Tumor expression of immune co-inhibitory ligands, such as PD-L1 and Galectin-9, have potential prognostic value in Hepatocellular Carcinoma (HCC). Circulating levels of these molecules, however, have hardly been studied. This study aims to assess the prognostic significance of circulating PD-L1 and circulating Galectin-9 in patients with resected HCC, and to compare their prognostic significance to the intra-tumoral expression of these same molecules. Archived tissues and stored peripheral blood samples from 81 patients who underwent HCC resection or liver transplantation, with curative intent, were used. Immunohistochemistry was performed to determine intra-tumoral expression of PD-L1 and Galectin-9, while ELISA was used to quantify their respective circulating levels. High circulating PD-L1  and high circulating Galectin-9  levels were both associated with improved HCC-specific survival. Surprisingly, there was no correlation between circulating levels of PD-L1 and Galectin-9 and their intra-tumoral expression levels. In fact, circulating levels of PD-L1 and Galectin-9 were predictive of HCC-specific survival independently of intra-tumoral levels and baseline clinicopathologic characteristics. Combined analysis of circulating levels and intra-tumoral expression of PD-L1  and Galectin-9  resulted in more confident prediction of survival. In conclusion, circulating PD-L1 and Galectin-9 levels prognostically differentiate resected HCC patients, independently of their intra-tumoral expression. Combining circulating and intra-tumoral expression levels of PD-L1 or Galectin-9 further improves the prognostic values of these immune biomarkers. Only 20% of patients with HCC are diagnosed early enough to be candidates for curative treatments such as resection, local ablation or liver transplantation2. Once advanced disease is diagnosed, HCC is incurable and overall survival can be only modestly extended with sorafenib3.Worldwide over half a million people die from HCC4 have brought forth new therapeutic strategies against cancer5. Currently, the most successful immunotherapeutic strategies are those that are designed to overcome immune resistance mechanisms by using antibodies that abrogate co-inhibitory receptor-ligand interactions, the so-called negative immune checkpoint inhibitors6. Recent approval of anti-CTLA-4 and anti-PD-1 antibodies for the treatment of advanced melanoma, non-small cell lung cancer, renal cell carcinoma and bladder cancer has made it clear that immunotherapy is the new wave of anti-cancer treatments. Immunotherapy clinical trials are now ongoing in many cancers, including HCC7, and it is likely that these therapies will be approved in the future in other cancer types as well. In view of the high costs and occasional severe toxicity of these novel therapies, immune specific biomarkers that can predict which patients will benefit are urgently needed. Two such recent promising immune biomarkers in HCC are PD-L1 and Galectin-9 .Developments in our understanding of tumor immunologyin vitro disruption of this interaction restores the function of tumor-derived effector T-cells8. PD-L1 is known to be expressed by HCC cells13. We have previously shown that tumor PD-L1 protein expression is a promising prognostic biomarker in HCC14. In addition, tumor PD-L1 protein expression has shown promise as a predictive biomarker to identify cancer patients that respond to anti-PD1 immunotherapy16.PD-L1 is a ligand that binds PD-1, a co-inhibitory receptor expressed on activated T cells. Binding of the ligand PD-L1 to its receptor PD-1 transduces a negative signal into T-cells inhibiting their activation. In HCC the PD-L1/PD-1 interaction impairs effector T-cell function and 17. Gal-9 causes T-cell inhibition and apoptosis through its binding to the co-inhibitory receptor TIM-3 and blockade of the interaction between Gal-9 and TIM-3 reinvigorates ex vivo responses of T-cells of HCC and melanoma patients to tumor antigens18. Humanized antagonistic antibodies against TIM-3 are currently in preclinical development19. In addition, binding of Gal-9 to CD44 enhances the differentiation of immunosuppressive T regulatory cells20. A direct anti-metastatic role for Gal-9 has also been described22. We and others have demonstrated that Gal-9 is also expressed by HCC tumor cells and that Gal-9 protein expression is a potential prognostic biomarker in HCC24.Gal-9 is a glycan-binding protein and an important modulator of T-cell function25, lung cancer26, gastric cancer27, pancreatic cancer28 and HCC29. Elevated levels of circulating Gal-9 have been observed in metastatic colon cancer30 and in benign inflammatory liver diseases such as chronic hepatitis-C and active chronic hepatitis-B infection32. However, no study has investigated the relationship of circulating Gal-9 to cancer survival before.However, in addition to cell-bound expression, soluble forms of PD-L1 and Gal-9 exist in the circulation. These circulating forms of PD-L1 and Gal-9 have been poorly studied in cancer patients. Circulating levels of PD-L1 have been examined in renal-cell cancerOne may hypothesize that circulating forms of PD-L1 or Gal-9 would correlate with their tumor tissue expression status, since release from tumor cells, or from the tumor microenvironment, may be the source of these molecules in the circulation. In that case, circulating PD-L1 and Gal-9 may act as preferred biomarkers given the easier accessibility of peripheral blood when compared to tumor tissue. On the other hand, these physiologic molecules may have functions in the circulation that are independent of the immune interactions ongoing in the tumor microenvironment. Thus, the potential for the circulating levels of PD-L1 and Gal-9 to act as independent prognostic biomarkers, or independent predictors of immunotherapy treatment efficacy, exists. Thus, the aims of our study were to examine how circulating levels of PD-L1 and Gal-9 compare to tissue expression of these molecules and whether circulating levels have the potential to replace tissue expression, or add to tissue expression, as potential immune biomarkers in HCC patients undergoing surgical resection.Archival blood samples (59 serum samples and 22 plasma samples) and formalin fixed paraffin-embedded tissue samples from 81 patients who underwent hepatic resection or liver transplantation for HCC at Erasmus MC-University Medical Center between January 2007 and March 2013, were used for this study. All patients had undergone procedures with curative intent.Medical ethical approval for this study was obtained from the Medical Ethics Committee of Erasmus MC (MEC-2015-237). All patients gave informed consent for the use of their blood and tissue in medical research. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.33 in the laboratory of Dr. Haidong Dong . The dynamic range of this ELISA is: 86–3670 pg/ml. Limit of detection: 84 pg/ml. Recovery from heparin plasma: 84–105%. Soluble Gal-9 was performed using an ELISA kit according to the manufacturer’s protocol . This ELISA kit has been used in several published studies35. The dynamic range of this ELISA is 7.8–500 pg/ml. Limit of detection 3.3 pg/ml. Recovery from serum: 91–104%. Recovery from heparin plasma: 87–102%. All samples were tested in duplicate and mean values were used for analysis.Sera or plasma samples were drawn pre-operatively  and were stored at −80 °C. Soluble PD-L1 was measured using a validated ELISA14. In brief, three 0.6 mm cores were taken from the tumorous areas and two 0.6 mm cores were taken from the surrounding tumor free liver tissue of each tissue block. The tumorous areas with vital tissue were marked by an experienced pathologist using archived H&E glass slides. The TMAs were made using an automated tissue-arrayer ATA-27 .TMAs were constructed as previously described14. In brief 4 µm thick sections were mounted on Superfrost PlusTM slides. The sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.3% H2O2 for 15 minutes. Antigen retrieval was performed in a microwave for 10 minutes using the appropriate antigen retrieval buffer. After serum block, primary antibodies were applied at 4 °C overnight. The primary antibodies were PD-L1 clone 405.9A1136 , and Gal-9 goat polyclonal31 (R&D systems). HRP-conjugated anti-mouse or anti-goat IgG polymer secondary antibody  was then applied for 1 hour, followed by diaminobenzadine (DAB) as the chromogen detection method. The slides were stained with haematoxylin followed by dehydration. Negative controls consisted of omission of the primary antibody and appropriate positive control tissues included in the TMAs were used to evaluate specificity of all antibodies.Immunohistochemistry and scoring was performed as previously describedScoring was performed by 2 independent investigators blinded to clinical outcome and differences resolved by mutual agreement. Only cytoplasmatic staining was observed for both antibodies. Intensity of tumor cell and hepatocyte staining was scored in a scale from zero to three. Intra-core heterogeneity of staining intensity of tumor cells or hepatocytes was rarely observed, thus percentages of positive tumor cells were not analyzed. Average values of the scores of the different cores were used for analysis. Patients with any evaluable PD-L1 or Gal-9 staining on their tumor cells were considered to have high expression, while patients with complete absence of staining were considered to have low expression.2 tests or the T-test as appropriate. For the correlations between the circulating and intra-tumoral immune biomarkers the Spearman Correlation test was performed. Sensitivity analysis was performed in relation to sample source (serums versus plasma), type of surgery (resection versus liver transplantation) and the use of the TNM classification system. The statistical analysis was performed using the SPSS© 21 software.All analyses were performed in duplicate. Survival curves were estimated by the Kaplan-Meier method. Survival was calculated from the date of surgery to the date of event (recurrence or death), or date of last follow up. The patients who died from causes other than HCC were censored at their time of death. The log-rank test was used to assess differences between survival curves of different groups, while for biomarkers with three linearly associated levels the linear trend for factor levels was used. Optimal high vs low values were established by examining a grid of cutoffs and choosing the cutoff with the lowest −2 log likelihood. For multivariate analysis, the Cox proportional Hazard regression analysis was used. The associations between clinicopathologic parameters with immune biomarkers were examined using the χ−1  showed a non-significant trend toward predicting HCC-specific mortality. Number of lesions, tumor differentiation, vascular invasion and cirrhosis did not predict HCC-specific mortality in our cohort. Tumor size >3 cm also predicted HCC recurrence . Circulating PD-L1 and Gal-9 were not associated with any of the above clinicopathologic characteristics with the exception of a positive association between circulating PD-L1  and cirrhosis (p = 0.045).Pre-operative circulating PD-L1 and Gal-9 levels were studied in 81 HCC patients who underwent hepatic resection or liver transplantation. Plot diagrams of circulating PD-L1 and Gal-9 levels can be seen in Supplementary Fig. −1 (p = 0.060) showed non-significant trends to association with HCC-specific survival.Median pre-operative circulating PD-L1 concentration was 383 pg/ml (intra-quartile range (IQR) 206–774 pg/ml), and median pre-operative circulating Gal-9 concentration was 21 pg/ml (IQR 3–44 pg/ml). With an optimal cutoff of 700 pg/ml, high circulating PD-L1 was associated with improved HCC-specific survival , and with a cutoff of 42 pg/ml high circulating Gal-9 was also associated with improved HCC-specific survival . The respected Kaplan-Meier curves are shown in Fig. In the majority of patients PD-L1 and Gal-9 were measured in sera, but from 22 patients only plasma samples were available. When the patients with plasma samples were excluded from the analysis, the Kaplan-Meier survival curves for the remaining 59 patients are similar to Fig. 14. Specifically, 58 patients from the previous cohort of 154 resected HCC patients had available stored peripheral blood, and were thus included in the current study. An additional 23 patients, unique in the present study, had available stored peripheral blood and thus had their corresponding tumor tissue immunohistochemically stained for PD-L1 and Gal-9. Examples of PD-L1 and Gal-9 immunohistochemical staining of tumor tissues are shown in Supplementary Fig. 14. The respective relationships of intra-tumoral PD-L1 and Gal-9 with HCC recurrence can be seen in Fig. −1 (p = 0.004) were independent predictors of HCC-specific survival.The intra-tumoral expression of PD-L1 and Gal-9, in combination with other immune inhibitory molecules, has been studied by our group before, in an overlapping cohort−1 (p = 0.050). In the case of PD-L1, both intra-tumoral PD-L1 (p = 0.056) and circulating PD-L1 (p = 0.067) showed a non-significant trend towards independently predicting HCC-specific survival, together with AFP > 100 µgl−1 (p = 0.039). In addition, there was no correlation between circulating levels and expression levels of PD-L1 or Gal-9 on hepatocytes in the surrounding tumor free liver tissue.Next we examined if pre-operative circulating levels of PD-L1 and Gal-9 reflected intra-tumoral expression. There was no correlation between intra-tumoral PD-L1 and circulating PD-L1  or between intra-tumoral Gal-9 and circulating Gal-9 . Box-and-whisker plot graphs supporting the above lack of association can be seen in Supplementary Fig. −1 and cirrhosis, are independent predictors of HCC-specific survival  or liver transplantation (n = 23) with curative intent, while Finkelmeier et al., included patients from all stages of HCC of which the majority were treated by local ablation or palliative therapy with sorafenib. Only 21% of their patients were treated with resection (n = 26) or liver transplantation (n = 19). It is entirely possible that circulating PD-L1 may have different roles in a setting of a small amount of resectable disease versus an advanced palliative setting with a large tumor burden. In the early curative setting, high levels of circulating PD-L1 may represent adaptive immune resistance of the tumor against successful immune attack, resulting in increased expression and release of PD-L1 by cells in the tumor in response to pro-inflammatory cytokines secreted by infiltrating immune cells. In this setting high levels of circulating PD-L1 would be associated with good prognosis. In the advanced metastatic setting the anti-tumor immune system may be overburdened and no longer able to suppress tumor growth. In this setting high levels circulating PD-L1 may reflect increased tumor load and therefore be associated with worse prognosis. Indeed, in the Finkelmeier study circulating PD-L1 levels were positively associated with more advanced disease, while this was not the case in our cohort . Similarly, in advanced renal cell carcinoma and advanced lung cancer high circulating PD-L1 levels were positively correlated with disease stage and with worse prognosis26. On the other hand, no correlation between circulating PD-L1 levels and outcome was found in advanced pancreatic cancer28. Such contrasting observations may not be surprising since such differences have been similarly observed in the setting of intra-tumoral PD-L1 expression where the prognostic significance of PD-L1 differs depending on the cancer type and stage. An additional difference with the Finkelmeier et al. study is the fact that in our study 54% of patients did not have cirrhosis and 25% of patients had no identifiable underlying liver disease. In contrast, in the Finkelmeier et al. study, all patients had an identifiable underlying liver disease and all patients were assigned a Child-Pugh score, indicating that all patients had cirrhosis. Thus, our opposite results may also be due to the inclusion of many HCC patients without underlying liver disease in our study. Finally, the use of different ELISA assays could be another explanation for the differing results although, despite using different assays, both studies observed similar median circulating PD-L1 levels  and used similar cutoff values (700 vs 800 pg/ml).No study has yet examined the prognostic significance of circulating Gal-9 in any cancer. On the other hand, the prognostic role of circulating PD-L1 has been examined in a few cancer types before25, suggesting that expression of soluble and membrane-bound PD-L1 are differentially regulated. Preliminary data have shown involvement of matrix metalloproteinases in shedding of membrane-bound PD-L138. More recently it has been shown that tumor cells can produce soluble PD-L1 by alternative splicing39. Moreover, PD-L1 can be released from tumor associated macrophages and dendritic cells. It has been shown by us and by other research groups that tumor associated macrophages and dendritic cells express PD-L1 in the tumor microenvironment in HCC40. Myeloid cells, especially mature dendritic cells, have been shown to release soluble PD-L1 in vitro41 and it has been recently shown that circulating activated macrophages in ovarian cancer patients also express high levels of PD-L143. Finally, another possible source of soluble PD-L may be circulating tumor cells, which have been shown to express PD-L1 in breast cancer patients44. Whatever the source of circulating PD-L1 may be, the circulating ligand is known to retain its PD-1 binding domain and immunosuppressive properties39. Regarding the form of soluble PD-L1, recent studies have shown that PD-L1 can be released from tumor cells in the form of exosomes. Interestingly, pre-treatment levels of circulating exosomal PD-L1 were higher in melanoma patients that failed to respond to pembrolizumab therapy than in non-responders, but this difference was not observed for total circulating PD-L145. In head and neck cancer, levels of PD-L1 carried by exosomes correlated with disease activity but total plasma PD-L1 levels did not46. Therefore, the form of circulating PD-L1, either expressed in circulating exosomes or in non-bound form, may additionally explain why high circulating PD-L1 levels are associated with worse or better prognosis depending on the cancer setting. Another theoretical possibility for differences between studies is the use of differing ELISA systems. It is important to note however that Frigola et al.25, found that high circulating PD-L1 is associated with worse patient survival in Renal Cell Carcinoma, results opposite to ours, using the same ELISA and the same laboratory where our assays were performed. Thus, it is likely that biologic factors, rather than technical factors, account for the differences in results between our study and that of Frigola et al., as we discuss above.An additional consideration that may influence the prognostic role of PD-L1 has to do with the source and the particular form of the circulating ligand. One hypothesis is that PD-L1can be actively shed from membrane-bound PD-L1 expressing tumor cells and/or hepatocytes. Soluble PD-L1 has indeed been shown to be released into cell culture supernatants by several, but not all, membrane PD-L1 expressing tumor cell lines23. Gal-9 does not have a signal peptide, and its secretion must involve a non-classical pathway. Cleavage from the cell surface by matrix-metalloproteinases has been suggested as a secretion pathway47, while other studies showed that cancer cells can secrete Gal-9 via exosomes48. Therefore, circulating levels of these molecules are probably not passive reflections of tissue expression. In addition, while their expression in tumor tissues probably determines their local effects, their circulating counterparts may exert systemic effects, e.g. by inhibiting systemic immunity or, in case of Gal-9, prevent formation of distant metastasis by hampering extravasation of tumor cells into other tissues22. This means that future studies on the role of PD-L1 and Gal-9 as prognostic or predictive markers should take both intra-tumoral and circulating levels into consideration to maximize the potential for biomarker optimization.Like PD-L1, Gal-9 is not only expressed on tumor cells, but also at high levels on tumor-associated macrophages in HCC24. We have previously studied the prognostic value of intra-tumoral expression of PD-L1 and Gal-914. Our current results regarding intra-tumoral expression of PD-L1 and Gal-9  indicating that sample size may be another explanation for the difference, and that perhaps our results are not that discrepant with the results of the Fujita et al., study. In addition, it is important to keep in mind that in multivariate analysis the association of circulating PD-L1 and Gal-9 with survival was independent of the presence or absence of cirrhosis. Thus cirrhosis was not a confounder of PD-L1 or Gal-9 levels in regards to patient survival.We found an association between circulating PD-L1 and the presence of cirrhosis. A similar relationship was suggested by Finkelmeier Our study has several strengths. It is the first study to examine the role of circulating Gal-9 in HCC and one of only two studies, to date, to examine the role of circulating PD-L1 in HCC. In addition, few studies have studied circulating levels of these molecules in other cancers, thus this is a fairly unexplored field of research. Most importantly both the intra-tumoral expression and the circulating levels of these molecules were compared. The observation that intra-tumoral and circulating levels independently contribute to prognostication, is novel. Testing for the expression of these molecules in the surrounding tumor free liver tissue also allowed us to show that it is not the expression on hepatocytes in the diseased liver that determines circulating levels of PD-L1 and Gal-9. Our study has also limitations. One limitation of our study is the use of both serum and plasma samples for our analysis. While we show in Supplementary Fig. In summary, pre-operative circulating levels of PD-L1 and Gal-9 do not correlate to their expression in tumor tissue, but have prognostic value in resected HCC patients independently of their expression in tumor tissue. Combined circulating levels and intra-tumoral expression of PD-L1 and Gal-9 provide more confident prognostic immune biomarker profiles compared to each separately.SUPPLEMENTARY INFO


For the first time, we estimated perspectives for using a new 2D carbon nanotube (CNT)–graphene hybrid nanocomposite as a base element of a new generation o optical nanodevices. The 2D CNT–graphene hybrid nanocomposite was modelled by two graphene monolayers between which single-walled CNTs with different diameters were regularly arranged at different distances from each other. Spectra of the real and imaginary parts of the diagonal elements of the surface conductivity tensor for four topological models of the hybrid nanocomposite have been obtained. The absorption coefficient for p-polarized and s-polarized radiation was calculated for different topological models of the hybrid nanocomposite. It was found that the characteristic peaks with high intensity appear in the UV region at wavelengths from 150 to 350 nm (related to graphene) and in the optical range from 380 to 740 nm irrespective of the diameter of the tubes and the distance between them. For waves corresponding to the most intense peaks, the absorption coefficient as a function of the angle of incidence was calculated. It was shown that the optical properties of the hybrid nanocomposite were approximately equal for both metallic and semiconductor nanotubes. The spin degeneracy is already taken into account in the above equations by the factor 2, η is a phenomenological parameter characterizing electron scattering processes.where , the known substitution P(k)→ was used, where  is the Hamiltonian. The detailed description for the calculation of the matrix elements of the momentum operator is given in [xx is similar to the spectrum of graphene, while the spectrum of σyy has complex and multiple peaks. As previously shown [To calculate the elements of the impulse matrix given in . The Hamly shown , a complly shown ).Special attention should be paid to the peak of great intensity observed in the wavelength range of 800–830 nm for models with tube  . One canA) for four types of the tubes with an intertube distance of 13 hexagons, and The calculation results of the absorption of electromagnetic waves of the CNT–graphene hybrid nanocomposite are presented in The analysis of the diagrams in XZ-plane; 2) the wave vector lies in the YZ-plane. YZ-plane, and at angles of 85–90° for the irradiation in the XZ-plane. The absorption reaches values of 45–50% at these angles of incidence. Thus, one can say that the optical properties of the composite do not explicitly depend on the type of the tubes.For wavelengths corresponding to the most intense peaks, a diagram of the dependence of absorption coefficient  on the aIn summary, it can be concluded that the new 2D CNT–graphene hybrid nanocomposite is very promising for optoelectronic devices. In particular, the established regularities of change in absorbance as a function of the angle of incidence of the electromagnetic wave allows us to suggest the possibility of using the CNT–graphene film as a polarizer for electro-optical and magneto-optical thin film modulators. The advantages of such polarizers are a wide spectral range and low loss.


AbstractPorcellionidespruinosus, a terrestrial isopod, was chosen as a non-target species to investigate such possible effects. Two different experiments were conducted to study two different neem-derived plant protection products, i.e., NeemAzal T/S (1% azadirachtin) which is a commercial product registered in the EU, and neem leaf extract from dried neem leaves (1%).The latter simulates the plant protection product, is domestically produced, and widely used by farmers in India and other tropical and subtropical countries. Findings are consistent with previous results obtained with other non-target organisms, i.e., neither of the tested neem products have adverse effects on the mortality of P.pruinosus. However, further research on a wider range of soil organisms is needed to prove the safety of neem-based products as biological control agents and to be part of integrated pest management.Neem-based products have gained major attention over the last few years due to their wide range of applications in pest management, and have been in the focus of biological plant protection research in the past decade. Yet, there is limited information available to understand the side effects of these neem-derived pesticides on non-target species in soil. Therefore,  Azadirachtaindica)-based biological insecticides requires more information about their possible side effects on non-target organisms. Such organisms are the woodlice species, important decomposers of organic material in agricultural areas. It was demonstrated that neither a commercial product of neem nor a domestic neem leaf extract had any adverse effect on a ubiquitous woodlice species, Porcellionidespruinosus.The safe use of neem (Azadirachtaindica A. Juss) which has been known in the Indian sub-continent for more than 4000 years  (Lepidoptera), one of the most important cabbage pests in agriculture. Extensive research is being conducted with neem and its derived products against target pests. Aphisgossypii (Glover) and found that the aqueous neem seed extract is efficient in nymph mortality and reducing the survival period and fecundity of the cotton aphid.Neem extracts and products have been used against different orders of insects such as Daphniamagna  and Hyalellaazteca . Nicrophorusvespilloides (Herbst 1783), a common burying beetle, feeding on a variety of animal carcasses. Nicrophorusvespilloides were fed with cockchafers which were previously fed with neem-treated leaves. They found that it had no negative impact on the reproduction nor they found any impacts on the morphology of N.vespilloides.The effect of neem-derived products on non-target organisms has also been studied. However, information on the effect of neem-based pesticides and formulations on non-target organisms of soil biota are still limited. Isopoda, Oniscidae) are ubiquitous saprophagous members of the soil fauna  and Enchytraeusalbidus  an annelid, commonly known as white worm, using avoidance as test parameter. For both the species, patterns of antagonism were found when exposed to dimethoate and atrazine, synergism for lindane, dimethoate, and atrazine, synergism for lindane and dimethoate exposures and concentration addition for cadmium and zinc occurred, while the exposure to cadmium and dimethoate showed dissimilar patterns   . ES can  society . Soil fu society . If the  society , or heav society  that esp society .A small-scale terrestrial ecosystem containing soil collected from an agricultural field in Central Portugal was used to evaluate the effects of the combination of the herbicide glyphosate and the insecticide dimethoate in another study. The application of dimethoate led to a decrease in feeding activity in all concentrations tested. The mortality of isopods exposed to dimethoate in single and binary exposures was high. Exposure to dimethoate decreased the acetylcholinesterase activity of isopods .Porcellionidespruinosus. We selected P.pruinosus as a non-target organism, because it is ubiquitous and it occurs in anthropogenic environments, where pest control is applied. They play a vital role in the fragmentation and decomposition process of leaf litter, thereby causing changes in soil quality and soil services  (t (1833) .Preparation of neem leaf extract: For neem leaf extract, air-dried neem leaves were obtained from local growers in India, Maharashtra, Konkan Division, Mumbai Suburban area. A stock concentration of 1% was prepared by soaking 1g of crushed dried neem leaves in 100 ml distilled water overnight and then filtered using a non-sterile filter paper. Different working concentrations  of neem leaf extract were prepared from 1% stock solution using distilled water in the laboratory and were used on the same day. Generally the applied dosage used by local growers in India has a maximum concentration of 1%. In this experiment, we tried to model the concentration used by the local growers in the field conditions.Preparation of azadirachtin: NeemAzal T/S (Trifolio-M GmBH), a commercial product containing 1% azadirachtin, registered in the EU, was used. A stock concentration of 1% azadirachtin was prepared  by diluting 1 ml NeemAzal T/S in 100 ml of distilled water which equals to 0.01% azadirachtin. It was further diluted to get the 0.0005, 0.001, 0.0025, 0.005, 0.0075 and 0.01% azadirachtin concentrations respectively and was used on the same day. The registered dosage of azadirachtin ranges from 0.0025 to 0.005%, depending on the plant culture in the EU.A control with only distilled water was used for both experiments. The working concentrations and distilled water were sprayed using a hand sprayer under laboratory conditions.Experimentaldesign: Five adults of Porcellionidespruinosus were placed in glass Petri dishes (13 cm in diameter), with 1 g of commercial horticultural soil (pH = 7.0) and approximately 1 g of fresh potato as a food source. Two milliliters of different working concentrations of neem leaf extract and azadirachtin were sprayed using a hand sprayer. After spraying, the Petri dishes were kept in the dark, checked after time periods of 1, 24, 48, 72, 96, and 120 hours post-application of neem leaf extract and azadirachtin respectively, and mortality data was recorded. The mortality data obtained after 120 hrs was subjected to statistical analysis using R software (R Core Team 2017). Logistic regressions were fitted  to check the effect of the two different products on isopod mortality. To test whether the concentrations have significant effect on mortality, chi-squared tests were performed on model deviances. Prior to running the tests model diagnostic plots were investigated to assess homoscedasticity and residual normality  as studied by Nicrophorusvespilloides, which co-occurs with the forest cockchafer Melolonthahippocastani  and also feeds on the carcasses of M.hippocastani. In their study, they fed N.vespilloides with dead M.hippocastani which were previously fed with neem-treated leaves. They neither observed any impact on the mean larval weights nor on the morphology of N.vespilloides. Still, they authors did not dismiss the possibility of azadirachtin passing through the food chain and affecting other non-target organisms.While there are numerous literature references available on the effect of neem and neem-derived products on target organisms, some of the studies reported data on non-target organisms as well. For instance, Philosciamuscorum  and did not find any negative effects on P.muscorum. The results of our experiments were found to be similar and this experiment for the first time investigated the effects of two different neem products on this non-target isopod species, i.e., Porcellionidespruinosus.P.pruinosus.From our results it can be concluded that neither NeemAzal T/S nor neem leaf extracts pose any risk to the terrestrial isopod species studied in the tested concentrations. However, further research is needed to test the possible effect of various neem products on the members of the soil fauna. Also, it can be concluded that NeemAzal T/S and domestic neem leaf extract do not differ in respect to their mortality effects on PD and AMP designed and conducted the experiments. MS analyzed the data. PD wrote the manuscript. AMP, FT, GT, and MS gave their feedback. All authors read and approved the manuscript.


Complexation of cis-Pd(tmeda)(NO3)2 with L1 at a 1:1 or 3:2 ratio produced [Pd(tmeda)(L1)](NO3)2 (1a). The reaction mixture obtained at 3:2 ratio upon prolonged heating, produced a small amount of [Pd3(tmeda)3(L1)2](NO3)6 (2a). Complexation of Pd(NO3)2 with L1 at a 1:2 or 3:4 ratios afforded [Pd(L1)2](NO3)2 (3a) and [(NO3)2@Pd3(L1)4](NO3)4 (4a), respectively. The encapsulated NO3– ions of 4a undergo anion exchange with halides  to form [(X)2@Pd3(L1)4](NO3)45a–7a. The coordination behaviour of ligand L1 and some dynamic properties of these complexes are compared with a set of known complexes prepared using the regioisomeric ligand bis(pyridin-3-ylmethyl)pyridine-3,5-dicarboxylate (L2). Importantly, a ligand isomerism phenomenon is claimed by considering complexes prepared from L1 and L2.The complexation study of  N-donor ligands) is particularly advantageous for the construction of a variety of metallocages (NO3)2  variation was also carried out to successfully prepare a series of complexes [Pd(tmeda)(L1)](BF4)2 (1b); [Pd(tmeda)(L1)](ClO4)2 (1c); [Pd(tmeda)(L1)](OTf)2 (1d). The complexes 1a–d were prepared by mixing the corresponding metal component Pd(tmeda)(Y)2 with the ligand L1 where Y = NO3−, BF4−, ClO4− and OTf−. The metal components were prepared in situ by reacting Pd(tmeda)(Cl)2 with AgY in DMSO-d6 followed by separation of the precipitated AgCl.The addition of one equivalent of 1a was characterized by various NMR techniques  as compared to the free ligand L1. The peak positions of Hf and Hg remained unchanged which indicated that the central pyridine ring is not involved in the complexation. The 1H NMR spectra of compounds 1b, 1c and 1d are very much comparable to that of 1a (NO3)2 to a clear solution of two equivalents of ligand L1 in DMSO-d6 produced a turbid mixture. However, a clear yellow solution was obtained upon stirring the mixture at 90 °C for 5 min. The progress of the complexation reaction was monitored by 1H NMR spectroscopy. We targeted a Pd3L’3L2-type complex [3(tmeda)3(L1)2](NO3)6 ](NO3)2 (1a) and uncomplexed cis-Pd(tmeda)2+. The reaction was allowed to continue where upon the integration ratios of the peaks corresponding to Hf and Hg were lower than expected and that of Hd was higher than expected. In addition, a new peak was observed at around 9.15 ppm 2+ remaining in solution. The peaks assigned to Hf and Hg of 2a are shifted downfield compared to those of Hf and Hg in 1a whereas other signals of 2a merged with the respective signals of 1a. The observed downfield shift of the Hf signal in 2a is complexation-induced and found to have a unique position. The downfield shift of the Hg signal in 2a could not be induced by complexation and is best described by considering an anisotropy effect of the nearby carbonyl groups the ligand strand. The Hg signal of 2a, however, merged with the Hd signal of 1a.The addition of three equivalents of  complex , i.e., (NO3)2  variation was also carried out to successfully prepare a series of complexes [Pd(L1)2](BF4)2 (3b), [Pd(L1)2](ClO4)2 (3c), and [Pd(L1)2](OTf)2 (3d). These complexes were prepared by complexation of Pd(Y)2 with the ligand L1 where Y = BF4−, ClO4−, and OTf−. It is important to note that Pd(Y)2 solutions were prepared by reacting PdI2 with AgY and the precipitated AgI was removed by filtration. Following this procedure, the presence of iodide as impurity could not be ruled out but its presence was found to not influence the formation of the targeted complex. In contrast, the presence of chloride remaining as impurity when PdCl2 was reacted with AgY to prepare Pd(Y)2, contaminated Pd(Y)2 and produced upon complexation with L1 complexes 3a–d along with some other products. The choice of PdI2 is on the basis of our previous experience from related cages (NO3)2 (3a) and [(NO3)2@Pd3(L1)4](NO3)4 (4a) in the mixture  with the ligand L1 at a 3:4 ratio resulted in the formation of only mononuclear complexes 3b–d (depicted in a later Scheme) and the unutilized proportion of Pd(Y)2 remained in solution. Thus, the counter ion nitrate has a determining role by acting as template for the cavities in the formation of the trinuclear complex 4a. It is proposed that the anion is essential to avoid charge repulsion between the metal centers in the ensuing cavities. However, the anions should be of fitting sizes only to get accommodated in the cavities so that discrete architectures are formed. Larger anions such as BF4−, ClO4− and OTf− are not accommodated in the cavities and not helpful as templates. The Pd(NO3)2 sample prepared from PdCl2 and AgNO3, contained chloride as impurity and resulted in a mixture of products along with the targeted 4a (3)@Pd3(L1)4](NO3)4 (6a’) and [(Cl)2@Pd3(L1)4](NO3)4 (6a) and their proportion was found to depend on the amount of chloride as impurity. The influence of chloride on the product composition is discussed in a later section.A solution containing three equivalents of commercially acquired Pd as compared to the free ligand L1. The signal of Hf also got downfield-shifted indicating that the central pyridine ring is involved in the complexation.The complex of peaks  featured4a confirmed the formation of a trinuclear complex ]2+, [Pd3(tmeda)3(L1)2]6+, [Pd(L1)2]2+, and [(NO3)2@Pd3(L1)4]4+ are shown in 3(tmeda)3(L1)2]6+ could not be prepared exclusively, we looked into the energetics of the system. The overall Gibbs free energies (∆G) and the enthalpies (∆H) for the formation of the trinuclear complex [Pd3(tmeda)3(L1)2]6+ considering its formation from 1 equivalent of [Pd(tmeda)(NO3)2] and 2 equivalents of [Pd(tmeda)(L1)]2+ were found to be not feasible  and endothermic , respectively (L1)2](NO3)2 (3a) was found to be stable at room temperature for days in DMSO-d6  got reduced to palladium(0). In another experiment, Pd(NO3)2 was added to a solution of 3a at a 2:1 ratio where upon complex-to-complex conversion was observed at room temperature or upon heating to afford 4a (3)2 a complete formation of 4a was observed within 20 min at rt or 5 min at 90 °C  to match the stoichiometric requirement for the formation of 3a. Although the formation of 3a was observed, it remained only as a minor product, and the added ligand L1 was partially consumed. Thus, the unbound ligand remained in its free state along with 4a. No further change was observed after 30 min 2@Pd3(L1)4](NO3)4 (4a) was prepared by mixing Pd(NO3)2 with ligand L1 at a 3:4 ratio (4)2, Pd(ClO4)2 or Pd(OTf)2 with L1 at a 3:4 ratio did not afford the analogous trinuclear complexes; rather the corresponding mononuclear complexes were formed and the uncomplexed palladium(II) remained in solution   in four separate experiments using DMSO-d6 as the solvent. The anion exchange processes were monitored by 1H NMR spectroscopy of the samples (NO3)@Pd3(L1)4](NO3)4 (6a’) and [(Cl)2@Pd3(L1)4](NO3)4 (6a). With further addition of TBACl, the proportion of 6a increased at the cost of 4a and 6a’ to finally yield compound 6a as the only product. Similarly, the addition of TBABr initially produced a mixture of 4a, [(Br)(NO3)@Pd3(L1)4](NO3)4 (7a’) and [(Br)2@Pd3(L1)4](NO3)4 (7a) with complex 7a as the exclusive final product. In the case of TBAF, initially there was no change observable except a slight broadening of the signals for Hb and Hc. Further addition of TBAF led to the formation of [(F)2@Pd3(L1)4](NO3)4 (5a) along with minor impurities. However, the addition of TBAI to 4a showed no changes and hence iodide encapsulation did not happen. The 1H NMR spectra for the mixtures of products formed at intermediate and final stages are provided in 4a, 5a, 6a and 7a are shown in 1H NMR spectra of these anion-encapsulated complexes are influenced by coordination of ligand L1 with palladium(II) and interaction of the encapsulated anion with the endohedrally oriented hydrogens of the cages.Each of the two cavities of cage  earlier –23. Hali4−, ClO4− or OTf− could not support the formation of the double-decker cage even though the required amount of palladium(II) was available. The addition of TBAI to any of these solutions containing Pd(Y)2 and 3b, 3c or 3d, respectively, did not lead to double-decker cages indicates that I− is not suited for the cavity. However, addition of TBANO3, TBAF, TBACl and TBABr produced the corresponding anion encapsulated double-decker cages as shown in steps (iv), (v) and (vi) of 1H NMR spectra for the conversion of 3b to corresponding products 4b, 5b, 6b and 7b are shown in Is iodide not capable of replacing the preexisting nitrate in a competition or iodide is not suited at all for the cavity irrespective of any competition? The following argument might answer this question. The complexation reaction shown in steps (ii) of 1H NMR spectral analysis of the two nitrate anions incorporating compound 4a discussed in an earlier section revealed downfield shifts of particular signals as compared to the free ligand L1 and the Δδ values were 1.42, 0.82 and 1.02 ppm for the signals of Ha, Hb, and Hf, respectively. A similar comparison for the (i) two F− encapsulated compound 5a: Δδ = 1.75, 0.99 and 1.78 ppm for Ha, Hb, and Hf, respectively), (ii) the two Cl− encapsulated compound 6a: , and (iii) the two Br− encapsulated compound 7a:  are in line with the expectation. Although fluoride (F–) could replace NO3– in 4a to afford complex 5a, the complex 5a was found to be unstable and it decomposed within a few hours. Thus the 1H NMR and ESIMS spectrum of 5a were recorded from freshly prepared samples. The complexes 4a, 6a and 7a are quite stable and no decomposition was observed. Detailed characterization data of 4a–7a form a variety of NMR techniques are provided in The 6a and 7a were detected. Also, one of the mixed halide–nitrate encapsulated complexes, i.e., 7a’ was also detected.The molecular compositions of the halide encapsulated complexes were confirmed by recording ESIMS data for the systems. The double halide encapsulated complexes 6a ](NO3)2 (1a), [Pd(L1)2](NO3)2 (3a), [(NO3)2@Pd3(L1)4](NO3)4 (4a), and [(X)2@Pd3(L1)4](NO3)45a–7a fulfill the definition of ligand isomerism when compared with the reported complexes [Pd(tmeda)(L2)](NO3)2 (8a), [Pd(L2)2](NO3)2 (10a), [(NO3)2@Pd3(L2)4](NO3)4 (11a), and [(X)2@Pd3(L2)4](NO3)412a–14a, respectively. We have demonstrated ligand isomerism in Pd2L4-type cages [3L4-type double-decker coordination cages for the first time.As discussed in the introduction section “The definition of ligand isomerism includes metal complexes (at least two) having the same molecular formula but are composed of different structural isomers of the ligand.” The complexes prepared in this work namely [Pd(tmeda)(NO3)2 with L1 produced the trinuclear complex [Pd3(tmeda)3(L1)2](NO3)6 (2a), the ligand L2 did not afford the targeted [Pd3(tmeda)3(L1)2](NO3)6 (9a). The complex [Pd(L1)2](NO3)2 (3a) was unstable when heated in DMSO medium whereas the corresponding complex [Pd(L2)2](NO3)2 (10a) was stable under comparable conditions. The addition of two equivalents of L1 to a solution of [(NO3)2@Pd3(L1)4](NO3)4 (4a) produced only a small amount of [Pd(L1)2](NO3)2 (3a) and the added ligand remained in solution. On the other hand, addition of the required amount of L2 to a solution of [(NO3)2@Pd3(L2)4](NO3)4 (11a) resulted in complete transformation to complex Pd(L2)2](NO3)2 (10a). The F− encapsulated complex [(F)2@Pd3(L1)4](NO3)4 (5a) decomposed within a few hours whereas [(F)2@Pd3(L2)4](NO3)4 (12a) was stable for a few hours. These differences are ascribed to the positional exchanged functionalities in the ligands L1 and L2. Probably, the coordination ability of the central pyridine ring is better than that of the terminal pyridine rings in case of L1. However, in the mononuclear complexes of L1 the central pyridine remained uncomplexed, which may be due to the formation of metallomacrocyclic rings. This behavior is not observed in the case of mononuclear complexes of L2. Thus, the mononuclear complexes of L1 are reluctant to form  and prone to decomposition. As far as trinuclear complex formation is concerned the central pyridine ring of L1 is in a relatively favorable situation, thus the complex 4a could form and 3a was a kinetic product.The complexation behavior of cis-protected palladium(II) and bare palladium(II) components with the new tridentate ligand L1. A variety of counter anions were employed to broaden the scope of the choice of metal components. Mononuclear complexes with PdL’L composition could be prepared easily, however, Pd3L’3L2-type trinuclear complexes were obtained in only small amounts. Also, mononuclear complexes of PdL2 and trinuclear complexes of Pd3L4-type compositions were prepared easily. The choice of the counter anion did not influence the formation of mononuclear complexes whereas the counter anion displayed a template role for the formation of trinuclear complexes, especially for Pd3L4-type complexes. The anions helped to screen the charge repulsion between the palladium(II) ions. The complexation behavior of palladium(II) components with the ligand L2 have been reported earlier +.[Pd(tmeda)(L1)](NO3)2 (1a): To a solution of cis-Pd(tmeda)(NO3)2 , in 3 mL of DMSO ligand L1  was added. The reaction mixture was stirred at room temperature for 10 min to obtain a clear yellow solution. The product was precipitated by the addition of ethyl acetate (10 mL), separated by centrifugation, washed with acetone (4 mL) and dried under vacuum to afford complex 1a . 1H NMR  δ 9.90 , 9.35 , 8.67 , 8.54–8.52 , 8.18 , 7.85 , 5.91–5.29 ; 13C NMR  δ 164.50, 154.60, 151.91, 147.21, 140.92, 133.03, 132.24, 129.41, 127.31, 63.41, 62.31.[Pd(L1)2](NO3)2 (3a): To a solution of ligand L1  in 3 mL of DMSO, Pd(NO3)2  was added and the reaction mixture was stirred for 10 min at room temperature to give a clear yellow solution. The product was precipitated by the addition of 10 mL of ethyl acetate. The pale yellow precipitate was separated by centrifugation, washed with acetone and dried under vacuum to afford complex 3a . 1H NMR  δ 9.92 , 9.30 , 8.75 , 8.55–8.53 , 8.31 , 7.82 , 5.63 ; 13C NMR  δ 162.43, 154.57, 151.59, 147.83, 141.30, 133.97, 132.31 129.57, 127.38, 63.86; ESIMS (m/z): 866.10 [3a − 1NO3]+; 402.05 [3a − 2NO3]2+.[(NO3)2@Pd3(L1)4](NO3)4 (4a): To a solution of ligand L1  in 2 mL of DMSO, Pd(NO3)2  in 1 mL of DMSO was added and the reaction mixture was stirred for 5 min at 90 °C to give a clear yellow solution. The product was precipitated by the addition of 10 mL of ethyl acetate. The pale yellow precipitate was separated by centrifugation, washed with acetone and dried under vacuum to afford the complex 4a . 1H NMR  δ 10.54 , 9.74 , 9.66 , 8.58 , 8.26 , 7.95–7.93 , 5.45 ; 13C NMR  δ 162.40, 155.05, 153.19, 149.91, 141.88, 138.39, 133.83, 128.66, 127.68, 64.84; ESIMS (m/z): 982.04 [4a − 2NO3]2+; 634.03 [4a − 3NO3]3+; 460.03 [4a − 4NO3]4+.File 1Experimental procedures, NMR, ESIMS data, and theoretical study.


The EC50 values of the PD-L1 inhibitors were 6.46 ng/ml (5.48–7.61 ng/ml), 6.15 ng/ml (5.24–7.21 ng/ml) and 7.64 ng/ml (6.52–8.96 ng/ml) for atezolizumab, avelumab, and durvalumab, respectively. In conclusion, a functional assay evaluating antibodies targeting PD-1 inhibition in vitro revealed that pembrolizumab is a slightly more effective PD-1 blocker than nivolumab, and that PD-L1 antibodies are superior to PD-1 antibodies in reverting PD-1 signaling.Inhibitors of PD-1 signaling have revolutionized cancer therapy. PD-1 and PD-L1 antibodies have been approved for the treatment of cancer. To date, therapeutic PD-1 inhibitors have not been compared in a functional assay. We used an efficient T cell reporter platform to evaluate the efficacy of five clinically used PD-1 inhibitors to block PD-1 signaling. The half maximal effective concentrations (EC However, PD-1, which is upregulated on activated and exhausted T cells, also limits productive immune responses against pathogens and cancer cells. PD-1 signaling is induced upon binding of its ligands, PD-1 ligand-1 (PD-L1) and PD-1 ligand-2 (PD-L2). The expression of PD-L2 is mainly restricted to professional antigen presenting cells (APCs) like macrophages and dendritic cells (DCs), whereas PD-L1 is also expressed in non-hematopoietic tissues and can thus be regarded as the major PD-1 ligand. Importantly, PD-L1 is also upregulated in the tumor microenvironment and is found in a large variety of tumor cells. Tumor infiltrating lymphocytes frequently express PD-1, providing a rationale for therapeutically disrupting the PD-1/PD-L1 interaction to improve anti-tumor responses. Currently, several PD-1 and PD-L1 antibodies are in clinical use for the treatment of various solid cancers and lymphomas, and blocking of the PD-1 pathway was shown to induce impressive response rates across a broad spectrum of tumor types50) provide valuable information on the efficacy of these antibodies. However, these values are not available for therapeutic PD-1 and PD-L1 antibodies, and the ability of these agents to block PD-1 signaling have not been compared in a functional assay. We have previously developed and described an efficient, robust and cost-effective fluorescent T cell reporter platform for the evaluation of immune checkpoint inhibitors (ICIs) targeting different inhibitory receptors, including PD-13.Although the clinical indications for which regulatory authorities have approved antibodies targeting PD-1 and PD-L1 only partially overlap, the mode of action of these drugs is blocking PD-1 signaling. Therefore, it is evident that the ability of these agents to disrupt the function of PD-1 critically impacts their clinical efficacy. As with other drugs, dose response curves and half maximal effective concentrations , while pembrolizumab (39.90 ng/ml) was slightly more effective , avelumab (12.69 ng/ml) and durvalumab (13.76 ng/ml)  and prembrolizumab (7.89 ng/ml) were also similar  Fig. . The EC5lar Fig. .Figure 350 values and the 95% CI from the functional assays and the binding assays are summarized in Table 50 values of the therapeutic PD-L1 antibodies were very similar and were significantly lower than the EC50 values of the therapeutic PD-1 antibodies  or modified IgG1 Fc sequences .Therapeutic antibodies targeting PD-1 inhibition have not been compared in clinical trials. Although analysis of published data on non-small-cell lung carcinoma (NSLC) patients did not reveal differences in the efficacy between PD-1 and PD-L1 antibodies, such studies have many limitations as pointed out by the authors25. Therapeutic PD-L1 antibodies also disrupt the binding of PD-L1 to CD80, and therefore PD-L1 antibodies could potentially also augment T cell responses by blocking this pathway.In addition to PD-1, PD-L1 also binds CD80, a molecule which has an important role as a costimulatory ligandPD-L1 blockers are used at much higher doses than PD-1 antibodies, while the results of our study reveal that PD-L1 antibodies have a superior blocking capacity. The serum half-life of IgG1 and IgG4 antibodies is 21 days and the reported half-lives of nivolumab, pembrolizumab, atezolizumab, and durvalumab were in this range, while a much lower half-life was reported for avelumab Table . It is p50 values measured in our in vitro assays26. Based on studies in mice, the concentration of these antibodies in the tumor microenvironment can be expected to be much lower than the Cmax values27. Information on the concentrations of PD-1 or PD-L1 antibodies in the tumors of treated patients is not available, and thus it is not known whether they are indeed higher than the EC50 values obtained in our in vitro assays. Nevertheless, the results of our study may provide a rationale for testing antibodies targeting PD-1 inhibition and in particular PD-L1 antibodies at lower doses. Various factors, such as hypoxia or difficulty in tumor stroma penetration, restrict the immunotherapy effects and measures to overcome these limitations; for instance, using antibody drug conjugates or prodrugs based disruption of hypoxia were shown to have utility in improving immune checkpoint blockade29.We show here, that the ICIs that are currently used to target the PD-1/PD-L1 interaction are extremely efficient blockers of PD-1 inhibition. Cmax values for pembrolizumab measured in sera of treated patients were more than three orders of magnitudes higher than the EC31. Selecting ICIs that are highly effective at blocking these pathways will greatly increase the prospects of such endeavors. Cellular reporter platforms such as the one used here are well-suited to discern ICIs with high potential.Currently, there are intense efforts to develop novel ICIs targeting inhibitory pathways like LAG-3, TIM-3, TIGIT, BTLA or VISTA32. The generation and validation of Jurkat E6.1 NF-κB::eGFP, and Jurkat E6.1 NF-κB::eGFP-PD1 reporter T cell lines have been previously reported3. T cell stimulator cells (TCS), which are BW cells engineered to stably express an anti-human CD3 single chain fragment have previously been described in detail4. TCS expressing high levels of PD-L1, PD-L2, CD86 and co-expressing PD-L1 and CD86 or no human costimulatory molecule (TCS-control) were generated by retroviral transduction. Surface expression was confirmed by flow cytometry. All cell lines were tested for the absence of mycoplasma using a method described recently33. The cells were stained with a panel of antibodies to authenticate them, and the reporter and stimulator cells were kept in culture for up to three months without perceptible loss of functionality. The following antibodies from Biolegend  were used: APC-conjugated PD-1 (#EH12.2H7), APC-conjugated PD-L1 (#29E.2A3), APC-conjugated CD86 (#IT2.2), PE-Cy7-conjugated CD3 (#UCHT-1), PE-conjugated PD-L2 (#24 F.10C12), PE-conjugated CD28 (#28.2), and the PE-conjugated isotype antibody control. A DyLight-649-conjugated goat-anti-mouse IgG (H + L) antibody  was used to detect the membrane-bound anti-CD3 fragment. An APC-conjugated antibody to mouse CD45  was used to exclude TCS in reporter assays.The Jurkat E6.1 cells and the BW5417 cell line, which is a murine thymoma cell line (short designation in this work: BW) were derived from inhouse stocks and cultured as previously describedThe PD-1 antibodies, nivolumab  and pembrolizumab , and the PD-L1 antibodies, avelumab , durvalumab , and atezolizumab  were used at the indicated final concentrations.Flow cytometry was performed on a FACSCalibur flow cytometer  using CellQuest software. Data were analyzed with FlowJo  and GraphPad Prism .4) and TCS (2 × 104) were cocultured in 96-well flat bottom plates for 24 h in the presence or absence of antibodies to PD-1  or PD-L1  at 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, and 0.98 ng/ml (two-fold dilution steps). Subsequently, reporter gene expression (eGFP) was analyzed by flow cytometry as previously described in detail3. Briefly, cells were harvested and TCS were excluded using mCD45 mAb. The geometric mean of the fluorescence intensity (gMFI) of viable reporter cells was used for further analysis. To estimate EC50 values, three independent PD-1 reporter stimulation experiments were performed in duplicate. For each stimulation experiment, reporter gene induction in response to stimulation in presence of PD-L1 (stimulation with TCS-CD86/PD-L1) was normalized to reporter gene expression in the respective control (stimulation with TCS-CD86) and expressed as fold induction of the (gMFI).Reporter cells (5 × 105) were incubated for 30 minutes at 4 °C with the PD-1 antibodies nivolumab or pembrolizumab at final concentrations of 3.16 µg/ml, 1 µg/ml, 316 ng/ml, 100 ng/ml, 31.6 ng/ml, 10 ng/ml, 3.16 ng/ml, 1 ng/ml, 316 pg/ml, 100 pg/ml, 31.6 pg/ml, and 10 pg/ml. All the PD-L1 antibodies, avelumab, durvalumab, and atezolizumab were incubated with PD-L1 expressing TCS at final concentrations of 10 µg/ml, 3.16 µg/ml, 1 µg/ml, 316 ng/ml, 100 ng/ml, 31.6 ng/ml, 10 ng/ml, 3.16 ng/ml, 1 ng/ml, 316 pg/ml, 100 pg/ml, 31.6 pg/ml and 10 pg/ml. Binding was detected using an APC-conjugated goat-anti-human IgG (Fc-specific) antibody (Jackson ImmunoResearch) measured via flow cytometry. Three independent PD-1 and PD-L1 antibody binding experiments were performed in triplicate. The geometric mean of the fluorescence intensity of viable reporter cells was used for further analysis.PD-1 expressing reporter cells .A 4-parameter logistic function was fitted to assess the relationship between log concentration (mg/ml) and binding (gMFI) or normalized stimulation. The fit was excellent with a pseudo-R² above 0.9. ECDataset 1


Cancer immunotherapy involves blocking the interactions between the PD-1/PD-L1 immune checkpoints with antibodies. This has shown unprecedented positive outcomes in clinics. Particularly, the PD-L1 antibody therapy has shown the efficiency in blocking membrane PD-L1 and efficacy in treating some advanced carcinoma. However, this therapy has limited effects on many solid tumors, suspecting to be relevant to PD-L1 located in other cellular compartments, where they play additional roles and are associated with poor prognosis. In this review, we highlight the advances of 3 current strategies on PD-1/PD-L1 based immunotherapy, summarize cellular distribution of PD-L1, and review the versatile functions of intracellular PD-L1. The intracellular distribution and function of PD-L1 may indicate why not all antibody blockade is able to fully stop PD-L1 biological functions and effectively inhibit tumor growth. In this regard, gene silencing may have advantages over antibody blockade on suppression of PD-L1 sources and functions. Apart from cancer cells, PD-L1 silencing on host immune cells such as APC and DC can also enhance T cell immunity, leading to tumor clearance. Moreover, the molecular regulation of PD-L1 expression in cells is being elucidated, which helps identify potential therapeutic molecules to target PD-L1 production and improve clinical outcomes. Based on our understandings of PD-L1 distribution, regulation, and function, we prospect that the more effective PD-L1-based cancer immunotherapy will be combination therapies. Cancer immunotherapy is a specific method to eliminate cancer cells by enhancing or modulating the host immune system. The immune checkpoint molecules regulate the immune balance, and the neutralization of immunosuppressive checkpoints can lead to cancer elimination. Among these immune checkpoints, the blockade of programmed death-protein 1 (PD-1) and its ligands 1 and 2 (PD-L1/2), an intrinsic negative checkpoint, leads to one of the most successful immunotherapies by enhancing T cell immune responses against tumor cells. Currently, the blockade of PD-1/PD-L1 can be achieved via three methods: (1) antibody blockade, (2) gene silencing, and (3) small-molecule pathway inhibition. The commercial PD-L1 antibodies have shown tremendous success, in particular, for advanced cancers such as melanoma and non-small lung cancers , which would affect the efficacy of PD-L1 based immunotherapy to some degree. Recent investigations are preferential to the blockade/suppression of mPD-L1 and extracellular sPD-L1, with lack of focus on other PD-L1 formats. Considering the roles of other PD-L1 formats in tumor immune resistance , the impDue to the difference in the distribution and functions of these PD-L1 formats, how to deal with all of them needs to be considered and developed comprehensively in order to improve the PD-L1 based cancer immunotherapy.Immunohistochemical (IHC) study of patient tumor tissues suggests that PD-L1 positive immune responses may appear on the tumor cell membrane (mPD-L1) and in the cytoplasm (cPD-L1). Given the transmembrane structure of PD-L1, the positive immune responses may partially be due to the obscure binding of antibody to specific domain of PD-L1 , 32. Meavia the same pathway  is often detected in sera/supernatants and its concentration is strongly associated with the expression level of PD-L1. Despite the obvious relation with mPD-L1, the generation of sPD-L1 is not clear. There are two possibilities: (1) the fragment from mPD-L1 cleaved by proteolytic enzymes, and (2) endogenous translated integrity protein or splice variant for secretion , 29, 35.The PD-L1 antibody is able to bind with PD-L1 on tumor/antigen presenting cell surfaces, thus reversing the negative immune regulation. With great success in clinic trials, the development of PD-L1 antibodies has attracted wide attention. Up to now, three PD-L1 antibodies were approved by FDA, as listed in Although PD-L1 based therapy could provide a specific and relative safe anti-cancer strategy, there are still several issues unsolved. The objective response (OR) to the treatment relies much on the expression of mPD-L1, showing an obvious correlation to PD-L1 positive cohort . Even thBased on the description of PD-L1 distribution, the antibody blockade is theoretically only efficient for mPD-L1 and sPD-L1, with limited influence on intracellular PD-L1 formats. Considering the translocation of cellular PD-L1, the antibody influence on intracellular PD-L1 would be associated with the pharmacokinetics. With the blocking antibody eliminated by proteolytic degradation or systemic clearance, the intracellular PD-L1 may transfer to the cell surface and resume the ability for immune escape. Therefore, we reasonably postulate the intracellular PD-L1s are the reservoir for mPD-L1, which may explain some failure in PD-L1 positive cohort as this translocated mPD-L1 requires more frequent antibody administration and higher dosage for efficient cancer immunotherapy.Despite that the positive PD-L1 expression is the premise of PD-L1 antibody treatment logically, it is not always in concordance to the PD-L1 positive and objective response in patients . This phThe idea of using gene silencing strategy to “switch off” PD-L1 translation, or PD-L1 upregulation, may be a new therapy. Gene silencing uses a small interfering RNA (siRNA) to knockdown PD-L1 directly in tumor cells or uses microRNAs (miRs) to regulate epigenetically. The gene silencing strategy can also be used in PD-L1 modulation on host immune cells, leading to the enhancement of immune responses to cancers.in vitro studies have demonstrated that cancer cells transfected with PD-L1 siRNA are more sensitive to T cell killing compared to control groups  and demonstrated that the interference of PD-1/PD-L1 interaction led to down-modulation of TCR via casitas B-lymphoma (Cbl)-b E3 ubiquitin ligase upregulation in CD8+ T cells, and sequentially enhanced anti-tumor immune responses , miR-138-5p, miR-142-5p, are able to bind directly to the 3′-UTR of PD-L1 mRNA, and downregulate PD-L1 expression  59–62).–62.59–62in vivo results  are vulnerable to RNase in the circulation. Therefore, suitable delivery systems are required for efficient transfection. Viral vector transfection methods are able to deliver genetic material to host cells with a high efficiency but their biosafety is a big concern. While non-viral transfection methods generally have a lower efficiency, there are considerably less safety concerns associated making them a hot topic in gene silencing research. Non-viral transfection methods utilize carriers such as calcium carbonate/phosphate , 68, inen beings .PD-L1 expression is mainly regulated via MAPK (RAS/RAF/MEK/ERK) and PI3K/Akt pathways, and can be controlled by many intracellular and extracellular signals . Inhibitvia TTP mediated increase of PD-L1 mRNA stability  signaling is able to block HIF-1α accumulation in hypoxic cells, and sequentially prevent hypoxia-induced PD-L1 expression and diminish immune resistance . The indApart from antibody and siRNA approaches, commercialized chemical inhibitors can still benefit PD-L1 based treatments by regulating the relevant expression pathways. The elucidation of PD-L1 regulation mechanisms provides appealing therapeutic regimens in overcoming the related cancer immune resistance. Based on this knowledge, the upstream regulatory molecules are ideal targets for screening inhibitors. Note that the inhibitors mentioned here are only small chemical molecules, the majority form of regulatory inhibitors. Small molecule inhibitors competitively bind to the enzyme/receptor, and can be as effective as other form of inhibitors, such as antibodies and oligonucleotides. A variety of marketed chemical inhibitors are able to downregulate cellular PD-L1 expression, directly targeting regulatory signaling pathways associated with inducible PD-L1. In addition, the sPD-L1 can be reduced in expression treated with matrix metalloproteinase inhibitor (MMPI) .Another possible inhibitor vemurafenib, a commercialized competitive enzyme inhibitor, is approved by FDA for the treatment of late stage melanoma with BRAF V600E mutation  obstruction of cellular biological functions, and (2) abolishing drug resistance. The PD-L1 modulation involves MAPK and PI3K/AKT pathways, which participate in regulating other biological responses such as proliferation and survival. Notably, the suppression of AKT would upregulate Caspase cascade and induce apoptosis. The suppression of PKD2, which regulates IFN-γ induced PD-L1 expression, is able to abolish P-glycoprotein associated multidrug resistance (MDR). Taken together, the inhibition of signal pathways may result in chemo-immune combination therapy with much better efficacy.The deficiency of signaling pathway inhibition is that it may take time to reduce PD-L1 expression . TherefoAs it stands direct comparison between antibody blockage and chemical inhibition is not viable due to lack of good evidence. However, just from the view of total inhibition of PD-L1 production and function, chemical inhibition may be more effective although the side effects of chemical inhibitors could be more significant than antibodies.Based on the above review, it seems that none of the current three treatment methods is a perfect regime to adequately inhibit PD-L1 production and function for effective immunotherapy. We think that it is important to consider controlling intracellular PD-L1 formats in future immunotherapy. The intracellular PD-L1 formats will not be affected if the treatment utilizes only antibody to block the membrane format. However, for PD-L1 positive cancer cells, (of a patient) antibody blockade will be the most effective and direct way to restore T cell immunity. Therefore, we believe the future treatment strategy will probably combine these methods as a combinational therapy.Clinicaltrials.gov, 16 clinical trials (14 under recruiting) using combination therapies with Atezolizumab and the chemical inhibitors were submitted in 2017  the information of PD-L1 distribution in cancer cells, and (2) PD-L1 mediated innate/adaptive immune resistance. This postulation that how PD-L1 distribution and adaption in individual patient guides the choice of an optimal combination regimen is illustrated in Clinicaltrials.gov from January to May 2019 on PD-L1 antibody-based combination therapies are listed in The current clinical trials of PD-L1 antibody broaden the applicable range with other therapies and enhance the therapeutic efficacy. The clinical trials documented in The PD-1/PD-L1 immune checkpoint interaction is an arch-important regulator in tumor immune escape. The blockade or downregulation of PD-L1 in both immune cells and tumor cells is beneficial to breaking down the negative immune regulation and evoking enhanced immunity against tumor. Emerging data on PD-L1 biology demonstrate its relevance to other behaviors of cancer cells such as drug resistance and metastasis, highlighting its multiple roles in cancer development, additionally to immune regulation. Current treatment strategies mainly focus on PD-L1 expressed on the cell membrane (mPD-L1), other formats of intracellular/extracellular PD-L1 are not well-studied and understood. Therefore, these should be an important direction of related research. The methods of gene knockdown and chemical inhibition of signaling pathways have been proved to be efficient in inhibiting intracellular PD-L1 production but not effective for serum PD-L1 format (sPD-L1). They may also be ineffective for PD-L1 already expressed on the cell surface. Based on these understandings, we postulate that the future direction of PD-L1 based cancer immunotherapy will lie in combined and personalized cancer therapies. Expectedly, the future combination approaches will strategically utilize antibody blockade, gene knockdown, and/or regulatory pathway inhibition based on patient PD-L1 distribution information, which will lead to more effective cancer immunotherapy.YW reviewed the literature and wrote the main part of first draft. WC contributed to the antibody advances. ZX and WG devised the main conceptual idea and polished the draft. All authors contributed to revisions of the manuscript, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The strong fluorescence background of Raman spectra, the upshift of the G peak (~23 cm−1), and the decrease of sheet resistance confirmed the successful intercalation of ionic liquid into the graphene layers. This intercalation control of the infrared emissivity of graphene in this work displays a new way of building an effective thermal camouflage system.Controllably tuned infrared emissivity has attracted great interest for potential application in adaptive thermal camouflage. In this work, we report a flexible multilayer graphene based infrared device on a porous polyethylene membrane, where the infrared emissivity could be tuned by ionic liquid intercalation. The Fermi level of surface multilayer graphene shifts to a high energy level through ionic liquid intercalation, which blocks electronic transition below the Fermi level. Thus, the optical absorptivity/emissivity of graphene could be controlled by intercalation. Experimentally, the infrared emissivity of surface graphene was found to be tuned from 0.57 to 0.41 after ionic liquid intercalation. Meanwhile, the relative reflectivity R All matter at a temperature above absolute zero emit infrared waves, the intensity of which is dependent on the temperature (T) and infrared emissivity (ε) of the surface materials . This isDue to linear band dispersion, graphene has a very broad band optical absorption/emissivity  ,10,11,12v/R0 of graphene with the sample on copperplate is increased from 1.0 to 1.15) confirms the decrease of emissivity. The in-situ Raman spectra with an electronic voltage above 3 V have a strong fluorescence background and an upshift of the G peak of about 23 cm−1, which indicates a strong doping effect of graphene layers with intercalation. In addition, the sheet resistance of graphene is reduced from 11 to 4 Ω/□ after intercalation due to the charge transfer between the ionic liquid and graphene layers. What is more, the intercalation process is fast and reversible; the film is light, flexible, and thin (<20 μm); and the four-layer architecture device architecture —ammonium bis (trifluoromethyl sulfonyl) imide, Sigma-Aldrich, Catalog No.727679, St. Louis, MO, USA) into multilayer graphene layers, we demonstrated a flexible thermal surface, which can electrically control the infrared emission without changing the surface temperature. Upon ionic liquid intercalation, the Fermi energy of graphene upshifts from the Dirac point to a high energy level, causing the emissivity of graphene to reduce from 0.57 to 0.41. The increase of the infrared relative reflectivity (Rture see a is compSample preparation: The multilayer graphene film with a thickness of about 50–70 nm used in this work is on nickel (Ni) foil . The supporting Ni foil was etched away in saturated iron chloride (FeCl3) aqueous solution , resulting in a free-standing multilayer graphene film on the solution surface  was used to apply voltage bias between two multilayer graphene layers to control the intercalation process. In-situ Raman measurements were conducted with a XperRam Compact Raman spectrometer , with excitation laser wavelength of 532 nm and laser power of 0.5 mW. The sheet resistance of the multilayer graphene under different intercalation voltage bias was measured through another Keithley 2636B source meter  using the four-point method. In order to analyze the change in the infrared emissivity, the reflection of the multilayer graphene device was measured with the device on a polished copper plate (thickness about 1 mm), which is described in detail below. The in-situ reflection measurement under voltage bias was carried out on a U-4100 spectrophotometer  with a wavelength range from 400 to 2000 nm.Thermal imaging: During the thermal image measurement, the multilayer graphene device was put on a polished copper plate (the emissivity was about 0.1), then the copper plate with the device was put on a hot plate 4, where εI is the emissivity used for thermal imaging, TI is the infrared temperature, and T is the real temperature measured by thermocouple. Thus, the integrated emissivity for different intercalation bias voltages could be determined from the thermal images. The integrated emissivity at different bias voltages is summarized in At thermodynamic equilibrium, the infrared emission energy is described by the Stefan−Boltzmann law, P = εσTV/R0) measurement of the multilayer graphene device on polished copper plate. The reflectance measurement shows that above 3 V there is an obvious increase of reflectance. This implies that the decrease of absorption/emissivity above 3 V, is consistent with Due to the ionic liquid intercalation, the Fermi level of graphene shifts to a higher energy level. The electronic transition below the Fermi level is suppressed due to Pauli blocking a, result−1), G (1580 cm−1), and 2D (2688 cm−1) modes, consistent with previous research [−1 with the bias voltage increase to 3 V. The increase of the G mode intensity is an indication of the doping effect through intercalation [−1 of the G mode implies the successful intercalation [The modulation of infrared emissivity is clearly due to the intercalation of the ionic liquid [DEME][TFSI] into the graphene layers. To further characterize the intercalation process of surface multilayer graphene, in-situ Raman measurement was carried out a. Figureresearch ,29,30. Tresearch . For an calation ,22. Whilcalation . The incThe sheet resistance under the intercalated bias voltage of multilayer graphene was also measured by the four-point resistivity method a. The weIn addition, we noted that the change of the infrared temperature of the surface multilayer graphene started at the edge and then propagated across the device at the very beginning. However, the intercalation process induces defects on sthe urface multilayer graphene, which was demonstrated by the increase of D peak intensity in the in-situ Raman spectra b. Thus, In summary, we demonstrated a flexible multilayer graphene-based device with tunable infrared emissivity. Upon ionic liquid intercalation, the Fermi level shifts to a higher energy level, which blocks optical transition below the Fermi level. Thus, the reflectivity/absorption of graphene increased/reduced, which further reduces the infrared emissivity. Actually, the infrared emissivity of the surface multilayer graphene could be tuned between 0.57 and 0.41 through ionic liquid intercalation/deintercalation. The in-situ Raman and resistivity measurements present a reversible intercalation of anions/cations of the ionic liquid into graphene layers and heavy doping of the graphene layers. These results demonstrate a new strategy for a wide range of applications, such as thermal camouflage, infrared sources, and thermal management.


GC) is one of the most common malignancies. Immunotherapy is a promising targeted treatment. The immune regulatory programmed death‐1 (PD‐1)/programmed death‐ligand 1 (PD‐L1) axis has been used as a checkpoint target for immunotherapy. Currently, considerable discrepancies exist concerning the expression status of PD‐L1 and its prognostic value in GC. We aimed to evaluate the expression rates of PD‐L1 in GC, and further assess its relationship with mismatch repair (MMR), and human epidermal growth factor receptor 2 (HER2) status. We retrospectively collected 550 consecutive cases of GC in Fudan University Shanghai Cancer Center from 2010 to 2012. PD‐L1, MMR protein, and HER2 status were detected by immunohistochemistry (IHC). Fluorescence in situ hybridization was further used in HER2 IHC 2+ cases. Cases with at least 1% membranous and/or cytoplasmic PD‐L1 staining in either tumor cells (TCs) or tumor‐infiltrating immune cells (TIICs) were considered as PD‐L1 positive. The correlation between clinicopathological parameters, HER2, MMR, and PD‐L1 expression status was determined using chi‐squared tests. About 37.3% cases (205/550) showed PD‐L1 expression in TCs and/or TIICs. 17.3% cases (95/550) showed PD‐L1 expression in TCs, 34.5% (190/550) cases showed PD‐L1 expression in TIICs. There were 45 deficient MMR (dMMR) cases (8.2%), which showed higher rates of PD‐L1 expression compared with MMR‐proficient carcinomas . HER2 was positive in 66 (12.0%) cases. The expression of PD‐L1 occurred more frequently in HER2‐negative group than HER2‐positive cohorts . The survival analysis revealed that PD‐L1 was not associated with prognosis. This study evaluated the association between the PD‐L1 expression and a specific subgroup (dMMR and HER2‐negative) in a large Asian cohort of GC. GC patients with dMMR and HER2‐negative status exhibited higher PD‐L1 expression rates. Our finding indicated that MMR and HER‐2 status might be potential biomarkers for anti‐PD‐L1 therapy.Gastric cancer ( Gastric cancer (GC) is one of the most prevalent cancer in the world Immunotherapy using immune checkpoint inhibitors is a rapidly growing modality for the treatment of human cancers The study was approved by The Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center. Written informed consent was obtained from all participants included in the study. Five hundred and fifty cases of surgically resected gastric cancer were collected from the files of Department of Pathology, Fudan University Shanghai Cancer Center. All the patients had not undergone previous chemotherapy treatment. All the cases were reviewed by two pathologists and the histological diagnoses were confirmed without discrepancy. Clinical findings, including age, gender, tumor location and size, therapy, and clinical outcome were obtained from the medical record, pathology report, or discharge summary. The follow‐up information was conducted via medical records plus telephone interview, and the following information was obtained as follows: follow‐up duration, disease‐free survival (DFS), and overall survival (OS).μm‐thick tissue sections with the automated immunohistochemical stainer . The primary antibodies used in the study included anti‐PD‐L1 , anti‐MLH1 , anti‐PMS2 , anti‐MLH2 , anti‐MLH6 , and anti‐ erbB‐2 (HER2) . Omission of primary antibody and substitution by non‐specific immunoglobins were used as negative controls. The appropriate specificity and sensitivity of the antibody against PD‐L1 staining were determined using human placenta as the positive control. Appropriate positive controls were run concurrently for all antibodies tested. PD‐L1 expression showed membranous staining and/or cytoplasmic staining. The proportion of immunostained cells was evaluated among tumor cell and tumor‐infiltrating immune cells. The patients with at least 1% PD‐L1 staining of tumor cells or immune cells were considered positive. The cases showed preserved nuclear expression of 4 MMR proteins were considered MMR‐proficient (pMMR). HER2 expression was graded using a score scale of 0 to 3 IHC was performed on 4 Fluorescence in situ hybridization (FISH) studies were performed on HER2 IHC2+ cases. Representative sections were incubated in a humidified chamber using HER2 Probe  according to the manufacturer's protocol . For gene amplification assessment, the total number of HER2 and chromosome 17 signals was counted in at least 10 high power fields. The HER2/chromosome 17 ratios which was equal or greater than 2.2 was considered as gene amplification.P < 0.05. SPSS  version 18.0 was used to analyze all data.The chi‐squared and the Fisher test were used to test the difference between groups. The survival difference between groups was assessed by the Kaplan–Meier method. Differences were considered to be significant at P = 0.001, TIICs P = 0.004, TCs and/or TIICs P = 0.001), with bigger size (≥5 cm)  and solid‐type histological features . PD‐L1 expression was less common in the GCs with submucosal invasion . And GCs in stage II, stage III, and IV showed higher PD‐L1 expression than GCs in stage I . Clinicopathological characteristics of GC are summarized in Table PD‐L1 was detected in the tumor cells and/or tumor‐infiltrating immune cells with variable intensities and proportions, but not in non‐neoplastic gastric epithelium  present positive PD‐L1 staining  in our cohort. As shown in Table % in our P = 0.020, Table HER2 was positive in 66  cases, among which 16 cases were positive for PD‐L1 (24.2%). However, in HER2‐negative group, 39.0% (189/484) of tumors were positive for PD‐L1 in TCs and/or TIICs. The expression rate of PD‐L1 in HER2 negative GCs was significantly higher than that in HER2 positive GCs  than most previous studies. In our study, the positive expression of PD‐L1 was observed in 37.3% cases. PD‐L1 was detected in 15% to 75% of GC patients according to previous literatures  = 0.52, P < 0.001) (data was not shown). The reason for the difference in positivity rate of PD‐L1 was that PD‐L1 expression was heterogeneous, there is a chance that PD‐L1 positive tumor areas not be taken into TMA as tissue cores were drilled at random from the tissue blocks. In our study, in order to minimize the intratumoral heterogeneity, each case had two cores taken into TMA, one from the center and the other from the invasive front. It has recently been reported that PD‐L1 expression was frequently discordant between surgical specimens and matched biopsy specimens because of the heterogeneity of PD‐L1 expression Our study was conducted to investigate the correlation between PD‐L1 expression and clinicopathological features of GC patients. This cohort included a considerable larger number of GC patients  in a large Asian cohort of GC patients. Our study demonstrated that PD‐L1 expression in GC is significantly correlated with dMMR and HER2‐negative status. We also found that PD‐L1 expression was not related to patients’ prognosis. PD‐1/PD‐L1 checkpoint inhibitors might become a novel therapy strategy to HER2‐negative patients.The authors declare that they have no conflict of interest.


We found that the vertical conductivity of the heterojunction can be tuned by ∼5 times under different twist configurations, and the highest/lowest conductivity occurs at a twist angle of 0°/30°. Density functional theory simulations suggest that this conductivity change originates from the transmission coefficient difference in the heterojunctions with different twist angles. Our work provides a guidance in using the MoS2/graphene heterojunction for electronics, especially on reducing the contact resistance in MoS2 devices as well as other TMDCs devices contacted by graphene.Van der Waals heterostructures stacked from different two-dimensional materials offer a unique platform for addressing many fundamental physics and construction of advanced devices. Twist angle between the two individual layers plays a crucial role in tuning the heterostructure properties. Here we report the experimental investigation of the twist angle-dependent conductivities in MoS 2 over graphene.Twisting vertically stacked individual layers of two-dimensional materials can trigger exciting fundamental physics and advanced electronic device applications. Here, the authors report five times enhancement in vertical heterojunction conductivity on rotating MoS One of the crucial structural parameters in a vdW heterostructure is its twist angle, a new degree of freedom for modulating its properties. Indeed, many fascinating twist angle-dependent properties have been investigated including the strong twist angle-dependent resistivity of Gr/graphite contact4, the quantum transport in Graphene/BN superlattice7, resonant tunneling in Gr/BN/Gr8, interlayer excitons in MoSe2/WSe29, and band evolution in MoS2/graphite10, just to mention a few. Among many investigations, unraveling the vertical conductivity of a vdW heterojunction is of fundamental importance; however, only limited knowledge has been established so far.Two-dimensional (2D) materials can be assembled into the so-called Van der Waals (vdW) heterostructures, offering an exotic platform for exploring many fundamental physics and important device applications2/graphene (MoS2/Gr) heterojunctions under various lattice twist configurations. We found that vertical conductivities of these heterojunctions are strongly twist angle-dependent. When varying the twist angle from 0° to 30°, conductivities monotonically decrease and the modulation depth (maximum/minimum) is ∼5. Considering that MoS2/Gr heterojunction has great potential in various devices applications12 and even batteries13, graphene can also form excellent contact to MoS217; our results provide a guidance in using the MoS2/Gr heterojunction for electronics.Here, we report the experimental investigation of vertical conductivities of MoS2/Gr heterojunctions were fabricated by an epitaxial growth technique19 . Figure 2 triangle domains on graphene. These domains have obviously preferred orientations and similar sizes, suggesting the epitaxy nature of MoS2 domains. Figure 2 and graphene substrate are clean and free of contaminations. The height of as-grown MoS2 triangle domains is ∼0.855 nm, corresponding a monolayer thickness21. Raman and photoluminescence (PL) spectra also indicate that the monolayer MoS2 domains are of high qualities  imaging 22. Note that in Fig. In this study, the MoSern Fig. , the hex2 domains and the graphene substrates underneath, we rotated these as-grown MoS2 domains via AFM-tip manipulation technique. This manipulation process is like that reported in our previous work23. As illustrated in Fig. 2 triangle from its corner to actuate its rotation. By controlling the tip moving direction and length, we thus can rotate the MoS2 domains on graphene with any deterministic twist angles, as illustrated in Fig. 2 domain under different twist angles are likely to be different if considering the interlayer coupling; however, due to the probing limit (no more than 60 pm) of our AFM, these height variations were not observed in our experiments.To achieve different twist angles between the MoS2/Gr heterostructures with deterministic twist angles are available, we thus measured their vertical conductivities to investigate the twist-angle dependence. In our experiments, the conductivity was directly measured under the C-AFM mode. During C-AFM scanning, a metal-coated AFM tip was placed in direct contact with the sample surface under a controlled load of ∼23 nN 24. The bias applied to the measurement circuit was fixed to be 1.5 V, and a 110-MΩ resistor was connected into the circuit to prevent current overload to our C-AFM holder, which has a measuring range of ±20 nA and a noise level of 1.5 pA, as illustrated in the inset of Fig. 2 triangle to reduce the feedback noise.Since MoS2/Gr heterojunction with a twist angle of 28.03°. The mapping can be clearly divided into two different regions: the bright region from the bare graphene surface with higher conductivities and the dark region from the MoS2/Gr heterojunction with lower conductivities. To quantitatively extract the conductivity meanwhile eliminating the metal at the two different regions, we thus inverted this current mapping into a statistical current distribution chart as illustrated in Fig. x-axis of the chart is current and the y-axis is the counts, which is from an area of 500 nm × 500 nm, corresponding to 256 × 256 points. From the statistics, we can clearly see two Gaussian peaks whose centers reflect the statistic currents Ig and Ij flowing through the graphene and the junction, respectively. Because the resistance of the graphene is much smaller than the connected 110-MΩ resistor, Rg = V/Ig just reflects the system resistance and barely shifts. In order to facilitate comparison, we used Q = Ij/Ig to normalize the current distribution statistics.Figure 2/Gr heterojunctions with different twist angles θ. The intensity comes from the scale of heterojunction and graphene area we chose to make statistics. It can be clearly seen that the two peaks are moving apart to each other when the MoS2/Gr heterojunction is twisted from 0° to 30°. As illustrated in Fig. 2/Gr heterojunctions with different twist angles, which shows an obvious 60° period. . Error bar in Fig. 2 peaks, which can characterize the dispersion of the heterojunction resistance. From the results, we can see that the dispersion of the heterojunction resistance has no clear correlation with twist angles. In addition to the measurements of these MoS2/Gr heterojunctions fabricated by AFM-tip-facilitated rotation, we also measured a polycrystalline MoS2/Gr heterojunction  is the Fermi–Dirac distribution. T(E) is the transmission coefficient. It can be seen that the tunneling current is dominated by the transmission coefficient under a certain tip-to-sample bias.As observed above, vertical conductivities across the MoS2/Gr heterojunctions, we thus performed density functional theory (DFT) simulations  chamber. S  and MoO3  sources were loaded in two separate inner tubes and placed at zone-I and zone-II, respectively. Substrates were load in zone-III. During the growth, Ar/O2 (gas flow rate: 75/3 sccm) are used as carrying gases and temperatures for the S-source, MoO3-source, and substrates are 115 °C, 530 °C, and 930 °C, respectively. Thin graphene flakes were mechanically exfoliated from both HOPG and Graphenium graphite  and placed on SiO2 substrates.The MoSPL and Raman characterizations were performed in a Horiba Jobin Yvon LabRAM HR-Evolution Raman system using a 532-nm laser excitation wavelength. SAED was performed in a TEM (Philips CM200) operating at 200 kV.AFM imaging was performed by Asylum Research Cypher S with AC160TS tip. In C-AFM mode, we used ASYELEC-01 tip with Ti/Ir coating and the holder 901.730. The bias was applied from sample to tip with a current limit of ±20 nA and noise of 1.5 pA.28. Succinctly, NEGF-DFT calculates density matrix by NEGF as ΓL,R are the self-energy of the left (L) and right (R) leads. Furthermore, the conductance e is the electron charge and h is the Planck constant. In our model. We choose multilayer graphene for both left lead and right lead, considering the lattice match problem. The structure has mirror symmetry centered on the plane of molybdenum atoms. The structure parameters have been chosen as: superlattice constant: 1.2816 nm; thickness of MoS2: 0.3227 nm (0°) and 0.3174 nm (30°); interlayer spacing of MoS2/Gr heterojunction: 0.3110 nm (0°) and 0.3131 nm (30°); interlayer spacing of graphene: 0.34 nm, all same with ref. 29.We used DFT within the Keldysh non-equilibrium Green’s function (NEGF) formalism2 was treated as a limited barrier; thus, the transmission coefficient has a simple relation with the twist angle, described as a, b are pending constants, T is transmission, and Eg is the bandgap. Since we already have two transmissions hot maps for two twist angles, i.e., 0° and 30°, which have been used to determine a and b in the equation. In order to obtain the relation between the tunneling gap and the twist angle, band-folding technique has been used.In WKB calculations, MoSSupplementary Information