Circulating tumor cells participate in the formation of microvascular invasion and impact on clinical outcomes in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide. Although the treatment strategies have been improved in recent years, the long-term prognosis of HCC is far from satisfactory mainly due to high postoperative recurrence and metastasis rate. Vascular tumor thrombus, including microvascular invasion (MVI) and portal vein tumor thrombus (PVTT), affects the outcome of hepatectomy and liver transplantation. If vascular invasion could be found preoperatively, especially the risk of MVI, more reasonable surgical selection will be chosen to reduce the risk of postoperative recurrence and metastasis. However, there is a lack of reliable prediction methods, and the formation mechanism of MVI/PVTT is still unclear. At present, there is no study to explore the possibility of tumor thrombus formation from a single circulating tumor cell (CTC) of HCC, nor any related study to describe the possible leading role and molecular mechanism of HCC CTCs as an important component of MVI/PVTT. In this study, we review the current understanding of MVI and possible mechanisms, discuss the function of CTCs in the formation of MVI and interaction with immune cells in the circulation. In conclusion, we discuss implications for potential therapeutic targets and the prospect of clinical treatment of HCC.

New classification system for radical rectal cancer surgery based on membrane anatomy.

BACKGROUND: Total mesorectal excision along the "holy plane" is the only radical surgery for rectal cancer, regardless of tumor size, localization or even tumor stage. However, according to the concept of membrane anatomy, multiple fascial spaces around the rectum could be used as the surgical plane to achieve radical resection. AIM: To propose a new membrane anatomical and staging-oriented classification system for tailoring the radicality during rectal cancer surgery. METHODS: A three-dimensional template of the member anatomy of the pelvis was established, and the existing anatomical nomenclatures were clarified by cadaveric dissection study and laparoscopic surgical observation. Then, we suggested a new and simple classification system for rectal cancer surgery. For simplification, the classification was based only on the lateral extent of resection. RESULTS: The fascia propria of the rectum, urogenital fascia, vesicohypogastric fascia and parietal fascia lie side by side around the rectum and form three spaces (medial, middle and lateral), and blood vessels and nerves are precisely positioned in the fascia or space. Three types of radical surgery for rectal cancer are described, as are a few subtypes that consider nerve preservation. The surgical planes of the proposed radical surgeries (types A, B and C) correspond exactly to the medial, middle, and lateral spaces, respectively. CONCLUSION: Three types of radical surgery can be precisely defined based on membrane anatomy, including nerve-sparing procedures. Our classification system may offer an optimal tool for tailoring rectal cancer surgery.

Repurposing of the FGFR inhibitor AZD4547 as a potent inhibitor of necroptosis by selectively targeting RIPK1.

Necroptosis is a form of regulated necrosis involved in various pathological diseases. The process of necroptosis is controlled by receptor-interacting kinase 1 (RIPK1), RIPK3, and pseudokinase mixed lineage kinase domain-like protein (MLKL), and pharmacological inhibition of these kinases has been shown to have therapeutic potentials in a variety of diseases. In this study, using drug repurposing strategy combined with high-throughput screening (HTS), we discovered that AZD4547, a previously reported FGFR inhibitor, is able to interfere with necroptosis through direct targeting of RIPK1 kinase. In both human and mouse cell models, AZD4547 blocked RIPK1-dependent necroptosis. In addition, AZD4547 rescued animals from TNF-induced lethal shock and inflammatory responses. Together, our study demonstrates that AZD4547 is a potent and selective inhibitor of RIPK1 with therapeutic potential for the treatment of inflammatory disorders that involve necroptosis.

Thymosin ß4, a potential marker of malignancy and prognosis in hepatocellular carcinoma.

BACKGROUND: The lack of effective early diagnostic markers is an obstacle in clinical diagnosis and treatment of hepatocellular carcinoma (HCC). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an increasing popular approach for identification of clinically relevant parameters including biomarkers. PATIENTS AND METHODS: 540 subjects, including 274 HCC, 119 liver cirrhosis, 89 hepatitis, and 58 healthy volunteers were enrolled. MALDI-TOF MS was used to select potential novel biomarkers from serum of HCC patients. Its clinical application was evaluated by experiments and clinical data analysis. RESULTS: We identified Thymosin ß4 (Tß4) in serum by MALDI-TOF MS. The expression of Tß4 was detected up-regulating in HCC cells and tissues which enhanced motility of HCC cells. More important, the level of serum Tß4 was significantly elevated in HCC patients. The AUROC showed the optimum diagnostic cut-off was 1063.6 ng/mL, ROC and 95% CI of Tß4 (0.908; 0.880-0.935) were larger than that of serum AFP (0.712; 0.662-0.762; p < 0.001). The sensitivity (91.3% vs 83.1%) and specificity (81.2% vs 20.3%) of serum Tß4 were higher than alpha-fetoprotein (AFP). In AFP-negative HCC, the sensitivity could reach to 80.5%. ROC analysis showed serum Tß4 had a better performance compared with AFP in distinguishing early-stage and small HCC. Tß4 is correlated with TNM stage (p = 0.016) and vascular invasion (p = 0.005). Survival analysis indicated the survival time of Tß4 positive patients was shorter (p < 0.001). Cox analysis suggested Tß4 could be an independent factor for HCC prognosis. CONCLUSION: Tß4 may serve as a novel biomarker for HCC diagnosis and prognosis.

Gliosarcoma: The Distinct Genomic Alterations Identified by Comprehensive Analysis of Copy Number Variations.

Gliosarcoma (GSM), a histologic variant of glioblastoma (GBM), carries a poor prognosis with less than one year of median survival. Though GSM is similar with GBM in most clinical and pathological symptoms, GBM has unique molecular and histological features. However, as the rarity of GSM samples, the genetic information of this tumor is still lacking. Here, we take a comprehensive analysis of DNA copy number variations (CNV) in GBM and GSM. Whole genome sequencing was performed on 21 cases of GBM and 15 cases of GSM. CNVKIT is used for CNV calling. Our data showed that chromosomes 7, 8, 9, and 10 were the regions where CNV frequently happened in both GBM and GSM. There was a distinct CNV signal in chromosome 2 especially in GSM. The pathway enrichment of genes with CNV was suggested that the GBM and GSM shared the similar mechanism of tumor development. However, the CNV of some screened genes displayed a disparate form between GBM and GSM, such as AMP, BEND2, HDAC6, FOXP3, ZBTB33, TFE3, and VEGFD. It meant that GSM was a distinct subgroup possessing typical biomarkers. The pathways and copy number alterations detected in this study may represent key drivers in gliosarcoma oncogenesis and may provide a starting point toward targeted oncologic analysis with therapeutic potential.

CHMFL-26 is a highly potent irreversible HER2 inhibitor for use in the treatment of HER2-positive and HER2-mutant cancers.

Oncogene HER2 is amplified in 20%-25% of human breast cancers and 6.1%-23.0% of gastric cancers, and HER2-directed therapy significantly improves the outcome for patients with HER2-positive cancers. However, drug resistance is still a clinical challenge due to primary or acquired mutations and drug-induced negative regulatory feedback. In this study, we discovered a potent irreversible HER2 kinase inhibitor, CHMFL-26, which covalently targeted cysteine 805 of HER2 and effectively overcame the drug resistance caused by HER2 V777L, HER2 L755S, HER2 exon 20 insertions, and p95-HER2 truncation mutations. CHMFL-26 displayed potent antiproliferation efficacy against HER2-amplified and mutant cells through constant HER2-mediated signaling pathway inhibition and apoptosis induction. In addition, CHMFL-26 suppressed tumor growth in a dose-dependent manner in xenograft mouse models. Together, these results suggest that CHMFL-26 may be a potential novel anti-HER2 agent for overcoming drug resistance in HER2-positive cancer therapy.

[Intra-annual high resolution stable isotope records of tree ring: Methods, progress and prospects]. / æ ‘æœ¨å¹´è½®å¹´å† é«˜åˆ†è¾¨çŽ‡ç¨³å®šåŒä½ç´ è®°å½•:方法、进展和展望.

Stable isotope ratios of tree ring can effectively record climate and environmental changes during tree growth and the physiological responses of trees to such changes. Intra-annual high resolution stable isotope ratios of tree ring can provide more detailed climatic and environmental information, reveal the physiological and ecological response mechanism of trees to seasonal climatic variation, and thus with great potential in the study of paleoclimate and global change ecology. Based on the intra-annual high resolution stable isotope ratios related literature since 1990, we reviewed the research progress of intra-annual high resolution tree ring stable isotope records in the aspects of sample stripping method, chemical extraction method of α-cellulose and application, and further proposed the potential and future development direction of intra-annual high resolution tree ring stable isotope records.

Laparoscopic lateral lymph node dissection in two fascial spaces for locally advanced lower rectal cancer.

BACKGROUND: The procedure for lateral lymph node (LLN) dissection (LLND) is complicated and can result in complications. We developed a technique for laparoscopic LLND based on two fascial spaces to simplify the procedure. AIM: To clarify the anatomical basis of laparoscopic LLND in two fascial spaces and to evaluate its efficacy and safety in treating locally advanced low rectal cancer (LALRC). METHODS: Cadaveric dissection was performed on 24 pelvises, and the fascial composition related to LLND was observed and described. Three dimensional-laparoscopic total mesorectal excision with LLND was performed in 20 patients with LALRC, and their clinical data were analyzed. RESULTS: The cadaver study showed that the fascia propria of the rectum, urogenital fascia, vesicohypogastric fascia and parietal fascia lie side by side in a medial-lateral direction constituting the dissection plane for curative rectal cancer surgery, and the last three fasciae formed two spaces (Latzko's pararectal space and paravesical space) which were the surgical area for LLND. Laparoscopic LLND in two fascial spaces was performed successfully in all 20 patients. The median operating time, blood loss and postoperative hospitalization were 178 (152-243) min, 55 (25-150) mL and 10 (7-20) d, respectively. The median number of harvested LLNs was 8.6 (6-12), and pathologically positive LLN metastasis was confirmed in 7 (35.0%) cases. Postoperative complications included lower limb pain in 1 case and lymph leakage in 1 case. CONCLUSION: Our preliminary surgical experience suggests that laparoscopic LLND based on fascial spaces is a feasible, effective and safe procedure for treating LALRC.

[Accuracy of implantation between two immediate implantation methods in mandibular molars].

PURPOSE: To compare the accuracy of implant placement between modified and traditional immediate implant placement in mandibular molar regions. METHODS: Twenty-four patients were selected for immediate implantation in the molar area including 24 implantation sites. Preoperative cone-beam CT(CBCT) was conducted and then digital software Simplant 18.0 was used to design the ideal three-dimensional position of the implants. In the experimental group, the implant socket was prepared first according to reference of the remaining natural teeth, then the implant was implanted after minimally invasive extraction. Twelve patients in the control group underwent immediate implantation by traditional immediate implant procedures. Minimally invasive extraction, then socket preparation, and final implanting were performed. All patients underwent CBCT after surgery. Implant sites designed prior to surgery and actual implant sites differences between modified and traditional immediate implant placement were measured by Simplant 18.0 and compared with SPSS 17.0 software package. RESULTS: In the experimental group and control group, the measured average deviation were as follows, the angle was (4.492±0.912)° and (7.255±1.307)°, respectively; The horizontal error of the implant shoulder was (0.379±0.083) mm and (1.229±0.270) mm, respectively; The measuring horizontal error of the implant apex was (1.263±0.267) mm and (2.183±0.264) mm, respectively; The calculative horizontal error of the implant apex was (1.324±0.203) mm and (2.709±0.383) mm, respectively; Depth error of the implant apex was (0.663±0.123) mm and (1.533±0.155) mm, respectively, which were significantly lower than those of the control group. CONCLUSIONS: Compared with the traditional method, modified immediate implantation can improve the accuracy of implantation in mandibular molars.

Induction of core symptoms of autism spectrum disorder by in vivo CRISPR/Cas9-based gene editing in the brain of adolescent rhesus monkeys.

Although CRISPR/Cas9-mediated gene editing is widely applied to mimic human disorders, whether acute manipulation of disease-causing genes in the brain leads to behavioral abnormalities in non-human primates remains to be determined. Here we induced genetic mutations in MECP2, a critical gene linked to Rett syndrome (RTT) and autism spectrum disorders (ASD), in the hippocampus (DG and CA1-4) of adolescent rhesus monkeys (Macaca mulatta) in vivo via adeno-associated virus (AAV)-delivered Staphylococcus aureus Cas9 with small guide RNAs (sgRNAs) targeting MECP2. In comparison to monkeys injected with AAV-SaCas9 alone (n = 4), numerous autistic-like behavioral abnormalities were identified in the AAV-SaCas9-sgMECP2-injected monkeys (n = 7), including social interaction deficits, abnormal sleep patterns, insensitivity to aversive stimuli, abnormal hand motions, and defective social reward behaviors. Furthermore, some aspects of ASD and RTT, such as stereotypic behaviors, did not appear in the MECP2 gene-edited monkeys, suggesting that different brain areas likely contribute to distinct ASD symptoms. This study showed that acute manipulation of disease-causing genes via in vivo gene editing directly led to behavioral changes in adolescent primates, paving the way for the rapid generation of genetically engineered non-human primate models for neurobiological studies and therapeutic development.

Pharmacologically inhibiting phosphoglycerate kinase 1 for glioma with NG52.

Inhibition of glycolysis process has been an attractive approach for cancer treatment due to the evidence that tumor cells are more dependent on glycolysis rather than oxidative phosphorylation pathway. Preliminary evidence shows that inhibition of phosphoglycerate kinase 1 (PGK1) kinase activity would reverse the Warburg effect and make tumor cells lose the metabolic advantage for fueling the proliferation through restoration of the pyruvate dehydrogenase (PDH) activity and subsequently promotion of pyruvic acid to enter the Krebs cycle in glioma. However, due to the lack of small molecule inhibitors of PGK1 kinase activity to treat glioma, whether PGK1 could be a therapeutic target of glioma has not been pharmacologically verified yet. In this study we developed a high-throughput screening and discovered that NG52, previously known as a yeast cell cycle-regulating kinase inhibitor, could inhibit the kinase activity of PGK1 (the IC50 = 2.5 ± 0.2 µM). We showed that NG52 dose-dependently inhibited the proliferation of glioma U87 and U251 cell lines with IC50 values of 7.8 ± 1.1 and 5.2 ± 0.2 µM, respectively, meanwhile it potently inhibited the proliferation of primary glioma cells. We further revealed that NG52 (12.5-50 µM) effectively inhibited the phosphorylation of PDHK1 at Thr338 site and the phosphorylation of PDH at Ser293 site in U87 and U251 cells, resulting in more pyruvic acid entering the Krebs cycle with increased production of ATP and ROS. Therefore, NG52 could reverse the Warburg effect by inhibiting PGK1 kinase activity, and switched cellular glucose metabolism from anaerobic mode to aerobic mode. In nude mice bearing patient-derived glioma xenograft, oral administration of NG52 (50, 100, 150 mg· kg-1·d-1, for 13 days) dose-dependently suppressed the growth of glioma xenograft. Together, our results demonstrate that targeting PGK1 kinase activity might be a potential strategy for glioma treatment.

Osimertinib successfully combats EGFR-negative glioblastoma cells by inhibiting the MAPK pathway.

Glioblastoma (GBM) patients have extremely poor prognoses, and currently no effective treatment available including surgery, radiation, and chemotherapy. MAPK-interacting kinases (MNK1/2) as the downstream of the MAPK-signaling pathway regulate protein synthesis in normal and tumor cells. Research has shown that targeting MNKs may be an effective strategy to treat GBM. In this study we investigated the antitumor activity of osimertinib, an FDA-approved epidermal growth factor receptor (EGFR) inhibitor, against patient-derived primary GBM cells. Using high-throughput screening approach, we screened the entire panel of FDA-approved drugs against primary cancer cells derived from glioblastoma patients, found that osimertinib (3 µM) suppressed the proliferation of a subset (10/22) of EGFR-negative GBM cells (>50% growth inhibition). We detected the gene expression difference between osimertinib-sensitive and -resistant cells, found that osimertinib-sensitive GBM cells displayed activated MAPK-signaling pathway. We further showed that osimertinib potently inhibited the MNK kinase activities with IC50 values of 324 nM and 48.6 nM, respectively, against MNK1 and MNK2 kinases; osimertinib (0.3-3 µM) dose-dependently suppressed the phosphorylation of eukaryotic translation initiation factor 4E (eIF4E). In GBM patient-derived xenografts mice, oral administration of osimertinib (40 mg· kg-1 ·d-1, for 18 days) significantly suppressed the tumor growth (TGI = 74.5%) and inhibited eIF4E phosphorylation in tumor cells. Given the fact that osimertinib could cross the blood-brain barrier and its toxicity was well tolerated in patients, our results suggest that osimertinib could be a new and effective drug candidate for the EGFR-negative GBM patients.

Sequential release of immunomodulatory cytokines binding on nano-hydroxyapatite coated titanium surface for regulating macrophage polarization and bone regeneration.

Inflammation occurs when the material is implanted into the body. As one of the important immune cells in the regulation of inflammation, macrophages are able to remove pathogens and necrotic cells, and polarize to different phenotypes to regulate inflammatory response for tissue regeneration. Therefore, it is known that the sequential release of immunomodulatory cytokines from the surface of titanium (Ti) implants can regulate the polarization of macrophages and promote osseointegration of implants. In order to control the switch of macrophage phenotypes at desired time, we fabricated hydroxyapatite (HAp) nanotube arrays coating on Ti surface, by acid-etching, alkali-heating and HAp coating sequentially. Then we loaded the interleukin-4 (IL-4) encapsulated by poly (lactic-co-glycolic acid) (PLGA) on the bottom of the nanotube and the interferon-γ (IFN-γ) encapsulated by sodium hyaluronate (SH) on the top of the nanotube. Based on the physical and chemical properties of PLGA and SH and the spatial distribution of loaded cytokines, we hypothesized that the programmed release of IFN-γ and IL-4, which made the phenotypic transition of macrophages at a specific time, so as to regulate inflammation and promote osteogenic repair. Our hypothesis created a new type of drug sustained release system, which has high research value for improving the osseointegration of implants.

Cellular prion protein transcriptionally regulated by NFIL3 enhances lung cancer cell lamellipodium formation and migration through JNK signaling.

Tumor invasion and metastasis are the major causes of treatment failure and mortality in lung cancer patients. In this study, we identified a group of genes with differential expression in in situ and invasive lung adenocarcinoma tissues by expression profiling; among these genes we further characterized the association of the upregulation of PRNP, the gene encoding cellular Prion protein (PrPc), with lung adenocarcinoma invasiveness. Immunohistochemistry on clinical specimens showed an association of PrPc expression with invasive but not in situ lung adenocarcinoma. Consistently, the expression of PrPc was higher in the highly invasive than in the lowly invasive lung adenocarcinoma cell lines. Knockdown of PrPc expression in cultured lung adenocarcinoma cells decreased their lamellipodium formation, in vitro migration and invasion, and in vivo experimental lung metastasis. Phosphorylation of JNKs was found to correlate with PrPc expression and the inhibition of JNKs suppressed the PrPc-induced up-regulation of lamellipodium formation, cell migration, and invasion. Moreover, we identified the nuclear factor, interleukin 3 regulated (NFIL3) protein as a transcriptional activator of the PRNP promoter. Accordingly, NFIL3 promoted lung cancer cell migration and invasion in a PrPc-dependent manner. High NFIL3 expression in clinical specimens of lung adenocarcinoma was also associated with tumor invasiveness. Overall, our observations suggest that the NFIL3/PrPc axis, through regulating lamellipodium formation and cell mobility via JNK signaling, plays a critical role in lung cancer invasiveness and metastasis.

Chitosan oligosaccharide inhibits skull resorption induced by lipopolysaccharides in mice.

BACKGROUND: Low-molecular-weight chitosan oligosaccharide (LMCOS), a chitosan degradation product, is water-soluble and easily absorbable, rendering it a popular biomaterial to study. However, its effect on bone remodelling remains unknown. Therefore, we evaluated the effect of LMCOS on lipopolysaccharide (LPS)-induced bone resorption in mice. METHODS: Six-week-old male C57BL/6 mice (n = five per group) were randomly divided into five groups: PBS, LPS, LPS + 0.005% LMCOS, LPS + 0.05% LMCOS, and LPS + 0.5% LMCOS. Then, the corresponding reagents (300 µL) were injected into the skull of the mice. To induce bone resorption, LPS was administered at 10 mg/kg per injection. The mice were injected three times a week with PBS alone or LPS without or with LMCOS and sacrificed 2 weeks later. The skull was removed for micro-computed tomography, haematoxylin-eosin staining, and tartrate-resistant acid phosphatase staining. The area of bone damage and osteoclast formation were evaluated and recorded. RESULTS: LMCOS treatment during LPS-induced skull resorption led to a notable reduction in the area of bone destruction; we observed a dose-dependent decrease in the area of bone destruction and number of osteoclasts with increasing LMCOS concentration. CONCLUSIONS: Our findings showed that LMCOS could inhibit skull bone damage induced by LPS in mice, further research to investigate its therapeutic potential for treating osteolytic diseases is required.

Anti-tumor activity and mechanism of oligoclonal hepatocellular carcinoma tumor-infiltrating lymphocytes in vivo and in vitro.

Objective: To explore a method for culturing hepatocellular carcinoma and tumor-infiltrating lymphocytes (HCC-TIL) and investigate the mechanism of TIL in killing tumors. Methods: The distribution of regulatory T cells (Treg) in HCC was detected by immunohistochemistry. Conventional TIL and oligoclonal TIL were isolated by the traditional method of enzyme digestion combined with mechanical treatment for whole HCC and micro HCC tissue block culturing method. MTT was used to compare the killing activity of TIL. Flow cytometry was used to analyze the proportion of CD8+ T cells and Treg cells in TIL. Tumor-bearing mice were established, and TIL adoptive immunotherapy was performed. Results: Treg cells were mainly distributed in the stroma of HCC. In vitro experiments showed oligoclonal TIL had higher cytotoxicity to tumor cells which negatively correlated with the proportion of Treg cells. In vivo experiments showed oligoclonal TIL had a higher anti-tumor effect. IFN-γ in peripheral blood and the positive rate of intratumoral lymphocytic infiltration in oligoclonal TIL group were both higher. TGF-ß and IL-10 in peripheral blood and the positive rate of intratumoral FoxP3 and IL-17 were both lower than those in conventional TIL group. Conclusion: The oligoclonal TIL culture method could obtain TIL with higher purity, and cytotoxicity to tumor cells was associated with Treg cells. The oligoclonal TIL had cytotoxicity to autologous HCC cells and significant inhibitory effect on the growth of transplanted tumors. The mechanism might be associated with the inhibition of Treg cells proliferation, increase of IFN-γ secretion, and decrease of TGF-ß, IL-10, and IL-17 secretion.

Survival Mechanisms and Influence Factors of Circulating Tumor Cells.

Circulating tumor cells (CTCs) are cancer cells shed from either the primary tumor or its metastases that circulate in the peripheral blood. The CTCs are regarded as the source of tumor recurrence and metastasis and speculated as the indicators of residual tumors, thereby indicating a poor prognosis. Although CTCs play a vital role in tumor metastasis and recurrence, little is known about the underlying survival mechanisms in the blood circulation. The accumulating evidence has revealed that CTCs might survive in the peripheral blood by overcoming the mechanical damage due to shear stress, resistance to anoikis, evasion of immune destruction, and resistance to chemotherapy. The present review addresses the putative survival mechanisms underlying the formation and migration of CTCs according to their biological characteristics and blood microenvironment. In addition, the relationship between CTCs and microenvironment is illustrated, and the influencing factors related to the interactions of CTCs with various components in the peripheral blood are reviewed with respect to the platelets, immune cells, cytokines, and circulating tumor microemboli (CTM). Furthermore, the recent advances in the new treatment strategies targeting the survival mechanisms of CTCs are also discussed.