Blast transformation of chronic myelogenous leukemia (CML) to T lymphoblastic lymphoma/acute lymphoblastic leukemia (T‐LBL/ALL) is rare, and the molecular mechanism is still unclear.A 28‐year‐old woman who developed T‐ALL with coexpressing both p210 and p190 BCR‐ABL transcripts five years after the initial diagnosis of CML in chronic phase. The proliferation of bone marrow was extremely active with blast cells over 20%. Chromosome analysis revealed t and t. Flow immunophenotyping showed that blasts expressed CD4, CD7, CD11b, CD38, CD34, CD33, and cCD3.It is the first T‐cell blast of CML case with coexisting p210 and p190 as well as additional chromosome translocations. Through review this case and previous reports, we will reveal that CML patients with T‐lymphocyte transformation depend on potential molecular and pathological mechanism. The natural course of CML consists of three stages: chronic phase (CP), accelerated phase (AP), and blast crisis (BC). CML‐BC, the late stage of CML, usually means the worsening of malignancy and poor prognosis. In the CML‐BC, the majority progressed to acute myeloid leukemia (AML) and the rest to lymphoid blast. Almost all patients with lymphocytic blast were B‐ALL and rarely involved in T‐lineage.The BCR‐ABL fusion gene, derived from chromosomal translocation t9;22), is considered to be a molecular marker of CML. According to the different breakpoint site, the BCR‐ABL transcripts are divided into three subtypes of M‐bcr, m‐bcr, and μ‐bcr, encoding protein p210, p190, and p230, respectively. M‐bcr (p210) is expressed in 95% CML and a few of Ph + ALL, m‐bcr (p190) is almost all expressed in Ph + ALL, and μ‐bcr (p230) is rare, mainly present in chronic neutrophilic leukemia. CML with coexpressing p210 and p190 is rare, which may be associated with progress of ALL. In addition to t, CML‐BC is usually accompanied by additional chromosomal abnormalities (ACAs).2 smear image, cytogenetic karyotype 46, XX, tq34;q11)  and a few B acute lymphoblastic leukemia (B‐ALL), rarely involving T‐lineage in clinical practice. Up to now, only a limited number of CML cases with T‐cell blast 52 cases) were reported. cases weMost blasting CML patients (about 70%) have additional chromosomal abnormalities, which were associated with worse effect for therapy, the adverse prognosis, blasting more easily, and shorter overall survival (OS).Chronic myelogenous leukemia with T‐cell blast crisis is easily confused with Ph + de novo T‐ALL/LL, especially when patients lack a history of CML or only involve extramedullary organs. It is of great practical significance to differentiate T‐cell blast crisis of CML from primary T‐lymphocyte disease timely and accurately, because of differences in treatment options and prognosis between the two entities. Based on traditionally clinical presentations, the former is frequent in adults and usually has a history of CML, extramedullary involvement alone, and without cell receptor rearrangement. Conversely, younger, lack of the natural course of CML, bone marrow involvement, and cell receptor gene rearrangements tend to be diagnosed as Ph + primary T‐LBL/ALL. Ichinohasama et alCurrently, there is no standard treatment for the rapid progression of CML to T‐LBL/ALL. Most of the patients who received combined chemotherapy for T‐LBL/ALL died after several months of treatment. One case reported that imatinib was used to treat CML to T‐LBL/ALL with a remission period of more than 2 years.4In summary, our report broadens the spectrum of T cell of CML‐BC and provides useful molecular evidence to support the presence of m‐bcr transcript, and chromosome 11p15 is crucial for T‐LBL/ALL from blast crisis of CML.Yuchao Yang and Weixiao Ren performed molecular analysis. Jianmei Chang and Li Guoxia performed cytogenetic analysis. Jianlan Li performed morphological analysis. Fanggang Ren performed flow cytometry analysis. Hongwei Wang helped revise the manuscript. Ting Xia and Zhifang Xu collected the case information and drafted the manuscript. All authors have reviewed and agreed upon the manuscript content.The authors have no ethical disclosures to declare.


Currently used biomarkers for immunotherapy are inadequate because they are only based on tumor properties. In view of microenvironment changes by tumors, host immunity should be considered, which may result in identifying more accurate and easily detectable biomarkers for daily clinical practice. Here, we assessed serum immune‐modulating factor levels for the response to anti‐PD‐1 antibodies during the first cycle in non‐small cell lung cancer (NSCLC) patients.Serum was collected from patients with advanced NSCLC treated with nivolumab or pembrolizumab at several time points during the first cycle. We applied the enzyme‐linked immunosorbent assays (ELISAs) and multiplex assays to measure the levels of immune modulators.A total of 40 patients treated with nivolumab and 26 patients treated with pembrolizumab were studied. By ELISA, serum perforin, but not granzyme B, was measured in all samples. By multiplex assay, 10 immune modulators, including granzyme B, were measured in some, but not all, samples. Serum baseline perforin levels were strongly associated with increased progression‐free survival (PFS) and overall survival (OS) times. Sequential changes in perforin levels during the first cycle were weakly associated with the clinical outcome.Serum baseline perforin levels may be used to predict the prognosis of NSCLC patients treated with anti‐PD‐1 antibody therapy.To identify a useful predictive marker for anti‐PD‐1 antibody therapy, using blood samples might be helpful.Serum baseline perforin levels were closely associated with prognosis with anti‐PD‐1 antibody therapy in non‐small cell lung cancer. This study demonstrates immune‐modulator changes in serum during the first cycle of anti‐PD‐1 antibody therapy in non‐small cell lung cancer. Serum baseline perforin levels were found to be closely associated with clinical outcome with anti‐PD‐1 antibody therapies. Cytotoxic lymphocytes (CLs), represented by cytotoxic T cells and natural killer cells, effectively protect against transformed malignant cells or infected cells through the process of cell death, including apoptosis. CLs produce cytotoxic factors to target cells through two direct main mechanisms: granule exocytosis pathways and death ligand‐mediated pathways.Programmed cell death 1 (PD‐1) has been previously reported to be isolated from a murine T cell hybridoma and a murine hematopoietic progenitor cell line in which apoptotic cell death is easily induced.+ T cells, and releases perforin, granzyme B, and cytokines, which exhibit cytotoxic activity against tumor cells.+ T cells respond more actively than those in patients who do not respond. Eventually, the serum concentration of cytotoxic immune modulators may increase more in responders than in nonresponders. Based on this hypothesis, we measured the serum concentrations of perforin, granzyme B, and other immune modulators as biomarkers of the response to PD‐1 blockade in non‐small cell lung cancer (NSCLC) patients. In particular, we focused on the changes during the first cycle of anti‐PD‐1 antibody treatment so that we might be able to identify biomarkers at the earliest time point.PD‐1 blockade by anti‐PD‐1 antibodies restores the function of exhausted T cells, reactivates the cytotoxicity of CD8Patients who were eligible for inclusion in the study had histologically or cytologically confirmed stage IIIB or IV NSCLC. Patients with known epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) translocation were allowed to have received anti‐PD‐1 antibody therapies. Patients had to be 20 years of age or older. Tumor tissue obtained before treatment was used in biomarker analyses. The research protocol was approved by institutional review boards at each participating institution. All participants gave written informed consent before enrollment.Patients received nivolumab at a dose of 3 mg/kg of bodyweight every two weeks and pembrolizumab at 200 mg per bodyweight every three weeks. Both drugs were administered intravenously. Patients were treated until disease progression or discontinuation of treatment due to unacceptable toxicity, investigator or patient decision to withdraw. Adverse events (AEs) were monitored and graded according to National Cancer Institute Common Terminology Criteria for adverse events, version 4.0. Treatment was interrupted for grade 3 AEs or severe drug‐related AEs until toxicity resolved to less than grade 1.g for five minutes and stored in 330 μL aliquots at −80°C. Blood samples were collected on days 1, 2, 8, and 15 for nivolumab and on days 1, 2, 8, 15 and 22 for pembrolizumab. For nivolumab, blood samples were collected before nivolumab administration on days 1 and 15. For pembrolizumab, blood samples were collected before pembrolizumab administration on days 1 and 22.Peripheral blood samples were collected into tubes (Terumo Venogect II), mixed well, and then stored for 5–10 minutes at room temperature. Samples were centrifuged at 1000 × Tumor response was assessed by the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. The tumor response was evaluated by a physician every six weeks to three months by using computed tomography (CT). The best overall response was defined as the best tumor response from the start of the treatment to the time of disease progression or death. The best overall response was assessed after three months of follow‐up. The end of the follow‐up period for this study was 31 July 2018.Tumor PD‐L1 protein expression was assessed using immunohistochemistry (IHC) in formalin‐fixed tumor samples at SRL Inc. . A monoclonal anti‐PD‐L1 antibody 22C3 pharmDx  was used and stained by an automated stainer . The stained slides were assessed by pathologists at each institution. The tumor proportion score (TPS) was evaluated on samples with at least 100 viable tumor cells in specimens with membranous PD‐L1 expression. TPS was calculated as the percentage of PD‐L1‐positive tumor cells.elisaanalysis.com).We analyzed the serum levels of soluble granzyme B, perforin, and soluble CD137 by ELISA according to the manufacturer's protocol. Granzyme B and perforin ELISA kits were purchased from Mabtech, Inc. . CD137 ELISA kit was purchased from Thermo Scientific Fisher . All samples were measured in duplicate. For granzyme B and CD137, serum was diluted 1:1. For perforin, serum was diluted 1:4–1:20. The data were calculated using a four‐parameter logistic curve , the following 10 factors were measured at Filgen : CD137, PD‐L1, Fas ligand, glucocorticoid‐induced tumor necrosis factor (TNF)‐related protein (GITR), granzyme B, interferon (INF)‐γ, interleukin (IL)‐2, IL‐6, IL‐10, and TNF‐α. A total of 10 serum samples at four‐time points of patients treated with nivolumab and six serum samples at five‐time points of patients treated with pembrolizumab were measured. Serum was diluted 1:1, and all samples were measured in duplicate.P‐values less than 0.05 were considered statistically significant. Statistical analysis was performed using GraphPad Prism, version 8 (GraphPad Software), R (http://www.R-project.org.) or EZR (version 1.42).Kaplan‐Meier analysis of PFS and OS was performed based on perforin levels, with differences between each pair of groups being assessed with the log‐rank test. Hazard ratios (HRs) and associated 95% confidence intervals (CIs) were assessed by a Cox proportional‐hazards regression model. All EGFR mutation was found, and we considered an EGFR tyrosine kinase inhibitor treatment to be preferable. The clinical characteristics of the patients are summarized in Table From November 2016 through March 2018, we enrolled 41 patients for nivolumab treatment. One patient was excluded before the treatment started because an From March 2017 through March 2018, we enrolled 27 patients for pembrolizumab treatment. One patient was excluded from the analysis because of unnatural death. The clinical characteristics of the patients are summarized in Table Using ELISA, we assessed serum perforin and granzyme B levels during the first cycle of nivolumab or pembrolizumab in advanced NSCLC. Serum granzyme B was challenging to measure by ELISA. Serum perforin was measured in all samples.A total of 10 immunomodulators were measured simultaneously using Human Magnetic Luminex Assays. Granzyme B was included in the 10 immunomodulators. Samples that showed different types of efficacy were chosen Table . With niCD137 levels were measured by ELISA in 20 cases treated with nivolumab and eight cases treated with pembrolizumab. The cases were different from the cases identified by the multiplex assay. Two cases treated with nivolumab and one case treated with pembrolizumab were difficult to measure. Further analysis revealed that CD137 levels were not associated with the efficacy and prognosis Fig .First, we evaluated the correlation between the baseline concentration and efficacy with perforin levels. In patients treated with nivolumab, we excluded one patient because her target lesion was not assessable due to her death. For partial response (PR), stable disease (SD), and progressive disease (PD), there were no significant differences between groups Fig . The PR vs. PD. The calculated optimal cutoff levels were 5.45 ng/mL with nivolumab (area under the receiver operating characteristic curve (AUC), 0.703), and 6.63 ng/mL with pembrolizumab   .P = 0.007). The median OS was 14.9 months  in the high concentration group versus 2.0 months (95% CI: 0.13 to not reached) in the low concentration group .Using the cutoff levels, cases were separated into high and low concentration groups. The high concentration group was beyond the cutoff value. For patients treated with nivolumab Fig , the medP = 0.03). Median OS was not reached (95% CI: 7.9 to not reached) in the high concentration group versus 2.1 months (95% CI: 0.57 to not reached) in the low concentration group .For patients treated with pembrolizumab Fig , the medBecause of the reported correlation between PD‐L1 TPS and the response to immune check point inhibitors as anti‐PD‐1 antibody therapy,EGFR mutation or ALK rearrangements show low efficacy in general.EGFR mutation or ALK rearrangement is associated with perforin levels. With nivolumab, we observed that perforin levels were lower in EGFR mutant cases than in EGFR wild type cases, but the difference was not statistically significant (P = 0.1152)  Fig . With peWhen exhausted, T cells are activated by anti‐PD‐1 antibody therapy, perforin, granzymes, and other cytokines are released.The sequential changes in perforin levels were not different between the PR, SD, and PD groups with nivolumab or pembrolizumab treatment Fig . In the The benefit of anti‐PD‐1 antibody therapies with respect to PFS or OS was evident in baseline perforin levels , whereas partial deficiency of perforin induces late‐onset HLH or hematological malignancies.Our study showed that there were differences between nivolumab and pembrolizumab with regard to (i) the optimal cutoff values for the baseline concentration; and (ii) the benefit on prognosis in sequential changes. As we have noted, there are limited studies about the serum perforin concentration associated with malignancies. The differences might be because of (i) the differences between nivolumab and pembrolizumabPD‐L1 expression in the tumor assessed by IHC is closely associated with prognosis.EGFR mutations and ALK rearrangements have low response rates to anti‐PD‐1/anti‐PD‐L1 antibodies.+ tumor‐infiltrating lymphocytes. Our study showed that nivolumab treatment produced lower perforin levels in mutated EGFR cases; however, these levels were not significantly different between patients with or without EGFR mutations. This observation implies that factors other than EGFR mutations might be involved in regulating the CD8+ T cell activation loop associated with perforin. Moreover, we should note that in the present study, one patient who had low baseline perforin levels and EGFR mutations had a good prognosis. NSCLC with tyrosine kinase receptor mutations may have aberrant CD8+ T cell activation.Tumors with + T cells become reinvigorated and release immune modulators, which induce antitumor activity. CD8+ T cells in responsive patients might be more likely to release immune modulators than those in nonresponsive patients. In support of this hypothesis, we found that some sequential changes in perforin levels after nivolumab or pembrolizumab treatment contributed to a favorable prognosis. However, by calculating the optimal cutoff levels in efficacy with a Cox regression analysis, the values of these sequential changes were considered either to have low predictive power or no association with prognosis. Thus, the baseline perforin level is more appropriate than sequential perforin changes to predict the prognosis of NSCLC patients treated with anti‐PD‐1 antibody therapy.By PD‐1/PD‐L1 blockade, exhausted CD8There are certain limitations to this study. First, this was a small cohort, and we could not conclude the best cutoff levels of perforin to determine prognosis. Nevertheless, we were able to show that increased baseline perforin levels were strongly associated with prognosis. Second, we studied 11 immune modulators, which were highly selective and therefore had only limited results. More relevant candidate factors may exist to predict the clinical outcome of anti‐PD‐1 antibody therapy.In summary, this study investigated serum immune modulators during the first cycle of anti‐PD‐1 antibody treatment in NSCLC. Measuring serum perforin levels before treatment may predict the clinical outcome of NSCLC patients treated with anti‐PD‐1 antibody therapy, which might be beneficial.Dr Nakahara reports grants and personal fees from Ono Pharmaceutical Co. Ltd., Bristol‐Myers Squibb K.K. and Eli Lilly Japan K.K., grants from Takeda Pharmaceutical Co., Ltd. and personal fees from AstraZenaka K.K., Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd. and Nippon Boehringer Ingelheim Co., Ltd. outside the submitted work. Dr Fukuoka reports personal fees from Eisai Co., Ltd. and Chugai Pharmaceutical Co., Ltd. outside the submitted work.All other authors have nothing to declare.Figure S1 Serum baseline CD137 levels and efficacy of nivolumab and pembrolizumab.  Baseline levels of CD137 are plotted by efficacy. All cases were measured by ELISA. The results are the medians, and the whiskers are the minimum to maximum. (A) Nivolumab and (B) pembrolizumab. (A) PR, n = 6; SD, n = 6, PD, n = 6. Data were analyzed by ANOVA. PR versus SD, P = 0.9983; PR vs. PD, P = 0.4388; SD versus PD, P = 0.4692. (B) PR, n = 4; PD, n = 3. Data were analyzed by Student's t‐test. PR versus, P = 0.1470. (C‐G) Sequential changes in CD137 levels with nivolumab or pembrolizumab treatment. The Y‐axis shows the ratio of the serum perforin concentration at each time point divided by the baseline serum perforin concentration. (C–E) Nivolumab and  pembrolizumab.  PR, (D) SD,  SD. (C) n = 7, (D) n = 7, (E) n = 7, (F) n = 6, (G) n = 5.Figure S2. Receiver operating characteristic (ROC) curves of baseline concentration of perforin predicting the clinical outcome by anti‐PD‐1 antibody therapies. (A) Nivolumab and (B) pembrolizumab. MCT: misclassification‐cost term.Click here for additional data file.


The remitting seronegative symmetrical synovitis with pitting edema syndrome primarily occurs in elderly individuals to represent symptoms of edema, pain, and joint swelling. It could be misdiagnosed in elderly maintenance hemodialysis patients, as hemodialysis patients often present with pain and joint swelling induced by hypervolemia, inflammation, amyloidosis, and/or chronic kidney disease. Here, we describe a maintenance hemodialysis patient with remitting seronegative symmetrical synovitis with pitting edema syndrome.A 71‐year‐old man on maintenance hemodialysis who complained of continuous pain and swelling of joints was diagnosed with remitting seronegative symmetrical synovitis with pitting edema syndrome on his clinical findings that revealed tenosynovitis at the joint without joint erosions and no elevation of anti‐cyclic citrullinated peptide antibody and rheumatoid factor. After administration of prednisolone, systemic edema, and pain improved in 2 days.Remitting seronegative symmetrical synovitis with pitting edema syndrome should be considered as a differential diagnosis in hemodialysis patients with edema and/or arthralgia. CCPcyclic citrullinated peptideCRPC‐reactive proteinEORAelderly onset of rheumatoid arthritisHbhemoglobinHLAhuman leukocyte antigenIgimmunoglobulinMMP‐3matrix metalloproteinase‐3NAnot availablePMRpolymyalgia rheumaticaRFrheumatoid factorRS3PEremitting seronegative symmetrical synovitis with pitting edemaVEGFvascular endothelial growth factorWe report a case of the RS3PE syndrome in a maintenance hemodialysis patient. RS3PE should be considered as a differential diagnosis in hemodialysis patients with edema and/or arthralgia.McCarthy described the “RS3PE” syndrome in 1985.A 71‐year‐old man was admitted to our hospital following 5 weeks of sudden onset of continuous pain and swelling of the shoulder, wrist, and knee joints, bilaterally. The patient had been treated on an out‐patient basis by an orthopedic surgeon for high fever and arthritic symptoms, which were not improved with antibiotics and acetaminophen. The patient had a past medical history of bilateral renal pelvic and bladder cancer and had undergone bilateral nephroureterectomies and total cystectomy at ages 64, 66, and 70 years, respectively; followed by undergoing maintenance hemodialysis for 5 years. Pathological findings in bilateral nephroureterectomy without chemotherapy revealed urothelial carcinoma, high grade and pT2. Remained bladder was removed by cystectomy due to mucosal epithelial cancer after induction of hemodialysis.9/L and 14.3 mg/L respectively. Blood and urine cultures were negative. Antinuclear antibody, anti‐CCP antibody, and RF were negative. The MMP‐3 level was markedly increased to 677.9 ng/mL  was initiated, systemic edema, CRP level (decrease to 1.16 mg/dL) and pain improved over 2 days. Edema and joint pain were fully recovered, and the patient was discharged from hospital 9 days after the start of prednisolone treatment. No complications were identified during hospitalization, with no recurrence of the symptoms of peripheral edema and joint pain, with no recurrence for 6 months post‐treatment initiation. The dosage of prednisolone was gradually reduced, as maintenance hemodialysis was continued.RS3PE syndrome is characterized by the following criteria: abrupt onset, marked pitting edema of the hands and/or feet, onset >60 years, good response to steroids, seronegative for RF and CCP, and absence of radiographic evidence of joint erosions. RS3PE is associated with VEGF, HLA‐A2, HLA‐B7, and HLA‐DRB1, as well as parvovirus and streptobacillus infections. Moreover, RS3PE can occur in association with paraneoplastic syndromes, such as leukemia and lung, breast, and bladder carcinoma.Differential diagnoses of RS3PE syndrome include PMR and elderly onset of EORA. Although RS3PE syndrome develops mainly in males involving the distal extremities with extensor tenosynovitis,Only two cases of RS3PE syndrome were reported in hemodialysis patients at 9 and 23 years after the start of hemodialysis.An excellent response with RS3PE syndrome to glucocorticoids treatment (0.3 mg/kg/day) is reported, with symptoms typically resolving within 1 week, regardless of hemodialysis status, with a relapse rate after treatment of 8.76%. The risk of relapse is higher for malignancy patients,We propose that RS3PE syndrome should be considered as a differential diagnosis among elderly hemodialysis patients with edema, pain, swelling of joints, and distal extremities, rather than attributing these symptoms to hypervolemia, chronic inflammation, amyloidosis, and/or mineral and bone disorder associated with chronic kidney disease.In conclusion, although RS3PE syndrome is rare, RS3PE syndrome should be considered as a differential diagnosis in maintenance hemodialysis patients presenting with edema and/or arthralgia.The authors declare no conflict of interest.


A–VI metal monochalcogenides (MMCs) have been surged recently due to their distinctive crystalline symmetries, exotic in‐plane anisotropic electronic and optical response, earth abundance, and environmentally friendly characteristics. In this article, the recent research advancements in the field of anisotropic 2D MMCs are reviewed. At first, the unique wavy crystal structures together with the optical and electronic properties of such materials are discussed. The Review continues with the various methods adopted for the synthesis of layered MMCs including micromechanical and liquid phase exfoliation as well as physical vapor deposition. The last part of the article focuses on the application of the structural anisotropic response of 2D MMCs in field effect transistors, photovoltaic cells nonlinear optics, and valleytronic devices. Besides presenting the significant research in the field of this emerging class of 2D materials, this Review also delineates the existing limitations and discusses emerging possibilities and future prospects.The family of emerging low‐symmetry and structural in‐plane anisotropic two‐dimensional (2D) materials has been expanding rapidly in recent years. As an important emerging anisotropic 2D material, the black phosphorene analog group IV A–VI metal monochalcogenides (MMCs) are presented, with a focus on their unique wavy crystal structures, their exotic physical properties and applications. The in‐depth synthesis and characterization methods as well as applications related to the anisotropic response of 2D MMCs in emerging electronics and photonics are concretely summarized.Herein, the recent research advances in 2D phosphorene‐analog in‐plane structural anisotropic group IV In particular, beyond graphene materials such as hexagonal boron nitride (h‐BN), transition metal dichalcogenides (TMDs), multinary layered chalcogenides, perovskites, and MXenes have opened up a new horizon in 2D material research. These 2D materials possess intriguing optical, electronic, mechanical, and optoelectronic properties, and are being explored for rich physics and many emerging scientific applications. However, completing the intensive research on semimetal graphene and beyond graphene semiconducting 2D materials, group V element black phosphorene (BP) discovered as a 2D material in 2014, leading to many discoveries of novel physical phenomena. The puckered or wavy lattice structure with reduced crystal symmetry (D2h) than graphene (D6h) and TMDs make it more interesting for exhibiting the novel physical phenomena. Moreover, layer‐dependent tunable bandgap, high carrier mobility, and strong in‐plane anisotropy make it promising for next‐generation emerging electronic and photonic applications. The electronic bandgap of BP varies from 0.33 to 2.0 eV, when the thickness reduced to a monolayer. An ultrathin layer of BP revealed an extraordinary electrical hole mobility ≈5 × 103 cm2 V−1 s−1 at room temperature, which is much higher than the TMDs, making it suitable for advanced electronic applications. Most importantly, in‐plane anisotropic optical and electrical response along x‐direction (armchair) and y‐direction (zigzag) was directly visualized and studied, which added a new dimension. Besides this, unique structural in‐plane anisotropic nature in BP played a critical role in designing multifunctional and tunable 2D novel electronic, optoelectronic, and photonic devices. In spite of a series of exotic novel physical phenomena, rapid ambient degradation of phosphorene is a critical issue for its practical implementations.Since the successful isolation of graphene,1 reseA–VI metal monochalcogenides (MMCs), with chemical formula MX  has been surged as a star 2D material  with low crystal symmetry Cυ2, in which the inversion symmetry is further broken, which makes them possible to observe new order parameters  and polarization properties. Many exotic phenomena have been predicted on a monolayer MMCs, including valley physics, spontaneous polarization and bulk photovoltaic effect, piezo‐phototronic, giant piezoelectricity, ferroelectricity, multiferroics of ferroelectricity, and ferroelasticity. However, valley selective dichroism, and more than 90% room temperature valley polarization (VP) degree in SnS provide a completely novel platform for valleytronics. In addition, the giant optical second harmonic generation (SHG) in MMCs is also promising in nonlinear optoelectronic applications.Most interestingly, beyond graphene 2D materials, BP's isostructural and isoelectronic group IVl Figure 1 due toA–VI metal monochalcogenide materials (MMCs) are reviewed. The intriguing physical  and optical properties are initially highlighted and discussed. Then the various methods that have been employed for the synthesis of such materials, including mechanical and liquid phase exfoliation, as well as vapor phase deposition techniques are demonstrated. Besides presenting the potential and significance of 2D MMCs in various electronic applications it will also delineate the existing limitations and discuss emerging possibilities and future prospects.In this article, the latest advances in the field of the emerging 2D group IV22.1A–VI MMCs is isostructural with orthorhombic black phosphorene. The chemical formula of MMCs is the MX, where M is group IV metals  and X is chalcogens (S and Se). M and X atoms are alternate with each other and form a puckered or wavy layer structure of zigzag (y) and the armchair (x) plane  with different electronegativity lowers the crystal symmetry compared to other 2D crystals. In particular, the bulk structure belongs to the space group Pnma(D2h16), while in the monolayer, the inversion symmetry is broken placing them in the D2υ7 space group. The MXs monolayer has four atoms per unit cell  rows of alternating elements. The corresponding first Brillouin zone, as well as the high‐symmetry points along armchair (x) and zigzag (y) directions, are indicated in Figure 10ns2np2 and ns2np4, where n is the periodic number of the element. MMCs show strong covalent bonding in the 2D plane and a strong interlayer force owing to the lone pair electrons, which generate a large electron distribution and electronic coupling between adjacent layers. The MMCs are layered p‐type semiconductors with 1:1 stoichiometry, with most studied examples being SiS, SiSe, GeS, GeSe, SnS, and SnSe. In particular, SnS has typical acceptor states, which are formed by Sn vacancies (VSn). On the other hand, sulfur vacancies (Vs) can also be formed, under appropriate Sn‐rich conditions, as well as substitutional oxygen at sulfur sites (OS), leading, in all cases, to p‐type conductivity. Stereochemically, active lone pair electrons in group IV metals electronic configuration, ns (n = 5 or 4), play a pivotal role in the structural distortion, resulting in the anisotropic crystal and layered structure.The monolayer crystal structure of group IVsphorene.31 Thee Figure . and [011¯] planes, well agreed with the theoretical predictions. While, the perfect rhombus lattice fringes in SAED pattern further confirmed the single crystallinity of the NSs. Finally, the NSs exhibited the typical characteristic Ag and B3g Raman modes , with the thicker NSs to show stronger and sharper Raman modes compared to the thinner counterparts. This confirmed the better crystalline quality of the thicker NSs, which was consistent with the edge morphology obtained from AFM images.In another work, Tian et al.172 had Figure . For thed Figure . The obtd Figure ,j. TEM ad Figure  with d s Zhao et al. have realized the synthesis of single‐crystal SnSe NSs on mica substrates in a controlled manner. A 15.8 nm thick SnSe NS having orthogonal lattice fringes with well‐defined lattice spacings (0.30 nm) and intersect angle (92°) of the crystal planes was identified, which further confirmed the orthorhombic SnSe crystal structures. In PVD method, the temperature mainly controls the evaporation quantity of bulk SnSe, while the pressure affects the nucleation and growth process. With the evaporation temperature and pressure fixed at 500 °C and 70 Torr, the deposition temperature was varied (from 340 to 390 °C), and as a result controlled synthesis of SnSe NSs was achieved. A few years ago, Xu et al. have followed a synthesis process similar to that reported by Zhao et al., however the sublimation and the deposition temperatures were kept higher. In particular, the sublimation, deposition temperature of SnSe, and argon flow rate was set at 650 °C (in 17 min), 350–450 °C, and 200 standard cubic centimeters per minute (sccm). Following this process, SnSe NSs with a uniform surface were grown. However, a distribution of thickness (10–210 nm) was found , which can be controlled through the growth conditions. Interestingly, the rectangular NSs showed clear orthogonal lattice fringes with two similar lattice spacings of 0.30 nm. The measured angle (94°) between two crystallographic planes is well matched with the orthorhombic SnSe crystal structure. The observed orthogonal structure together with the orthogonal symmetry  during the growth.The synthesis of few‐layer 2D SnSe via PVD has been reported by various groups.61 Zhay Figure  confirme Recently, Wang and Pang used a proper proportion of SnO2 and Se precursors to synthesize 2D SnSe or SnSe2 NSs with lateral sizes of few micrometers. Specifically, the Sn:Se stoichiometry was varied by changing the weight of Se from 50 to 500 mg. It was revealed that the shape of as‐grown sample switches from square to truncated triangle .180 Ree Figure ,e. As a e Figure . On the 2 powders are heated to react with Se vapor, the reaction forms SnSe or SnSe2 via the following reaction paths2 NSs are formed.In particular, when the bulk SnO have introduced a novel bottom‐up synthesis method to obtain SnS monolayers. In particular, a molten droplet of Sn was exposed to an anoxic atmosphere containing a sulfur source at 350 °C. As a result, the surface forms a sulfide skin in a self‐limiting Cabrera–Mott reaction. Then liquid metals are employed to exfoliate ultrathin SnS sheets and to transfer them onto the desired substrates. Using this method, SnS nanosheets with large lateral dimensions can be attained, with a highly crystalline orthorhombic structure , and thioacetamide, are used as group VI elements. Both reactants are mixed together with organic solvents and heated up to the reaction temperature. Subsequently, the obtained products are redispersed into an organic solvent and centrifuged to isolate the required nanosheets. In 2013, Li et al. have adopted one‐pot synthesis as a WCS method to synthesize ≈300 nm wide and 2 ML (≈1 nm) thick SnSe nanosheets. In another report, Schaak and co‐workers have adopted one‐pot synthesis method to prepare GeS and GeSe nanosheets. The obtained GeS nanosheets were 5 nm thick with (2–4) µm times (0.5–1) µm average lateral dimensions. For GeSe a larger range of thicknesses was achieved (5–100 nm). Apart from the aforementioned approaches, much larger MMC flakes have been synthesized via WCS.Another bottom‐up approach is the WCS method. This method comprises hot injection, one‐pot synthesis, hydrothermal intercalation, and sequential deposition.183 AsTable  This is primarily due to the strong interlayer coupling energy (armchair/zigzag) in MMCs, which restricts the isolation of a single layer with high crystallinity. In this context, there are a few possible ways to overcome the different limitations. As far as the ME and PVD/CVD techniques, by proper choice of the deposition substrate and optimized protocols, one could make the lateral growth stronger than the vertical one, and as a result thinner and larger flakes can be attained. On the other hand, the postetching of mechanically exfoliated MMC flakes via chemical routes and/or laser processing can overcome the thickness limitation and give rise to large‐area monolayer flakes.4Since the isolation of single‐layer graphene a new era of 2D electronics has begun. The semimetallic nature with gapless band structure of graphene imposes important limitations in electronic, optoelectronic, and photonic applications. However, 2D MMCs exhibit a wide range of direct or indirect bandgaps. More important, MMCs are highly anisotropic in nature, as they exhibit in‐plane structural anisotropy along the armchair and zigzag crystalline directions. Such structural anisotropy is revealed in electronic, optoelectronic, and photonic response. In the following, we will present an overview of the very recent advances in electronic, optoelectronic, and photonic applications of MMCs, giving emphasis in VP and SHG.4.1 Following such first studies, in‐plane phosphorene‐analogous group IV–VI MMCs have received significant interest due to their anisotropic optical and electronic response. In wavy structured MXs, the effective mass, dielectric constant, and refractive index are entirely different along armchair compared to zigzag direction. Stereochemically active lone pair electrons in 5s  or 4s  and different electronegativity in chalcogens  play a crucial role in such anisotropy. This interesting feature critically affects the exotic optical and electronic behavior of such materials, therefore adds a new dimension to their optoelectronic properties and stimulates the development of angle‐resolved photonics and optoelectronics. Accordingly, understanding the effect of electrical and optoelectrical anisotropy in MMCs has evolved rapidly in the recent years.In‐plane anisotropy found in a layered BP open up a new horizon in 2D MMCs research for emerging nanophotonic and optoelectronic device applications.185 Fo Matsuda and co‐workers, in particular, introduced a highly polarization sensitive and broadband photodetector  and the light intensity. The transfer characteristics (Vg–I) of FET device exhibited a p‐type behavior  was also recorded at ±40 V. Moreover, current hysteresis appeared in the voltage sweeping, in which trap/defect states play a dominant role. The anisotropic crystal structure of GeS, causing its strong linear dichroism, was investigated through measuring of the photoresponse of the GeS photodetector with highly polarized light. It is found that the measured photocurrent (at λ ≈ 750 nm) strongly depends on the polarization angle (θ) of incident light  and hole (0.8 cm2 V−1 s−1) mobility at room temperature were measured, which are comparable with other metal dichalcogenides such as SnS2 and SnSe2. Furthermore, the photoresponsive behavior of the fabricated FET exhibited a deviation from the linear relationship . It is shown that a Schottky emission charge carrier transport mechanism takes place due to the large density of photoinduced charge carriers under illumination , as μα1m. Based on theoretical calculations, the respective masses are marmchair = 0.16m0 and mzigzag = 0.33m0, where, m0 is the free electron mass. Notably, the theoretically calculated mobility ratio (μarmchair/μzigzag) is well matched with the experimental value. This ratio was shown to increase from 1.85 to 3.15 upon decreasing the temperature from 300 to 60 K  and specific detectivity (3.04 × 108 Jones) were recorded, which is the highest value among recent reports. Furthermore, high photocurrent sensitivity to polarization angle was observed ‐grown few‐layer NS of 15 nm thickness used to fabricate a large‐area SnS FET device exhibited cyclic I–V characteristics with −50 V back gate voltage and ±5 V applied bias , is remarkably smaller than the theoretically calculated value. Such ferroelectric switching in multidomain SnS films was governed by the domain wall motion. In addition, an electrostatically tuned ferroelectricity has appeared =1d∫I(V)dV,where, d is the channel length.It is found that the electrical mobility and conductivity values were higher in zigzag direction, explained with the difference in the effective masses along the two principle axes. In particular, Vidal et al.58 ande Figure ,d. Furths Figure . This chd Figure  in SnS dd Figure . The tot have demonstrated anisotropy in the electronic behavior of LPE‐synthesized large‐area single‐crystalline multilayered SnS NSs. Four contacts were used to measure the anisotropy in conductivity of the NSs along armchair and zigzag crystallographic directions  was equal to ≈1.7, which is in very good agreement with reported values. Besides this, the conductivity along the (101) and (10Î) (ladder) crystal directions was also measured and found to be isotropic, namely, 2.2 and 2.3 S m−1, respectively ] and found to be highly angle‐dependent  + μz sin2(θ − φ), where μx and μz are the mobilities along the armchair (x) and zigzag direction (z), respectively. The calculated anisotropy in electrical field effect mobility along x and z direction (μx(max)/μz(max)) was ≈5.8, which is the highest value among other anisotropic materials, including SnS (1.7), ReS2 (3.1), and BP (1.5).Ab initio theoretical calculations offered a qualitative explanation for the angular dependence of the mobility, which was compared with experimental results. However, the theoretically predicted anisotropy in electrical mobility ratio is slightly lower (≈4.3) than the experimental value. Besides this, the electrical performance was probed in two‐terminal SnSe devices , high absorption coefficient, high carrier mobility, and low exciton binding energy, EB. A low EB is favorable for the efficient separation of photogenerated charge carriers. Besides this, in a solar energy device, the power conversion efficiency (PCE) largely depends on the band alignment between donor and acceptor materials. Another promising alternative property is the bulk photovoltaic effect, attributed to a nonlinear optical (NLO) response that yields net photocurrent in materials with net polarization. Owing to their outstanding light absorption properties, 2D TMDs  have been widely investigated for their application in solar energy conversion. In a similar manner, in‐plane anisotropic layered 2D MMCs have recently been investigated as potentially promising materials in photovoltaics.In a typical solar energy conversion device, the donor materials dominate the main physical processes, including light absorption and exciton transport. In such devices, 2D materials are widely used as in the active (donor) or in various interface/interconnecting layers for exciton generation or charge extraction.204 A  have designed and investigated a new photovoltaic system based on novel 2D MMCs. In particular, the photoresponse and the photovoltaic performance of GeS, GeSe, SnS, and SnSe monolayers were evaluated by means of theoretical quantum transport simulations. The corresponting EB values have been additionally estimated to be 0.68, 0.52, 0.25, and 0.21 eV for GeS, GeSe, SnS, and SnSe, respectively. These binding energies are much weaker compared with the respective ones calculated for SiC, GaN, and MoS2 monolayers , 2D MoS2 (ε = 7), and anisotropic BP. The higher dielectric constant of selenium‐based MMCs comply with lower EB values. The photovoltaic performance of monolayer MMCs was evaluted with a two‐probe device configuration  and EQE. It is important that the Rph vaules in MMCs are much higher than those of MoS2 phototransistors and three orders of magnitude higher than that of graphene detectors. As shown in Table  have reported a large photocurrent shift (≈100 µA V−2) in single‐layer Ge and Sn MMCs. Such shift was correlated with the large spontaneous effective 3D electric polarization of ≈1.9 C m−2 in Ge and Se MMCs.Dai and co‐workers41 havn Figure  and a las Figure –j. This  have systematically explored the electrical properties of bilayer GeSe with different metal electrodes. In particular, the interface geometry, electronic properties, band alignment, Schottky and tunneling barriers were calculated with Au, Ag, Al, Cu, Pt, and Ni, electrodes, respectively. Among such metals tested, Au, Pt, and Ni showed lower Schottky and tunneling barriers. In addition, a heterostructure of monolayer SnS with a bilayer GeSe has been constructed to investigate the resulting photovoltaic performance ; and the PCE of the bicomponent cell were much higher than those of the single‐layer ones. In particular, the obtained Jsc, Voc, FF, and PCE values were 37.62 mA cm−2, 0.76 V, 83.14%, and 23.77% for the bi‐component solar cell and 18.53 mA cm−2, 1.02 V, 88.15%, and 16.66% for the single perovskite cell, respectively. The significant rise of Jsc observed in the bi‐component cell can be attributed to the enhanced absorption, at higher wavelengths, by the GeSe layer. In another work, Michael et al. have investigated the performance of 2D piezo‐phototronic based solar cells. In such devices, an external strain was applied to monitor the piezo‐phototronic effect. The strain‐induced PCE of a monolayer SnS, SnSe, GeS, and GeSe can improve up to 18.2%, 16.6%, 7.0%, and 15.4%, respectively.The extended absorption of MMCs in the IR range was exploited by Hou et al.224 Ths Figure  comprisis Figure –g. Notabd Figure . It is s In particular, the photocatalytic water splitting for energy conversion and the electrode catalyst (cathode) activity for energy storage have been theoretically investigated. In photocatalytic water splitting and electrode catalytic activity, both the electronic structure and the optical properties of the semiconductor play an important role. When an incident light falls on a semiconducting catalysis material, an electron gets excited to CB and generate electron–hole (e–h) pairs. Subsequently, the electrons then take part in hydrogen evolution reaction (HER), while the holes cause an oxygen evolution reaction (OER), which are expressed as: H2: 2H+ + 2e− → H2 (HER) and O2: H2O + 2h+ → 1/2O2 + 2H + (OER), respectively. The minimum Eg required for these two processes to simultaneously take place is 1.23 eV. More interestingly, Zhang et al. have reported that an indirect bandgap material is more suitable for photocatalytic activity. Most of MMCs exhibit larger bandgaps (direct or indirect) than the minimum energy required for the water splitting reaction (>1.23 eV). In particular, the GeSe, SnS, and SnSe exhibited larger overpotential, which was behaving as a potential material for HER. On the contrary, SiS, SiSe, and GeS exhibit good photocatalytic activity in basic and acidic conditions. Apart from the photocatalytic activity, MMCs are predicted to be promising as cathode catalyst materials in Li‐O2 batteries. However, the impact of in‐plane anisotropy of MMCs in catalytic activity has not yet been investigated.Another important application of MMCs is their catalytic activity.228 In4.3valleytronics comes as the combination of the words valley and electronics and describes the ability to tune the carrier conduction in the valleys of a material's band structure. The idea was initially applied for the development of new quantum computing devices, which use the valley polarization as a means to store and/or carry the quantum information. Castro Neto and co‐workers theoretically predicted the valley properties and the optical selection rules in MMCs. In particular, the 2D form of SnS was studied via a combination of ab initio calculations and k·p theory. In monolayer SnS, two pairs of valleys were identified, placed along two perpendicular axes, which can be selected exclusively with linearly polarized light and can be separated using nonlocal electrical measurements. Thereafter, Park and co‐workers investigated the multistability of the direction of the puckering of monolayer MMCs using first principle calculations. It is found that the monolayer of SnS and GeSe exhibits two inequivalent valleys in momentum space, which are dictated by the puckering orientation along armchair or zigzag direction. These valleys can be excited selectively using linearly polarized light.The term  devices,234 wh demonstrated the direct access and identification of different sets of valleys in bulk in‐plane anisotropic SnS  represents the left (+) and right (−) circularly polarized PL intensity. Two peaks of SnS PL spectra were recorded at 817 nm (A) and 995 nm (B)  stand for the PL intensity under parallel polarization for the i valley. It is observed that the (A) peak maximizes at the polarization that minimizes the (B) peak and vice versa. Such 90° phase shift between the peaks signifies the selectivity of two valleys. The corresponding intervalley polarization degrees of the 817 (lying on ΓY axis) and 995 nm (lying on ΓX axis) peaks, calculated using the formula  Figure , respect) Figure ,d that wPintravalley, ΓX) and ΓY  directions were realized to be 95% and 96%, respectively. Such intravalley polarization values are among the highest polarization degrees reported, from experiments conducted at cryogenic temperatures.The intravalley polarization degrees in ΓX  of a bulk SnS (of 109 nm thickness) exhibited two photon emission peaks located at 1.16 and 1.204 eV  is the energy of the CBM and VBM of the ΓX and ΓY valleys, respectively. Considering that the two PL peaks arise from the band‐edge transitions, the polarized PL spectra were monitored and presented in Figure 2θ and sin2θ dependencies . The χn) corresponds to the elevated‐order nonlinearity in the medium. The generation of NLO response depends on the intrinsic crystalline symmetry, the microscopic transition dipole matrix, as well as on the specific frequency and orientation of the optical field applied. On top of them, a centrosymmetric or non‐centrosymmetric crystal structure plays a pivotal role due to the different order electric susceptibility tensors describing each case. To date, a large number of 2D materials including mono/multilayer MoS2, MoSe2, WS2, WSe2, hBN, GaSe, and InSe has been reported to exhibit the second and higher order harmonics generation. However, all of them belong to the centrosymmetric point group D3h comprising a single independent SHG susceptibility tensor element. On the other hand, the MMC monolayers belong to a non‐centrosymmetric point group and thus exhibit up to five independent SHG susceptibility tensor elements. Qian and Wang have theoretically investigated the optical second harmonic generation in monolayer MMCs. In particular, the quasiparticle band structure of a monolayer GeSe has been calculated using first principles, quasi‐atomic orbital method. Moreover, the second‐order susceptibility tensors in MMCs, MoS2, and hBN were estimated = χxyx(2)=χxxy(2)=−χyyy(2). On the other hand, the Cν2 exhibits five independent susceptibility tensor elements , χyyy(2),χyzz(2),χxyx(2)=−χxxy(2) and χzzy(2)=χzyz(2). The obtained magnitude of the calculated susceptibility tensor element at 3.22 eV was calculated to be 5.16 × 106 pm2 V−1 for GeSe, which is much higher than that of MoS2 (3.02 × 105 pm2 V−1) and hBN (6.38 × 104 pm2 V−1). Moreover, an independent SHG tensor element (χxyx(2)=χxxy(2)) with a substantial magnitude, which is much higher than the other three components of a GeSe monolayer, was predicted. The angular dependence of the SHG response that can be measured, for example, by rotating the crystal, and measuring the SHG polarization and intensity can provide important information on 2D materials anisotropy. Considering normal incidence geometry, the angular dependent SHG susceptibilities for the point group Cν2 and D3h had been calculated. The corresponding angulardependent polar plots of SHG are shown in Figure ω and the maximum χ(2) values. A significantly polarized colossal SHG response in monolayer GeSe with its maximum value located at an angle of 0° was observed. On the other hand, the polar plots of MoS2 monolayer precisely reflect the D3h symmetry. In the case of monolayer GeSe, the total susceptibility  ) contains an interband contribution (χintra(abc)), a modification due to the intraband motion (χinter(abc)), and a modulation due to interband motion (χmod(abc)). Such contributions were accounted by six tensor termsSHG is a NLO process describing the light–matter interaction where the induced polarization depends nonlinearly on the external electric field. SHG has been broadly applied for the materials analysis, sensors development, advanced spectroscopy, high‐resolution imaging, lasers, frequency conversion, electro‐optic modulators, and switches.243 Mas Figure  satisfyiχi(v)(2ω) and χi(r)(2ω) are the leading ones behind the giant SHG susceptibility in GeSe monolayer, governed by the interband and intraband Berry connection in the monolayer. These leading susceptibility terms are distributed at four spots in the vicinity of the first Brillouin zone and concentrated around the two valleys in the bandgap. Moreover, the authors have predicted SHG strength in the monolayer and trilayer GeSe and SnSe, which has very similar SHG strength and shape.Among such susceptibility terms, the 5A–VI MMCs and their emerging promising applications in electronics, optoelectronics, and nanophotonics. The in‐plane anisotropy along the armchair and zigzag directions makes these materials more important to explore the rich physics of 2D materials. Nevertheless, the experimental research on MMCs is still in a preliminary stage. Among the synthesis approaches, the mechanical exfoliation is fairly simple and easy to obtain low defect and highly crystalline nanoflakes. However, the low exfoliation yield, lateral size repeatability, and reproducibility in layer number, harshly restrict its application for large‐area electronics and optoelectronics. LPE is an effective method for bulk production and is widely used for the solution processable flexible organic and hybrid electronic applications. However, the control synthesis of MMCs with thickness uniformity in various solvents has not been investigated yet. Besides this, the choice of appropriate solvent for successful isolation into thinner layers is also an important topic of research. In contrast, the physical and/or chemical vapor deposition is no doubt the most promising route for large‐area 2D materials production for future electronic applications. At present, the synthesized MMCs are restricted only in few monolayers and therefore the isolation of single layer with good crystallinity remains a great challenge. In general, there is a huge room for further study on the controlled synthesis of electronic‐grade ultrathin, even atomically thin, MMC layer by means of top‐down and bottom‐up approaches.In this Review, we have presented the fundamental properties and recent advancements on the synthesis of layered 2D, in‐plane anisotropic, phosphorene analog group IVTo date, the optical and electrical anisotropic properties of orthorhombic MMCs have been investigated in few‐layer NSs, while the intriguing anisotropic optoelectronic response of a monolayer is only theoretically predicted. Such unique anisotropic response appeared due to the puckered crystal lattice structures of MMCs, resulting from the difference in the effective mass and refractive index along the armchair and zigzag directions, respectively. On top of that, the large absorption coefficient and the large spin–orbital coupling pave the way for the application of MMCs in solar energy harvesting and valley electronics. Apart from the current inability to isolate MMCs monolayers, the fast growing field of MMCs is also hampered by a huge gap between the materials’ fundamental studies and device applications. Furthermore, the highly anisotropic electrical and optical properties in MMCs integrated with 0D (quantum dots) or 1D organic and/or inorganic and perovskite semiconductor materials could open up a new path for next‐generation electronic applications. Overall, the very recent exciting achievements in the field of few‐layer MMCs showed a great potential for their application in next‐generation electronic, optoelectronic, and emerging nanophotonics, including valley electronics, solar cells, sensors, and nonlinear optical applications. However, the wealth of arising possibilities in fundamental research of 2D MMCs and the emerging new approaches to MMCs monolayer synthesis and functionalization prescribe a future where tuning of MMC band structure and subsequent electronic properties can be accomplished with a level of sophistication that we cannot presently imagine.Notably, puckered structured Ge and Sn‐based chalcogenides are p‐type semiconductors, which are desirable to form heterojunctions with n‐type TMDs to explore the interface physics at the nanoscale and the subsequent electronic properties. As a consequence, strong light–matter interaction and reduced dimensionality leads to the formation of quasi‐1D excitons and trions within in‐plane anisotropic MMCs, while quasi‐2D excitons and trions in isotropic TMDs materials. Therefore, heterostructures of in‐plane anisotropic/isotropic 2D materials can provide unique interlayer interactions between quasi‐1D and 2D excitonic species. This may find promising applications including high‐performance photoemitters and exciton–polariton lasers. Nevertheless, to date there are limited studies on such interlayer optical and electronic responses.258 FuNote added. We noticed that during the peer review process, a novel study on the PVD growth of monolayer SnS and the demonstration of room temperature purely in‐plane ferroelectricity and SHG was published by Nagashio and co‐workers.The authors declare no conflict of interest.


The aseptic meningitis caused by varicella zoster virus (VZV) reactivation was less described in the literature, most of which were detected by means of polymerase chain reaction. The authors presented 4 adult immunocompetent patients with acute aseptic meningitis with VZV infection diagnosed by next-generation sequencing (NGS).2O; range 165–400 mm H2O). White blood cell counts and protein levels were significantly elevated in CSF samples . The cytology of CSF demonstrated a lymphocytic pleocytosis, and most multinuclear cells. The culture of CSF was negative for all 4 cases, while T-cell spot test was positive for 2 cases, who were administrated with anti-tuberculosis treatment for suspicious tuberculous meningitis. NGS of CSF  detected specific sequences of VZV in the 4 cases within 72 h after admission. The inappropriate treatment were stopped while acyclovir were continued intravenously for 10–14 days. All patients recovered completely.Four patients were admitted to the hospital with headache and fever between March 2018 and August 2019. The median ages were 37 years (range 22–52 years). The median symptoms onset to clinic time was 3.5 days (range 3–6 days). Two patients had signs of meningeal irritation. Rash occurred after the meningitis symptoms in 1 patient . No other sign or symptom was reported. The brain Magnetic resonance imaging and electroencephalography were normal in all patients. Cerebrospinal fluid (CSF) samples were obtained at a median of 4 days (range 3–7 days) from the meningitis symptoms onset. Opening pressure of lumbar puncture after admission were high in these cases (median 256 mm HVZV is an infectious agent that causes aseptic meningitis in immunocompetent adults and could not be accompanied by skin manifestations. The NGS of CSF is a rapid detection for the identification and differentiation of meningitis in patients, which is of great importance for providing the rapid and accurate diagnosis and the targeted antimicrobial therapy for central nervous system infection. Aseptic meningitis is an inflammation of the meninges associated with acute onset of headache, fever and neck stiffness, with pleocytosis of the cerebrospinal fluid, and no growth on routine bacterial culture . The leaWe here described cases of 4 immunocompetent adults with aseptic meningitis due to VZV reactivation diagnosed by next-generation sequencing (NGS).On March 2018, a 52-year-old man was admitted to Northern Jiangsu People’s Hospital because of headache for 4 days and fever (37.6 °C) for 2 days. He had no other symptoms. The constant and progressive headache did not relieved after the routine treatment such as oral nonsteroidal analgesics. He had a history of chicken pox when he was 7 years old. He had symptomatic epilepsy since 8 years ago after a brain trauma, which occurred every 6 to 12 months. He had no sign of meningeal irritation, and vital signs were normal. No rash was seen at any stage  contained 147 × 10^6/L white blood cells (WBCs) ; CSF protein was 1.72 g/L   were continued intravenously for 10 days. The patient’s condition improved quickly. A repeat lumbar puncture on the ninth day after admission revealed improved WBCs (39 × 10^6/L) and protein (0.88 g/L) Table . Thus heOn November 2018, a 22-year-old man was admitted to Northern Jiangsu People’s Hospital because of fever (37.7 °C), severe headache, and nausea for 3 days. One day before admission, he noticed vescicles in a very limited region of the left dorsal skin. No other sign or symptom was reported . The cytology of CSF demonstrated WBCs with 200 × 10^6/L, and an increased protein concentration (0.57 g/L)  was continued intravenously for 10 days. The therapy was highly effective and the patient’s clinical condition rapidly improved. The CSF on the eighth day after the admission revealed WBCs with 130 × 10^6/L, and protein (0.40 g/L) Table . The pat2O). The CSF contained 478 × 10^6/L WBCs; CSF protein was 1.79 g/L  and protein (0.65 g/L) Table . Thus he2O, and the CSF contained 434 × 10^6/L WBCs , and normal protein (0.37 g/L)  and a progressive headache was admitted to Northern Jiangsu People’s Hospital. His disease began with headache and nausea 3 days earlier. No other symptom was developed. Neurological examination was moderate for neck stiffness and negative for Kernig signs  and VZV-R2 (GCGGTAGTAACAGAGAATTTCTT). The results showed that the read from Sanger sequencing was consistent with VZV genome  and herpes zoster. Varicella usually results in mild to moderate illness in mainly childhood or immunocompetent patients with disseminated vesicular rash. After primary infection, VZV remains latent in sensory cranial nerve ganglia or dorsal root ganglia, when reactivated, replicates along the course of the nerve and appears as a localized vesicular skin rash. Viral reactivation can cause a wide range of neurologic disease, most frequently manifesting as herpes zoster and post-herpetic neuralgia . Older aThe etiology of acute meningoencephalitis remains undiagnosed in approximately 60% of cases despite extensive clinical laboratory testing for infectious pathogens . Till no2O) in three patients, and it was also on the high side (165 mm H2O) for the other one. All cases showed elevation in CSF WBC, ranging from 147 to 478 × 106/L, and the CSF cytology indicated lymphocytic inflammation. It was regarded that mild elevation in CSF protein levels would been observed in aseptic meningitis. However, the relatively high CSF protein levels were reported seen in patients with VZV infection which were significantly higher than those seen in patients with enteroviral infection [Lumbar puncture revealed increased opening pressure of CSF (235–400 mm Hnfection , 13. ProThe difficulty in meningitis diagnosis is to distinguish whether it’s a viral or bacterial etiology, because this is crucial for treatment decisions. The treatment threshold is usually set low in clinical work, so that antibiotics, even anti-tuberculosis treatment, are often prescribed in cases of doubt, as were patients in this study. The patient of case No. 2 had vesicular rash before admission which made the diagnosis with aseptic meningitis relatively easy. However, the other 3 patients never showed cutaneous zosteriform lesions. Additionally, the T-cell spot test was positive for the second and fourth cases, meanwhile the protein levels in CSF elevated significantly for both these 2 patients, which implied possible tuberculosis infection, therefore anti-tuberculosis treatment as well as antibiotics were administrated. However, all the cases in our study were pathogen diagnosis within 72 h after admission. Once the sequences of VZV were detected by NGS, inappropriate treatment were stopped.NGS is a rapid and accurate approach for the molecular diagnosis with diseases compared to traditional clinical testing. It could dramatically reduce the diagnostic period to less than 3 days . PathogeVZV reactivation leading to aseptic meningitis in immunocompetent adults with or without cutaneous zoster is more common that previous regarded. Relatively high CSF protein levels could be observed in VZV meningitis. This study highlighted the feasibility of using NGS of CSF as a diagnostic tool for CNS infection. Unbiased NGS could facilitate identification of all the potential pathogens in a single assay theoretically, which is of great importance for providing the rapid and accurate diagnosis and the targeted antimicrobial therapy for CNS infection.Additional file 1. Details of NGS detection process and the results of Sanger sequencingAdditional file 2. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 1Additional file 3. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 2Additional file 4. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 3Additional file 5. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 4Additional file 6. Original images for results of Sanger sequencing detection of VZV from CSF specimen


Gardenia jasminoides, but its effects on the progression of HCC remain untested.As a typical hypervascular tumour, hepatocellular carcinoma (HCC) is predominantly grown through angiogenesis. Geniposide is a promising anti‐inflammatory compound found in The anti‐HCC effects of geniposide was investigated in cellular models and orthotopic HCC mice. Transcriptional regulation of the VEGF promoter was measured by dual‐luciferase reporter assay. The anti‐angiogenic action of geniposide was measured by tube formation assay. Both surface plasmon resonance techniques and human phospho‐kinase array analysis were utilized to validate the relationship between targets of geniposide and hepatocarcinogenesis.Geniposide exhibited significant disruption of HCC proliferation, invasion, angiogenesis and lung metastasis in orthotopic HCC mice. Geniposide inhibited secretion of VEGF by HCC and suppressed the migration of endothelial cells and the formation of intra‐tumour blood vessels, without cytotoxicity and independently of the transcription factor HIF‐1α. Direct inhibition of TLR4 by geniposide led to the shutdown of the TLR4/MyD88 pathway and STAT3/Sp1‐dependent VEGF production. However, LPS, an agonist of TLR4, restored STAT3/Sp1‐related VEGF production in geniposide‐inhibited HCC angiogenesis.The direct inhibitory effect of geniposide on TLR4/MyD88 activation contributes to the suppression of STAT3/Sp1‐dependent VEGF overexpression in HCC angiogenesis and pulmonary metastasis. This action of geniposide was not affected by stabilization of HIF‐1α. Our study offers a novel anti‐VEGF mechanism for the inhibition of HCC. HCChepatocellular carcinomaMyD88myeloid differentiation primary response 88Sp1specificity protein 1TLR4toll‐like receptor 4Progression of hepatocellular carcinoma is known to involve VEGF‐induced angiogenesis which is dependent on HIF‐1α.Geniposide exerts many anti‐inflammatory effects, but its effects on angiogenesis in hepatocellular carcinoma are untested.A novel anti‐VEGF mechanism, involving the TLR4/MyD88 pathway, to inhibit progression of hepatocellular carcinoma .Geniposide suppresses VEGF expression and angiogenesis in hepatocellular carcinoma, independently of HIF‐1α.Novel therapeutic targets for VEGF inhibition in hepatocellular carcinoma may improve pharmacological management of cancer.Geniposide, a potential and affordable anti‐VEGF compound, shows promise as clinical treatment for hepatocellular carcinoma.1VEGF family of ligands and receptors , a common and lethal form of cancer, results in an annual mortality of one million people worldwide. The high death rate reflects the unpredictable manifestations of HCC in the early stage and poor prognosis in the advanced stages of this condition  is an iridoid glycoside, abundantly available in the herb In the present study, we evaluated the inhibitory effect of geniposide on the development of HCC, in vivo and in vitro, including the proliferation, invasion, metastasis and angiogenesis of HCC.22.12 at 37°C. The normoxic condition refers to the standard condition (20% oxygen), whereas cells incubated in a nitrogen/carbon dioxide/oxygen chamber with 1% oxygen represented the hypoxic condition.Human HCC cell line PLC/PRF/5 cells and HUVECs were purchased from ATCC (USA). The luciferase reporter‐tagged MHCC‐97L cells were obtained as a gift by Prof. Man Kwan from the Department of Surgery, The University of Hong Kong. HUVECs were cultured in EGM‐2 complete medium . Both PLC/PRF/5 and MHCC‐97L were maintained in DMEM with 4.5% glucose, 10% FBS and 1% penicillin/streptomycin  in humidified condition containing 5% CO2.2British Journal of Pharmacology.All animal care and experimental protocols were approved by the Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong (Ref: 4251‐17). Animal studies are reported in compliance with the ARRIVE guidelines  and/or a single i.p. injection of LPS  at the beginning of a 5‐week treatment. LPS was utilized as an agonist of toll‐like receptor 4  at the end of the experiment.A model of orthotopic HCC‐implanted mice was established as described in our previous report .For the histological study, paraffin‐embedded sections (5 μm) were collected on the surface of the slides followed by de‐waxing in xylene and rehydrated by alcohol with gradient concentration from 100% to 70% . After the processing of rehydration, slides were incubated with haematoxylin and 0.25% eosin for 5 and 1 min, respectively . Afterwards, slides were immersed in Canada balsam  followed by the observation of haematoxylin and eosin‐stained HCC morphology with a BX43 light microscope  were immersed in 10‐mM citrate buffer  for antigen retrieval, followed by incubating with 10% goat serum for 30 min for blocking procedure. Then, sections were incubated for 12 h at 4°C with anti‐glypican‐3 , anti‐HIF‐1α , anti‐Ki67 , anti‐CD31 , anti‐MyD88 , anti‐Sp1  and anti‐STAT3 , respectively . Afterwards, slides were counterstained with corresponding secondary antibody Alexa Fluor 488 , Aleca Fluor 568  and DAPI , followed by mounting with fluorescent mounting medium . All the specimens were visualized by using the confocal microscope .The immuno‐related procedures used comply with the recommendations made by the 2.4https://david.ncifcrf.gov). In particular, GO analysis for target mining is composed of three ontologies, including biological process, molecular functions and cellular components. GlueGo‐based network pharmacology was constructed by Cytoscape (access at http://www.cytoscape.org/). To enhance the functionality of target prediction, cluster tree analysis was applied by SPSS Statistics  and RStudio . The correlation degree of –log10  from each identified geniposide‐regulated pathway was imported into SPSS for transforming all variables to Z scores by choosing the method of squared Euclidean distance, which aimed to yield equal metrics and weighting value. Next, the hierarchical cluster membership was defined by SPSS‐dependent weighted cluster analysis, followed by further identifying homogeneous biological terms in each cluster. Finally, the R programming visualization was performed by RStudio to circle‐hierarchically show the outcomes.Based on the analysis of human phospho‐kinase array, the underlying mechanism of geniposide‐induced HCC inhibition was predicted by bioinformatics analysis, as in our previous studies ; and the EF.STAT3C.Ubc.GFP was a gift from Linzhao Cheng , pN3‐Sp1FL (pN3 vector for hyperactivation of SP1) and EF.STAT3C.Ubc.GFP . In detail, the contributors of these specific plasmids were listed as follows: the pGL4.10‐VEGFprom was a gift from David Mu . PrimeScript RT master mix  was used to reverse transcription. Quantitative PCR was conducted by using SYBR Green Probe  and designed corresponding primers on a Light Cycler 480 PCR System . The sequences of isoform‐specific primers were shown as follows: VEGF‐A forward: CCTTGCCTTGCTGCTCTAC, VEGF‐A reverse: TTCTGCCCTCCTCCTTCTG; TLR‐4 forward: TTGTATTCAAGGTCTGGCTGG, TLR‐4 reverse: GCAACCTTTGAAACTCAAGCC; and β‐actin forward: GCTTCTCCTTAATGTCACGC, β‐actin reverse: CCCACACTGTGC CCATCTAC. Calculation of the relative transcriptional expression of the target genes was performed after standardization with the internal reference β‐actin transcript.Total RNA from HCC cells was extracted and purified by using TRIzol . Briefly, the in vitro HCC cells, including MHCC‐97L and PLC/PRF/5, were treated under normoxic or hypoxic condition after 24‐h geniposide or vehicle treatment followed by washing in the binding buffer. Both HCC cells were respectively stained with PE Annexin V and 7‐AAD for 15 min. The percentage of alive, apoptotic and necrotic cells were determined by flow cytometer .2.8n = 5 per group).In accordance with the manufacturer's instruction, concentrations of HCC‐secreted VEGF were detected by VEGF enzyme‐conjugated immunosorbent assay kit . Cell supernatant was transferred to a microplate that was coated with an anti‐VEGF antibody. Afterwards, HRP‐linked streptavidin was added in the plate and incubated for 30 min prior to the addition of TMB substrate solution. At the end of the experiment, the enzyme/substrate reaction was terminated by the stop buffer. VEGF concentration was determined at 450‐nm absorbance . Protein concentrations were determined by Bradford protein assay. Equal amounts of proteins were separated on SDS‐PAGE (CA) followed by electro‐transferring on the PVDF membrane . After finishing the electro‐transfer, membranes were immersed in the blocking buffer containing 5% BSA for 2 h prior to the incubation with primary antibodies, including anti‐TLR4 , anti‐MD2 , anti‐glypican‐3 , anti‐Sp1 , anti‐STAT3 , anti‐phospho‐STAT3 , anti‐HIF‐1α , anti‐phospho‐p38 MAPK , anti‐IκB‐α , anti‐p65  and anti‐MyD88 , respectively. HRP‐conjugated secondary antibodies, including anti‐rabbit IgG  and anti‐mouse IgG , were correspondingly used to the incubate the membranes for 2 h . Bands were visualized under the chemiluminescence system .2.10Dynabeads® Co‐immunoprecipitation Kit  was used for measuring the coupling of protein complexes. Briefly, after cells had undergone various treatments, cells were lysed by NP‐40 cell lysis buffer . The target protein was precipitated with antibodies that are covalently coupled to Dynabeads, which was followed with the elution (PBS in 0.1% Tween 20). The specific protein–protein complex was pulled down and separated by SDS‐PAGE. The chemiluminescence system  was applied for the visualization and quantification of the target proteins.2.11A co‐culture system was established to assess the cross‐biological activity of HCC‐induced HUVEC migration. HUVECs were inoculated in the donor chamber, whereas PLC/PRF/5 cells were seeded in the receiving chamber. Both cells were maintained in the serum‐deprived medium condition for 6 h. After then, HUVECs and geniposide‐treated PLC/PRF/5 were cultured in the complete medium under either normoxic or hypoxic condition at 37°C for 24 h. Next, the remaining cells located in the donor insert were removed by a cotton ball, while cells at the receiving chamber were fixed by 4% paraformaldehyde (PFA) for 2 h followed by staining with crystal violet. The number of migrated HUVECs was quantified by ImageJ  in five random fields of each well. Representative images were captured via a microscope .2.12−1) or recombinant VEGF (re‐VEGF) treatment  were prepared as the conditional media according to the published paper  and incubated in various conditional media for 6 h. Then, each well was washed with PBS followed by adding 4% PFA for 1 h at room temperature. After removing 4% PFA, the figures of tube formation were captured by a microscope . Quantification of tubular networks, including total branching length and number of tubes, was performed by ImageJ with Angiogenesis Analyzer plugin  in six independent wells.Each well of a 24‐well plate was coated with 300‐μl basement membrane matrix with reduced growth factor  for 30 min at 37°C prior to seeding with HUVECs with complete (EGM‐2) or conditioned medium. PLC/PRF/5‐culture medium (DMEM for 24 h) with or without either LPS (100 ng·ml2.13n = 5 per group).The determination of the activity of the VEGF promoter reporter was carried out according to the manufacturer's instruction . In brief, PLC/PRF/5 cells were transfected with pGL4.1 containing the VEGF promoter region  for 48 h. Afterwards, cells were lysed and then quantified the Firefly and Renilla luciferase values on a fluorescence plate reader . Firefly luciferase signal was analysed by the normalization of the Renilla luciferase signal  were extracted from PLC/PRF/5 cells with or without 24‐h geniposide treatment in the normoxic condition. The lysed proteins were transferred to the human phospho‐kinase array . Forty‐three phospho‐site‐specific antibodies were spotted onto the antibody array. Measurement of signal detection was conducted in line with instructions from the manufacturer. Signal intensities of spots on the array were analysed via ImageJ .2.15KD). In the Biacore X100 system, recombinant human TLR4  is covalently immobilized on the surface of a CM5 chip by a coupling buffer  from the Amine Coupling Kit . The coupling level was 1,300 RU. Then, a running buffer  was prepared prior to the dilution of geniposide. Afterwards, TLR4‐immobilized chip was treated with a running buffer containing the geniposide with different concentrations (0–12.8 μM) followed by the regeneration scouting by 10‐mM glycine–HCl (pH = 1.7). The injection and dissociation time was set to 3 and 15 min, respectively. The binding affinity of geniposide to TLR4 was analysed by BIAevaluation .The affinity determination of geniposide binding to TLR4 was measured by Biacore X100‐based surface plasmon resonance (SPR) technology with the BIA evaluation system . SPR assay is performed by an extremely sensitive biosensor that offers rapid screening of the alterations of refractive index on a molecular‐immobilized chip in response to another small molecule in a real‐time and label‐free manner. It is a powerful quantitative tool to monitor biomolecular interactions and provide specific kinetic and affinity determination, including the dissociation constant . The normality and homogeneity of variances were measured prior to statistical tests. For the comparison between two groups, Student's t test was used for normally distributed data, whereas Mann–Whitney U test was applied for data with non‐normal distribution. However, for the multiple comparisons (groups more than two), both the non‐parametric test  and one‐way ANOVA with Tukey's multiple comparisons test were used. Post hoc tests were run only if F achieved P < 0.05 in ANOVA and there was no significant variance inhomogeneity. Due to the reduction of unwanted variation, we used ‘fold‐matched control values’ to normalize the data of Western blotting and RT‐PCR. All the data are presented as mean ± SD. When the P value was less than 0.05, we considered it as evidence of a significant difference for all tests. P value for statistical significance was not varied later in Section The data and statistical analysis comply with the recommendations of the 2.17http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY . Notably, progressively decreased signal intensity was observed in mice treated with geniposide in comparison with the control group from Week 3 onwards, suggesting the slower growth of orthotopic HCC under geniposide treatment .We, first of all, identified the anti‐HCC potential of geniposide using an orthotopic HCC implantation model Figure . The modt Figure . More stt Figure . In addi−1 for 2 days) in treating animals .Consistent with the observation in bioluminescence images, the invasion, metastasis and proliferation of HCC were greater in mice without geniposide treatment. Firstly, orthotopic invasion of HCC exhibited an irregular and invasive edge in the growth front in the control mice, indicating that numerous HCC cells invaded into normal hepatic tissue. In contrast, a clear boundary was displayed in the mice on geniposide therapy Figure . Secondls Figure . Collect3.2−1 exhibited no cytotoxic and apoptotic effects on both MHCC‐97L and PLC/PRF/5 cells under normoxic or hypoxic condition for 24‐ or 48‐h incubation . Geniposide, up to 200 μg·ml−1, showed neither cytotoxic nor any proliferative effects on HUVECs, as analysed by MTT assay  were hierarchically clustered in the heatmap  = 4.783), GO Term B (−log10(q) = 4.625) and GO Term C (−log10(q) = 4.596), were statistically classified into Cluster 1 with high correlations (in red). More specifically, GO Term A (nucleus) was a single branch of Cluster 1. Besides, the GO Term B  and GO Term C (transcription) were conjugated together in another branch of Cluster 1, suggesting that the regulation of STAT (GO Term B + C) and nuclear target (GO Term A) may be responsible for the suppression of HCC by geniposide. Taken together, the inhibitory effect of geniposide on HCC angiogenesis was predictively mediated by STAT/NF‐κB/TLR‐related pathways with a potential nuclear target  by six residues, including ARG53, ARG55, ARG90, ARG132, LYS89 and LYS128 . The experimental data of steady‐state relative intensities (RU) fit well with a straight‐line regression model calculated by a four‐parameter logistic equation, with a positive correlation coefficient (R2 = 0.952). Moreover, the mRNA expression of TLR4 in both MHCC‐97L and PLC/PRF/5 cells, with or without geniposide treatment (200 μg·ml−1) was measured by RT‐PCR  co‐treatment . HCC signals were enhanced in mice co‐treated with LPS and geniposide, compared with those from the geniposide‐treated group on Week 5  under normoxic or hypoxic condition and neutralized after Sp1 silencing  in HCC mice only aimed to reverse the geniposide‐induced inhibition of TLR4, instead of endotoxin‐triggered HCC progression in this study (Kitamura et al., TLR4, a member of the toll‐like receptor family, is involved in the responses of the innate and adaptive immune systems (Arias et al., In conclusion, our study has demonstrated in vivo and in vitro activity, and the underlying mechanism, of geniposide against HCC. Geniposide exhibited potent inhibition of the proliferation, invasion, angiogenesis and lung metastasis of HCC. Suppression of HCC induced by geniposide was not associated with direct cytotoxicity in tumour cells but was related to a decrease of VEGF expression and HCC angiogenesis. Geniposide significantly blocked the transcription and production of VEGF from HCC cells. The inhibitory effect of geniposide on VEGF expression was independent of regulation by the HIF‐1α‐related pathway, but relied on suppression of Sp1 and STAT3 in HCC cells. Re‐expression of Sp1 and STAT3 could restore VEGF production in geniposide‐treated HCC cells. Further analysis showed that geniposide bound to TLR4 and repressed the TLR4/MyD88‐associated Sp1/STAT3 transcription activity. Re‐activation of the TLR4/MyD88 pathway by LPS, in vitro, restored the Sp1/STAT3 activities and VEGF production. Thus, geniposide may be a promising candidate for clinical use for HCC. Taken together, our study demonstrated that geniposide could be an inhibitor of angiogenesis in HCCby directly targeting the TLR4/MyD88‐regulated STAT3/Sp1 pathway followed by the suppression of VEGF transcription in a HIF‐1α independent manner, which provides a novel mechanism for VEGF inhibition in HCC management.Y.F. and N.W. conceived and designed the study. C.Z., N.W. and H.‐Y.T. did the experiments. C.Z., N.W., H.‐Y.T. and Y.F. wrote the manuscript. N.W., Z.Z., K.M., S.W.T., L. L. and Y.F. interpreted the data. All authors discussed and revised the manuscript and approved the final manuscript.The authors declare no conflicts of interest.BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and as recommended by funding agencies, publishers and other organizations engaged with supporting research.This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the