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


ID/IG) from ca. 0.09 to 0.22. The translocations of anionic and cationic monomers through graphene are significantly slower due to chemical interactions of monomers with the graphene defects. Interestingly, if micropatterned initiator-monolayers are used, the translocations of anionic monomers apparently cut the graphene sheet into congruent microscopic structures. The varied interactions between monomers and graphene defects are further investigated by quantum molecular dynamics simulations.Mass transport through graphene is receiving increasing attention due to the potential for molecular sieving. Experimental studies are mostly limited to the translocation of protons, ions, and water molecules, and results for larger molecules through graphene are rare. Here, we perform controlled radical polymerization with surface-anchored self-assembled initiator monolayer in a monomer solution with single-layer graphene separating the initiator from the monomer. We demonstrate that neutral monomers are able to pass through the graphene (via native defects) and increase the graphene defects ratio (Raman  Translocation of larger molecules through graphene holds potential for molecular sieving, however it is rarely observed. Here, the authors show the radical polymerization of vinyl monomers via their translocation through a single layer graphene to a monolayer initiator and additionally study the monomer-graphene interactions. In fact, a pristine graphene sheet is mostly impermeable to all atoms and molecules due to the unfavorable energy barriers of its closely spaced carbon atoms6. Even graphene prepared by chemical vapor deposition (CVD), which is expected to have Stone–Wales defects, is theoretically demonstrated to be impermeable to helium under ambient conditions7. As such, research has shown that graphene can be used as an effective barrier to oxidation of metal surfaces under certain conditions10, as well as barrier for the deposition of self-assembled monolayers11. However, in another scenario, ions and molecules have been observed to be able to transport through single-layer graphene (via intrinsic defects) promoted by external pressure or concentration gradient14. More interestingly, Geim and colleagues15 recently showed that, upon electronic potential, pristine graphene can be highly permeable to thermal protons under ambient conditions, and Geiger and colleagues16 reported a reversible proton transfer through graphene from the aqueous phase silanol groups of a substrate surface, which correlate principally to the pioneer work of voltage-driven ionic transport across suspended CVD graphene by Golovchenko and colleagues17 in 2010. These studies suggest that the permeability of single-layer graphene could be significant  if mass-transport is driven by an external force19.Graphene and other two-dimensional materials are promising candidates for next-generation separation membranes owing to their atomic thickness20: Can possibly also organic molecules such as monomers pass through single-layer CVD graphene? If so, what happens to graphene? Is the size and/or the charge of the transported monomers of importance? Since atomic-thin graphene is transparent to van der Waals21 and Coulomb forces22, it should be possible that the graphene-covered radical initiator monolayer24 can still interact with reactants  on the other side of graphene. Meanwhile, the living nature of controlled radical polymerization (CRP) requires a continuous transport of the monomers through the graphene membrane and steadily consumes monomers near the graphene sheet to surface-bound polymer brushes In this work, we show that neutral monomers of various sizes are able to smoothly pass through monolayer CVD graphene under the driven force of radical initiator in polymerization, although the native defects of graphene are largely increased due to the translocation. In contrast, the passage of charged monomers is severely interrupted by the graphene. Notably, the translocation of anionic monomers selectively cuts graphene into congruent microscopic patterns. These results indicate that the translocation of a large molecule through monolayer CVD graphene can be realized by applying an external driven force to the molecule, and the charge of the molecule has a greater impact on the translocation than the molecule size.2 wafer piece27 and covered by a monolayer of graphene (ca. 1 × 0.5 cm2) from CVD. The typical surface morphology of CVD graphene with micrometer size wrinkles is revealed by atomic force microscopy  mapping results suggest that the whole graphene sheet is rather homogeneous with low defect concentration , the ligand  for polymerization   readily gave poly(methyl methacrylate) (PMMA) brush layers as shown in Fig. −1) Fig. . The for−1) Fig. . Interes−1) Fig. , which a∗1) and without (∗2) initiator-SAM/polymer brush underneath, as confirmed by AFM  on graphene due to the translocation of MMA. Firstly, the ID/IG intensity ratio  increased from ca. 0.09 to ca. 0.22  was lifted from the substrate by the grown PMMA brush underneath.The graphene coverage was fully retained at both regions with   of graphene at this region (∗1) was unchanged   was converted as described above. Again, the SI-CRP of SPMA resulted in PSPMA brushes but apparently the graphene was cut into hexagons congruent to the initiator-SAM pattern Fig. , 17. Ram∗2, Fig. . Raman s∗1) Fig. . And theged Fig. , suggest−1 (without graphene) to 100 nm h−1 ethyl trimethylammonium chloride, METAC) was investigated. As for the anionic monomer, also the transport of METAC through the graphene was significantly slower as revealed by the slower polymer brush growth rate (decreased by 60% from 266 nm hSimilar translocation behavior was observed for another cationic monomer (4-vinylpyridine (4VP), Supplementary Fig. 35, to study the kinetic energy change ΔEK during the translocation process through the graphene nanopore of various edge terminations for the three monomers SPMA, MMA, and METAC  is found for the negatively charged SPMA  has been found for neutral MMA. To quantify the degree of distortion of the nanopore during the translocation process, the change in the pore size (DH) was calculated by computing the distance between diametrically opposed OH groups in the functionalized pore at each simulation time step, and then performing an average over the calculated distances , followed by METAC (DH ≈ 7.86–8.04 Å) and SPMA (DH ≈ 7.92–8.24 Å). In the case of ether termination10, both quantities, τ and ΔEK, smoothly decrease upon increasing the ether concentration, since the hole diameter gets larger and, hence, the monomers have less interaction with graphene defect edge to pass through  approachsee Fig. , 25. We ΔEK Fig. . In addiΔEK Fig. . For theThe combined experimental and simulation results indicate that electrostatic interactions between charged species (monomers) and the functionalized graphene nanodefects are dominating the translocation process. Moreover, the much higher energy loss experienced by the charged monomers may explain (i) the strong obstruction effect observed in the translocation of both cationic and anionic monomers, and (ii) the observed chemical cutting (in the case of anionic monomers), since the larger dissipated energy into graphene may lead to a larger degree of structural distortion of the nanodefects.38, which either enlarges the defects or completely breaks graphene depending on the charges of monomers. (ii) After vertical translocation, the monomers are able to transfer laterally underneath of graphene lattice, which accumulates as polymer brush under graphene and gradually delaminates graphene from substrate24. Both of these two processes are ensured by the atomic thinness and flexibility of graphene monolayer21 and strong Coulomb interactions between monomer and surface-bonded initiator in SI-CRP.We suggest that there are two possible passways for monomers transport at the graphene interface during SI-CRP Fig. : (i) Ver39 of two-dimensional materials into desired patterns or shapes for heterostructures40.In conclusion, we found that large molecules such as vinyl monomers of different sizes can pass through single-layer CVD graphene if driven by a proximal chemical reaction such as SI-CRP. Furthermore, the SI-CRP continues and the formation of polymer brushes indicate a constant flow of monomers through the graphene. Notably, we found that the transport process is more charge-selective than size-selective. For example, a neutral monomer could smoothly translocate to the other side of graphene, by enlarging the graphene native defects. However, the translocation of cationic and anionic monomers was significantly obstructed (60 and 65% in polymerization rates), due to the strong interactions between charged monomers and graphene defects. These results on the translocation of actual molecules through a single layer of a two-dimensional material are intended to trigger further studies on the transport phenomena through this exciting class of materials. An additional aspect of this work is the possibility of the chemical cutting with techniques used for nano- and micropatterned polymer brushestBuMA, OEGMA475, and 4VP were purchased from Sigma-Aldrich , purified before use by passing through a basic alumina column to remove the inhibitor. 3-SPMA, METAC, 2-BiBB, 3-aminopropyltriethoxysilane (APTES), PMDETA (99%), dichloromethane , DMSO, triethylamine (TEA), acetone (dry), and methanol  were used as received. For reactions and water contact angle measurements, bidistilled deionized water was used. Cu plate : 1-side polished, p-type (boron), total-thickness-variation (TTV) < 10 µm, 1–10 Ohm cm; 10 nm Ti adhesion layer; 200 nm Cu (purity >99.9%), root-mean-square roughness <10 nm. The copper plate was consecutively washed with portions of 3 M HCl (in methanol), methanol and ethanol under ultrasonication (2 min), and dried under a flow of argon. The cleaned copper plate was immediately used for reactions.MMA, 2O2:H2SO4, 1:3 v/v, 90 °C, 45 min; WARNING: Piranha solution reacts violently with organic matter!), washed extensively with bidistilled water, and dried with a stream of dry argon.Silicon wafer pieces with a 300 nm oxide layer were obtained from Wacker AG, Burghausen, Germany. To remove any grease or other contaminants, silicon substrates were cleaned with piranha solution  APTES solution in dry acetone and subjected to treatment with ultrasound for 45 min. After SAM formation, the samples were extensively rinsed with dry acetone and dried under argon atmosphere. The substrate was then immersed in dry DCM (20 mL) under a nitrogen atmosphere. TEA (0.4 mL) was added, followed by dropwise adding initiator BiBB  at 0 °C, and the reaction solution was allowed to stand for 24 h with stirring at 20 °C. The substrate was removed, washed with DCM, water, ethanol, and acetone, and then dried under a nitrogen stream. The quality of this surface-bonded initiator preparation method was confirmed by X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. The total surface layer thickness  was ca. 2.0 nm, as judged by ellipsometry.2 by UV illumination (200 W Hg (Xe) lamps, LOT-oriel, Germany) through a photomask. The samples were clamped with Cu TEM grids and irradiated for 45 min at a distance of ca. 10 cm.Patterned initiator was achieved on a selectively etching of as-prepared uniform initiator on SiO2, one side of the single-layer graphene (graphene supermarket) was coated with PMMA resist  and cured at 90 °C for 10 min. The other side of the sample was exposed to O2 plasma to remove the graphene on that side. The Cu substrate was etched away by an aqueous solution of ammonium persulfate (0.25 g mL−1) over a period of 2 h. After being rinsed thoroughly with deionized water, the PMMA-graphene film was transferred to a target substrate. The samples were naturally dried in air for 1 h and stored in high vacuum at room temperature for 24 h to enhance the adhesion of graphene with BiBB-SiO2 surface. PMMA was removed by thorough rinsing in acetone and cured in isopropyl alcohol.To transfer graphene to the initiator (BiBB) modified SiO2O and 0.5 mL methanol, 18.4 μL PMDETA; (iii) SPMA: 1 g monomer, 1 mL H2O and 0.5 mL methanol, 18.4 μL PMDETA.Polymerization solution preparation: (i) MMA: 1 mL monomer, 0.5 mL DMSO, 18.4 μL PMDETA; (ii) METAC: 1 mL monomer, 1 mL H2 was sandwiched with a copper plate  at a distance of D = 0.5 mm adjusted by two spacers29. A drop (ca. 20 µL) of monomer solution prepared above was introduced to the confined space between copper plate and graphene-BiBB-SiO2. The assembly was left for typically 1 h at room temperature. After reaction, the plate was separated and the substrate immediately washed with either fresh DMSO or methanol and water mixture. Finally, the substrates were exhaustively rinsed with acetone to remove all traces of monomer solution, and subsequently dried in a stream of nitrogen.The graphene-BiBB-SiO41 for the C, H, O, N, and S atoms. In all simulations, explicit water has been considered as parameterized in DFTB+ code, in which the interactions among water molecules are given by dipole–dipole as well as van der Waals interaction. In order to take into account van der Waals forces, dispersion corrections were included via Lennard–Jones potentials. The geometry optimization for the monomers and edge-terminated graphene was performed until the absolute value of the inter-atomic force lies below 10−4 atomic units. Periodic boundary conditions in all supercell directions were applied. We first generated a graphene pore defect with CH3 termination and, then, the influence of OH termination concentration is studied. The influence of ether termination on the monomers translocation process through an initially OH-passivated pore has also been analyzed . The Si peak at 520 cm−1 was used as a reference for wavenumber calibration. The peaks were fitted with a single Lorentzian line shape to determine peak position and full width at half maximum. The optical microscopy images were measured on Zeiss Axioskop 2 MAT microscope.AFM scans were recorded with an Ntegra Aura (NT-MDT) atomic force microscope with a SMENA head in the semicontact mode. The used probes have a typical curvature radius of 6 nm, a resonant frequency of 47–150 kHz, and a force constant of 0.35–6.10 N mSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


Proliferation of retinal pigment epithelium (RPE) is typically observed in limited ocular disorders, in connection with the local mechanism of RPE proliferation-mediated wound repair. Bilateral and diffuse type RPE proliferation is considered to be associated with paraneoplastic syndromes, such as a bilateral diffuse uveal melanocytic proliferation. However, other reported diseases that induce bilateral diffuse RPE proliferation are quite rare, especially for patients who are considered to have a non-malignant status.The bilateral eyes of a 47-year-old woman with bilateral ocular inflammation, presented united multiple small to medium white retinal lesions during the disease progress.Optical coherence tomography showed scattered serous retinal detachments, choroidal folds, choroidal thickening and diffuse RPE proliferation. As autofluorescence and angiography showed a “giraffe pattern”, bilateral diffuse uveal melanocytic proliferation was suspected. However, systemic investigations identified no malignancy. In consideration of the above findings, choroidal inflammation was thought to be the major cause of this condition.The patient was administered intensive systemic steroids. Over the next 2 months, the amount of steroid was tapered off.After administration, the bilateral diffuse RPE proliferation settled down. During the 2-year follow-up, there was no recurrence of ocular inflammation and diffuse RPE proliferation, or any other malignancy found.This finding demonstrates that bilateral diffuse RPE proliferation can be generated as a secondary phenomenon of choroidal inflammation in patients with a non-malignant status. BDUMP is a form of a paraneoplastic syndrome that causes proliferation of the melanocytes bilaterally, which causes visual impairment due to the rapidly progressing exudative retinal detachment and cataract formation. Although it has been reported that the proliferation of melanocytes in the choroid and RPE is considered to be caused by humoral factors produced from tumor, the details of the pathogenesis, pathophysiology and treatment method are still unclear. Other reported diseases that induce bilateral diffuse RPE proliferation are quite rare, especially for patients who are considered to have a non-malignant status.One of the typical diseases that has been reported to cause bilateral diffuse proliferation of RPE is bilateral diffuse uveal melanocytic proliferation (BDUMP).Here, we report bilateral diffuse RPE proliferation, which was a secondary phenomenon caused by an ocular inflammation that was predominantly found to be choroidal inflammation, in a patient considered to have a non-malignant status.2A 47-year-old woman visited an ophthalmology clinic in January 2017 with foggy vision and vision loss. Although her medical history included uveitis 15 years prior, she had no other ocular disease, systemic disease such as inflammatory diseases, or malignant disorders. At the first visit, the best corrected visual acuity (BCVA) was 20/30 and 20/40 in her right and left eye, respectively, while the intraocular pressure was 13 and 11 mmHg in her right and left eye, respectively. In the anterior segment findings, no keratic precipitate, 1+ cells and 1+ flare in the anterior chamber, and remarkable posterior synechia were detected in both eyes  followed by an oral administration (starting at 50 mg / day) led to improvement in the choroidal folds and choroidal thickening. However, the serous retinal detachment tended to remain. Two weeks after the onset of the disease, bilateral diffuse RPE proliferation was clearly observed. A fundus photo showed multiple, coalescing, circular depigmented plaques and pigmentation, which were considered to be caused by hyperplasia of the RPE Fig. A and B. Based on these findings, we considered BDUMP to be the most probable differential diagnosis of the disease that was associated with the hyperplasia of the RPE. As a result, we performed whole-body investigations using computed tomography, magnetic resonance imaging, positron emission tomography, and echo in order to try and detect the presence of malignant tumors in this patient. However, findings of these evaluations did not suggest the presence of any malignant tumors.During the 2-month steroid treatment, there was improvement of the choroidal folds, choroidal thickening and serous retinal detachment observed in the patient Fig. A–C. Corr3BDUMP is a representative disease that is associated with bilateral and diffuse proliferation of the RPE. The clinical features of this disease include:1.multiple circular or oval orange patches (decolorizing spots) at the level of the RPE,2.multifocal early hyperfluorescence corresponding patches,3.extensive thickening of the uvea with focal nodules,4.exudative retinal detachment, and5.rapid cataract progression.In addition, other typical findings include the recent OCT images of the BDUMP identified hyperplastic images of the RPE and the giraffe pattern observed by angiography and autofluorescence. a systemic investigation was repeated in this case for 2 years from the initial onset. However, there was no malignant tumor ever observed. Thus, the proliferation of RPE in this case was considered to differ from the pathological mechanism of BDUMP.When this case was compared to the typical findings for BDUMP, the common ophthalmological findings included bilateral diffuse hyperplasia of the RPE and serous retinal detachment. Therefore, we listed BDUMP as a differential diagnosis in this case. However, extensive thickening of the uvea with focal nodules and rapid progression of cataract, which are typical features of BDUMP, were not observed in this case. In addition, since BDUMP is considered to be a paraneoplastic syndrome and is often observed prior to a malignant tumor, The specific findings in these cases were the formations of local nevus-like elevated lesions, which suggested the proliferation of choroidal melanocytes. On the other hand, our current case did not exhibit any elevated lesions.In this case, we strongly suspected that inflammation played an important role in the pathogenesis of the proliferation of RPE, as OCT showed that the anterior segment inflammation was clearly observed and there was choroidal folds/choroidal thickening. Furthermore, IA showed that there was a filling delay of the choroidal large blood vessel, thereby indicating choroidal inflammation. Although VKH was considered as a differential diagnosis in this case, diffuse proliferation of the RPE and the presentation of the giraffe pattern in this case are not commonly seen in VKH. Moreover, the specific systemic features of VKH, such as aseptic meningitis and sensorineural hearing loss, were not seen in this patient. Therefore, VKH was excluded as a differential diagnosis. It has been reported in previous studies that pseudotumoral retinal pigment epithelial proliferation occurred with VKH. were all ruled out after ophthalmologic and systemic examinations. In addition, local proliferation of RPE induced diseases such as proliferative (diabetes) retinopathy, age-related macular degeneration, and retinal detachment were also ruled out based on the fundus findings.From the aspect of angiography, other differential diagnoses including idiopathic uveal effusion and blood diseases such as lymphoma/leukemia, which cause “giraffe pattern” changes, However, in our current case, steroid treatment was clearly effective on the choroidal inflammation, with a subsequent reduction of the hyperplasia of the RPE observed after the treatment. This strongly supported the theory that bilateral diffuse RPE proliferation was associated with the choroidal inflammation.From a treatment perspective, it has been shown to be difficult to improve the vision in BDUMP patients when just using steroid therapy.In conclusion, the findings described here support the hypothesis that diffuse RPE proliferation can be induced by ocular inflammation, even in patients without any malignant tumors. Furthermore, choroidal inflammation played a major role in generating bilateral diffuse RPE proliferation.Conceptualization: Koju Kamoi.Data curation: Miyuki Tanaka, Natsuko Nagaoka, Tomoka Ishida, Hiroshi Takase.Formal analysis: Miyuki Tanaka, Koju Kamoi.Investigation: Miyuki Tanaka, Koju Kamoi.Supervision: Koju Kamoi, Kyoko Ohno-Matsui.Writing – original draft: Miyuki Tanaka, Koju Kamoi.Writing – review & editing: Miyuki Tanaka, Koju Kamoi, Natsuko Nagaoka, Tomoka Ishida, Hisako Karube, Hiroshi Takase, Kyoko Ohno-Matsui.


Graphene is susceptible to morphological instabilities such as wrinkles and folds, which result from the imposition of thermo-mechanical stresses upon cooling from high temperatures and/ or under biaxial loading. A particular pattern encountered in CVD graphene is that of mosaic formation. Although it is understood that this pattern results from the severe biaxial compression upon cooling from high temperatures, it has not been possible to create such a complex pattern at room temperature by mechanical loading. Herein, we have managed by means of lateral wrinkling induced by tension and Euler buckling resulting from uniaxial compression upon unloading, to create such patterns in exfoliated graphene. We also show that these patterns can be used as channels for trapping or administering fluids at interstitial space between graphene and its support. This opens a whole dearth of new applications in the area of nano-fluidics but also in photo-electronics and sensor technologies. Owing to its atomic thinness, graphene is susceptible to wrinkles and folds. Here, the authors demonstrate controlled wrinkle formation in graphene under uniaxial tension and show that the obtained mosaic patterns can be used as channels for trapping or administering water and other fluids. Such corrugations are of paramount importance for the potential applications of graphene since they can modify the electronic structure and carrier transport, alter surface properties, create electron/hole puddle and induce pseudomagnetic field in bilayers4. The formation of wrinkles, ripples and crumples has been attributed to several physical causes, such as (a) the thermal vibration of the 2D lattice5, (b) edge instabilities, defects/ dislocations6, (c) the mismatch between the thermal expansion of the 2D material and its host substrate9 (d) the evaporation/removal of solvent and its surface tension10, and (e) the relaxation of pre-strained substrate and its surface potential13. Recently, it has been demonstrated that wrinkles in the form of delaminated folds in supported graphenes can be formed either by uniaxial compression or uniaxial tension beyond a certain critical load, depending on the mode of loading. In the first case, the wrinkling direction is perpendicular to the compression axis whereas in tension, wrinkles of the same pattern originate parallel to the loading direction due to Poisson’s contraction exerted laterally by the underlying polymer substrate14.Graphene and 2D materials exhibit interesting morphologies which have been classified as wrinkles, ripples and/or crumplesHerein we demonstrate that in-situ uniaxial tensile testing combined with Atomic Force Microscopy (AFM) can be employed to highlight the formation of complex morphologies in graphene flakes, such as the creation of mosaic patterns similar to those observed in CVD graphenes upon cooling from high temperatures (~1000 °C).15. Appropriate flakes deposited directly on the PMMA were located using the optical microscope and the exact thickness of each flake was identified by the corresponding Raman line of the 2D spectra.In the present study we used a 1-mm-thick sheet of poly-methylmethacrylate (PMMA) as substrate to simply support monolayer (1LG) and bilayer (2LG) graphene flakes that were prepared by mechanical cleavage from HOPG (High Order Pyrolitic Graphite) with the scotch tape method16 in both the loading and the transverse directions. At rest the flakes appear to be quite flat having only a small and smooth fluctuation of ±0.5 nm in the out-of-plane direction which corresponds to the surface roughness of the polymer substrate, in agreement with measurements conducted previously14  was adjusted under an AFM microscope (Dimension Icon Bruker), which allowed the visualization of the flake morphology under incremental uniaxial strain levels (in steps of 0.1%). Very importantly, a dual threaded leadscrew drives the jaws symmetrically in opposite directions, keeping the specimen centred in the field of view during the tensile loading. The graphene flakes were subjected incrementally to tensile strain and the AFM images were captured at various levels of loading. AFM images were acquired with a Scanasyst-air probe of stiffness ~0.40 N/m and the measurements of the topography of the flakes were selected by using the operating Peak Force Nano-mechanical mode. The critical strain for the out-of-plane wrinkling and the wrinkling pattern development beyond a critical strain has been experimentally observed and quantified by using the proposed methodology. The experimental findings for the flakes under pure uniaxial tension are presented in Fig. 00 was adved Fig. . As noteIt is interesting to note that, upon unloading, the lateral wrinkles previously created upon tension are still present since they have been detached from the substrate and due to the interface slippage, the system does not fully recover. In addition, the formation of new wrinkles now perpendicular to the loading direction is observed Fig. . The coe21 and has also been simulated by computational modelling21. It constitutes a unique form of failure initiation which is the main characteristic of compression  of such systems. On the contrary flakes fully embedded in a matrix have been found to exhibit the expected Eulerian sinusoidal mode of compression failure22 due to the presence of constraints for fold creation by the surrounding matrix.The formed wrinkles are in fact isolated folds which have been detached from the underlying substrate and control tests reveal that do not come from any undulations of the surface of the substrate  the graphene flake will be subjected to compressive strain, which can be of sufficient magnitude to induce longitudinal wrinkling, if exceeds the critical strain for wrinkling The threshold of fold formation in the lateral direction seems to be accompanied by interfacial failure or slippage of the whole flake giving rise to the formation of folds in the longitudinal direction Fig.  upon unlsee Fig. . Hence, 21; hence, it is possible to give an estimation of the minimum strain to apply to the substrate in order to create the mosaic morphology for the system at hand, which is approximately 1.8%. The proposed mechanism has been verified by independent mechanical experiments combined with Raman spectroscopy  stresses induced due to the mismatch of the thermal expansion coefficients between copper and graphene result in a compressive biaxial strain of 2–3% in the graphene at room temperature. Such residual strain is far higher than the critical strain required for graphene wrinkling (buckling) thus giving rise to a network of large out-of-plane wrinkles or folds8. It is interesting to note that the peripheral equi-biaxial strain creates circular mosaic morphologies in contrast to the square patterns obtained here. In our case, the final lateral size in the mosaic morphology is essentially determined by the density of wrinkles developed during the loading and the unloading stages. The higher dimensions of the domains developed in the 2LG flake is compatible to a lower wrinkles density, compared to 1LG, in agreement with theoretical predictions14. These features are strongly dependent on levels of adhesion between graphene and the underlying polymer; hence, the use of different substrates can be thus exploited to tailor the final morphology of the mosaic.The mechanically self-assembled mosaic pattern formed here is very similar to the island-like morphology of CVD graphene grown on a copper substrate24. These typical features of the mosaic could be of paramount importance since it may broaden graphene application in the areas of electronics, photonics, nano-fluidics and bio-engineering4. For instance, the strong coupling between localized deformation and the electronic structure that is expected at the junctions could be exploited by strain engineering. Furthermore, constructive use of the self-assembled wrinkle networks could be harnessed as surface texturing agent to direct cell alignment and morphology in tissue engineering25 or for the creation of nano-channels in nano-fluidic devices transporting liquids. In light of possible application, the stability of the wrinkles to cyclic loading, which is particularly important for the off/on controlled applications of the mosaic, has been also verified  for several days. The selection of bilayer mosaic for this feasibility study has been encouraged by recent results of a molecular dynamics study of pressure-driven water transport through graphene bilayers which suggests that, as the channel width increases, the water flow rate increases monotonically and also the increase of channel thickness leads to weaker water-graphene interaction and thus higher water molecule mobility29. The AFM images acquired immediately after the exposure reveal that triangular and polygonal blisters nucleate at most junctions  is a photoresist polymer and was spin coated on the top surface of a PMMA film at ∼2000 rpm creating a layer of ∼300 nm thickness. The samples were characterized by means of AFM (see below) and Raman spectroscopy, by InVia (Renishaw) microspectrometer with 2400 grooves/mm grating for the 785 nm laser excitation; Raman spectra were measured at and the laser power was kept at 1.2 mW on the sample to avoid laser-induced local heating. A ×100 objective with numerical aperture of 0.95 was used in all cases. Experimental data were fitted by using Lorentzian functions, one for the monolayer and four for the bilayer graphene.The tensile experiments were conducted using a micro-tensile tester (Deben MT 200) that could be adjusted under AFM for simultaneously applying strain and acquiring images under AFM. The increment step of strain was 0.1% for all cases. The AFM images were obtained using Dimension Icon microscope (Bruker) operating in Peak Force Tapping mode using ScanAsyst-Air probes . No data treatment apart from line subtraction (retrace) to remove the tilt has been performed. Wrinkle amplitudes and half wavelengths were measured from line profiles manually for each wrinkle. Medians of the thus obtained parameters were then used to characterize the wrinkles in each profile. Also, lateral size of the islands delimited by the wrinkles has been estimated by averaging the length of two crossed lines across the island measured manually from line profiles.Tensile experiments have been performed using the same set-up under the Raman microscope (InVia Renishaw microspectrometer) by using the 785 nm laser excitation. At each deformation step, several Raman spectra were acquired in different location of the 1LG flake under investigation (near the geometric centre) and experimental data were fitted by using one Lorentzian function.32.High humidity exposure experiments were performed at 90% RH and at RT in a custom-made climate chamberSupplementary Information


The new immunotherapy targeting the programmed cell death 1 (PD-1) receptor and its cognate ligand PD-L1 has renewed hopes of eradicating the most difficult human cancers to treat. Among these, there are the poorly differentiated and anaplastic thyroid cancers, unresponsive to all the therapies currently in use. In the present review we will summarize information regarding the expression of PD-L1 in the different thyroid cancer histotypes, its correlation with clinicopathological features, and its potential prognostic value. Then, we will evaluate the available data indicating the PD-1/PD-L1 axis as a promising target for thyroid cancer therapy.  BRAF and RAS oncogenes, and chromosomal translocations of the RET (REarranged during Transformation) and NTRK1 (Neurotrophic Tyrosine Kinase Receptor 1) genes, which lead to the activation of a common carcinogenic pathway, i.e., the MAPK/ERK signaling . ,63. 8,63 noticed ,37,64,67ild type . These fn (Treg) . From thn (Treg) . These vanalyzed . AltogetV600E and lymph node metastasis, but not with other clinicopathological features or DFI [Regarding PD-L2 expression in thyroid tumors, to the best of our knowledge, only two studies have been performed so far . One reps or DFI . The sams or DFI . In any Over the last decade, the considerable increase of our knowledge about mechanisms underlying the ability of cancer cells to elude the detrimental action of the immune system has led to a renewed interest for immune-based therapy in oncology, especially for hard-to-treat cancers, including the poorly differentiated and frankly anaplastic thyroid cancers ,81. In pPD-L1 and CTLA-4 genes, the canonical BRAF-like and RAS-like PTC were classified into 2 subgroups each: BRAF-IR (immunoreactive) and RAS-IR, characterized by up-regulation of immune-related genes and tumor infiltration by several immune cell subtypes, including T cells; and BRAF-ID (immunodeficient) and RAS-ID, characterized by low expression of immune-related genes and low infiltration of immune cells [The objective response rate to PD-1/PD-L1 directed therapies varies from 13 to 43% in different tumor types . The besne cells ,87. ConfV600E mutation and PD-L1 protein by IHC in tumor tissue, the patient was treated with vemurafenib (BRAF inhibitor) and nivolumab. Whereupon, the patient experienced a continued reduction of the metastatic lesions with complete radiographic and clinical remission of the disease 20 months after the beginning of nivolumab therapy [At present, no information from clinical trials with anti-PD-1/PD-L1 directed therapies enrolling patients affected by aggressive thyroid cancers is available. However, different preclinical studies demonstrated the efficacy of PD-1/PD-L1 axis blockade in restraining growth of ATC-derived cell lines injected in mice ,88,89. I therapy . In anot therapy . Of thes therapy . Very re therapy . Twenty- therapy . A parti therapy . At the  therapy . This isSince the activation of the MAPK signaling in thyroid cancer has been shown to negatively modulate the immune response in the tumor microenvironment, it is likely that targeted therapies directed against this pathway may revert the immune suppression due to abnormal kinase activation . For exaThe information so far available suggests that PD-L1 could represent a useful prognostic marker for risk stratification of thyroid cancer patients, and that anti-PD-1/PD-L1 directed therapies could be a valid option for patients affected by the most aggressive thyroid cancers, such as PDTC and ATC, unresponsive to the currently available therapies. Some issues, however, still remain to be addressed. In particular, the standardization of the IHC techniques, the interpretation of PD-L1 immunoreactivity in cancer tissues, and a more reliable characterization of biomarkers capable of predicting patients’ response to anti PD-1/PD-L1 therapies.