Interception Proximity Labeling for Interrogating Cell Efflux Microenvironment.

The difficulty in elucidating the microenvironment of extracellular H2O2 efflux has led to the lack of a critical extracellular link in studies of the mechanisms of redox signaling pathways. Herein, we mounted horseradish peroxidase (HRP) to glycans expressed globally on the living cell surface and constructed an interception proximity labeling (IPL) platform for H2O2 efflux. The release of endogenous H2O2 is used as a "physiological switch" for HRP to enable proximity labeling. Using this platform, we visualize the oxidative stress state of tumor cells under the condition of nutrient withdrawal, as well as that of macrophages exposed to nonparticulate stimuli. Furthermore, in combination with a proteomics technique, we identify candidate proteins at the invasion interface between fungal mimics (zymosan) and macrophages by interception labeling of locally accumulated H2O2 and confirm that Toll-like receptor 2 binds zymosan in a glycan-dependent manner. The IPL platform has great potential to elucidate the mechanisms underlying biological processes involving redox pathways.

OCTN2 enhances PGC-1α-mediated fatty acid oxidation and OXPHOS to support stemness in hepatocellular carcinoma.

BACKGROUND: The Metabolic reprogramming of tumor cells plays a vital role in the progression of hepatocellular carcinoma. Organic cation/carnitine transporter 2 (OCTN2), a sodium-ion dependent carnitine transporter and a sodium-ion independent tetraethylammonium (TEA) transporter, has been reported to contribute tumor malignancies and metabolic dysregulation in renal and esophageal carcinoma. However, the role of lipid metabolism deregulation mediated by OCTN2 in HCC cells has not been clarified. METHODS: Bioinformatics analyses and immunohistochemistry assay were employed to identify OCTN2 expression in HCC tissues. The correlation between OCTN2 expression and prognosis was elucidated through K-M survival analysis. The expression and function of OCTN2 were examined via the assays of western blotting, sphere formation, cell proliferation, migration and invasion. The mechanism of OCTN2-mediated HCC malignancies was investigated through RNA-seq and metabolomic analyses. Furthermore, xenograft tumor models based on HCC cells with different OCTN2 expression levels were conducted to analyze the tumorigenic and targetable role of OCTN2 in vivo. RESULTS: We found that gradually focused OCTN2 was significantly upregulated in HCC and tightly associated with poor prognosis. Additionally, OCTN2 upregulation promoted HCC cells proliferation and migration in vitro and augmented the growth and metastasis of HCC. Moreover, OCTN2 promoted the cancer stem-like properties of HCC by increasing fatty acid oxidation and oxidative phosphorylation. Mechanistically, PGC-1α signaling participated in the HCC cancer stem-like properties mediated by OCTN2 overexpression, which is confirmed by in vitro and in vivo analyses. Furthermore, OCTN2 upregulation may be transcriptionally activated by YY1 in HCC. Particularly, treatment with mildronate, an inhibitor of OCTN2, showed a therapeutic influence on HCC in vitro and in vivo. CONCLUSIONS: Our findings demonstrate that OCTN2 plays a critical metabolic role in HCC cancer stemness maintenance and HCC progression, providing evidence for OCTN2 as a promising target for HCC therapy.

Protein-Targeted Glycan Editing on Living Cells Disrupts KRAS Signaling.

The frequent mutation of KRAS oncogene in some of the most lethal human cancers has spurred incredible efforts to develop KRAS inhibitors, yet only one covalent inhibitor for the KRASG12C mutant has been approved to date. New venues to interfere with KRAS signaling are desperately needed. Here, we report a "localized oxidation-coupling" strategy to achieve protein-specific glycan editing on living cells for disrupting KRAS signaling. This glycan remodeling method exhibits excellent protein and sugar specificity and is applicable to different donor sugars and cell types. Attachment of mannotriose to the terminal galactose/N-acetyl-D-galactosamine epitopes of integrin αv ß3 , a membrane receptor upstream of KRAS, blocks its binding to galectin-3, suppresses the activation of KRAS and downstream effectors, and mitigates KRAS-driven malignant phenotypes. Our work represents the first successful attempt to interfere with KRAS activity by manipulating membrane receptor glycosylation.

Mechanism of cancer stemness maintenance in human liver cancer.

Primary liver cancer mainly includes the following four types: hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), hepatoblastoma (HB), and combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CCA). Recent studies have indicated that there are differences in cancer stem cell (CSC) properties among different types of liver cancer. Liver cancer stem cells (LCSCs), also called liver tumor-initiating cells, have been viewed as drivers of tumor initiation and metastasis. Many mechanisms and factors, such as mitophagy, mitochondrial dynamics, epigenetic modifications, the tumor microenvironment, and tumor plasticity, are involved in the regulation of cancer stemness in liver cancer. In this review, we analyze cancer stemness in different liver cancer types. Moreover, we further evaluate the mechanism of cancer stemness maintenance of LCSCs and discuss promising treatments for eradicating LCSCs.

68Ga-Labeled GX1 Dimer: A Novel Probe for PET/Cerenkov Imaging Targeting Gastric Cancer.

PURPOSE: To synthesize the dimer of GX1 and identify whether its affinity and targeting are better than those of GX1. To prepare 68Ga-DOTA-KEK-(GX1)2 and to apply it to PET and Cerenkov imaging of gastric cancer. METHODS: 68Ga-DOTA-KEK-(GX1)2 was prepared, and the labeling yield and stability were determined. Its specificity and affinity were verified using an in vitro cell binding assay and competitive inhibition test, cell immunofluorescence, and cell uptake and efflux study. Its tumor-targeting ability was determined by nano PET/CT and Cerenkov imaging, standardized uptake value (SUV), signal-to-background ratio (SBR) quantification, and a biodistribution study in tumor-bearing nude mice. RESULTS: 68Ga-DOTA-KEK-(GX1)2 was successfully prepared, and the labeling yield was more than 97%. It existed stably for 90 min in serum. The binding of 68Ga-DOTA-KEK-(GX1)2 to cocultured HUVECs (Co-HUVECs) was higher than that to human umbilical vein endothelial cells (HUVECs), BGC823 cells, and GES cells. It was also higher than that of 68Ga-DOTA-GX1, indicating that the dimer did improve the specificity and affinity of GX1. The binding of KEK-(GX1)2 to Co-HUVECs was significantly higher than that of GX1. Additionally, the uptake of 68Ga-DOTA-KEK-(GX1)2 by Co-HUVECs was higher than that of 68Ga-DOTA-GX1 and reached a maximum at 60 min. Nano PET/CT and Cerenkov imaging showed that the tumor imaging of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 was clear, and the SUV and SBR value of the tumor sites were significantly higher than those of the nude mice injected with 68Ga-DOTA-GX1, indicating that the probe had better targeting in vivo. Finally, the biodistribution showed quantitatively that when organs such as the kidney and liver metabolized rapidly, the radioactivity of the tumor site of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 decreased relatively slowly. At the same time, the percentage of injected dose per gram (%ID/g) of the tumor site was higher than that of other normal organs except the liver and kidney at 60 min, which indicated that the tumor had good absorption of the probe. CONCLUSION: GX1 was modified successfully, and the in vivo and in vitro properties of the GX1 dimer were significantly better than those of GX1. The imaging probe, 68Ga-DOTA-KEK-(GX1)2, was successfully prepared, which provides a candidate probe for PET and Cerenkov diagnosis of gastric cancer.

Endovascular treatment of cerebrovascular stenosis with stent for patients with ischemic cerebrovascular disease.

This study aimed to investigate the therapeutic effect of cerebrovascular stent implantation in southwest Chinese patients with ischemic cerebrovascular disease and underlying risk factors for stent restenosis.We made a retrospectively analysis of occurring risk, cerebrovascular lesion, stent implantation, complication treatment, and prognosis of 54 patients with ischemic cerebrovascular disease in our department.A total of 85 stents were implanted into 54 patients, involving 44 of the internal carotid artery system, 34 of the vertebral-basal artery system and 7 of the subclavian artery system. All patients with stenosis were reduced by >70%, with all stenosis complete reduction in 5 (9%) patients and reduction of over 90% in 25 (46%) patients. A total of 50 patients were followed up for 28.5 (21-35) months. The stents in 42 patients exhibited satisfactory shape and location while restenosis occurred in 8 patients. Univariate analysis revealed that hyperlipidemia, hyperuricemia, surgery duration, and total length of hospital stay are significantly correlated with stent restenosis, and hyperlipidemia and hyperuricemia were proven to be independent risk factors for restenosis using logistic regression analysis.Cerebrovascular stent implantation and balloon inflation surgery can assist in abating angiostenosis and improving blood supplement effectively in patients with ischemic cerebrovascular disease. Besides, an overall evaluation, strict care, and regular check-up in perioperative period may reduce the occurrence of complications. Finally, several clinical parameters may need to be highly focused on in surgery for better prognosis.

188Re-labeled GX1 dimer as a novel dual-functional probe targeting TGM2 for imaging and antiangiogenic therapy of gastric cancer.

PURPOSE: The GX1 peptide (CGNSNPKSC) can specifically bind to TGM2 and possesses the ability to target the blood vessels of gastric cancer. This study intends to develop an integrated dual-functional probe with higher affinity, specificity and targeting and to characterize it in vivo and in vitro. METHODS: The dimer and tetramer of GX1 were prepared using cross-linked PEG and labeled with 99mTc. The best targeting probe [PEG-(GX1)2] was selected by gamma camera imaging in nude mouse models of gastric cancer. 188Re-PEG-(GX1)2 was prepared and characterized through cell binding analysis and competitive inhibition experiments, gamma camera imaging, MTT analysis and flow cytometry, BLI, immunohistochemistry, HE staining and biochemical analysis. RESULTS: PEG-(GX1)2 bound specifically to Co-HUVEC with higher affinity than GX1. 188Re-PEG-(GX1)2 had better ability to target gastric cancer in tumor-bearing nude mice and higher T/H ratios than 188Re-GX1. 188Re-PEG-(GX1)2 inhibited the growth of Co-HUVEC and induced apoptosis, and its effects were more robust than those of 188Re-GX1. BLI showed that 188Re-PEG-(GX1)2 inhibited tumor proliferation in vivo with a stronger effect than 188Re-GX1. Compared with 188Re-GX1, 188Re-PEG-(GX1)2 suppressed tumor angiogenesis and tumor cell proliferation and induced tumor cell apoptosis in vivo. The 188Re-PEG-(GX1)2 group did not cause visible changes in liver and kidney morphology and function in vivo. CONCLUSION: The dimer of GX1 was synthesized by using cross-linked PEG, and then 188Re-PEG-(GX1)2 was prepared. This radiopharmaceutical played both diagnostic and therapeutic functions, and gamma camera imaging could be utilized to detect the distribution of drugs in vivo during treatment. Through a series of experiments in vitro and in vivo, the feasibility of the drug was confirmed, and these results laid the foundation for the subsequent development and application of GX1.

Overexpression of LncRNA PSMG3-AS1 Distinguishes Glioblastomas from Sarcoidosis.

In clinical practices, glioblastomas (GBM) in some cases can be misdiagnosed as sarcoidosis. This study aimed to develop a biomarker to distinguish GBM from sarcoidosis. In this study, we found that PSMG3-AS1 was upregulated in plasma of GBM patients in comparison with that in sarcoidosis patients and healthy controls. Receiver operating characteristic (ROC) curve analysis showed that upregulation of PSMG3-AS1 effectively separated GBM patients from sarcoidosis patients and healthy controls. In GBM cells, overexpression of PSMG3-AS1 led to downregulated miR-34a and increased methylation of miR-34a gene. In addition, overexpression of PSMG3-AS1 reduced the inhibitory effects of miR-34a on GBM cell proliferation. In conclusion, overexpression of PSMG3-AS1 distinguishes GBM patients from patients with sarcoidosis, and PSMG3-AS1 may promote GBM cell proliferation by downregulating miR-34a through methylation.

Mesenchymal stem cells promote pancreatic adenocarcinoma cells invasion by transforming growth factor-ß1 induced epithelial-mesenchymal transition.

Mesenchymal stem cells (MSCs) could be ideal delivery vehicles for antitumor biological agents in pancreatic adenocarcinoma (PA). While the role of MSCs in tumor growth is elusive. Inflammation is an important feature of PA. In this study, we reported that MSCs pre-stimulated with the combination of TNF-α and IFN-γ promote PA cells invasion. The invasion of PA cell lines were evaluate by wound healing assay and transwell assay in vitro and liver metastasis in nude mice. We observed MSCs pre-stimulated with the combination of TNF-α and IFN-γ promoted PA cells invasion in vitro and in vivo. Consistent with MSCs promoting PA cells invasion, PA cells were found undergo epithelial-mesenchymal transition (EMT). We demonstrated that MSCs pre-stimulated with both of TNF-α and IFN-γ provoked expression transforming growth factor-ß1 (TGF-ß1). MSCs promoting EMT-mediated PA cells invasion could be reversed by short interfering RNA of TGF-ß1. Our results suggest that MSCs could promote PA cells invasion in inflammation microenvironment and should be cautious as delivery vehicles in molecular target therapy.