N,N′-Dihexyl-3,4,9,10-perylenedicarboximide (PDI-C6) semiconducting molecules’ ink. We demonstrated the high resolution (about 50 µm) and accurate printing of organic ink on bare chemical vapor deposited (CVD) graphene. PDI-C6 forms nanocrystals onto the graphene’s surface and transfers charges via π–π stacking to graphene. While the doping from organic molecules was compensated by oxygen molecules under normal conditions, we demonstrated the photoinduced current generation at the PDI-C6/graphene junction with ambient light, a 470 nm diode, and 532 nm laser sources. The local  photoresponse of 0.5 A/W was demonstrated at a low laser power density. The methods we developed open the way for local functionalization of an on-chip array of graphene by inkjet printing of different semiconducting organic molecules for photonics and electronics.In this work, we report a novel method of maskless doping of a graphene channel in a field-effect transistor configuration by local inkjet printing of organic semiconducting molecules. The graphene-based transistor was fabricated via large-scale technology, allowing for upscaling electronic device fabrication and lowering the device’s cost. The altering of the functionalization of graphene was performed through local inkjet printing of  As fap-type  or n-typ [n-type  dependinficiency . Despiteficiency .Organic molecules can be deposited either by thermal evaporation from solid-state or by a solution process. The former gives less impurity from the deposited organic crystals but demands vacuum equipment and the necessity of photolithography processes ,6,8. SolThis work reports on the maskless method of hybrid photosensitive junction fabrication in a graphene field-effect transistor (GFET) based on the inkjet printing of semiconducting organic ink. While graphene serves as a conductive channel, the non-covalent functionalization with semiconducting organic molecules alters the optical sensitivity in normal conditions to the visible wavelengths. We showed the one-step technique of graphene-channel functionalization by PDI molecules and in-plane junction formation by local printing of organic inks. The suggested method of junction formation in GFET is a prospective approach for a non-covalent functionalization of an array of graphene-based transistors by different organic molecules for photonic and electronic device fabrication.2 atmosphere. The graphene was then patterned via oxygen plasma etching . Using e-beam assisted evaporation, we deposited the 10 nm Ti and 100 nm Au metallization. The details of the fabrication process can also be found elsewhere [GFETs were fabricated using a chemical vapor deposited (CVD), single-layer graphene. To assist in transfer, a thin layer of poly(methyl methacrylate) (PMMA) was spin-coated on top of the graphene/copper. A previously published, high-throughput transfer technique of graphene transfer was utilized in this work . In a nulsewhere . Prior tN,N′-Dihexyl-3,4,9,10-perylenedicarboximide (PDI-C6)  semiconducting organic molecules for ink preparation. The inks of 0.7 mg of organic molecules were prepared in 6 mL of toluene/IPA (50/50%) solution. Toluene (0.36 D) was used as a less polar solvent for PDI. As far as the viscosity of toluene is small (0.56 cP), we added IPA (2.04 cP) to prepare the ink. For ultrasound treatment of the ink, the ultrasound bath Bandelin Sonorex was used for 30 min. Nevertheless, PDI had sedimented, and the top layer of the solution was taken for inkjet cartridge filling. The ink was filtered using a filter with pore size of 200 nm  before loading to the cartridge.We used the following For inkjet printing, Dimatix Materials Printer DMP-3000  inkjet printer was used. The printer operates via piezoelectric jetting cartridges with 21.5 µm nozzle diameters. In order to obtain a controllable process, basic printing parameters, such as waveform, frequency, jetting voltage, and drop spacing, were adjusted. For low-viscous ink (<2 cP), the waveform of pulse supplied on piezoelectric nozzles is the main parameter to control the formation of the stable droplets. To ensure stable conditions during the process and proper layer formation, the substrate and the cartridge worked at room temperature. All the experiments with organic inkjet printing were performed in cleanroom facilities.2 and laser power of 0.5 mW. A Solver Pro atomic force microscope (AFM)  was used to study the morphology of pristine graphene, and later, its modification with organic molecules.We measured the electrical characteristics via a semiconductor parameter analyser, IPPP 1/5 . For transfer current-voltage characteristic measurements, we used the liquid gate configuration with phosphate-buffered saline and AgCl reference electrode. Optical characterization was performed with HRM-300  with 5×–50× objectives. Raman spectra were recorded on a Centaur HR Raman spectrometer  with a 100× objective at 532 nm wavelength  with a beam spot of ≈1 μmTo evaluate the photocurrent response across the GFET, we used the same Raman laser with 0.5 mW maximum power delivered through 50× fixed lens while the sample moved on the motorized stage. The steps between measured points were 1 µm with 20 s delay between measurements.To conduct the photocurrent measurements, 470 nm light-emitting diode (LED) was used . An LED was placed at a distance of 10 cm above the structures. The experiments were performed in the room light and in the light-protected box with and without LED illumination.The PDI-based ink was prepared in order to fulfil the demands for viscosity and surface tension for stable printing a and to −1 (in-plane ring “breathing”), 1354 cm−1 (out-of-plane C–C stretching), 1462 cm−1 (ring deformation), and 1581 cm−1 (in-plane C–C stretching), respectively [−1 and the 2D peak at 2700 cm−1, respectively, whereas for the “PDI on graphene” sample, their locations were at 1592 and 2699 cm−1. The charges in deposited organic molecules induced doping of graphene, and we observed a clear redshift of the G band up to 8 cm−1 [2D/IG of the bare graphene (0.5) and the one for graphene covered with a PDI adsorbate layer (0.3) further confirm the π–π stacking of PDI on graphene, and the chemical doping of graphene as well [Before the characterization of printed layers, the post-processing was done by temperature annealing on a hot plate at 150 °C for 15 min to evaporate the solvent matrix. The results of the Raman measurements for PDI-C6 modified graphene are presented in ectively ,25. As fo 8 cm−1 ,27. The  as well . The sli as well . The emi2 substrate after single droplet printing. The film deposited inside the drop is not uniform because of differences in evaporation temperatures of several components of the ink that led to clustering during droplet drying. We observed an increase in resistance of the graphene channel of about 2–3 times  when it is irradiated with a 470 nm light source. The output curves are non-linear after the organic molecules’ deposition due to the difference of barrier height at the interface of metal with bare graphene, and graphene modified by organic molecules [R) for 470 nm LED was calculated as R = Iph/P (where P—is an incident light power), which is equal to 0.15 A/W at 0.2 V bias voltage (To investigate the efficiency of the photodetectors, we performed characterization under an ambient and an LED light. The results are shown in olecules . The max voltage b.In conclusion, we have developed robust and scalable technology for the functionalization of arrays of graphene field-effect transistors by inject printing. We prepared the semiconducting organic ink and studied the deposition of organic molecules onto the graphene surface. We demonstrated the resolution of printing is consistent with current imaging techniques. This technology is very flexible and can provide the modification of an array of GFET on the same substrate with different organic molecules that can be tuned for specific wavelengths. The drastic changes in the electrical properties of functionalized graphene with organic molecules were shown. The in-plane photosensitive junction was developed based on partial coverage of the graphene channel. The junction is sensitive to visible light, with a photoresponse up to 0.5 A/W for 532 nm light at 30 nW light intensity. We suggested that the photovoltaic effect is responsible for photocurrent generation in a junction created by light-induced doping of graphene from organic molecules.


In addition, we demonstrate further control of charge transport by breaking the conjugation of their π systems which lowers their conductance, and via heteroatom doping of fullerene, which introduces transport resonances and increase their conductance. Supported by our combined density functional theory (DFT) calculations, a promising future of tunable full-carbon electronics based on numerous sub-nanoscale fullerenes in the large family of carbon allotropes is anticipated.Full-carbon electronics at the scale of several angstroms is an expeimental challenge, which could be overcome by exploiting the versatility of carbon allotropes. Here, we investigate charge transport through graphene/single-fullerene/graphene hybrid junctions using a single-molecule manipulation technique. Such sub-nanoscale electronic junctions can be tuned by band gap engineering as exemplified by various pristine fullerenes such as C All-carbon electronics holds promise beyond the conventional silicon-based electronics, but it remains challenging to manufacture them with well-defined structures thus tunability. Tan et al. control charge transport in single-molecule junctions using different fullerenes between graphene electrodes. Although the CNTs and carbon nanoribbons5 have been in the fabrication of computing circuits6, the ability of CNTs to conduct charge is sensitive to atomic structural topology as well as the presence of edge defects that are hard to control due to structural uncertainties and unavoidable dangling bonds in the low-dimensional carbon materials8. Therefore, there is an urgent need to develop the potential of defect-free zero-dimensional (0D) fullerenes and atomically defined carbon allotropes for carrying out band gap engineering and constructing all-carbon electronics at angstrom scale based on the well-established fullerene science10.The high operational speed of all-carbon electronics60 junctions were fabricated by contacting fullerenes with a scanning tunneling microscope (STM) or using electron-beam lithography to gold electrodes13. Recent developments suggest that fullerene-based junctions could exhibit a pronounced Seebeck effect16 and that charge transport could be precisely manipulated depending on the contact area between the molecule and electrodes17, for example, through chemical modification of the fullerene with planar anchor groups such as pyrenes, which bind to the graphene via van der Waals interactions16. An even simpler strategy involves the bridging of graphene electrodes via the overlap of graphene and fullerene π orbitals, which could be achieved using a single-molecule junction techniques22, whose electrode gap can be controlled with angstrom precision.Early pioneering single-C18. It is found that the fullerene can be bridged between two electrodes of the chemical vapor deposition (CVD) graphene via van der Waals interaction to form the single fullerene junctions. More importantly, the conductance of the graphene/single-fullerene/graphene junctions can be controlled by inserting different fullerenes with a variety of energy gaps. The charge transport can be further tuned through heteroatom doping, which introduces desirable transport resonances. These results demonstrate the exquisite controllability of all-carbon electronics via band gap engineering.Here we fabricate and investigate charge transport through a series of hybrid graphene/single-fullerene/graphene junctions with pristine fullerene cores using mechanically controlled break junction (MCBJ) technology60, C70, C76, and C90 are synthesized using our low-pressure benzene–oxygen diffusion combustion approach (See “Methods” for more details), and charge transport through the graphene/single-fullerene/graphene junctions are investigated using the MCBJ technique with CVD graphene-coated copper wire as electrodes.As shown in Fig. 60 show clear plateaus around 2.0 × 10−5G0 , which represent the conductance of single-C60 junctions bridged between the two graphene electrodes, while there is only the exponential decay of tunneling in the absence of fullerenes. Furthermore, it is found that the molecular plateaus shift to higher conductances as the sizes of the fullerenes increase, such that the conductance of C90 is almost half an order of magnitude higher than that of C60. To further investigate the conductance variations among single-C60 junctions, the corresponding 2D histogram of the graphene/single-C60/graphene measurement . In the presence of fullerenes in the solution, a fullerene molecule can be trapped between the graphene electrodes via van der Waals interactions to form a graphene/single-fullerene/graphene junction, which is further connected with the external circuit with 100 mV bias voltage to monitor the current across the junction. As demonstrated in Fig. ent Fig.  construc60, C70, C76, and C90/graphene are located at 2.0 × 10−5G0, 2.2 × 10−5G0, 2.5 × 10−5G0, and 5.1 × 10−5G0 respectively, demonstrating that there is a >250% conductance enhancement from C60 to C90. This result shows that the conductance of the single-C60 junctions with graphene electrodes is lower than that of gold electrodes25, which agrees with the previous report that the π–π interaction is enough to construct molecular junctions, but the conductance is reduced26. Data of the closing process of graphene/single-C60/graphene junctions are given in Supplementary Fig. −5G0) is slightly larger than that of the opening process because of the contribution of pure tunneling27. As shown in Fig. 60 with ~7 angstroms to C90 with ~9 angstroms. We also analyzed the most probable displacement of junctions with different sizes of fullerene 2 with the same atomic numbers (See the Methods section). Figure 120/graphene junctions is 1.3 × 10−5G0 2/graphene . Figure z direction. In the simulations, to avoid edge effects, the graphene sheets are assigned periodic boundary conditions in the x direction. The cross-plane current is injected from lead 1 and collected from leads 3 and 4. Figure 60, C70, C76, and C90 as a function of the electrode Fermi level. In the experiments, the Fermi level is influenced by imperfections in the graphene and contact with copper. Several transmission spectra due to different orientations and locations were calculated and a Boltzmann distribution based on the total energy of the system was used to compute the average conductance 2 are in Fig. 50H10 where the conductance of C60 is larger than that of C50H10 due to the significant difference in their HOMO–LUMO gaps. This theoretical result supports the experimental measurements, where C60 is more conductive and shows that the broken conjugation of C50H10 due to the connection to ten hydrogen atoms leads to a decrease in conductance. To highlight the effect of doping, Fig. 59N)2 vs. Fermi energy and reveals that the presence of nitrogen creates two transport resonances around −0.5 eV within the HOMO–LUMO gap of C120. Consequently, over a large energy window, the conductance is significantly larger than that of the parent compound before nitrogen doping. We further analyze the corresponding frontier orbitals HOMO-2, HOMO-1, HOMO, and LUMO as plotted in Fig. To investigate the effect of conjugation and doping, the average conductance values  with graphene on the surface  was bent into a curved surface, then fixed on a flexible substrate  with epoxy glue. Parallel to it, in the opposite side, another electrode treated in the same way was fixed on the substrate. With the help of a microscope, the bent part of the two copper wires are placed in the middle of the substrate by the “head-to-head” configuration, and they are adjusted close enough (10–20 μm) but without touching  protocol to measure the tunneling decay constant in the pure solvent (decane). As shown in Supplementary Fig. G0. The conductance–displacement curves from both opening and closing processes were recorded, and several individual traces are shown in Supplementary Fig. We also add the control experiments using “graphene STM-BJ” protocol .35, with a generalized gradient approximation (GGA-PBE) exchange-correlation functional, and a double-ζ polarized atomic-orbital basis set for carbon. The cutoff energy was 200 Ry, and the force tolerance was 0.02 eV/Å. To compute their electrical conductance, the fullerenes were each placed between two graphene electrodes. For each structure, the transmission coefficient T(E) describing the propagation of electrons of energy E from lead 1 to lead j  was calculated using Gollum the quantum transport code36, which utilizes the DFT mean-field Hamiltonian and overlap matrices from SIESTA and computes T(E) via the following formula:E) is the retarded Green’s function of the molecule in the presence of the electrodes and j(E). Γj determines the broadening of transmission resonances due to the contact between the molecule and electrode j. The finite-temperature conductance is obtained from the transmission coefficient via the following formula:h is the Planck’s constant; e is the charge of an electron; EF is the Fermi energy, T is the temperature, and kB is Boltzmann’s constant. If the total energy of a molecule i with a certain location and orientation on the electrodes is Ei, then the probability of obtaining such a molecular location and orientation is Gi is the corresponding conductance, then the Boltzmann average of the conductance is Geometrical optimizations were performed by using the standard Kohn–Sham self-consistent density functional code SIESTAFor each molecule, the transmission coefficient was computed for a range of contact geometries between graphene and the fullerene, examples of which are shown in Supplementary Fig. In the experiments, the copper can modify the density of states in the graphene, primarily by shifting the Fermi energy of the graphene relative to the Dirac point. To account for this, we plot conductance values Fig.  as a funAlso, the four-terminal simulation is used, because if the leads are terminated close to the fullerene, then multiple scattering between the lead edges and the fullerene will occur, which creates additional structure in the transmission curves vs. energy. In the experiment, there is no such termination and therefore the four-terminal calculation is closer to the experiment.42. The flame was maintained under a pressure of 10–20 torr in the homemade set-up with the burner that consisted of two concentric tubes with the same center. Oxygen was fed to the inner tube, while benzene flowed through the space between the two concentric tubes. The synthetic conditions were optimized to give an optimal yield of typical C60 in the soot and to keep the diffusion flame stable if possible. The optimized gas flow rates are 0.55 L/min for O2, 1.10 L/min for C2H2, and 1.0–1.1 L/min for vaporous benzene. Both C60 and C70 are the major products with minor components of C50H10, C76, and C90, subject to high-performance liquid chromatography (HPLC) separation.The fullerene-containing soot was synthesized by means of an acetylene–benzene–oxygen diffusion combustion in our homemade set-up60 in the absence of any solvent, based on previous literature44. This method was originally designed for the preparation of a well-mixed homogeneous powder by vigorously vibrating a stainless-steel capsule that contains the sample and stainless-steel balls with a frequency of 2800 cycles/min. A mixture of C60 and 20 molar equivalents of KCN powder was vigorously vibrated for 30 min under nitrogen. The major product is dimer of C60 with unchanged C60. After separation using HPLC on a 5PBB column, the purified C120 was afforded 2 according to previous literature49. We optimized the synthesis conditions to increase the yield of (C59N)2. As shown in Supplementary Fig. Heterofullerene C50H10, C60, C70, C76, and C90 from the toluene-extracting solutions of the crude soot. In each stage, the C50H10-, C60-, C70-, C76-, and C90-containing fractions were collected and followed by concentration and HPLC until the purified sample with 99.9% purity were obtained. Supplementary Figs. 50H10, C60, C70, C76, and C90 for the multi-stage HPLC isolation. All the separations were performed at room temperature using toluene as the eluent. After several cycles of separation, the purified samples were obtained. The hydrofullerene (C50H10) and pristine fullerenes  can be identified by mass spectrometry -C9050 . Our X-ray crystallographic experiment further identified the precise structure of C1(30)-C90 involved.The fullerene-containing carbon soot was extracted with toluene by ultrasound and followed by filtration. The compositions of the toluene-extracting solutions were very complex and typically contain lots of polycyclic aromatic hydrocarbons, fullerenes, and fullerene derivatives. Several stages of HPLC with different columns were conducted to separate and purify the sample of CMass spectra were recorded by a Bruker HCT ion trap instrument interfaced by an atmospheric pressure chemical ionization (APCI) source. In the APCI, ion–molecule reactions were occurred to generate the ions at ambient conditions, and then the ions were transferred into the mass analyzer by a vacuum interface. An oven with temperature tunable in the range of 200–350 °C was connected to the APCI source for solvent evaporation and possibly sample decomposition.Supplementary InformationPeer Review File


The NTRK2 genetic locus encodes neurotrophin membrane receptors that play an important role in normal neural tissue plasticity, growth, and survival. One NTRK2-encoded protein is TrkB-FL, which can regulate multiple pathways relevant to cancer. A second NTRK2 gene mRNA isoform encodes TrkB-T1, a receptor that has a different cytoplasmic domain encoded in a mRNA with a unique 3′ terminal exon.Tumors from The Cancer Genome Atlas (TCGA) and other studies were classified according to the expression of a single form of NTRK2 mRNA, TrkB-T1, identified by its unique 3′ terminal exon. Analysis of differentially expressed genes in TrkB-T1 high expressers was done to determine if tumors enriched for TrkB-T1 mRNA were a uniform group independent of anatomic site.n = 96) revealed gene expression differences consistent with the high TrkB-T1 tumors being more neural-like. The upregulated genes in the TrkB-T1 RNA high expressers also showed enrichment of pathways involved in retinol metabolism, hedgehog signaling, and the Nfe2l2 response, among other pathways. An examination of oral, esophagus, and lung SCCs  showed induction of the same pathways among tumors that expressed high levels of TrkB-T1 mRNA. Proteins associated with regulation of the sonic hedgehog pathway, and the Nfe2l2 response, Tp63, and Keap1 and p62/SQSTM1 proteins, showed differential expression in larynx, oral and lung high TrkB1-T1 expresser SCCs. Unexpectantly, the relationship of high level TrkB-T1 expression to patient outcomes was SCC anatomic site specific. High TrkB-T1 mRNA levels in laryngeal SCC correlated with poor survival, but the opposite was true for lung SCC. This may be because pathways enriched in the TrkB high expressers, like those involving oncogenes NFE2L2, PIK3CA, and SOX2, are known to have SCC anatomic site-specific effects on progression.The mRNA for TrkB-T1 is the most abundant NTRK2 gene mRNA in all squamous cell carcinomas (SCCs) in the TCGA database. Comparison of larynx SCC high TrkB-T1 RNA expressers to low expressers  contains supplementary material, which is available to authorized users. Squamous cell carcinoma (SCC) is a cancer identified histologically, with the cells resembling, and possibly derived from, the flattened epithelial cells of skin or mucosal epithelium. Nearly 95% of head and neck carcinomas (HNC), 30% of lung cancers, and 50–90% of esophageal cancers, depending on geographic site, are SCCs –3. The tThe intact, nonmutated TrkB neurotrophin receptor is thought to play a role in progenitor neural cell migration, survival, and differentiation –10. In oAccurate measurement of NTK2 gene expression can be difficult. The TrkB receptor protein is differentially glycosylated and there are multiple forms produced from differentially spliced NTRK2 gene mRNA . While mThe work here exclusively focuses on the measurement of the two most common forms of NTRK2 gene RNAs: TrkB-FL and TrkB-T1. This work highlights that TrkB-T1 is the most abundant form of the mRNA in all tumor types examined as has been shown for many normal cell types in the body . The eashttps://www.tcpaportal.org/tcpa/download.html. Level 4 replica-based normalization was used to minimize batch effects.The Cancer Genome Atlas (TCGA) gene expression (RNAseq V2), RSEM (RNA-Seq by Expectation-Maximization), CNA copy number amplification and clinical data for the 518 lung adenocarcinoma (LUAD), 501 lung SCC (LUSC), 284 oral SCC (OSCC), 96 laryngeal SCC (LASC), 90 esophageal adenocarcinoma (ESAD), and 97 esophageal SCC (ESSC) samples, data originally curated by TCGA Research Network were obtained from the Broad Institute TCGA GDAC Firehose repository via Firebrowse (http://fbdev/api-down.html). The Cancer Proteome Atlas (TCPA) reverse-phase protein array analysis data  of 353 LUAD, 322 LUSC, 206 OSCC, and 79 LASC was downloaded from the MD Anderson TCPA website  https://. Exon expression measurement is based on exon quantification file and gene expression is based on normalized gene expression values in RSEM. Gene expression for NTRK2 gene, TrkB-T1 RNA isoform was derived based on the exon 16 quantification, while TrkB-FL RNA was proportional to exons 23 and 24 and TrkB-SHC RNA was proportional to exon 19 . These ehttps://brb.nci.nih.gov/BRB-ArrayTools/index.html), then class comparison was performed using a t test with and without permutation of class labels to allow determination of FDR on all RNAs with at least 80% of the values available. Heat map of DE for OSCC was generated for the class comparison including 1140 genes FDR < 0.005.Gene expression datasets were categorized into ‘TrkB-T1 high expressers’ and ‘TrkB-T1 low expressers’ based on TrkB-T1 mRNA levels, with the cutoff at the mean level. DE genes were then identified between high and low TrkB-T1 mRNA expressers, as explained below. For all groups there were substantially more subjects in the low-expresser groups. For esophageal adenocarcinoma (ESAD) this scheme produced less than 15 DE genes, so the top 30 and lowest 30 TrkB-T1 expressers were compared instead to produce a larger DE gene list. All mRNA data were log2 transformed using BRB Array Tools V 4.5 (http://amp.pharm.mssm.edu/Enrichr/) [adj ≤ 0.05).” Enrichr calculates this value by using a corrected Fisher exact test. KnowEnG is a second pathway analysis tool [http://software.broadinstitute.org/gsea/index.jsp), and all were used [The Enrichr tool (nrichr/)  was applsis tool . It usesere used . The entFor the comparison to Genotype Tissue Expression (GTEx) Project of normal tissue transcriptomes, Enrichr was used under the category of Cell Types .To determine transcription factors associated with high TrkB-T1 expression, promoter sequence of enriched DE genes were scanned using the position weight matrix method from the TRANSFAC and JASPAR transcription factor binding motifs , 45.To analyze and visualize mutations, copy number alteration and gene expression across all the samples in the head and neck SCC dataset cBioPortal was used , 47. In P < 0.05 considered significant. For clinical parameters of high and low TrkB-T1 RNA tumor patients, either Chi Squared or Fisher Exact test when appropriate or in the case of age, the Student t test was used to determine statistical significance.Survival curves were generated using Cutoff Finder Web application, though all categorizations of the group pairs were based on separation of the two groups into high expressers and low expressers, with the cutoff at the mean level for TrkB-T1 or other RNA . LogistiTCGA cohorts of cancer patients provide large datasets of measured mRNA levels in tumors. The mRNA encoding TrkB-T1 contains a unique terminal exon, exon 16, and poly A site providing the encoded protein with a distinct carboxy terminus and serving as a means of identification of this RNA. RNA containing exon 16 comprise over 97% of the RNA coming from the NTRK2 gene in both normal oral mucosa and OSCC. The same was true for laryngeal SCC (LASC) and esophageal and lung tumors studied  pathways. A comparison of the DE genes in LASC TrkB-T1 high versus low expressers revealed a number of pathways enriched among the KEGG pathways as shown in Fig. The next step was to determine if transcriptome patterns associated with high TrkB expression indicated similar pathways were enriched in tumor sites distinct from the head and neck. Again, the same pathways were seen to be enriched in high TrkB-T1 expressers versus low expresser ESSCs and LUSCs . This analysis revealed that the Nfe2l2 pathway involved in cellular response to oxidizing insults was enriched most highly in all 4 high TrkB-T1 expresser SCCs tested, LASC, OSCC, ESSC and LUSC, and not in the high TrkB-T1 adenocarcinomas Fig. 3)..3).Fig. All these DE analyses were based on TGCA datasets done with RNAseq analysis. To corroborate this approach, an independent RNA expression dataset from a different OSCC database with gene expression measured by a different approach, DNA microarray hybridization, was used (GSE30784) . ResultsGlobal protein measurement of TCGA samples by the TCPA reverse-phase protein array analysis  revealedp = 0.0037).Some gene and gene expression changes associated with TrkB-T1 high level expression. cBioPortal was used to examine genes whose mutation, amplification and mRNA level are associated with head and neck SCC and are suspect drivers of SCC at that site . This liHigh level expression of TrkB-T1 is associated with poor survival of LASC but not LUSC.p < 0.04, Fig. We initially examined LASC patient samples because they showed a large number of tumors with very high levels of TrkB-T1 mRNA, ensuring sufficient number to do survival analysis. An examination of patient outcomes revealed that high expressers of TrkB-T1 had reduced overall survival Additional file 2:Table S1. Clinical characteristics of cancer patients with high and low TrkB-T1 mRNA expression levels. (XLSX 18 kb)Additional file 3:Table S2. Of the 194 genes whose expression was enriched >3x in high TrkB-T1 mRNA expresser OSCCs, 73 are also highly enriched in normal neurological tissue. (XLSX 14 kb)Additional file 4:Figure S2. KEGG pathways that are elevated in high expresser TrkB-T1 OSCC samples from the TCGA database. Negative log10 of the adjusted P value. All identified with ENRICHR program. Samples were divided into two groups above and below the mean TrkB-T1 mRNA level with the exception of ESAD where only the top 30 samples in each group were included to determine the DE gene list. Shown are values for A. OSSC, B. ESSC, C LUSC and D LUAD. ESAD showed no enriched pathways. (PDF 335 kb)Additional file 5:Figure S3. KEGG pathways that are enriched in TrkB-T1 high expressers in an earlier study of OSCC gene expression, (GSE30784) [p value. TrkB-T1 levels are average of 3 probe sets of the specific for TrkB-T1, 221795_at, 221796_at and 214680_at of the Affymetrix Human Genome U133 Plus 2.0 Array. (PDF 65 kb)SE30784)  measuredAdditional file 6:Table S3. Gene Set Enrichment Analysis of pathway enrichment in TrkB-T1 high expresser SCCs. (XLS 19 kb)Additional file 7:Figure S4. Composite view of mutation and amplification events for genes previously identified as potential drivers of HNSCC using cBioPortal with addition of NTRK2. Samples are sorted by increasing NTRK2 mRNA level, as shown in the bottom bar which is a schematic of NTKK2 mRNA levels in each sample. The percentage on the left is frequency of mutations and amplifications. Shown on the right is the correlation between TrkB mRNA level and the level of the named mRNAs. (PNG 647 kb)Additional file 8:Table S4. Correlation of TrkB-T1 mRNA level and oncogene mRNA and or gene copy number for TCGA datasets. (DOCX 12 kb)


To analyze the prognostic value of programmed death factor ligand 1 (PD-L1) in colorectal cancer.Electronic databases, such as PubMed, Web of Science, Embase, and Cochrane library, were searched to identify studies evaluating the PD-L1 expression and overall survival (OS) in these patients. Afterwards, the relevant data were extracted to perform the meta-analysis.P = 0.04), indicating that high expression of PD-L1 was significantly correlated with poor prognosis of colorectal cancer. Apropos of clinicopathological features, the merged odds ratio (OR) exhibited that highly expressed PD-L1 was firmly related to lymphatic invasion  and advanced stage , but not correlative with patients’ gender, microsatellite instability, or tumor location.A total of 3481 patients were included in 10 studies. The combined hazard ratio (HR) was 1.22  contains supplementary material, which is available to authorized users. Among the most common cancers worldwide, colorectal cancer ranks third, accounting for 10% of all tumor cases . AccordiProgrammed cell death protein 1 (PD-1), a sort of inhibitory checkpoint molecule, was discovered and named by Japanese scholar Ishida in 1992 . It beloCurrent theories on the expression of PD-L1 in colorectal cancer and tumor prognosis are limited and controversial. Some studies have manifested the palpable connection between PD-L1 expression and overall survival rate of colorectal cancer patients –18, but Two authors independently searched PubMed, Web of Science, Embase, and Cochrane Library for published literature on PD-L1 and colorectal cancer. Publication time of the included articles ranges from the time when the database was established until August 2018. All publications are in English. Search strategies are  AND (“PD-1” OR “PD-L1” OR “programmed death 1” OR “programmed death ligand 1” OR “programmed cell death ligand 1” OR “programmed death 1 ligand 1” OR “B7-H1” OR “CD274”).The clinical and pathological data of all cases are complete, and all were diagnosed as colorectal cancer by pathological examination;Detecting the PD-L1 expression in colorectal cancer tissues by immunohistochemical staining;The literature provides the relationship between PD-L1 expression and overall survival (OS) in patients with colorectal cancer;The literature provides the relationship between PD-L1 expression and clinicopathological features, such as primary tumor size, clinical stage, and differentiation;The literature provides sufficient information to estimate the hazard ratio (HR).The following are the inclusion criteria:The included literature is not an original study;The data contained in the research is wrong, or the quality of the incorporated literature is low;The included literature is based on animal or cell experiments;Cannot use the data provided in the literature to calculate the hazard ratio (HR) associated with PD-L1;The included literature did not analyze the expression of PD-L1 in tumor cells.The following are the exclusion criteria:Two authors independently screened and extracted data found on inclusion and exclusion criteria and discussed together or adjudicated by third parties in case of disagreement. For the lack of information, we contacted the original author as much as possible. Extracted contents included author, publication year, country, positive threshold, follow-up period, baseline and clinicopathological information of patients, hazard ratio (HR), and 95% confidence interval (95%CI) related to PD-L1 expression.Methodological quality assessment of the included data was carried out using the Newcastle-Ottawa Scale (NOS). Scores of the NOS are split into three aspects: object selection, inter-group comparability, and outcome measurement. The highest rating is 9 points, and the study with more than 6 points is considered as a high-quality one .χ2 test, and then we utilized the fixed-effect model or the random-effect model according to heterogeneity. All the analyses above were presented by Revman 5.3 software.We implemented the meta-analysis based on the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Checklist   and advanced stage , but not correlative with patients’ gender, microsatellite instability, or tumor location  exhibited that highly expressed PD-L1 was firmly related to lymphatic invasion . In addition, the results of the Asian group , the non-Asian group , and the tumor stages I–IV group  were still statistically significant, but other subgroup analyses failed to arrive at such a statistically significant conclusion , there was no publication bias in the included literature involving PD-L1 and OS . Nevertheless, there was inevitable heterogeneity among the retrospective studies included in this meta-analysis . In order to make the conclusion more persuasive and scientific, we adopted the subgroup analysis to explore the heterogeneity sources. And as the findings suggested, the heterogeneity of each subgroup decreased in varying degrees, indicating that these factors have certain degrees of influence. Among them, the non-Asian group had the minimum heterogeneity (I2 = 12% < 50%), which implied the essential role the regional or ethnic differences played on engendering heterogeneity.This meta-analysis demonstrated that PD-L1 expression could be utilized as an independent factor in judging the prognosis of colorectal cancer  leads to lymphatic invasion , and theTo mention first, all the included articles were retrospective studies, whose bias could not be eliminated, so the consequences were generally stable. Also, it should be noted that all the articles included are in English, meaning the lack of research especially those negative studies in non-English speaking countries, which leads to the absence of representativeness and the production of bias.Secondly, it has been reported that the expression of PD-L1 in tumor cells is a critical factor in making the monoclonal antibody against PD-1/PD-L1 effective. However, in every article, the threshold of PD-L1 positive is different and brings about a tremendous impact on the experimental results. According to published articles, patients with PD-L1 positive can obtain a better outcome in the treatment with immunocheckpoint inhibitors as the threshold increases . So, uniFurthermore, the latest experiments indicate that cancer patients  with intThis study analyzed all the available interrelated information in the published literature and exhibited that the expression of PD-L1 was significantly correlated with the overall survival rate of colorectal cancer. The more the PD-L1 was expressed, the worse prognosis the colorectal cancer patients would undergo. Concerning clinicopathological features, the expression of PD-L1 was bound up with lymphatic invasion and tumor stage, but not gender, microsatellite instability, or tumor differentiation. In other words, the expression of PD-L1 could be utilized as an independent factor in judging the prognosis of colorectal cancer, and patients with advanced cancer or lymphatic invasion were more likely to express PD-L1. This conclusion may lay a theoretical foundation for the application of PD-1/PD-L1 immunoassay point inhibitors but still need to be verified by sizeable well-designed cohort studies.Additional file 1:Table S1. PRISMA 2009 Checklist. (DOC 53 kb)Additional file 2:Figure S1. Forest plots assessing the relationship between PD-L1 and clinicopathological characteristics: (a) gender; (b) grade; (c) lymphatic invasion; (d) microsatellite instability; (e) mucinous properties; (f) stage; (g) the involvement of regional lymph nodes; (h) tumor differentiation; (i) tumor location; (j) the situation of primary tumor; (k) vascular invasion. (PNG 220 kb)Additional file 3:Figure S2. Subgroup analysis of heterogeneity sources: (a) Asian; (b) non-Asian (c) stages I–IV; (d) follow-up more than 5 years; (e) follow-up less than 5 years (f) postoperative adjuvant chemotherapy; (g) sample size ≥ 200; (h) sample size < 200. (PNG 287 kb)Additional file 4:Figure S3. Detection of publication bias in subgroup analysis: (a) gender; (b )lymphatic invasion; (c) microsatellite instability; (d) stage; (e) the involvement of regional lymph nodes; (f) the situation of primary tumor; (g) tumor location; (h) tumor differentiation; (i) vascular invasion. (PNG 99 kb)Additional file 5:Figure S4. Sensitivity analysis of subgroup analysis: (a) gender; (b) lymphatic invasion; (c) microsatellite instability; (d) stage; (e) the involvement of regional lymph nodes; (f) the situation of primary tumor; (g) tumor location; (h) tumor differentiation; (i) vascular invasion. (PNG 147 kb)


However, this resistance was attenuated with IL-8 inhibition. Further studies proved that IL-8 derived from GCMSCs induced PD-L1 expression in GC cells via c-Myc regulated by STAT3 and mTOR signaling pathways. Our data indicated that blocking IL-8 derived from GCMSCs may overcome the immune escape induced by PD-L1 in GC cells and provide a potential strategy to enhance the immunotherapy efficiency in GC.The expression of PD-L1 in tumor cells is one of the main causes of tumor immune escape. However, the exact mechanism for regulating PD-L1 expression in gastric cancer (GC) cells remains unclear. Our previous studies have shown that mesenchymal stem cells (MSCs) exert broad immunosuppressive potential, modulating the activity of cells either in innate or adaptive immune system to promote tumor progress. This study aims to investigate whether GCMSCs regulate the PD-L1 expression in GC cells and explore the specific molecular mechanism. The results have shown that GCMSCs enhanced PD-L1 expression in GC cells resulting in the resistance of GC cells to CD8 Although remarkable achievements in surgical and other therapies have been obtained, there is still a poor 5-year survival rate among GC patients2. In recent years, immunotherapy is a major breakthrough in cancer therapy and clinical trials with PD-1/PD-L1 antibodies have shown unprecedented responses in GC.Gastric cancer (GC) is the fourth most common malignant tumor, and the second leading cause of cancer-related death worldwide4. Furthermore, PD-L1 expression was associated with the resistance to anticancer therapies and the poor prognosis7. Takahashi et al. reported that in metastatic GC patients, high serum levels of sPD-L1 were correlated with worse overall survival on the first-line chemotherapy8. Böger et al. found that PD-L1 expression was not only an independent survival prognosticator but also correlated with distinct clinico-pathological patient characteristics9. C-Myc, serving as a well-known oncogene, is thought to be involved in tumor initiation and development. Casey et al. proved that Myc bound directly to the promoters of the PD-L1 genes in mouse T cell acute lymphoblastic leukemia10. Sato et al. reported that STAT3 was required for PD-L1 up-regulation in prostate cancer cell lines and osteosarcoma cell lines11. AKT/mTOR signaling pathway represents a convergence point for many oncogenes activation and is also associated with PD-L1 regulation in non-small cell lung cancer12. It has been reported that INF-γ played a vital role in PD-L1 induction in many cancers and then caused their resistance to NK cells14. Mimura et al. also found that PD-L1 expression could be regulated by INF-γ in GC15. However, the specific molecular mechanism for regulating PD-L1 expression in GC cells remains unclear.PD-L1 has been reported to be overexpressed in several malignant tumors and the mechanisms of PD-L1 regulation was multifaceted, such as genomic aberrations, mRNA stability, transcriptional control, protein stability and oncogenic signaling16. Our previous studies proved that bone marrow MSCs (BMMSCs) served as primary cellular components contribute to the tumor progress and mainly via secretory cytokines or exosomes19. Further, we isolated GCMSCs from GC tissues which had a stronger tumor promoting effect compared with BMMSCs. Kim et al. also found that GCMSCs contributed to the formation and progress of GC20. In addition, we found that GCMSCs exert broad immunosuppressive potential, which increased the proportion of regulatory T cells and decreased that of Th17 cells in peripheral blood mononuclear cells (PBMCs)21. However, the exact mechanism still remains unclear.Mesenchymal stem cells (MSCs) with multiple differentiation potential and immune modulating function, are one of the most important cell components of tumor microenvironment (TME)+ T cells cytotoxicity. In brief, our data indicated that inhibition of IL-8 derived from GCMSCs may overcome the immune escape induced by PD-L1 in GC cells and provide a potential strategy to enhance the efficacy of PD-L1 antibody immunotherapy in GC.This study aims to investigate whether GCMSCs regulate the PD-L1 expression in GC cells and explore the specific molecular mechanism. The results have shown that IL-8 derived from GCMSCs induced PD-L1 expression in GC cells via c-Myc regulated by STAT3 and mTOR signaling pathways. Moreover, IL-8 inhibition weakened GCMSCs protective effects on GC cells against CD822. In brief, fresh tissues were cut into about 1 mm3-sized pieces and adhered to 35 mm cell culture dishes  and were cultured in Dulbecco’s Modified Eagle Medium  with 10% fetal bovine serum  at 37 °C in humid air with 5% CO2. The culture medium was refreshed every three days. When the confluence of fibroblast-like cells reached about 80%, the cells were further expanded up to five passages and used for the subsequent experiments.GC tissues were obtained from GC patients of Jiangsu Cancer Hospital Affiliated to Nanjing Medical University and the Affiliated People’s Hospital of Jiangsu University. The procedure was approved by the Ethics Committee of Jiangsu University. The informed consents were obtained from all subjects. GCMSCs were isolated from human GC tissues as previously described2. After about 14 days, the adherent cells were trypsinized and passaged up to five passages for use.BMMSCs were isolated from healthy donors and this procedure was approved by the Ethics Committee of Jiangsu University. The BM cells were diluted with equal volume of phosphate-buffered saline (PBS) and separated over a gradient of 1.077 g/ml Ficoll , washed with PBS and cultured in DMEM with 10% FBS at 37 °C in humid air with 5% CO2.The human GC cell lines  were obtained from the Chinese Academy of Sciences Type Culture Collection Committee Cell Bank . MGC-803 and BGC-823 were cultured in DMEM with 10% FBS. SGC-7901 and HGC-27 were cultured in RPMI 1640 (Gibco) with 10% FBS at 37 °C in humid air with 5% COg for 5 min to remove cells, then filtered through a 0.22 μm membrane  and stored at −80 °C until use.BMMSCs or GCMSCs were cultured in cell culture flasks and the culture medium was refreshed when the confluence reached about 70%. After 48 h, conditioned medium was harvested and centrifuged at 1000Cells and tissues were lysed in RIPA buffer containing protease inhibitor cocktail (Vazyme). The protein concentration of cell lysates was quantified by a BCA Protein Assay kit (Cwbio). The primary antibodies were anti-PD-L1 , anti-c-Myc , anti-p-S6 , anti-S6 , anti-p-STAT3 , anti-STAT3 , anti-p-AKT , anti-AKT , and anti-GAPDH . HRP-conjugated secondary antibody  and ECL reagent (Millipore) were used for detection of immunoreactive proteins captured by ImageQuant LAS 4000 detection system.PD-L1 staining used standard protocols with human monoclonal antibody: PE-anti-PD-L1 (BD Biosciences). Data were acquired by a flow cytometer  and flow imaging analysis .Tissues were first formalin-fixed and paraffin-embedded, then dewaxed in xylene, rehydrated with gradient ethanol and treated in citrate buffer for antigen retrieval. Then samples were stained with rabbit anti-human PD-L1 antibody  at 4 °C overnight, followed by incubation in secondary biotinylated anti-rabbit antibody for 30 min at 37 °C, and finally visualized with DAB solution and counterstained with haematoxylin. Each stained sample was evaluated by three senior pathologists and five sights were selected typically.The concentration of sPD-L1 in culture supernatants was measured by ELISA kit  according to the manufacturer’s instructions. The concentration of sPD-L1 was calculated based on standard curve provided with the kit.Cells were incubated overnight at 4 °C with the anti-rabbit antibody PD-L1 . Secondary antibodies  were applied for 1 h at 37 °C. Then cells were mounted with hoechst and images were captured with a structured illumination microscopy .A cytokine array was performed in each conditioned medium after 24 h of culture. Human cytokine array panel  was used according to the manufacturer’s instructions.+ T cells was following the procedure described by Wölfl et al.23. Briefly, the subpopulation of naïve CD8+ T cells were purified from PBMCs from healthy donors using human naïve CD8+ T cells isolation kit (Miltenyi Biotec). Monocyte-derived dendritic cells were generated followed by stimulation with GC cell lysates and then co-incubated with naïve CD8+ T cells. Next, CD8+ T cells were separated and co-cultured with GC cells at the ratio of 5:1. PD-L1 neutralizing antibody was added to the culture system with a concentration of 2 μg/ml (eBioscience). After 24 h, CD8+ T cells and dead cells were discarded and the survival GC cells were stained with crystal violet, imaged and counted. Three experiments were independently performed.Antigen-specific priming of CD8P < 0.05 is considered to be significant.Data were shown as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to analyze the data and We chose PD-L1 positive GC tissues and detected the expression of PD-L1 in corresponding adjacent normal tissues. The results have shown that GC tissues expressed higher PD-L1 than corresponding adjacent normal tissues Fig. , b, whicDifferentiation assay in vitro showed that GCMSCs differentiated into adipocytes and osteocytes successfully Figure . ImmunopFurther, we investigated the mechanism of GCMSC-CM regulating PD-L1 expression in GC cells. The results have shown that GCMSC-CM could up-regulate c-Myc and PD-L1 expression in GC cells and the PD-L1 level decreased after JQ1, a BET domain inhibitor added, which inhibits BRD4 that acts as co-factors for Myc transcription Fig. . Next, MTo further elucidate GCMSCs contribute to PD-L1 expression mainly through secreting soluble cytokines, we compared the level of PD-L1 in SGC-7901 which was co-cultured with GCMSCs or treated with GCMSC-CM. The results have shown that the influence of GCMSCs and GCMSC-CM on PD-L1 up-regulation in SGC-7901 was similar Figure . For the24. This study showed that GCMSC-CM increased the phosphorylation and activation of STAT3, either IL-6 or IL-8 neutralizing antibody reversed this phenomenon , and Rapamycin (mTOR inhibitor) to further determine the signaling pathways involved in GCMSC-CM-mediated up-regulation of PD-L1 expression in GC cellls.PD-L1 has been reported to be overexpressed in several malignant tumors and was associated with the resistance to anticancer therapies and the poor prognosis10. So we speculated that c-Myc plays a role in GCMSC-CM-mediated PD-L1 up-regulation in GC cells. We used JQ1, a novel small molecule that inhibits actions of BRDs and mainly down-regulates the expression of c-Myc to resist cells proliferation34. Therefore, we inhibited c-Myc expression by JQ1 and found that GCMSC-CM could up-regulate c-Myc and PD-L1. At the same time, the PD-L1 level decreased after the inhibition of c-Myc. So, we thought that c-Myc played an important role in GCMSC-CM-mediated PD-L1 up-regulation in GC cells.C-Myc, serving as a well-known oncogene, is thought to be involved in tumor initiation and development. Casey et al. proved that Myc could regulate the antitumor immue response through PD-L1Moreover, we tested the concentration of sPD-L1 in culture supernatants of SGC-7901 and MGC-803. The concentration of sPD-L1 in SGC-7901 GCMSC-CM group was higher than that in control or BMMSC-CM group. However, the concentration of sPD-L1 in culture supernatants of MGC-803 was still under the test line after GCMSC-CM treatment, probably because MGC-803 itself expressed very low PD-L1.+ T cells. PD-L1 can deliver an inhibitory signal to PD-1 expressing CD8+ T cells, leading to suppression of the immune response by inducing functional exhaustion of CD8+ T cells. PD-L1 antibody was used to block the interaction between PD-L1 and PD-1 to improve cytotoxic effect of CD8+ T cells. However, PD-L1 antibody had little effect on the survival cell number in GCMSC-CM group and that might be because GCMSC-CM induced the up-regulation of PD-L1 in GC cells so that a certain dose of the antibody could not work and eventually, caused the immune escape of GC cells. Besides, compared with GCMSC-CM and GCMSC-CM + anti-PD-L1 groups, the survival cell number was decreased in GCMSC-CM + anti-IL-8 + anti-PD-L1 group. These results indicated that GCMSCs promotes GC cells against cytotoxic effect of CD8+ T cells through PD-L1 up-regulation in GC cells mediated by IL-8. This indicated that targeted inhibition of IL-8 may overcome PD-L1 antibody resistance in GC. In addition, with PD-L1 antibody added alone, SGC-7901 survival cells decreased slightly not the same as MGC-803 (no change), probably because MGC-803 expressed lower PD-L1 than SGC-7901.Cytotoxicity assay results have shown that the survival GC cell number in GCMSC-CM group was more than that in ctrl and BMMSC-CM groups, probably because GCMSC-CM protected GC cells against cytotoxic effect of CD8+ T cells cytotoxicity. Combination with IL-8 inhibition enhanced the efficacy of PD-L1 antibody and promoted the cytotoxic effect of CD8+ T cells (Fig. In summary, this study have shown that GCMSC-CM enhanced PD-L1 expression in GC cells, and IL-8 played a key role in this process. GCMSCs-derived IL-8 induced PD-L1 expression in GC cells via c-Myc regulated by STAT3 and mTOR signaling pathways. In addition, IL-8 inhibition weakened GCMSCs protective effects on GC cells against CD8lls Fig. . Our datFigure S1Figure S2Figure S3Figure S4Figure S5supplementary figure legends


The programmed cell death ligand protein 1 (PD-L1) is a member of the B7 protein family and consists of 290 amino acid residues. The blockade of the PD-1/PD-L1 immune checkpoint pathway is effective in tumor treatment. Results: Two pharmacophore models were generated based on peptides and small molecules. Hypo 1A consists of one hydrogen bond donor, one hydrogen bond acceptor, two hydrophobic points and one aromatic ring point. Hypo 1B consists of one hydrogen bond donor, three hydrophobic points and one positive ionizable point. Conclusions: The pharmacophore model consisting of a hydrogen bond donor, hydrophobic points and a positive ionizable point may be helpful for designing small-molecule inhibitors targeting PD-L1. Under normal circumstances, the immune system can identify and remove tumor cells in the tumor microenvironment . HoweverPD-1 is a member of the B7 superfamily which consists of 288 amino acid residues and acts as an inhibitory receptor. PD-1 is one of the death receptors which have been identified as a subgroup of the tumor necrosis factor (TNF)-receptor superfamily, which can induce apoptosis via a conserved cytoplasmic signaling module called the death domain, including TNF-R1, Fas, DR3 (death receptor 3) and so on ,6. PD-L1Inhibitors of PD-1 may lead to the blockade of both the PD-1/PD-L1 pathway and the PD-1/PD-L2 pathway. However, inhibitors of PD-L1 can only block the PD-1/PD-L1 pathway, not the PD-1/PD-L1 pathway. Compared to PD-1 inhibitors, PD-L1 inhibitors can reduce the incidence of side effects resulting from immune disorders ,12,13. TIn 2015, the crystal holo-structure of hPD-1 (human PD-1) with hPD-L1 (human PD-L1) was solved by the team of Zak (Protein Data Bank (PDB) ID: 4ZQK). This result resolved the uncertainty brought by the mPD-1 (mouse PD-1)/hPD-L1 crystal structure . The cryIt is challenging to design small-molecule inhibitors targeting the surface in protein–protein interactions because of the flexibility of proteins ,29. The We built seven pharmacophore models based on the crystal structure of peptide-71 and PD-L1 .Based on the selectivity score, sensitivity and specificity, Pharmacophore03 is the best choice. Pharmacophore03 is chosen as the best mainly because it seems that it is more important to effectively remove the inactive compound; in the case of the current example, most PD-L1 inhibitors are macromolecules. Compared to Pharmacophore06, Pharmacophore03 not only predicts active small molecules but also has good predictive power for active peptides, although the two pharmacophores have the same specificity and sensitivity to small-molecule active compounds. Pharmacophore03 was selected as the pharmacophore model (Hypo 1A) for the peptide targeting of PD-L1. Hypo 1A consists of one hydrogen bond donor, one hydrogen bond acceptor, two hydrophobic points and one aromatic ring point .The specificity and sensitivity were calculated to evaluate the quality of the generated model. The resulting sensitivity was 0.741, and the specificity was 0.993 .The AUC value of the model was 0.906, and Hypo 1A was thought to have the ability to distinguish active molecules from inactive molecules .We built 10 pharmacophore models based on the crystal structure of BMS-1001 and PD-L1. The fit value is a predictor of the activity of the compound and reflects the degree of matching of the compound to the pharmacophore model .Model02 was selected as the pharmacophore model (Hypo 1B) with the highest correlation. Hypo 1B consists of one hydrogen bond donor, three hydrophobic points and one positive ionizable point .Specificity and sensitivity were calculated to evaluate the quality of the generated model Hypo 1B. The resulting sensitivity was 0.709, and the specificity was 1 .The AUC value of the model was 0.985, and Hypo 1B was thought to have the ability to distinguish active molecules from inactive molecules .We docked BMS compounds to PD-L1 (PDB ID: 5NIX) based on CHARMM using CDOCKER in DS. The interaction energy of BMS-1166, BMS-1001, BMS-37, BMS-202, BMS-8, BMS-200, BMS-242 and PD-L1 were 76.614, 67.602, 65.688, 63.355, 59.252, 70.329, 65.587 respectively.The computational molecular modelling studies were carried out using Discovery Studio .50 of peptide-71 is 7 nM, indicating that it is more active than peptide-57  curve, sensitivity and specificity were used to evaluate the quality of the pharmacophore hypothesis. Sensitivity is defined as the power of a model to identify positives, and specificity is defined as the power of a model to determine negatives. These attributes were calculated as follows to validate the pharmacophore model:http://dude.docking.org/) at random. The 27 active compounds used as positives were acquired from Integrity [The decoy consists of positives and negatives. The 148 angiotensin-converting enzyme inhibitors used as negatives were selected from the database  value is the area under the ROC curve, which often ranges from 0 to 1. The model is thought to be better if the AUC value is closer to 1.Scientists at Bristol–Myers–Squibb (BMS) have discovered a series of nonpeptidic small-molecule PD-L1 inhibitors, and the activities of these compounds were tested in a homogeneous time-resolved fluorescence (HTRF) binding assay ,26,27. IThe decoy set and ROC curve were used to evaluate the quality of the pharmacophore hypothesis. The decoy set including 260 molecules was constructed from 150 negatives, which were angiotensin-converting enzyme inhibitors selected from the DUDE (A Database of Useful Decoys Enhanced) database at random, and 110 active molecules selected from Reaxys.In addition to BMS-1001, which was used to generate the pharmacophore models, the BMS series contains six other small molecules: BMS-8, BMS-37, BMS-200, BMS-202, BMS-242, and BMS-1166. Additionally, 6 other small molecules with known activity values were selected in Reaxys . “LigandAccording to reports , the hydThe crystal structures of PD-L1 and BMS-1166 (PDB ID: 5NIX), PD-L1 and BMS-1001 (PDB ID: 5NIU), PD-L1 and BMS-200 (PDB ID: 5N2F), PD-L1 and BMS-37 (PDB ID: 5N2D), PD-L1 and BMS-202 (PDB ID: 5J89), and PD-L1 and BMS-8 (PDB ID: 5J8O) were downloaded from the Protein Data Bank (PDB). Water molecules were deleted, and hydrogen atoms were added to the protein. The “display receptor–ligand interactions” and “analyze ligand poses” protocols were used to analyze the interactions between PD-L1 and the BMS compounds.The “pharmacophore comparison” protocol was used to map and align the two models generated based on peptides and small molecules, respectively. The root mean squared error (RMSE) is an indicator of matching pharmacophore features.Hypo 1A consists of one hydrogen bond donor, one hydrogen bond acceptor, two hydrophobic points and one aromatic ring point. The hydrophobic points were located at NMePhe7 and Val6, the aromatic ring point was located at Trp10, the hydrogen bond donor was located at Leu12, and the hydrogen bond acceptor was located at Asp5 . PeptideWe can observe that Leu12 on peptide-71 interacts with Glu58 on PD-L1 via hydrogen bonds, and an intramolecular hydrogen bond can be generated between Asp5 and Tyr8 on peptide-71. Val6 on peptide-71 interacts with Ile54 on PD-L1 via hydrophobic bonds, Trp10 on peptide-71 interacts with Ala121 on PD-L1 via hydrophobic bonds, and NMePhe7 on peptide-71 interacts with Met115 on PD-L1 via hydrophobic bonds. Intramolecular hydrogen bonds can be generated between Arg13 and Trp8 and between Scc14 and Ser7 on peptide-57. Trp10 on peptide-57 interacts with Arg113, Met115, and Tyr123 on PD-L1 via hydrophobic bonds. In conclusion, Ile54, Arg113, Met115, Ala121, and Try123 of PD-L1 may be important in the hydrophobic interactions between peptides and PD-L1.The binding surface of the peptide consists of hydrophobic regions and hydrophilic regions, and the hydrophobic interactions are essential for the binding of peptides to PD-L1 . AccordiThe model consists of one hydrogen bond donor, three hydrophobic points and one positive ionizable point. According to the interactions between PD-L1 and its ligand, we can observe that the group of the ligand corresponding to the hydrophobic features interacts with Ile54, Tyr56, Val68, Met115 and Ala121 of PD-L1 via hydrophobic interactions, and the group of the ligand corresponding to the hydrogen bond donor interacts with Asp122 and Lys124 of PD-L1 via hydrophobic interactions. In addition, the group corresponding to the positive ionizable point interacts with Asp122 . The outBy analyzing the interactions between PD-L1 and BMS compounds, we observed that Ile54, Tyr56, Val68, Met115 and Ala121 of PD-L1 are important in generating hydrophobic bonds between PD-L1 and molecules; that Asp122 and Lys124 of PD-L1 are helpful in forming hydrogen bonds between molecules and PD-L1; and that the positive ionizable located at Asp122 of PD-L1 may be essential for the interactions between molecules and PD-L1. This outcome was consistent with Hypo 1B, which consists of one hydrogen bond donor, three hydrophobic points and one positive ionizable point.214, and the three hydrophobic points were located at Phe1, NMeNle3, and Trp10.An attempt was made to map peptide-57 and peptide-71 with Hypo 1B. Peptide-57 could be well mapped with the model . The posThe two models were superimposed using the pharmacophore comparison in DS, and the RMSE of Hypo 1A and 1B was 2.58. There were some common features between the two models; two hydrophobic points and one hydrogen bond donor could almost be matched. However, a hydrophobic point and a positive ionizable point of Hypo 1B and an aromatic ring point and a hydrogen bond acceptor of Hypo 1A were not in the same location .50 value is 9 nM. When peptide-57 was mapped with Hypo 1B, the positive ionizable point was located at Arg13 (There were some differences between Hypo 1A and Hypo 1B. First, a hydrophobic point of Hypo 1B and an aromatic ring point of Hypo 1A were not in the same location. The aromatic ring point of Hypo 1A located at Trp10 of peptide-71 interacted with Met115 and Ala121 of PD-L1 via hydrophobic interactions, and it was thought to play the same role as that of the hydrophobic point . The hydat Arg13 . This ouIn this study, the critical chemical features of PD-L1 inhibitors were found via pharmacophore models. Two pharmacophore models, Hypo 1A and Hypo 1B, were built based on small molecules and peptides, respectively. Hypo 1A consists of two hydrogen bond donors, three hydrophobic points and one positive ionizable point. Hypo 1B consists of one hydrogen bond donor, one hydrogen bond acceptor, two hydrophobic points and one aromatic ring point. The reliability of the pharmacophore models was validated by ROC curves and a decoy set. Hydrophobic features located in the same hydrophobic pocket are essential for both peptide and small-molecule inhibitors. The hydrogen bond donors of two models could be mapped. Though the positive ionizable point only exists in the pharmacophore model based on small-molecule inhibitors, it may be important for both small-molecule and peptide inhibitors. The similarity between Hypo1A and Hypo1B means that pharmacophore models consisting of a hydrogen bond donor, a hydrophobic point and a positive ionizable point may be helpful in designing small-molecule inhibitors targeting PD-L1.