The hepatocyte growth factor (HGF): MET axis is a ligand-mediated receptor tyrosine kinase pathway that is involved in multiple cellular functions, including proliferation, survival, motility, and morphogenesis. Aberrancy in the HGF/MET pathway has been reported in multiple tumor types and is associated with tumor stage and prognosis. Thus, targeting the HGF/MET pathway has become a potential therapeutic strategy in oncology development in the last two decades. A number of novel therapeutic agents—either as therapeutic proteins or small molecules that target the HGF/MET pathway—have been tested in patients with different tumor types in clinical studies. In this review, recent progress in HGF/MET pathway-targeted therapy for cancer treatment, the therapeutic potential of HGF/MET-targeted agents, and challenges in the development of such agents will be discussed. Hepatocyte growth factor (HGF) and its receptor MET regulate multiple normal cellular functions, including proliferation, survival, motility, and morphogenesis. HGF/MET signaling appears to be a critical pathway in a variety of malignancies, and thus it has become a potential target for cancer therapeutics .One of the key considerations in developing anticancer drugs is their selectivity against cancer cells while sparing normal cells. Among a group of biologically important targets for cancer intervention, MET is a receptor tyrosine kinase (RTK) that has low activity in normal tissues but is dysregulated in many tumor types . HGF, alMET mutations in patients with hereditary papillary renal cell carcinomas [MET mutations in many other cancers continue to be discovered, such as in gastric, head and neck, liver, ovarian, non-small cell lung, and thyroid cancers [Evidence directly linking MET to cancer has come from the original discovery of missense rcinomas . Somatic cancers ,11,12,13 cancers . In tumo cancers . CollectResearch on anticancer drugs targeting HGF/MET signaling in solid tumors has been under preclinical and clinical investigation for the past two decades ,20,21,22(1)i.e., HGF) vs. targeting the receptor Mechanism of action: targeting the ligand ((2)vs. therapeutic proteinsModality of therapeutic agents: small molecules (3)Treatment strategies: monotherapy and combination therapy(4)Value and clinical implications of biomarkers for the HGF/MET pathway(5)Challenges and perspectives on the development of HGF/MET therapeuticsIn this review article, we will discuss the recent progress and advances in HGF/MET pathway-targeted therapy for cancer treatment, the therapeutic potential of HGF/MET-targeted agents, and challenges in the development of such agents, focusing on the following aspects:Based on their mechanisms of action, the HGF/MET-targeting agents can be generally categorized into agents that either target the HGF ligand or those that target the MET receptor. For agents targeting the ligand, they can be further categorized into either HGF activation inhibitors that prevent the cleavage of pro-HGF into the active form of HGF or HGF inhibitors that block the direct binding of HGF to the MET receptor. For agents targeting the MET receptor, they can further be categorized into either MET antagonists that bind the receptor without activation of downstream signaling or MET tyrosine kinase inhibitors (TKIs) that target the adenosine triphosphate (ATP)-binding site of the receptor and block receptor transphosphorylation .The activation of HGF from its inactive precursor pro-HGF is a critical step in HGF functioning . This prHGF inhibitors bind and neutralize HGF and block HGF from binding to the MET receptor, thus preventing the downstream activation of the pathway. To date, only a few monoclonal antibodies (mAbs) that directly bind against HGF, including ficlatuzumab, HuL2G7, and rilotumumab, have been studied in clinical trials.i.e., partial response) was observed in patients with non-small cell lung cancer (NSCLC) from these early trials [p = 0.03) [Ficlatuzumab (AV-299) is a humanized anti-HGF antibody currently in phase 2 development. Clinical data from phase 1 trials have shown that ficlatuzumab was well tolerated as a monotherapy and in combination with the epidermal growth factor receptor (EGFR) inhibitors erlotinib and gefitinib ,41,42. P = 0.03) . These oRilotumumab (AMG 102) is the first HGF ligand inhibitor to reach phase 3 development. Its antitumor effects have been evaluated in the last decade as a monotherapy or in combination with other anticancer therapies in a variety of tumor types ,48,49,50HuL2G7 (TAK-701) was one of the earliest mAbs developed that effectively neutralizes HGF. There was one phase 1 study conducted in advanced nonhematologic malignancies, in which HuL2G7 displayed a favorable safety profile with some indication of activity .Activation of the HGF/MET pathway requires the dimerization of the MET receptor upon the binding of the active form of HGF, which leads to kinase autoactivation. MET antagonists compete with HGF for MET binding, resulting in the degradation of MET and subsequent inactivation. Several MET antagonists have been discovered, including onartuzumab, CE-355621, DN-30, and LA480 ,54,55. WOnartuzumab is the only monovalent MET antibody that has been tested in clinical studies to date. Phase 1 data had shown that onartuzumab was well tolerated when administered alone or in combination with bevacizumab in patients with cancer . A phaseAnother approach for inhibiting the MET pathway is through MET kinase inhibitors, which target MET intracellularly. Several small-molecule MET kinase inhibitors have entered clinical development over the past decade, including selective MET kinase inhibitors , AMG 337, and INC 280) and nonselective MET kinase inhibitors , cabozantinib (XL 184), and foretinib).vs. 11.2 months, hazard ratio (HR) = 0.89, p = 0.4). Numerical trends toward improved PFS and ORR were also seen favoring the treatment arm [vs. 3.8 months, HR = 0.38, p = 0.01) was observed in patients with high tumor MET expression [Tivantinib (ARQ 197) is the first non-ATP-competitive agent that selectively targets the MET kinase. In clinical trials, treatment with tivantinib has been generally well tolerated and has shown some clinical activity in patients with cancer ,66,67,68ment arm . In a phpression . Two folMET-amplified solid tumors [MET-amplified gastric/esophageal adenocarcinoma and other solid tumors.AMG 337 is a selective MET kinase inhibitor that is currently in phase 2 clinical development. Preliminary results showed that AMG 337 has potential as a therapeutic agent for treating d tumors . This inINC 280 is another highly selective, oral small-molecule MET inhibitor. In a phase 1 study, it was well tolerated in patients with advanced MET-dependent solid tumors . Dose exOther types of MET inhibitors in clinical development are nonselective, multikinase inhibitors. For example, crizotinib targets MET and ALK ; cabozanMET amplification, suggesting that crizotinib may be a potential agent for the treatment of MET-amplified NSCLC [Crizotinib earned accelerated approval by the United States Food and Drug Administration (US FDA) in 2011 for the treatment of ALK-positive NSCLC. Subsequently, the drug was granted regulatory approval in 2013 based on its superiority on PFS and ORR compared with chemotherapy . Recent ed NSCLC .in vivo growth of a MET-positive tumorigenic subpopulation of giant cell tumor of bone stromal cells, and thus it may be an effective therapeutic option for the targeted elimination of a tumorigenic stromal subpopulation in patients with nonresectable or recurrent giant cell tumor of bone [vs. 4.0 months, HR = 0.28, p < 0.0001) and ORR improvement (28% vs. 0%) [Cabozantinib strongly inhibited the self-renewal potential and  of bone . Cabozan vs. 0%)  and is cSimilar to cabozantinib, foretinib, golvatinib, and MGCD265 are dual MET/VEGF2 inhibitors. In addition, foretinib also inhibits PDGFR, RON, FLT-2, FLT-3, AXL, and TIE2 with low potency. BMS-777607 is a dual inhibitor of MET and RON. Early-phase clinical data for these multikinase inhibitors have demonstrated favorable safety profiles either as a single agent or in combination with chemotherapy ,80. ClinBased on the modality of therapeutic agents, HGF/MET-targeting agents can be classified as either therapeutic mAbs or small molecules. Both mAbs and small-molecule HGF/MET inhibitors have shown clinical benefits in patients with cancer, and they are different in terms of pharmacological properties and their underlying mechanisms of action.mAbs are large proteins, and are difficult to penetrate cell membrane; they mainly target HGF in the extracellular circulation system or target the MET receptor on the cell surface. While small molecules have the advantage of crossing the cell membrane to interact with targets inside a cell, they are usually designed to interfere with the enzymatic activity of the MET tyrosine kinase. The mAbs are highly specific for their target, while the TKIs vary from being highly specific class I molecules to class II molecules that have multikinase activity and are less potent against MET.Interestingly, for HGF/MET inhibitors that are currently under clinical evaluation, all agents targeting circulating HGF or the extracellular MET receptor are mAbs, while all agents targeting the MET tyrosine kinase are small molecules. However, there appears to be no clear difference in the spectrum of indications between mAbs and small molecules. The major target indications for HGF/MET inhibitors are solid tumors, including gastric cancer, NSCLC, HCC, and thyroid cancer .Although mAbs can only act on extracellular targets, they are generally more specific for the targets than small-molecule inhibitors and thus are expected to have better safety profiles than small molecules. This has been demonstrated by rilotumumab, ficlatuzumab, HuL2G7, and onartuzumab as these molecules were well tolerated in patients with cancer up to the highest doses administered (either 20 mg/kg once every 2 weeks (q2w) or 30 mg/kg once every 3 weeks (q3w)). There were no dose-limiting toxicities (DLTs) reported for any of these mAbs . The mosIn contrast, the adverse effects associated with small-molecule inhibitors appeared to be compound-specific, and DLTs have been observed with small-molecule MET kinase inhibitors ,81,82,83Multiple clinical trials, ranging from phase 1 to phase 3, of HGF/MET axis-targeting agents are ongoing or have been completed. Some studies have generated negative results, whereas others have generated intriguing signals of potential efficacy. To date, no agent has generated conclusive data in a large enough sample set to garner a marketing indication from a global health authority. However, few observations have begun to emerge.With respect to efficacy, a target-specific agent might not have therapeutic advantage over a dual- or multi-target agent, since most tumors are more frequently linked to defects of more than one target. To date, the only US FDA-approved MET pathway-related agents, crizotinib and cabozantinib, are dual- or multi-tyrosine kinase inhibitors, indicating that blocking MET jointly with other cancer-related pathways might be more beneficial for cancer treatment.MET protein level, MET gene amplification, and MET mutational status. Within each selection strategy are various cutoffs, reagents, and methodologies to define an overall biomarker “positive” or “negative” status. As an example, in a review of the high-profile study of onartuzumab in NSCLC that was recently reported to be negative [For any agent to succeed in this space, proper patient selection is of paramount importance . Variousnegative , it has negative ,90, althnegative .Furthermore, mAbs and small-molecule HGF/MET inhibitors differ in other pharmacological properties . Rilotumvs. ~500 Da for small molecules), mAbs have limited tissue distribution and usually have a similar volume of distribution (Vd) as plasma volume. In contrast, small molecules have different chemical- and protein-binding properties and can extensively distribute into tissues and have a much larger volume of distribution than mAbs (®) which are independent of tissue penetration, while others target receptors , which may rely on their specific ways to reach target tissues to exert pharmacological activities. Furthermore, mAbs normally have much longer circulating time in the blood than small molecules, which compensates for the slow process of drug distribution.Unique pharmacokinetic characteristics were observed for mAbs and small-molecule HGF/MET inhibitors . Due to han mAbs . Thus, t+ enters the endosomes, resulting in increased IgG binding to FcRn at a pH lower than the physiological value. FcRn-bound IgG is returned to the central compartment, and unbound IgG proceeds to the lysosome and undergoes proteolytic degradation. Therefore, IgG elimination is likely dominated by affinity for the FcRn receptor.The half-lives of mAbs are about 9–18 days, which renders a q2w or q3w dosing regimen possible. Generally, the vast majority of mAbs is eliminated by catabolism . Collecti.e., crizotinib, cabozantinib and foretinib) are predominantly metabolized by cytochrome P450 (CYP) enzymes [i.e., crizotinib) could be CYP450 inhibitors or inducers [In contrast, the half-lives of small-molecule MET inhibitors were in a range of hours, requiring once daily (QD) or twice daily (BID) dosing regimens. The small-molecule MET inhibitors  for the treatment of NSCLC ,85. AlthAnother combination approach being evaluated in patients with cancer is MET inhibitors plus other targeted therapies, including VEGF, EGFR, and ALK.Recent data from a series of experiments in animal models suggested that inhibition of MET and VEGF simultaneously not only reduced tumor size but also reduced invasion and metastasis, thereby improving the treatment effect with either VEGF or MET inhibitors alone ,98. The Furthermore, for the treatment of HCC, the HCC samples with high levels of phosphorylated MET were found to be resistant to adjuvant sorafenib treatment. The dual blockade of MET and VEGFR2 by cabozantinib has significant antitumor activities in HCC, and the activation of MET in HCC may be a promising efficacy-predicting biomarker .Research in the last decade has revealed that crosstalk between the MET and EGFR pathways on cell’s survival, proliferation and migration  may playEML4-ALK fusion [Another potential target combination is MET/ALK inhibition. Recent phase 3 results have shown striking activity of the dual MET/ALK inhibitor crizotinib in patients with NSCLC carrying an K fusion . AlthougK fusion .MET gene can promote tumor resistance to EGFR inhibition, suggesting drug resistance to EGFR inhibition can be mediated through parallel activation of downstream signal transducers. From a mechanism point of view, MET amplification may contribute to resistance to EGFR inhibition through increased MET heterodimerization with human EGFR-related protein 3 (HER3) and activation of the PI3K/AKT cell survival pathway. Thus, dual EGFR–MET inhibition may overcome this resistance [Drug combinations have been continuously evaluated for the reduction of drug resistance. It was found in a phase 1 trial evaluating the combination of the selective MET inhibitor tivantinib plus erlotinib that amplification of the sistance . Preclinin vitro and in vivo. The decrease in MET was found to be correlated with higher sensitivity to paclitaxel and cotreatment of KFr13Tx cells with MET inhibitors sensitized the tumor cells to paclitaxel. The study demonstrated miR-31-dependent regulation of MET for chemoresistance of ovarian cancer, raising the possibility that combination therapy with a MET inhibitor and paclitaxel will increase paclitaxel efficacy [Furthermore, the MET/HGF axis plays a major role in the development of chemoresistance so that HGF/MET inhibitors may enhance sensitivity of cancer cells to the chemotherapy. For instance, ovarian cancer is one of the most aggressive female reproductive tract tumors. Paclitaxel is widely used for treatment but chemotherapeutic resistance is often acquired. It was found that miR-31 was downregulated in KFr13Tx cells and re-introduction of miR-31 resensitized them to paclitaxel both efficacy .MET-amplified cancer cells by reactivating key survival pathways. Experiments were conducted to treat MET-amplified GTL-16 and MKN-45 gastric cancer cells with the highly selective MET inhibitor PHA-665752, and it was found to abrogate MAPK and PI3K/AKT signaling. The results illustrate that highly targeted MET tyrosine kinase inhibition leaves MET oncogene-addicted cancer cells vulnerable to HER kinase-mediated reactivation of the MEK/MAPK and PI3K/AKT pathways, providing a rationale for combined inhibition of MET and HER kinase signaling in MET-amplified tumors that coexpress EGFR and/or HER-3 [HER kinase activation confers resistance to MET tyrosine kinase inhibition in MET oncogene-addicted gastric cancer cells. It was hypothesized that collateral growth factor receptor pathway activation can overcome the effects of MET inhibition in or HER-3 .In vitro, HGF reduced expression level of apoptosis-inducing factor (AIF) and cisplatin sensitivity in NSCLC cells. The effect of HGF was via the MET receptor and the downstream effector, focal adhesion kinase (FAK). It was found that expression level of AIF inversely correlated with the expression of positive NSCLC markers, such as dihydrodiol dehydrogenase (DDH), MET, short oncostatin M receptor (OSMR), matrix metalloproteinase (MMP)-1, and HER2/neu, which were closely associated with drug resistance, tumor recurrence, metastasis, and poor prognosis. Addition of HGF inhibited AIF expression level in HER2/neu-silenced cells, indicating that both HGF and HER2/neu affect drug resistance by regulating AIF expression level in NSCLC [Patients with advanced stages of NSCLC were frequently detected with HGF upregulation. in NSCLC .In summary, the choice of monotherapy or drug combination therapies is dependent on drug mechanisms of action, target indications, and mechanisms of drug synergy or resistance.MET signaling has been shown to mediate a variety of cellular processes related to invasive growth. Under normal physiological conditions, these signals are crucial for mediating related biological processes, such as tissue patterning during embryogenesis, organ homeostasis, and wound repair. Since tumorigenesis may involve the dysregulation of the MET signaling pathway, changes in HGF or MET signaling are expected to be potential biomarkers for understanding cancer development and the effect of drugs.MET gene amplification, copy number, and mutation, as well as markers related to the crosstalk of related pathways.Most biomarkers studied to date were related to its functions in cancer development. The commonly measured biomarkers are circulating HGF and MET levels, MET protein expression levels in tumor tissues, and An increase in circulating HGF was found in drugs targeting the MET receptor. For instance, circulating HGF was evaluated as a pharmacodynamic biomarker of MET inhibition by onartuzumab in patients with advanced cancers or with NSCLC. Administration of onartuzumab resulted in a rapid and sustained rise in HGF. It was hypothesized that onartuzumab displaced HGF binding to the MET receptor, resulting in increased circulating HGF levels. The elevation in HGF was independent of dose, drug exposure, dose duration, or tumor type and was only restricted to patients who received onartuzumab rather than those who received placebo. It was found that neither higher baseline nor elevation of HGF levels upon treatment was associated with baseline tumor burden, number of liver metastases, tumor response, or stage of the tumor at diagnosis. Evaluation of the relationship between changes in HGF levels and PFS and/or OS in MET-positive patients was inconclusive . In contIn addition, HGF has been evaluated as a potential biomarker in clinical studies for ficlatuzumab and rilotumumab, two mAbs targeting the HGF ligand. An elevation of total HGF (free plus bound) was observed with the treatment of ficlatuzumab or rilotumumab in clinical studies ,47,48,50Taken together, although high HGF levels might indicate enhanced activation of the MET signaling pathway in cancer development, these data should be cautiously interpreted due to their limited predictive value for clinical response.The current understanding is that high MET protein expression levels in tumor tissues may be associated with poor patient survival in selected cancer types.vs. erlotinib alone. However, mRNA expression of MET, EGFR, amphiregulin, epiregulin, and HGF did not predict survival in patients treated with onartuzumab [In a phase 2 study of onartuzumab, patients whose NSCLC tissue scored as positive for MET protein by immunohistochemistry (IHC) experienced a significant benefit from treatment with onartuzumab plus erlotinib rtuzumab . Furtherrtuzumab . Similarrtuzumab . HoweverMET mRNA expression was higher in CD138(+) than in CD138(−) cells (p = 0.0009). Low MET mRNA expression characterized patients with better response  compared to other patients (p = 0.0006). Patients with high MET mRNA expression displayed worse PFS (p = 0.0029) and OS (p = 0.0023) compared to those with low MET mRNA levels. Patients with both high MET mRNA expression and high β2-microglobulin level (>5.5 mg/L) had further worse median PFS (p < 0.0001) and OS (p < 0.0001) [Nevertheless, MET mRNA expression showed an association with survival in newly diagnosed patients with multiple myeloma treated with bortezomib-based induction therapy.  0.0001) .MET gene mutations, leading in most cases to a constitutively active form of the molecule, and amplification of the MET gene locus with overexpression of the receptor on the cell surface. On the other hand, transcriptional deregulation with overexpression of the receptor on malignant cells is probably the most common mechanism.The most characterized aberrancies are MET gene represents a rare event in cancer, but it might play a causal role in the development of certain cancers or lead to enhanced activation of MET kinase activity and confer tumorigenic properties in cells. High MET gene copy number was more frequently identified in cancers . MET gene amplifications might be associated with constitutive activation of the MET receptor and an oncogenic addiction to the MET signaling pathway [MET gene copies (9.8%) had a short PFS [Mutation of the  pathway . Furtherhort PFS .MET gene amplification, copy number, and mutation appear to be relatively conservative biomarkers, but they may be associated with rare events in cancer development.In summary, circulating HGF and MET levels do not appear to be predictive biomarkers for clinical response or disease status. MET protein expression levels in tumor tissues may be prognostic biomarkers of survival in selected cancer types. Evaluation of the predictive values of HGF/MET biomarkers for clinical benefit is an emerging area. Some recent examples are provided here.in vitro effects of onartuzumab on cell-associated and circulating MET and circulating HGF were evaluated, and results suggested that onartuzumab stably associates with MET on the surface of cells without inducing MET internalization or shedding. There was no observed effect of onartuzumab on circulating HGF levels in xenograft tumor-bearing mice or on endogenous HGF in cynomolgus monkeys; the findings were similar to those in clinical studies. It was also observed in animals that binding of MET to onartuzumab in circulation resulted in increasing MET serum concentrations due to reduced clearance of the MET-onartuzumab complex, which was also observed in the clinical setting. These preclinical findings indicated that the circulating level of HGF and MET might not be useful biomarkers for onartuzumab [The rtuzumab  and thatIn another example, genetically engineered mouse models were used to evaluate the role of HGF overexpression in the acceleration of HCC progression. For both liver regeneration and spontaneous HCC development, there is an inclusive requirement for MET expression, and when HGF induces autocrine activation, the tumor displays sensitivity to a small-molecule MET inhibitor. The finding indicated that HGF is a driver of hepatitis B virus (HBV)-induced HCC progression and may serve as a biomarker for MET-targeted therapy. Genomic analysis showed that an upregulated HGF signature is associated with poor prognosis in HBV-positive patients with HCC . Future MET gene amplifications. Assessing MET expression levels by IHC will most likely be applicable to the majority of indications; gene amplification is rare (<10% in NSCLC and gastric cancer), and activating mutations are limited to few and infrequent tumor types. IHC assays to determine HER2 status in breast and gastric cancer have been in clinical practice as companion diagnostic assays and have taught us a great deal about the benefits and challenges of such an approach [Alterations in the MET receptor might be associated with transcriptional overexpression, gene amplification, somatic or germline mutations, and/or ligand-dependent autocrine/paracrine loops. Most clinical evidence to identify predictive or prognostic values of HGF/MET biomarkers for survival was primarily based on retrospective analysis of clinical samples. Strategies in clinical studies to identify MET-driven tumors to date have focused primarily on measuring protein levels of MET, evaluating tumors for the presence of activating mutations, or assessing tumors for the presence of approach .Recent examples of identifying potential biomarkers that could be associated with clinical outcome in various cancers are presented in MET gene amplification, copy number and mutation, as well as markers related to crosstalk of related pathways, are commonly evaluated biomarkers. There was no universal observation in different types of cancers, and the association between biomarkers and clinical efficacy endpoints might vary with cancer types or disease stage. Therefore, specific and in-depth biomarker evaluations are needed for each cancer development.The information presented indicates that circulating HGF and MET levels, MET protein expression levels in tumor tissues, and While the benefits of biomarker selection are clear, the path to achieving the benefits is challenging. Putative biomarkers often have to be evaluated in the pivotal trial of a novel agent, at a time when many critical operating characteristics of the biomarker are unknown. There are also a variety of ways to test any given marker. Even if a novel agent makes it to the marketplace, the methodology by which a marker was assayed in a clinical trial may not mimic that which is available in regional medical communities. This leads to challenges not only in making the correct test of the marker available but also with the potential incorrect selection of patients when alternative tests are used. In addition, many drug researchers are evaluating different testing approaches for the same marker. Should multiple tests ultimately be approved for a given target, this will lead to significant challenges in cross-referencing the test developed for one agent for use with another agent, as the ability to test for the same marker with multiple different tests is constrained by both tumor tissue availability and cost.Although promising initial results have been reported in patients with lung, gastric, prostate, and papillary renal cancers treated with HGF/MET-targeted antagonists, optimal patient selection, diagnostic and biomarker development, and the identification and testing of optimal therapy combinations are critical for success. The wealth of basic information, analytical reagents, and model systems available concerning HGF/MET oncogenic signaling will continue to be invaluable in meeting these challenges and moving expeditiously toward more effective disease control. In this section, we discuss several challenges and perspectives on the development of HGF/MET therapeutics.in vivo antitumor activity of rilotumumab was evaluated in xenograft models in which HGF was expressed in either a paracrine or autocrine fashion, demonstrating that rilotumumab could inhibit the growth of autocrine HGF-driven glioblastomas (U-87 and U-118) and paracrine-regulated leiomyosarcoma [in vitro functional studies were performed to determine effective concentrations for inhibiting cell proliferation and migration and for enhancing the antitumor effects of chemotherapeutic agents [Selection of relevant animal species is the first issue that needs to be addressed in preclinical evaluations due to species cross-reactivity. For instance, a tissue cross-reactivity study was conducted for rilotumumab with normal tissues from mice, rats, rabbits, cynomolgus monkeys, and humans; only the patterns of tissue cross-reactivity in cynomolgus monkeys and humans were similar . This crosarcoma ,129,130.c agents . The resr = 0.91, p = 0.0008) was observed between simulated xenograft TGI driven by human pharmacokinetics and clinical response. However, TGI observed at maximum tolerated doses in mice was not correlated with clinical response . Agents that lead to greater than 60% TGI in preclinical models, at clinically relevant exposures, are more likely to lead to responses in the clinic [It is not uncommon that a drug showing activities in a xenograft model fails to replicate the effect in humans. Xenograft models are widely used in the discovery of anticancer drug candidates. However, the limited predictive value of certain models for discerning clinical activities has been recognized as an area of improvement. It has been reported that pharmacokinetic/pharmacodynamic modeling could be used to improve predictive capability because it may provide a better understanding of the relationships between drug concentration and antitumor activities for each agent in dose-ranging xenograft or allograft experiments and because it can simulate tumor growth inhibition (TGI) in xenografts at clinically relevant regimens with predicted human pharmacokinetics. A significant correlation ; HER2 overexpression was observed in 10% of cases and associated with increased survival (p = 0.041); and MET overexpression was observed in 4% of cases and associated with worse survival (p < 0.001). With multivariate analysis, only MET overexpression retained significance (p = 0.006). In contrast, pS6 and pERK levels were observed in 73% and 30% of tumors, respectively, with no association with survival. These findings implied that FGFR, HER2, and MET overexpression could be useful biomarkers only for small portion of patients. If a study enrolled unselected patients with gastric cancer, the effect of drugs targeting these specific pathways would be difficult identify due to the lack of clinical response in >90% of patients. In the development of increasingly precise therapeutics whose administration is driven by the presence of a predictive biomarker, the variability in tumor biology and prevalence of target biomarkers that exists across populations should be considered.One of the major challenges in developing targeted therapy agents is the identification of a true signal from a noisy background. For instance, the prognostic significance of HER2 and MET protein expression level, arcinoma . Tissue Variability in global treatment practices can ultimately impact the development of targeted therapeutics. These variabilities translate to complexities across a range of steps in bringing a new drug from the research setting to patient treatment centers and the commercial marketplace.MET amplification seems to be more often associated with CIN tumors. Given that various molecular categories are likely more common in different geographic regions , and various MET-targeting agents may be more efficacious against some MET abnormalities than others , these factors now have to be considered when developing a MET-targeting agent for gastric cancer. This affords the opportunity for optimizing delivery of the correct agent to the correct patient while simultaneously having significant impact on clinical trial conduct, such as what tissue analysis must be conducted, what geographies a trial should be conducted in, and what statistical assumptions should be employed.In the case of MET-targeting agents, gastric cancer is a lead indication for the class and provides a good case study of these challenges. Historically, gastric cancers are broadly classified based on histological characteristics using the Lauren and/or World Health Organization classification schemes. However, classification as one type or another does not significantly impact the clinical management of patients with gastric cancer or the development strategies of novel agents. Recently, a molecular classification has been suggested from analyses published by The Cancer Genome Atlas (TCGA) project . The molVariability in local treatment practices also impacts the development of targeted agents. Again, gastric cancer illustrates this well. Gastric cancer is highly prevalent in Asian countries, such as China and Japan, and less common in Western countries. In countries such as Japan, patients with resectable disease undergo wide so-called D2 resections followed by the administration of systemic cytotoxic chemotherapy. In the West, it is more common to administer chemotherapy or chemotherapy with concurrent radiation therapy prior to surgery and to follow resection with additional chemotherapy.vs. non-Asian countries. For example, in the case of the ERBB2-targeting monoclonal antibody trastuzumab, a phase 3 global study comparing chemotherapy with trastuzumab vs. chemotherapy with placebo for patients with HER2-positive advanced gastric cancer suggested differences in the OS achieved by Asians (OS HR = 0.82) compared to non-Asians  [In patients with metastatic disease, patients in Japan receive more lines of systemic therapy than those in the West. These differences in treatment practice seem to be responsible for the differential outcomes observed in the development of targeted therapies in Asia ericans) . A similericans) . WhethererbB1 (the protein product of which is EGFR) are known to be driver mutations in a subset of patients with NSCLC; these mutations are much more prevalent in Asian patients (30%–40%) than in Western patients (15%), and highly effective TKIs targeting mutant EGFR are in widespread clinical use. MET signaling is widely recognized to be an escape pathway to EGFR–TKIs, for which reason there is great interest in combining anti-MET with anti-EGFR therapies in EGFR-mutated NSCLC. Therefore, the increased prevalence in Asian patients may significantly impact where and how the trials are conducted.The potential regional difference in biology of a disease may affect biomarker evaluation. For instance, mutations in Development of anticancer therapeutics targeting the HGF/MET pathway has become a potential strategy in pharmaceutical research in the last two decades. A number of novel therapeutic agents, either therapeutic proteins or small molecules, have been tested in patients with different tumor types in clinical studies. Promising results have been obtained in the treatment of selected cancers. Accurate identification of the sensitive patient population and treatment with the right drug or drug combination is critical for success in the clinic.


In vitro, savolitinib inhibits MET phosphorylation with nanomolar potency, which correlates with blockade of PI3K/AKT and MAPK signaling as well as MYC down-regulation. In vivo, savolitinib causes inhibition of these pathways and significantly decreases growth of MET-dependent xenografts. To understand resistance mechanisms, we generated savolitinib resistance in MET-amplified NSCLC cell lines and analyzed individual clones. We found that upregulation of MYC and constitutive mTOR pathway activation is a conserved feature of resistant clones that can be overcome by knockdown of MYC or dual mTORC1/2 inhibition. Lastly, we demonstrate that mechanisms of resistance are heterogeneous, arising via a switch to EGFR dependence or by a requirement for PIM signaling. This work demonstrates the efficacy of savolitinib in NSCLC and characterizes acquired resistance, identifying both known and novel mechanisms that may inform combination strategies in the clinic.Lung cancer is the most common cause of cancer death globally with a significant, unmet need for more efficacious treatments. The receptor tyrosine kinase MET has been implicated as an oncogene in numerous cancer subtypes, including non-small cell lung cancer (NSCLC). Here we explore the therapeutic potential of savolitinib , a potent and selective MET inhibitor, in NSCLC.  MET as a key oncogenic driver of several human cancers, including small cell (SCLC) and non-small cell lung cancers (NSCLC) [and genetic background, which has opened the door to personalized medicine approaches. Recent molecular characterization of patient samples demonstrates that NSCLC arises from alteration of a relatively small subset of genes [MET copy number (CN) gain and exon 14 skipping, which together account for ~6.5% and 3.6% of driver mutations in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) cases, respectively [MET CN gain in 2-22% of patients, while others have observed MET overexpression in a high percentage of patients [The hepatocyte growth factor (HGF) receptor MET is a receptor tyrosine kinase (RTK) important in numerous biological functions ranging from embryonic development to tissue repair . When HG (NSCLC) . Until t (NSCLC) . Accordiof genes –6, incluectively . Additiopatients .Given the prevalence of MET aberrations across multiple cancer types, it is not surprising that MET has been a target of significant clinical interest and drug discovery efforts for several years. Two small-molecule multi-kinase inhibitors with MET inhibitory activity have been FDA approved: cabozantinib and crizotinib. The first of these, cabozantinib, is a multi-kinase inhibitor targeting RET, VEGFR2, KIT, TIE2, AXL and the FLT family of kinases in addition to MET  and was In vitro and in vivo, we find that savolitinib inhibits MET, PI3K/AKT and MAPK signaling and downregulates MYC expression in NSCLC models. We further show that acquired savolitinib resistance occurs through reactivation of downstream kinase signaling and is driven by aberrant mTOR activation, MYC over-expression and context-specific reliance on EGFR signaling. Lastly, we uncover a novel role for PIM kinases in acquired savolitinib resistance and show that PIM inhibition restores savolitinib sensitivity both in vitro and in vivo. Together, this study demonstrates the preclinical efficacy of savolitinib in NSCLC and elucidates both known and novel mechanisms of acquired MET inhibitor resistance, identifying possible patient stratification and drug combination strategies to combat potential savolitinib resistance in the clinic.Recently, the highly-selective and potent small-molecule MET inhibitor savolitinib  has been described , and worMET CN gain leading to MET dependence can predict MET small-molecule inhibitor sensitivity of tumor cells [in vitro interrogation, we determined savolitinib GI50 values for more than 900 cell lines present in the Sanger Cell Line Panel [In vitro, we found that those cell lines most sensitive to savolitinib were highly-amplified for MET, harboring ten or more copies  in 1.38% and 2.98% of LUAD and LUSC tumors, respectively, while high-level MET gain (CN >3) was present in 1.97% of LUAD and 1.39% of LUSC samples  and EBC-1 (LUSC) showed nanomolar savolitinib sensitivity, in agreement with previous work [versus other MET inhibitors, we performed a five-day proliferation assay comparing savolitinib to the selective MET inhibitors PHA-665752, JNJ-38877605 and SGX-523 as well as crizotinib, a multi-kinase inhibitor approved for clinical use. Savolitinib was more potent than all other molecules tested, with GI50 values of 4.20 nM and 2.14 nM in H1993 and EBC-1 cells, respectively , ~31% ± 10.49% or ~61% ± 6.82% (s.e.m.) TGI at 1, 3 and 30 mg/kg doses, respectively.  derived from a NSCLC lymph-node metastasis. The HLXF-036LN tumor model harbors 3.7 copies of MET and displays robust basal MET phosphorylation, indicating that MET is activated in this model  or human MYC and treated each with a range of savolitinib doses following RFP/MYC induction. Strikingly, MYC — but not RFP — overexpression partially rescued the effects of savolitinib treatment, even at 300 nM Figure ; MYC ovede novo dependence on EGFR signaling is a common means to circumvent MET inhibition [EGFR, thus, enhanced EGFR expression in resistant clones was independent of EGFR amplification . All EGTo determine if EGFR activation is a more general mechanism of savolitinib resistance in NSCLC, we assessed EGFR activation and EGFR inhibitor sensitivity in savolitinib-resistant EBC-1 cells. Indeed, resistant EBC-1 cells show increased basal EGFR phosphorylation that is unchanged following treatment with 100 nM savolitinib for up to 24-48 hours , suggestTo identify EGFR-independent mechanisms of resistance, we screened a panel of 36 small-molecules targeting multiple RTKs and downstream signaling kinases/targets. Data for a single concentration of each compound are shown in Figure Screen results were confirmed by treating resistant clones with a nine-point dose response curve of AZD2014, AZD8055 or PF-04691502, ranging from ~11.1 μM to ~1.7nM, in the presence or absence of 100 nM savolitinib, followed by viability measurements. Parental cells were treated with a nine-point dose curve of savolitinib or each of the mTORC1/2 inhibitors as single-agents only. All mTORC1/2 inhibitors showed single-agent activity in parental cells and in clone 3 and clone 6, reducing viability by ~90% and ~50-70%, respectively. In contrast, mTORC1/2 inhibitors were inactive in clone 11 Figure . CombinaConcurrent with small-molecule screening, we measured the phosphorylation status of a focused target panel using a phospho-protein array. We plated parental H1993 cells and clones in drug-free medium for 24 hours, treated with 0.1% DMSO or 2 μM savolitinib for four hours and applied the lysates to the arrays. MET phosphorylation was strongly downregulated in all four cell lines following savolitinib treatment Figure . InteresFurther analysis of the AKT/mTOR/S6 axis revealed additional clone-specific differences in the activation state of this pathway. First, we found that savolitinib treatment of parental H1993 and clone 11 cells strongly reduced phosphorylation of AKT and the direct AKT substrate PRAS40, a negative regulator of the mTORC1 kinase complex  Supplem. Thus, iAs further evidence of clone-specific savolitinib resistance mechanisms, we found that neither AKT nor PRAS40 phosphorylation were reduced by savolitinib treatment of clone 3 and clone 6 cells , and botde novo dependence on PIM kinase signaling, a novel mechanistic finding in the context of MET inhibitor resistance. Lastly, during preparation of our manuscript, An et al. reported that PIM signaling can mediate MET inhibitor resistance by regulating BCL2 translation [Using a candidate approach to identify non-canonical mTOR activators, we tested PIM kinase inhibitors. The PIM family can promote tumorigenesis , and PIMnslation , corroboversus grown in vivo. Thus, we first determined whether savolitinib resistance is maintained in vivo by implanting H1993 clone 11 cells sub-cutaneously into nude mice and treating tumor-bearing mice with a range of savolitinib doses. Indeed, H1993 clone 11 tumors are insensitive to savolitinib over a 100-fold range of doses, including the clinically-relevant dose of 30 mg/kg , phospho-ribosomal protein S6  and MYC proteins. Interestingly, the relative levels and modulation of all three proteins in both tumor models are strikingly consistent with our s Figure ; total Es Figure , as we oo Figure . S6 phoso Figure . Lastly,o Figure . Furtherin vivo, we next tested whether our in vitro findings could be extended in vivo by testing the effects of AZD1208 and AZD2014 treatment, alone or in combination with savolitinib, on the growth of H1993 clone 11 tumors. Nine days after sub-cutaneous implantation of H1993 clone 11 cells, tumor-bearing nude mice were randomized into treatment groups of ten mice per group. Treatment began the following day with each drug — AZD1208 , AZD2014  and savolitinib  — given alone or in combination with savolitinib. After 17 days of dosing, treatment with the pan-PIM inhibitor AZD1208 alone or savolitinib alone resulted in statistically-insignificant, tumor growth inhibition (TGI) of ~12% ± 9.6% s.e.m. relative to the vehicle-treated group  that potentially represent models of intrinsic savolitinib resistance. While almost nothing is known about the genetics of CS-1 cells, a more detailed analysis of the mutational landscape of OE33 cells revealed, among other variations, a striking HER2 (ERBB2) amplification of approximately 14 copies [Tumor cells harboring hibition . Our ana4 copies . It is t4 copies –45. ValiMET CN gain but savolitinib GI50 values below 1.0 μM (EW-11 and MV411). The precise mechanism(s) conferring such savolitinib sensitivity are not readily apparent for these cell lines; however, one study looking at determinants of sensitivity to another MET inhibitor, PHA-665752, found that while MET amplification was a strong predictor of MET inhibitor sensitivity, high levels of MET phosphorylation in the absence of MET amplification can also predict ‘intermediate’ sensitivity to PHA-665752 [MET CN gain is a strong predictor of savolitinib sensitivity, MET CN-independent factors can influence savolitinib sensitivity. Inclusion of such criteria will be critical to the success of savolitinib clinical trial design and patient stratification strategies in the future.Conversely, we found two cell lines with low or no A-665752 . In sum,MET or additional MET CN gains can drive resistance [MET mutation as a resistance mechanism. Exome sequencing of 45 genes, including MET, confirmed the absence of MET mutations but did reveal MET CN gains in some resistant clones; however, the biological significance of such MET gains is unclear given that MET is equally inhibited in all resistant clones irrespective of MET CN.Previous studies of acquired resistance to MET inhibitors have shown that activating mutations in sistance –49. HowePTEN deletion [PIK3CA mutations [PTEN loss or PIK3CA mutation, suggesting that an uncommon or novel mTOR activation mechanism may underlie savolitinib resistance.TKI resistance can arise through aberrant activation of the PI3K/AKT/mTOR axis in several cancer types, including NSCLC, often through deletion  or activutations ; howeverAdditionally, we observe MYC upregulation in all resistant H1993 clones and in a resistant EBC-1 population, suggesting that MYC may be a critical node upon which more diverse mechanisms converge. Indeed, we demonstrate that constitutive, elevated MYC expression actively promotes resistance to savolitinib in H1993 and EBC-1 cells. Interestingly, during preparation of this manuscript, Shen and colleagues reported that MYC can promote acquired resistance to another MET inhibitor, SGX-523 . Alternafunctionally assessing the relationships between gene products and drug resistance can reveal actionable, perhaps unexpected, vulnerabilities in drug-resistant cells regardless of gene expression, copy number, mutation or activation status [A switch to MET signaling can drive acquired resistance to EGFR inhibitors, such as erlotinib  and gefin status .in vitro. Interestingly, when we assessed the in vivo efficacy of combining the PIM inhibitor with savolitinib, in savolitinib resistant H1993 clone 11 tumors we observed similar anti-tumor activity as seen with savolitinib alone in H1993 parental tumors. This demonstrates that inhibition of PIM concurrent with MET inhibition resensitizes savolitinib-resistant tumors in vivo as well.Lastly, we demonstrate a role for PIM kinases in savolitinib resistance. Importantly, and to the best of our knowledge, our study and that of An et al  are the Intriguingly, clone 11 is the only clone sensitive to EGFR or PIM inhibition in the presence of savolitinib, leading us to hypothesize that that this particular clone may utilize a novel signaling pathway involving EGFR and PIM to circumvent MET inhibition. In support of this notion, Siu and colleagues previously demonstrated in prostate cancer models that PIM-1 inhibition upregulates MIG6 , a negatThe treatment of NSCLC with evermore potent and selective therapies is a boon to patients and a testament to our increasing knowledge of oncogenesis. Our study demonstrates savolitinib efficacy in preclinical NSCLC models representing adenocarcinoma, squamous cell carcinoma and metastatic disease. We uncover acquired resistance mechanisms in common between clones as well as clone-specific mechanisms, including identification of PIM as a novel mediator of resistance, laying the groundwork for combination strategies to combat savolitinib resistance that may arise in the clinic. Importantly, we illustrate the heterogeneity of potential resistance mechanisms within a single population of tumor cells, underscoring the need for careful molecular characterization of tumors from relapsed patients.2 in a humidified atmosphere. Savolitinib-resistant cells were generated by sequentially increasing the savolitinib concentration during culture until cells grew in the presence of 2.0 μM compound.NCI-H1993 (H1993) and NCI-H2009 (H2009) were purchased from ATCC . EBC-1 cells were purchased from the Japanese Collection of Research Bioresources (JHSF). All lines were authenticated by STR analysis  and used within six months of receipt. Cells were cultured in RPMI-1640 (Sigma-Aldrich) containing 10% FBS at 37°C/5% COMET CN frequency in NSCLC. MET CN gain was classified using both focality and amplitude, and samples were subsequently divided into either low-level (CN >2 but ≤3) or high-level (CN>3).Gene copy number (CN) data for LUAD and LUSC cohorts (TCGA) were used to estimate Cell line screening was performed as previously described .Preparation of protein lysates and immunoblot analyses were performed as described previously . Please 50 calculations were performed as described in [Drug treatment, cell viability measurements, curve fitting and ICribed in . Please All cell viability measurements were performed as described in  using Ce6 cells suspended in 0.2 mL 1:1 matrigel and randomized based on tumor volume; dosing began when tumors reached 120-275 mm3. For HLXF-036LN PDX studies , tumor fragments were passaged into NSG mice (The Jackson Laboratory) and allowed to grow to an average size of 250.0 mm3 and then randomized. Savolitinib was formulated in acidic CMC-Na 0.5% (pH = 2.1) and dosed orally once daily at the indicated concentrations for all studies. For pharmacodynamic analysis, tumors were lysed in ten volumes of lysis buffer containing protease and phosphatase inhibitors and dounce homogenized. Samples were centrifuged at 20,000g for 10′ at 4°C and the supernatants transferred to a new microfuge tube three times. Lysates were quantified by BCA assay and subjected to immunoblotting. All animal studies were performed according to AstraZeneca Institutional Animal Care and Use Committee guidelines.For H1993 and EBC-1 xenografts: 6-7 week-old female Balb/c nu/nu mice  were maintained in a controlled, specific pathogen-free environment at 20-25°C, 40-70% humidity and a photoperiod of 12 hours light-to-dark. Each mouse was injected subcutaneously in the right lateral flank with 5×10® RTK Signaling Antibody Array exactly as instructed by the manufacturer . Chemiluminescence was captured on an ImageQuant LAS 4000 instrument (Fuji). Densitometry analysis was performed using the ImageQuant TL Array software, V8.1 .Cells were incubated overnight without savolitinib, treated with 2 μM savolitinib for four hours and then lysed and subjected to the PathScanMYC: s9130 and s9129; non-targeting control #4390843) were reverse-transfected at 5 nM final concentration with Lipofectamine RNAimax (Life Technologies). MYC transcript levels were measured 72 hours later by qRT-PCR  - Taqman probes: GAPDH #1404051, MYC Hs00153408_m1 (Life Technologies). For viability experiments, cells were transfected with siRNA, exposed to DMSO or savolitinib 24 hours later, and viability was assessed after three days.Ambion Silencer Select siRNAs , selection with 0.5 μg/ml puromycin (Sigma-Aldrich) three days post-infection followed by culture in RPMI-1640 medium containing tetracycline-free serum and 0.5 μg/ml puromycin. For proliferation assays, 2,000 cells/well were plated in 96-well plates +/− 1 μg/ml doxycycline and treated with DMSO or savolitinib 24 hours later; viability was measured after five days. For immunoblot analyses, 5×10


Escherichia coli. A synthetic pathway of maleate is constructed in E. coli by combining the polyketide biosynthesis pathway and benzene ring cleavage pathway. The metabolic engineering approach used to fine-tune the synthetic pathway drastically improves maleate production and demonstrates that one of the rate limiting steps exists in the conversion of chorismate to gentisate. In a batch culture of the optimised transformant, grown in a 1-L jar fermentor, the amount of produced maleate reaches 7.1 g L−1, and the yield is 0.221 mol mol−1. Our results suggest that the construction of synthetic pathways by combining a secondary metabolite pathway and the benzene ring cleavage pathway is a powerful tool for producing various valuable chemicals.Maleate is one of the most important dicarboxylic acids and is used to produce various polymer compounds and pharmaceuticals. Herein, microbial production of maleate is successfully achieved, to our knowledge for the first time, using genetically modified  E. coli by combining polyketide biosynthesis and benzene ring cleavage pathways.Maleate is an important stock chemical for the production of polymer compounds and pharmaceuticals. Here the authors design a synthetic pathway for maleate in  Escherichia coli and yeasts have been widely studied in the past few decades3. These clean processes using renewable feedstocks as the raw materials are expected to provide solutions to the problems of global warming and exhaustion of fossil fuels. Metabolic engineering enables the construction of synthetic pathways for the production of valuable compounds using metabolically optimised microorganisms. Using rational approaches based on metabolic engineering and synthetic biology, various genetically modified microbes have been created to produce fuels, bulk chemicals, amino acids and pharmaceuticals6.Bioprocesses using conventional microorganisms such as 10. Maleic anhydride is easily obtained by heating maleic acid at 135 °C. The market volume for maleic anhydride was estimated to have reached 1,807,000 tons in 2007, which is much larger than the estimated volumes of other four-carbon dicarboxylic acids, such as succinic acid and fumaric acid 11. Maleic anhydride is chemically synthesised via catalytic oxidation of n-butane in the presence of vanadium phosphorus oxide under high temperature (>400 °C)12. Despite the high-global demand for maleic acid and maleic anhydride, there have been no reports on microbial production of maleic acid using renewable feedstocks.Maleic acid is one of the most valuable organic acids, and its derivative, maleic anhydride, can be converted into various polymer materials and pharmaceuticals17. Succinic acid can be easily converted to other valuable chemicals, such as 1,4-butandiol, and used for the production of the biodegradable plastic polybutylene succinate18. Fumaric acid is a starting unit for polymerisation and esterification reactions in the production of unsaturated polyester resins and food or beverage additives15. Production of these organic acids is usually achieved in microorganisms via the tricarboxylic acid (TCA) cycle. Some other organic acids, including 3-hydroxypropionic acid, malic acid and adipic acid, are also mainly synthesised via the TCA cycle21. Thus, extending the TCA cycle is the most common strategy to produce industrially important organic acids using microorganisms.Organic acids, including succinic acid, fumaric acid, malic acid and 3-hydroxypropionic acid, have been categorised as a key group among the top platform chemicals23 , we successfully produce maleate with high productivity, compared to the initially tested condition. Although a maleate synthetic pathway is successfully constructed in E. coli and the maleate production drastically increased, the rate and level of production are lower than those reported for other compounds produced by genetically engineered microbes. In the synthetic pathway of maleate created here, 2 mol of pyruvate are also produced. Recycling of pyruvate to maleate would be one of necessary steps to further increase the maleate production for industrial application in the future.In the present study, we attain the production of industrially valuable maleate using genetically engineered 27, we successfully produced 3HBA from glucose using genetically engineered E. coli, and the concentration of 3HBA reached more than 2 g L−1 in the test tube culture. To create an E. coli strain capable of producing gentisate from glucose, we used the 3HBA-producing E. coli strain which is the derivative strain of CFT5. Our base strain, CFT5, is a modified E. coli strain, with the genes involved in the reaction of conversion of phosphoenolpyruvate (PEP) to pyruvate completely inactivated. In this strain, the phosphotransferase (PTS) system was replaced with a combined system of galactose permease  and glucokinase (Glk), while the two genes encoding pyruvate kinase (pykF and pykA) were completely inactivated27.To construct a synthetic pathway for maleate production from renewable carbon sources, the first step is the reaction converting chorismate to 3HBA Fig. . In our cgl3026 from Corynebacterium glutamicum, 3hb6h from Rhodococcus jostii RHA1, and xlnD from Pseudomonas alcaligenes NCIMB 9867. Each gene encoding 3HB6H was introduced downstream of the hyg5 gene, which encodes the enzyme converting chorismate to 3HBA   Fig. .hbzF from P. alcaligenes NCIMB 9867 was adopted as the gene encoding MPH29. Using M9Y medium supplemented with gentisate (500–600 mg L−1), the gentisate-degrading ability of each strain (CFMP1–5) was evaluated. In this experiment, the additional gentisate was added to the medium after 4 h cultivation. Figure mps from Pseudomonas putida (sgp1) and CFMP4 carrying mps from Rhodococcus sp. strain NCIMB 12038 (mps2) produced 262 and 264 mg L−1 maleate, respectively. Surprisingly, CFMP1 carrying mps from Pseudaminobacter salicylatoxidans (mps0) assimilated gentisate more quickly than the other strains; however, the maleate production was not confirmed , the cell growth of CFT5 was evaluated. Figure 600) and the initial growth rate slightly decreased. The specific growth rate (μ) in the cultures with 0, 0.5, 1.5, 3 and 6 g L−1 maleate was 0.69, 0.64, 0.64, 0.64 and 0.65 h−1, respectively, between 2 and 4 h of cultivation. We also investigated whether E. coli assimilates maleate. Figure E. coli strain. In addition, no significant change was observed in the maleate concentration, indicating that the E. coli strain does not have the ability to assimilate maleate. These results suggested that E. coli was a suitable candidate host for maleate production from renewable feedstocks.Maleate is not usually produced in microbial metabolic pathways, unlike fumarate, which is the geometric isomer of maleate and one of the important compounds in the TCA cycle. Therefore, the tolerance of E. coli strains and those degrading gentisate to maleate were successfully created. We then continued with the construction of a synthetic pathway of maleate from glucose. First, four different MA modules  were combined with the two different GA modules , yielding eight different transformants. The MA and GA modules were tandemly cloned into pTrcHisB  were observed at ~3.2 min in the GC–MS spectra of the culture supernatant of CFM1; however, those were not detected in the culture supernatant of the control strain, CFM0, carrying the empty vector . Specific peaks derived from maleate–bis(trimethylsilyl) ester derivatives  was detected after 144 h of cultivation. To evaluate whether other by-products were formed, we assessed the amounts of produced organic acids  was detected after 144 h of cultivation, whereas CFMt2 carrying the GAMA module did not produce maleate  concentration used was optimised at 0.1 mM in our preliminary experiments , which improve the condensation of erythrose-4-phosphate (E4P) and PEP and transketolase (TktA), which enhances the E4P availability, were used as the candidates33. In this study, we used modified AroF and AroG, whose feedback inhibition was deregulated 35. The second group included genes whose expression levels increased in the salicylate production by CFT5, as we have previously reported27. Phosphoenolpyruvate carboxylase (Ppc), malic enzyme B (MaeB) and phosphoenolpyruvate synthase (PpsA) were selected as the candidates. The third group included genes involved in the glucose uptake system in CFT5. The genes encoding Glk and GalP were integrated into the genome of CFT5, whereas the glucose uptake PTS system was completely inactivated27. These two genes were also used as the candidates. The fourth group included genes needed to increase the carbon flux from chorismate to maleate, and hyg5 and the GA module were adopted as the candidates. All these genes were individually cloned into pSAK, which is a low-copy-number vector, and introduced into CFMt1.To further improve the maleate production, we tried to identify and improve the rate limiting step in the synthetic pathway of maleate production, focusing on several genes, which were classified into four categories Fig. . The firmps2, hbzF, hyg5 and 3hb6h. However, the maleate production by transformants carrying the genes classified in the other groups decreased. Figure −1 after 48 and 72 h of cultivation, respectively. In the case of CFMtS09, maleate and 3HBA were simultaneously produced until 24 h of cultivation, after which time the produced 3HBA was converted to maleate between 24 and 48 h of cultivation . These results should strongly support that maleate was produced mainly from glucose.To further improve the maleate production, we tried to enhance the carbon flux from chorismate to 3HBA by introducing pZAM01, which is a medium-copy-number vector carrying the ly Table . Althougly Table . We also−1. Figure −1 after 114 h of cultivation. However, the bacterial growth rate decreased after 24 h of cultivation  improved the maleate production and glucose uptake rate. In the test tube culture, CMtS091 (overexpressing two hyg5 genes in addition to the MAGA module) showed the highest yield and production rate among all transformants, which were 22.1-fold and 47.3-fold higher than those in CFM00, respectively. The maximum level of produced maleate was achieved in the culture of CMtS101 (overexpressing two hyg5 genes and one 3hb6h gene in addition to the MAGA module), which was 49-fold greater than that of CFM00. These results indicate that our platform strain CFT5 is versatile and can be used to synthesise valuable chemicals by extending the chorismate pathway. In this study, to increase the maleate production, three copies of the same gene involved in the production of 3HBA (hyg5) were introduced into three different vectors, and two copies of the same gene involved in the production of gentisate (3hb6h) were introduced into two different vectors. A total of seven genes were introduced into our base strain CFT5 using three vectors with different copy numbers in order to increase the carbon flux from glucose to maleate. The intracellular pool of chorismate is increased in CFT5 without additional introduction of genes enhancing the biosynthesis pathway of aromatic amino acids, whereas several genes, such as the feedback-deregulated aroF, aroG, aroB, aroL, tktA and ppsA, must be overexpressed when using general strategies designed to enhance this pathway41. Thus, our CFT5 strain, whose carbon flux to chorismate was increased without introduction of any genes, may have the advantage in creating strains carrying multiple genes involved in the production of aromatic compounds of interest.In the production of aromatic compounds and their derivatives, the bottleneck mainly occurs in the shikimate pathway. There are numerous reports describing deregulation of the feedback inhibition, enhancement of the shikimate pathway, and increased accumulation of PEP and E4P, carried out to increase the production of target compoundshyg5 genes and one 3hb6h gene in addition to the MAGA module) was performed. The concentration of maleate reached 7.1 g L−1 after 114 h of cultivation under optimised DO conditions. The yield of maleate from the carbon sources was 0.221 mol mol−1. Under optimised conditions, DO was shifted from 1.0 to 3.0 p.p.m. after 24 h of cultivation. In the synthetic pathway of maleate from chorismate, 2 mol of oxygen are essential to cleave the benzene ring and form maleylpyruvate and are considered one of the key factors. However, in the case of DO maintained at 3.0 p.p.m. throughout the cultivation, the initial bacterial growth until 24 h of cultivation was inferior to that observed under the optimised DO conditions (DO shifted from 1.0 to 3.0 p.p.m. after 24 h of cultivation) and under conditions of DO maintained at 1.0 p.p.m. during the entire culture. Although the accumulation of intermediates such as gentisate was not confirmed, the production of maleate also decreased compared to that obtained under the other two conditions  in the case of DO maintained at 1.0 p.p.m. during the entire culture.Using a 1-L jar fermentor, a batch culture of CMtS101 , has been widely researchedhyg5 from S. hygroscopicus ATCC 29253, 3hb6h from R. jostii RHA1, mps2 from Rhodococcus sp. strain NCIMB 12038, and hbzF from P. alcaligenes NCIMB 9867 were selected as the suitable genes. The carbon flux from chorismate to gentisate was a significant step in the pathway. In the constructed maleate synthetic pathway, 2 mol of pyruvate are released to produce 1 mol of maleate  were used for gene cloning. Polymerase chain reaction (PCR) was performed using the KOD FX Neo DNA polymerase  and the appropriate primer pairs. Each gene was assembled with the respective plasmid using Gibson Assembly . Plasmids were transformed into bacterial strains using a Gene Pulser II . Where applicable, 100 µg mL−1 ampicillin, 50 µg mL−1 kanamycin, and 15 µg mL−1 chloramphenicol were added to media for selection.The strains, plasmids and oligonucleotides used in this study are summarised in Supplementary Tables cgl3026 from Corynebacterium glutamicum, 3hb6h from Rhodococcus jostii RHA1, and xlnD from Pseudomonas alcaligenes NCIMB 9867, optimised for the Escherichia coli codon usage, were obtained from a commercial source (Invitrogen)    (Novagen), and the resulting plasmids were designated pETM1.pETM1 was constructed as follows. The All of the transformants constructed in this study are listed in Supplementary Table 2HPO4·12H2O, 17.1 g; KH2PO4, 3 g; NH4Cl, 2 g; MgSO4·7H2O, 246 mg; CaCl2·2H2O, 14.7 mg; FeSO4·7H2O, 2.78 mg; thiamine hydrochloride, 10 mg; L-phenylalanine, 100 mg; L-tyrosine, 40 mg; and L-tryptophan, 40 mg.  For the culture of CFT5 derivative strains, 10 mM sodium pyruvate was added to the medium to encourage bacterial growth in the initial phase. When needed, ampicillin, kanamycin and/or chloramphenicol were added to the medium to a final concentration of 100, 50 and 15 µg mL−1, respectively. Each preculture was seeded to 5 mL of M9Y medium in a test tube at an initial OD600 of 0.05. Tube-scale cultures were incubated at 37 °C in a shaker at 180 r.p.m. IPTG (0.1 mM) was added to the culture medium at OD600 of 0.5.M9Y medium was used for maleate production in 5-mL test tube-scale cultures. M9Y minimal medium contains (per litre): glucose, 20 g; yeast extract, 5 g; NaCl, 0.5 g; Na4OH was automatically added to the medium. DO was maintained at 1.0–3.0 p.p.m. by automatically controlling the agitation speed from 200 to 800 r.p.m. and supplementing with air (pre-warmed to 37 °C) when needed. In this experiment, 0.1 mM IPTG was added to the medium 2.5 h after the initiation of cultivation. For cultures supplemented with 10 mM sodium pyruvate, the amounts of consumed glucose and sodium pyruvate were used for yield estimation, which was calculated as Yield (mol mol−1) = /.Batch-scale cultures were performed in a 1.0-L jar fermentor with a 400-mL working volume. M9Y medium supplemented with 1% yeast extract was used for maleate production at this scale. The culture medium (400 mL) in the jar fermentor was inoculated with 20 mL of a preculture. To maintain the pH at 7.0 during cultivation, NHRhodococcus sp. strain NCIMB 12038 (Mps2) was performed as follows. pETM1 was introduced into the BL21 (DE3) pLysS strain of E. coli, and the resultant transformant was named BL21MP. This transformant was grown in Luria–Bertani medium to an optical density (OD600) of 0.5 at 37 °C, and then the cells were incubated for an additional 30 min at 25 °C. Protein expression was induced by the addition of IPTG to a final concentration of 0.5 mM. After incubation for an additional 24 h at 25 °C, the cells were collected by centrifugation. The cell pellet was resuspended in 50 mM phosphate buffer containing 150 mM NaCl (pH 8.0) and lysed by sonication, followed by Mps2 purification using a TALON metal affinity resin  according to the manufacturer’s protocol.Expression and purification of gentisate 1,2-dioxygenase from m/z of 185 and 140, respectively. Thus, maleylpyruvate was successfully synthesised from gentisate using Mps2.The enzymatic synthesis of maleylpyruvate from gentisate was conducted as follows. A reaction mixture (1 mL) containing 26 mM gentisate and 123 μM purified Mps2 in 50 mM Tris-HCl buffer pH 7.5) was incubated for 2 h at 30 °C. The reaction mixture, as well as samples of culture supernatants, were analysed using HPLC. Supplementary Fig. .5 was in600 with a UVmini-1240 spectrophotometer . The concentration of glucose in the culture supernatant was measured using the Glucose CII test  following the manufacturer’s protocol.Cell growth was monitored by measuring OD−1. The injection volume was 1 μL with a split ratio of 1:10. The amounts of produced maleate, gentisate and 3HBA were quantified as follows. The oven temperature was initially held at 150 °C for 1 min, then raised to 240 °C at 12 °C min−1, raised further to 300 °C at 120 °C min−1, and finally held at 300 °C for 3 min. The total running time was 10 min. The other settings were as follows: interface temperature, 250 °C; ion-source temperature, 200 °C, and electron impact ionisation at 70 eV. Dried residues of maleate, gentisate and 3HBA were derivatised for 60 min at 80 °C in 50 µL of N-methyl-N-trimethylsilyltrifluoroacetamide and 20 µL of pyridine prior to the analysis46. Sorbitol was used as the internal standard.GC–MS was performed using a GC−MS-QP2010 Ultra instrument (Shimadzu) equipped with a CP-Sil 8 CB-MS capillary column . Helium was used as the carrier gas to maintain a flow rate of 2.1 mL ming for 20 min. The culture supernatant was analysed by HPLC (Shimadzu) using a 5C18-PAQ column . The column was operated at 30 °C with a flow rate of 1.2 mL min−1. A dual-solvent system was used, consisting of solvent A  and solvent B (acetonitrile). The gradient was initiated at 100% A (0–3 min), gradually shifted to a 50:50 mixture of A and B (3−6 min), which was kept between 6 and 7 min, and subsequently shifted to 100% A (7−13 min). Product concentrations were determined using an ultraviolet absorbance detector  at 210 nm.The maleate concentration was determined in a culture supernatant, which was separated from the culture broth by centrifugation at 21,880×g for 20 min. The concentrations of organic acids as by-products were determined using an organic acid analysis system (Shimadzu) consisting of an HPLC instrument equipped with a Shim-pack SPR-H column. The column was operated at 48 °C with a flow rate of 0.8 mL min−1. CDD-10A was used as the detector. p-Toluenesulphonic acid at a 5 mM concentration was used as the mobile phase, and 20 mM bis-Tris containing 5 mM p-toluenesulphonic acid and 100 μM ethylenediaminetetraacetic acid was mixed immediately before the detection to enhance the sensitivity.Concentrations of organic acids were also determined in culture supernatants, which were separated from the culture broth by centrifugation at 21,880×The data that support the findings of this study are available from the corresponding author upon reasonable request. The NCBI accession codes for the proteins used in this study are summarised in Supplementary Table Supplementary InformationPeer Review File


Cracking due to temperature and restraint in mass concrete is an important issue. A temperature stress testing machine (TSTM) is an effective test method to study the mechanism of temperature cracking. A synchronous closed loop federated control TSTM system has been developed by adopting the design concepts of a closed loop federated control, a detachable mold design, a direct measuring deformation method, and a temperature deformation compensation method. The results show that the self-developed system has the comprehensive ability of simulating different restraint degrees, multiple temperature and humidity modes, and closed-loop control of multi-TSTMs during one test period. Additionally, the direct measuring deformation method can obtain a more accurate deformation and restraint degree result with little local damage. The external temperature deformation affecting the concrete specimen can be eliminated by adopting the temperature deformation compensation method with different considerations of steel materials. The concrete quality of different TSTMs can be guaranteed by being vibrated on the vibrating stand synchronously. The detachable mold design and assembled method has greatly overcome the difficulty of eccentric force and deformation. Cracking due to temperature and restraint in mass concrete, such as concrete dams, highway pavement, and bridge decks, can be easily observed. It is a critical concern to study the mechanism of temperature cracks through the testing method ,2. To in−6/°C, to eliminate the thermal effect on the specimen. Currently, two types of the concrete temperature control methods are provided, primarily. One type uses a fluid-cooled temperature control formwork, which controls the temperature history of concrete by adjusting the temperature of the flowing fluid in the formwork. The other type uses an air environmental chamber, where the concrete specimen is placed into the small environmental chamber, and the temperature of the concrete specimen is affected by adjusting the temperature and flowing of the air in the environmental chamber [Both ends of the specimen for cracking frame are held by two cross-heads . The cro chamber ,6. The c2); 2) of the steel shaft. In reality, creep and damage could affect the concrete so that Researchers have carried out many tests for concrete cracking with the cracking frame ,9,10,11.To achieve a certain restraint condition, the TSTM was developed by Gierlinger and Springenschmid . Its schThe deformation measurement method. It is important to determine the value of shrinkage, elastic modulus, and creep. Several methods have been adopted, such as monitoring the displacement of the movable grip ,15,16,17Temperature control method. Some devices without thermal regulation systems have been designed to meet the purpose of studying the shrinkage property ,24,25. FConcrete quality. Concrete is poured into the mold of the TSTM directly and a vibrating rod is used to guarantee the concrete quality. When multi-TSTMs are employed, the homogeneity of all samples is hardly to be guaranteed by vibrating the sample separately with a handheld vibration rod. The representation and applicability of the test results will be influenced.The TSTM has been developed by many researchers in recent years and the development history of the TSTM has been reported by Staquet . The desA certain number of concrete samples should be conducted to guarantee that the test results are representative and acceptable. To develop a multi-TSTM system, some designs and methods could be developed.To improve the shortcomings of the TSTM, a synchronous closed loop federated control TSTM system is developed and will be introduced in detail as follows.Referring to The walk-in environment simulation laboratory may simultaneously accommodate a plurality of TSTMs therein, and four TSTMs have been placed in the same environmental simulation atmosphere, which can achieve synchronous control of multiple machines. The system is, thus, named the synchronous closed loop federated control TSTM system, and the system has some obvious advantages. First, multi-concrete specimens of temperature stress tests can be conducted synchronously under the same environmental conditions, so that the discretization errors of results can be decreased, enabling the testing results to be more representative and have a broader application scope. The test set parameters of four TSTMs, such as the restraint degree, can be the same or different according to the test requirements. Many parallel tests can be carried out and the test efficiency is improved greatly. Second, no obvious fluctuation of the concrete temperature appears before or after the concrete specimen demolding, which is hardly to be realized by a fluid-cooled formwork . FinallyThe concrete specimen casting mold is designed to be detachable from the TSTM host part to modify the problems in The fixed ends are placed on the working platform. Eight height-adjustable ball supporting points and two bottom formwork supports are designed on the platforms, which can allow the concrete to move on it. The movable cross-head and the steel shaft are connected by the sliding sleeve of which only the longitudinal direction (X direction) is allowed. The detachable mold design has some advantages. First, multi-concrete specimens can be vibrated on the vibrating stand synchronously, so the quality of multi-concrete specimens can be guaranteed. Parallel tests could be conducted so that results are more representative and test efficiency is greatly improved. Second, formworks can be detached during the test, so that the influence of friction is eliminated. Finally, it is more convenient for the tester to adjust some operations during the experiment.The design program of the direct measuring deformation method is shown in −6/°C for 4J36 invar) and α’ (12 × 10−6/°C for 45# steel). The steel shaft is made from 4J36 invar and its length is L1. The parts of the left fixed end, the right fixed end, and the snap joint are also 4J36 invar and the total longitudinal length is L2. The fixed cross-head, the moveable cross-head, and the load cell are made from 45# steel, wherein the total length is L3. Referring to All components of the TSTM are in the varied environmental atmosphere, and the temperature deformation is inconsistent as the material thermal expansion coefficients of the concrete and the steel are different. The deformation of the concrete may include the contribution of the temperature deformation caused by the steel shaft and other steel components. A compensation method of temperature deformation is proposed to eliminate the impact of the temperature deformation of the steel components on the concrete specimen deformation. Two different steel materials, 4J36 invar and 45# steel, are adopted. The thermal expansion coefficient is α  to evaluate the concentricity of the TSTM.To evaluate the effect of the temperature deformation compensation method, the temperature deformation test is designed. The parts of the steel shaft, cross-head, and the snap joint in The temperature stress test of concrete is conducted and the TSTM performances, such as simulating the different restraint degrees, multiple temperature regulation modes, and closed-loop control of multi-TSTMs, are verified. Four concrete specimens numbered as TSTM-A, TSTM-B, TSTM-C, and TSTM-D are poured into the TSTMs, correspondingly. The thin film is placed between the concrete and the formwork, and the lubricant is used for reducing the friction and making the demolding easier.Test Start TimeThe method to determine the beginning of the test has been discussed by the researchers. Altoubat  states tAfter 25 h, the formworks are demolded as the concrete strength has increased and the direct measuring deformation method is adopted by the upper side of the deformation sensor, as described in Multi-TSTM SettingsFour TSTMs are all tested in the same environmental atmosphere, for which the air relative humidity (RH) is 80%. TSTM-A is set as the free specimen and the reference of TSTM-C. The compensation cycle of TSTM-C begins when the increment value of the free specimen strain exceeds 2 με (2 μm for 1 m measured distance) and the restraint degree of TSTM-C is 50%. TSTM-D is set as 100% restrained, which is implemented by the motor adjusting the concrete to its original position when the concrete strain exceeds 2 με.TSTM-B is also set as an almost free specimen, of which the restraint degree is 5%. To measure the elastic modulus (E), a compressive load about 45 kN (before 90 h) and 60 kN (after 90 h) is applied on the specimen to generate deformation every 12 h, which is called “the active method of elastic modulus determination” . E is caThe method called standard method  is also Temperature ModesTo evaluate the temperature control ability of the TSTM system, three temperature modes are designed. No. 1, from four hours to 70 h, the mode of simulating the semi-adiabatic condition for concrete is designed and the phase is named as the temperature following phase, which is implemented by controlling the environment temperature the same as the internal temperature of the concrete. No. 2, to simulate the temperature history in the dam by water cooling, a designed cooling temperature curve is input and this phase is named as the design cooling phase (70–135 h). No. 3, the cold wave weather case, which is common in dam engineering, is designed by cooling down the environment temperature at the rate of 1 °C/h to obtain a crack (after 135 h).The specific processes of the temperature results are shown in top-dave ranges between −2 με and 3 με in all loading processes. The difference between the top deformation value and the average deformation value is quite small, which shows that only adopting the top side deformation measuring device during the test is proper to achieve the aim of directly measuring the concrete deformation. After the centricity assessment test, the sample is pulled to failure, and the failure pattern is shown in The concrete specimen was pushed and pulled by the actuating motor to verify the eccentric degree of the self-developed TSTM. The results in In addition, the shape of the load curve in −6/°C for the temperature ranging from −20 °C to 100 °C. When the temperature of the steel shaft varies from 20 °C to 60 °C, the concrete deformation varies by only 0.3 μm (the measuring distance is 1 m). However, the temperature deformation of concrete varies from 0.3 μm to 1.6 μm when the temperature increases from 60 °C to 70 °C.As shown in The concrete temperature is kept constant at 20 °C and the testing time is approximately one hour, so that the concrete deformation is only caused by the external temperature deformation. The temperature deformation of the steel shaft is approximately 150 μm as the temperature varies from 20 °C to 70 °C. However, the deformation magnitude of the concrete specimen is 1.6 μm, which is only approximately 1% of the steel shaft, and negligibly small. The temperature deformation of the TSTM, mainly contributed by the steel shaft, will transfer into the concrete if the compensation method was not adopted. Consequently, the motor will start, caused by the pseudo-deformation. The results indicate that the compensation method of the temperature deformation with different steel materials combined with each other can eliminate the vast majority of the effects of the external temperature deformation on the concrete specimen.Temperature Regulation ResultsDuring the temperature following phase, the concrete temperatures of four TSTMs develop synchronization in Temperature Stress Test Results of Multi-TSTMsFour TSTMs are operated synchronously with different setting conditions. The specimen of TSTM-D cracks first when the temperature is reduced to 25.67 °C. The stress and strain curves are drawn in The restraint degree of TSTM-C is 50% and TSTM-A is the reference free specimen. The cracking temperature of TSTM-C is 20.7 °C, which is 4.97 ˚C less than that of TSTM-D. The results show that the concrete cracks more easily under the strong restraint condition. The failure stress is 1.02 MPa and the failure strain is 115.75 με. The failure strain of TSTM-C is far greater than that of TSTM-D due to the different restraint degree. The creep strain is smaller than that of TSTM-D. The specific stress and strain curves are drawn in E ResultsTSTM-B is set as an almost free specimen to evaluate the elasticity modulus every 12 h. The E–time relationship is shown in The specimen is pulled to failure at the rate of 0.3 mm/min after the specimen of TSTM-C cracks; meanwhile, the concrete age is approximately six days. The stress-strain curve is drawn in The results indicate that the “active method of determining elastic modulus” may damage the sample. The micro-damage is not obvious in compression, but could be greatly affected in tension. The difference of E values derived from the active method and the standard method is small, so the active method is also an effective method. However, the active method is not recommended to be applied on the restraint specimen of the TSTM because the frequent tension and compression will influence the thermal and mechanical properties of the concrete under the effects of restraint and temperature. The E values of the active method and the standard method show little difference, indicating that the E value does not depend on the dimension of the sample , but couIt has the comprehensive ability of simulating different restraint degrees, multiple temperature and humidity modes, and closed-loop control of multi-TSTMs during one test period.The environment simulation laboratory system, including a walk-in environment simulation laboratory, allow for multi-concrete specimens of temperature stress tests to be conducted synchronously under the same environmental conditions. Additionally, no obvious fluctuation of the concrete temperature appears before or after concrete specimen demolding. The operation is more flexible during the test as the TSTM is no more restrained by the small chamber and fluid-cooled formwork. Finally, all parts of the TSTM are in the same environment, which reduces the disturbance caused by the external temperature.The deformation measuring design with embedded cross part can obtain more accurate deformation and restraint degree results with little damage.The compensation method of temperature deformation with different considerations of steel materials can eliminate the external temperature deformation’s effects on the concrete specimen.Some design concepts, such as the sliding sleeve on the steel shaft, height-adjustable ball supporting points, cross-head positioning fixture, and the mold assembly platform, have been taken into consideration so that the self-developed TSTM has greatly overcome the difficulty of eccentric force and deformation.The detachable mold design of TSTM guarantees that the concrete quality of different TSTMs is similar to the greatest extent by being vibrated on the vibrating stand synchronously.The active method should be used for determining E on a specific TSTM without any other application, such as restraint testing, because the frequent tension and compression will cause damage which will influence the mechanical properties in tension.The self-developed synchronous closed loop federated control TSTM system is an effective device for researching the mechanical and thermal properties, and it has the following characteristics: