The Diagnostic Accuracy of Liquid Biopsy in EGFR-Mutated NSCLC: A Systematic Review and Meta-Analysis of 40 Studies.

Epidermal growth factor receptor (EGFR) mutations are the most common carcinogenic driver mutations in non-small-cell lung cancer (NSCLC) patients, while invasive tissue biopsy has certain inherent defects. PubMed, Ovid Medline, Embase, and the Cochrane Library were systematically searched on January 4, 2020, using the keywords "liquid biopsy," "EGFR," and "NSCLC." The pooled sensitivity and specificity of EGFR mutations in paired tissue and blood were calculated. The accuracy was assessed by receiver operating characteristic curve. The meta-regression of the subgroup was performed to analyze the heterogeneity. Hazard ratio (HR) and 95% confidence interval (CI) were combined for evaluating the impact of EGFR mutation in tissue and liquid blood biopsy. A total of 40 studies with 5,995 patients were involved in the study. The pooled sensitivity was 68% (95% CI = 60-75%), and the specificity was 98% (95% CI = 95-99%). The diagnostic odds ratio was 88 (95% CI = 40-195), and the area under the curve was 0.91 (95% CI = 0.88-0.93). In the meta-regression, the sensitivity and specificity remain lower in the Asian studies than non-Asian studies (sensitivity: 66% vs. 73%, P = 0.04; specificity: 96% vs. 97%, P = 0.03, respectively). The EGFR mutation was associated with a better progression-free survival than wild type in both tissue (HR = 0.54, 95% CI = 0.34-0.85, P = 0.007) and blood (HR = 0.81, 95% CI = 0.71-0.92, P = 0.001) detection. Peripheral blood liquid biopsy had a better specificity for detecting EGFR mutation in NSCLC patients, while tissue biopsy still needs to be undertaken for negative blood biopsy patients due to its lower sensitivity.

The IL-33/ST2 pathway suppresses murine colon cancer growth and metastasis by upregulating CD40 L signaling.

Interleukin (IL)-33 is a member of the IL-1 family, participating in both helper T1 (Th1)- and Th2-type immune responses, but its ambiguous effects on tumor growth and related immune mechanisms remain unclear. Here, we report that recombinant mouse IL-33 (mIL-33) significantly inhibited colon cancer growth and metastasis to lung and liver in a murine CT26 or MC38 tumor-cell engraftment model. This effect could be associated with CD4+ T cells and CD40 L signaling, as depletion of CD4+ T cells or blocking CD40 L signaling in vivo partly abolished the antitumor function of IL-33. In addition, IL-33 treatment upregulated CD40 L expression on tumor-infiltrating lymphocytes, and promoted the activation of CD4+ T, CD8+ T and natural killer cells via CD40 L signaling. Furthermore, IL-33 was sufficient to induce the ST2 expression on CD4+ T cells, but not on CD8+ T and natural killer cells, indicating that IL-33 acted on CD4+ T cells via a positive-feedback loop. Our findings shed new light on the IL-33-mediated antitumor effects and mechanisms of Th1 action, and also suggest that IL-33 may serve as an activator to boost anticancer immune responses in singular or combinatory therapies.

Pharmacokinetics of midkine with different N-terminal structures in rats.

Midkine (MK) is a heparin-binding growth factor that functions in multiple physiological processes, making it a promising drug target for treating various diseases including osteoarthritis (OA). However, the lack of pharmacokinetic studies on MK limits further clinical research. As the N-domain of MK protein appears to be more important for its stability, this study aimed to investigate the pharmacokinetic profiles of recombinant human (rh)MK with different structures at the N-terminus via different administration routes in rats and guinea pigs. A single intramuscular (IM) injection of 1 mg/kg rhMK with or without extended sequences at the N-terminus expressed by E. coli or Pichia was administered to six male SD rats. rhMK concentrations in sequential tail blood samples were measured by ELISA. rhMK without extended N-terminal sequences expressed by Pichia had a greater area under the curve (AUC), slower clearance, and longer half-life in rats following a single IM injection than those of the other rhMK proteins. The AUC values for rhMK after IM and intra-articular (IA) administration were 1523.3 ± 35.2 h × ng/mL and 872.0 ± 36.1 h × ng/mL, whereas the apparent volumes of distribution (Vd/f) were 0.184 ± 0.067 L/kg and 11.6 ± 0.8 L/kg, respectively, suggesting that rhMK was distributed more locally after IA injection than after IM injection as Vd/f magnitude gives a general idea of extent distribution in the body and higher Vd/f represents more locally distribution. rhMK concentration in the articular cartilage was markedly higher than that in serum and reached the highest level at 3 days after a single IA injection in Hartley guinea pigs. As the dose increased from 10 to 50 mg/kg, the AUC increased in a greater-than-dose-proportional manner, suggesting that rhMK exhibits non-linear pharmacokinetics in rats after a single IM injection in this dose range. These results indicated that the N-terminal structure and administration route have substantial effects on the pharmacokinetics of rhMK in rats. Furthermore, rhMK was maintained in articular cartilage with minimal diffusion into the blood following IA injection in Hartley guinea pigs, providing a foundation for clinical research on the use of rhMK for OA treatment via IA delivery.

Midkine promotes articular chondrocyte proliferation through the MK-LRP1-nucleolin signaling pathway.

Osteoarthritis (OA) is the most common disease of joint tissues; unfortunately, there are currently no curative therapies available for OA. Chondrocytes, the only cell type residing in cartilage, secrete many types of collagen (the mainly one is type II collagen) and aggrecan, which are the main components of the cartilage matrix. Chondrocyte apoptosis can lead to OA degenerative progression. We previously indicated that recombinant human midkine (rhMK), as a chondrocyte growth factor has a significant reparative effect on cartilage injury animal models. However, the molecular mechanism of this restorative function remains under investigation. Herein, we focused on the molecular mechanism underlying the role of MK in promoting the proliferation of chondrocytes cultured in vitro. Chondrocytes from rats and OA patients were successfully isolated by the digestion of articular cartilage using type II collagenase, and their proliferation was evaluated by a CCK8 assay and flow cytometry. rhMK stimulated the proliferation of chondrocytes from both OA patients and rats. Furthermore, qRT-PCR, shRNA-mediated knockdown, Western blot and immunoprecipitation (IP) assays were performed to identify the receptor and key elements responsible for the role of MK in promoting chondrocyte proliferation. Low-density lipoprotein receptor-related protein 1 (LRP1) was identified as the dominant MK receptor in chondrocytes that, as a translocator, mediates the endocytosis of MK. After being transferred into chondrocytes, MK was shown to form a complex with nucleolin that interacts with the active form of K-Ras. Upon the activation of ERK1/2, cyclin D1 expression was upregulated, promoting the chondrocyte cell cycle. Our data reveal for the first time the role of the MK-LRP1-nucleolin signaling pathway in facilitating MK-induced chondrocyte proliferation, thus providing a strong theoretical foundation for the further use of MK in OA clinical therapy.

IL-1Ra protects hematopoietic cells from chemotoxicity through p53-induced quiescence.

The protection of constantly proliferating gut epithelia and hematopoietic tissues from cytotoxicity could improve conventional chemotherapy efficacy and widen its therapeutic window. Previously, we reported that, in mouse models, pretreatment of recombinant human IL-1 receptor antagonist (rhIL-1Ra) protected both types of vulnerable tissues from chemotherapeutics. Here, we showed that rhIL-1Ra treatment up-regulated the protein levels of phosphorylated p38, p53, and p21 and induced transient hematopoietic stem/progenitor cell (HS/PC) quiescence. Knockout of IL-1 receptor I (IL-1RI), p53, or p21 alleles and pharmacological inactivation of p38 mapped the rhIL-1Ra pathway in the induction of HS/PC quiescence. Therefore, rhIL-1Ra administration before but not after chemotherapy alleviated 5-fluorouracil-induced neutropenia. In addition, in vivo and in vitro cell proliferation assays revealed that the rhIL-1Ra treatment did not affect cancer cell proliferation or chemosensitivity. Lastly, we propose an IL-1/IL-1Ra pathway (IL-1RI → p38 → p53 → p21), which regulates HS/PC quiescence. The rhIL-1Ra may provide a new route for p53-based cyclotherapy, which spares normal cells but kills cancer cells during chemotherapy.-Ye, H., Qian, L., Zhu, S., Deng, S., Wang, X., Zhu, J., Chan, G. L., Yu, Y., Han, W. IL-1Ra protects hematopoietic cells from chemotoxicity through p53-induced quiescence.

Large-scale gene analysis of rabbit atherosclerosis to discover new biomarkers for coronary artery disease.

Atherosclerosis is the pathological basis of coronary artery disease (CAD) and causes high mortality. Thus, early detection is thought to be crucial in reducing the risk of CAD. Uncovering the mechanisms of the progression and regression of atherosclerosis will provide insights into discovering novel biomarkers to identify subjects at risk for CAD and improve prevention. We established atherosclerosis progression and regression in a rabbit model. Then, we extracted mRNA of the abdominal aorta from control, model and recovery groups to perform gene chip analysis. Candidate biomarkers were screened by large-scale gene analysis and validated in patients with CAD or with CAD recovery by ELISA. The differentially expressed genes in the progression and regression of atherosclerosis were mainly enriched in four clusters. Genes associated with inflammation and extracellular matrix were returned to normal or close-to-normal levels much earlier than genes associated with metabolism and sarcoplasmic proliferation, and they were maintained downregulated or upregulated after feeding a normal diet. We then selected four candidate biomarkers and found that lipoprotein lipase (LPL), bone morphogenetic protein 7 and somatostatin concentrations could indicate CAD diagnosis. In addition, LPL and macrophage cationic peptide 2 can be indicators of the prognosis of CAD. Molecular changes during the progression and regression of atherosclerosis in rabbits were revealed, and candidate regulators were identified. The identified factors could be used as novel biomarkers and targets for improving the diagnosis and prognosis of human CAD in the future.

Molecular dynamics based improvement of the solubilizing self-cleavable tag Zbasic-ΔI-CM application in the preparation of recombinant proteins in Escherichia coli.

Zbasic-ΔI-CM is a novel intein-based self-cleavable tag we developed to accelerate the soluble expression of recombinant proteins in Escherichia coli (E. coli). Previously we found that intein activity could be interfered by its flanking exteins, and thus reducing the production efficiency and final yield. In this work, we used CXC-chemokine 9 (CXCL9) as a model C-extein, which fusion with Zbasic-ΔI-CM showed high intein activity. When the fusion protein got soluble expression, CXCL9 was released immediately and purified directly from cell lysis supernatant. The results demonstrated that Zbasic-ΔI-CM tag had successfully mediated the efficient production of high-quality CXCL9 with reduced time and resources consumption in comparison with inclusion bodies expression. Molecular dynamics simulations suggested that the improved cleavage activity of Zbasic-ΔI-CM upon fusion with CXCL9 may be due to the higher dynamics of the first half loop and stabilization of the second half loop of intein. Our results proved that the self-cleavable Zbasic-ΔI-CM mediated soluble expression could be a feasible process for cytokines like CXCL9, thus of attractive potentials for production of therapeutic proteins using E. coli expression system.

Serum Triglyceride Lipase Concentrations are Independent Risk Factors for Coronary Artery Disease and In-Stent Restenosis.

AIM: Endothelial lipase (EL), hepatic lipase (HL), and lipoprotein lipase (LPL) are all triglyceride lipases and are associated with coronary artery disease (CAD). However, whether they can be simultaneous independent risk factors for CAD is unknown. In the present study, we investigated whether the three lipases can be independent risk factors simultaneously for CAD and whether combining these lipases could provide greater predictive power than high-density lipoprotein cholesterol (HDL-c) for the development of CAD. METHODS: Eighty-six patients with CAD and 65 healthy controls were enrolled in the study. Additionally, 38 patients who underwent one-year follow-up angiography after percutaneous coronary intervention with stent implantation were collected to investigate in-stent restenosis. Serum EL, HL, and LPL concentrations were measured and compared with other coronary risk factors. RESULTS: Serum EL and HL concentrations were both significantly increased in patients with CAD or in-stent restenosis, whereas serum LPL concentration was reduced significantly in patients with CAD. Multivariate logistic regression analysis indicated that the three lipases were simultaneous independent risk factors for CAD. However, only serum EL concentration was considered an independent risk factor for in-stent restenosis. Importantly, the receiver operating characteristic curve showed that the combined measurement of the three lipases displayed better predictive power than HDL-c or any one of the three lipases for CAD. CONCLUSIONS: Serum EL concentration was an independent risk factor for both CAD and in-stent restenosis. Moreover, the combined assessment of serum EL, HL, and LPL concentrations as multiple risk factors provided potent predictive power for CAD.

Non-platelet-derived CXCL4 differentially regulates cytotoxic and regulatory T cells through CXCR3 to suppress the immune response to colon cancer.

CXCL4 is mainly produced by activated platelets, and certain somatic cells and cancer cells also express CXCL4. However, the physiological function of non-platelet-derived CXCL4 is unclear. Previously, we reported that CXCL4 produced by cancer cells accelerated tumor growth by suppressing the antitumor activities of cytotoxic T lymphocytes (CTLs). To elucidate the mechanism of CXCL4 in tumor immunity, we compared the CTLs and regulatory T cells (Tregs) from CXCL4-/-, CXCR3-/- and C57BL/6 mice overexpressing CXCL4 via intramuscular electroporation. CXCL4 accelerated tumor growth in CXCL4-/- and C57BL/6 mice but not in CXCR3-/- mice. Furthermore, CXCL4 decreased CTLs proliferation and IFN-γ production and enhanced CTLs apoptosis and programmed death 1 (PD-1) expression. Conversely, CXCL4 promoted Tregs proliferation and TGF-ß production and downregulated PD-1 expression in Tregs. Notably, these effects of CXCL4 were both observed in the splenic and tumor-infiltrating CTLs and Tregs from wild-type but not CXCR3-/- mice. Thus, we revealed a negative immune regulatory function for non-platelet-derived CXCL4 through CXCR3 that cancer cells could hijack to evade the host immune system, suggesting that the CXCL4/CXCR3 axis may serve as a novel target for colorectal cancer immunotherapy.

CXCL4 mediates tumor regrowth after chemotherapy by suppression of antitumor immunity.

The recurrence of colorectal cancer after chemotherapy is the leading cause of its high mortality. We propose that elucidating the mechanisms of tumor regrowth after chemotherapy in tumor-bearing mice may provide new insights into tumor relapse in cancer patients. We firstly report the identification of a chemokine, CXCL4, that plays an important role in the molecular mechanism of cancer regrowth after chemotherapy. A syngenic transplantation tumor model was established with murine colon cancer CT26 cells and treated with 5-FU. Genome-wide gene expression analysis determined that CXCL4 was transiently upregulated in the tumor model. Systemic overexpression of CXCL4 accelerated cancer growth in vivo, but not in vitro. Conversely, the anti-CXCL4 monoclonal antibody (CXCL4-mab) retarded tumor-regrowth after 5-FU treatment in immune-competent mice, but not nude mice. The CXCL4-mab treatment increased the local expression levels of IFN-γ and Gran-b genes in the tumor-bed, and elevated the function of CTLs against CT26 cells. Thus, the colon cancer cells in responding to the cytotoxic stress of 5-FU produce a high level of CXCL4, which suppresses antitumor immunity to confer the residual cancer cells an advantage for regrowth after chemotherapy. Our findings provide a novel target for developing therapeutics aiming to increase antitumor immunity after chemotherapy.

DS147 improves pregnancy in mice with embryo implantation dysfunction induced by controlled ovarian stimulation.

The study examined the effect of DS147, the bioactive component of the traditional herbal recipe Bangdeyun, on pregnancy in mice with embryo implantation dysfunction induced by controlled ovarian stimulation (COS), and the underlying mechanisms. Female mice were superovulated by intraperitoneal injection of 7.5 IU of pregnant mare serum gonadotropin (PMSG) followed by an additional injection of 7.5 IU hCG 48 h later to establish embryo implantation dysfunction (EID) model. Pregnant mice were randomly divided into normal control group, COS group and DS147-treated groups. The pregnancy rate and the average implantation site were obtained on pregnancy day 8 (PD8). The side effect of 200 mg/kg of DS147 on naturally pregnant mice was also observed. Further, the uterine and ovarian tissue samples were collected on PD5 for measuring their weights, observing the development of the endometrium and ovary, and detecting the endometrial expression of MMP-2, TIMP-2, CD34 and angiogenin (ANG). The female mice treated with DS147 at doses of 100 to 800 mg/kg showed a higher pregnancy rate than those in COS group, and the highest pregnancy rate of 83.3% occurred in the 200 mg/kg DS147-treated group. Moreover, no obvious side effect was found in mice treated with 200 mg/kg DS147 on PD8 and PD16. The ovarian and uterine weights, and the expression levels of MMP-2, ANG and CD34 were significantly increased in DS147-treated groups when compared with COS group. The TIMP-2 expression level was much lower in DS147-treated mice than in COS mice and the ratio of MMP-2/TIMP-2 was much higher in DS147-treated group than in COS group, and even higher than normal control group. In all, these findings suggest that DS147 may improve pregnancy in mice with COS-induced EID by promoting matrix degradation and angiogenesis, and improving the development of corpus luteum and endometrial decidualization around the implantation window.

Ovarian stimulation leads to a severe implantation defect in mice.

The aim of the present study was to evaluate whether ovarian stimulation could induce embryo implantation dysfunction in mice and to explore the possible mechanisms involved. Ovarian stimulation was performed with intraperitoneal injections of 0, 2.5, 5.0, 7.5 and 10.0 IU pregnant mare serum gonadotrophin followed by the same dose of human chorionic gonadotrophin 48h later. A dose-dependent implantation defect in stimulated mice was demonstrated, which can be mainly explained by premature luteolysis and secondary endometrial changes induced by an imbalance in oestradiol and progesterone.