrated that PD-L1 has a critical role in regulatory T-cell (Treg) development and functional maintenance. Increases in FOXP3+Treg infiltration and PD-L1 expression have been revealed in gastric cancer tissues, colorectal carcinoma, and breast cancer. High PD-L1 expression and increased FOXP3+Treg infiltrates were both associated with high histological grade, and tumors with concomitant high expression levels of the two markers had the worst prognosis. Recently, it has been reported that PDL-1 blockade improves antitumor immunity and offers a promising cancer immunotherapy approach. The blockade of PD-L1 in non-small cell lung cancer might be one strategy to pursue for future immunotherapy.53 Several clinical studies have suggested that TILs have a critical role and a prognostic significance in certain human tumors. Recently, PD-L1 has also been found to be expressed in a broad range of cancers. The observation in our study that PDL-1 is overexpressed in NENs (G2–G3) indicates that tumor-related PD-L1 may be linked to a malignant potential and contribute to tumor progression by providing a protective mechanism against immune surveillance. The significant association between PD-L1 expression and grading confirms that tumor-related PD-L1 expression is indeed relevant to tumor development. In our study, we observed high levels of PD-L1 expression on the infiltrating immune cells of PD-NENs. Thus it is possible that the tumoral milieu promotes macrophages PD-L1 expression that, in turn, inhibits the function of TILs, favoring tumor progression. We recognize that 57 NENs cases, although rare tumors, are a small cohort, but the incidence of PD-L1-positive patients with grade G3 warrants a larger future study. PD-L1 expression could become the new gold standard for G3 NENs discrimination. To the best of our knowledge, this is the first report evaluating the characteristics and grading of PD-L1 tissue expression in patients with GEP-NENs, paving the way for a new and more efficient WHO classification. In fact, it is expected that a new WHO classification, to be published in 2017, should provide a new NEC category G4 (Ki67>55%), as recently proposed by Fazio et al.54 This subdivision, correlated with a timely diagnosis, may ensure the implementation of appropriate treatment and have a substantial impact on prognosis. Furthermore, based on these observations, pharmacological approaches using anti-PD-1 antibodies may become the logical choice for the treatment of G3 cases, currently burdened by a poor prognosis. Immunotherapy might be a valid alternative or support to other therapies in NENs. Moreover, PD-L1 might be a useful prognostic biomarker in GEP-NENs. At the moment, it seems that high-grade tumors might be the targets. In this context, it is important to carefully delineate those tumors that might better respond to this type of treatment.The expression of PD-L1 is significantly associated with the histopathological grade and poor clinical outcomes in various malignant tumors, such as gastric cancer, hepatocellular carcinoma, renal cell carcinoma, esophageal cancer, pancreatic cancer, ovarian cancer, and bladder cancer.32 or endoscopic resection25 from January 2006 to December 2016 were reviewed at the National Institute of Gastroenterology Research Hospital ‘Saverio de Bellis’. The following clinicopathological characteristics were collected for all patients: age, gender, primary site, tumor grade (according to the 2010 WHO classification), and metastasis , and a representative paraffin block from each specimen was chosen for IHC analysis. Tumor size was measured either grossly (obtained from pathology reports) or microscopically; no case measured >5 cm. The criteria for further classification and grading were based on both the morphological features of the tumor and the proliferation rate. On H&E and PAS mucin-stained sections, the cytological characteristics of cells, growth patterns, presence of ulcerations, level of infiltration in the organ, and infiltration of mucosa were evaluated. In addition, we evaluated perineural infiltration, vascular permeation, presence of necrosis, intratumoral and peritumoral lymphocyte infiltration, and lymph node metastases . Intratu55 In the present study, all PD-L1 staining results were scored as both the approximate percentage of positive tumor cells only in the membranous site and the intensity of overall staining. In particular, the expression of PD-L1 was scored: 0: (no staining) negative; 1: weak expression, but weaker than the placenta membrane, staining in <10% of tumor cells; 2: moderate expression in ⩾10% of tumor cells; and 3: strong more than placenta membrane staining in ⩾10% of the tumor cells. For the data assessment, our cases were considered to be positive for PD-L1 expression only if they had scores of 2+ or 3+  were evaluated on the NENs, using an automated autostainer . The Real Envision DAB Substrate Kit (DAKO) was used according to the manufacturer’s instructions. Human placenta was included as positive control. The staining pattern of both antibodies was as expected: cellular membranous/submembranous and cytoplasmic with occasional dots, corresponding to the PD-1/PD-L1 interaction sites. The E1L3N antibody clone was preferred for further evaluations primarily because of the better signal-to-noise results. Ki67 IHC was carried out again on all cases on paraffin-embedded sections using an Ab anti-Ki67 diluted 1:100  following the manufacturer’s instructions. All cases were revised and re-evaluated and then assigned a precise proliferation index number that encompassed the amplitude limit of the WHO range. All IHC stained sections were initially evaluated and scored by two pathologists; discrepancies in the interpretation of scoring were resolved by consensus. PD-L1 expression was evaluated both on tumor cells and inflammatory cells as intratumoral and peritumoral in location. PD-L1 expression can be observed as a cytoplasmic or membranous immunoreactivity. The proportion of PD-L1-positive cells was estimated as the percentage of total tumor cells; tumor cells typically showed membranous staining with a variable cytoplasmic staining component. In literature, tumors with ⩾1% of tumor cells stained either in membrane or cytoplasm were considered positive for PD-L1 regardless of the intensity of staining.2+ or 3+ . Moreovet-test or Fisher’s exact test and χ2 test. All P-values were determined by two-sided tests and P-values<0.05 were considered significant. Statistical analyses were performed with StataCorp. 2007 Stata Statistical Software: release 10 .Correlations among the PD-L1 expression score of tumor cells, immune-infiltrating cells, and grading were statistically analyzed by