N6-methyladenosine enhances the expression of TGF-ß-SMAD signaling family to inhibit cell growth and promote cell metastasis.

TGF-ß-SMAD signaling pathway plays an important role in the progression of various cancers. However, posttranscriptional regulation such as N6-methyladenosine (m6A) of TGF-ß-SMAD signaling axis remains incompletely understood. Here, we reveal that insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) is low expression as well as associated with poor prognosis in clear cell renal cell carcinoma (ccRCC) patients and inhibits proliferation as well as promotes metastasis of ccRCC cells. Mechanistically, IGF2BP2 systematically regulates TGF-ß-SMAD signaling family, including TGF-ß1/2, TGF-ßR1/2 and SMAD2/3/4, through mediating their mRNA stability in an m6A-dependent manner. Furthermore, the functional effects of IGF2BP2 on ccRCC cells is mediated by TGF-ß-SMAD signaling downstream effector SMAD4, which is identified three m6A sites in 5'UTR and CDS. Our study establishes IGF2BP2-TGF-ß-SMAD axis as a new regulatory effector in ccRCC, providing new insights for developing novel therapeutic strategies.

Identification of a novel peptide targeting TIGIT to evaluate immunomodulation of 125I seed brachytherapy in HCC by near-infrared fluorescence.

Introduction: Hepatocellular carcinoma (HCC) has very poor prognosis due to its immunosuppressive properties. An effective measure to regulate tumor immunity is brachytherapy, which uses 125I seeds planted into tumor. T cell immune receptors with immunoglobulin and ITIM domains (TIGIT) is highly expressed in HCC. The TIGIT-targeted probe is expected to be an effective tool for indicating immunomodulation of 125I seed brachytherapy in HCC. In this study, We constructed a novel peptide targeting TIGIT to evaluate the immune regulation of 125I seed brachytherapy for HCC by near-infrared fluorescence (NIRF). Methods: Expression of TIGIT by immunofluorescence (IF) and flow cytometry (FCM) in different part and different differentiated human liver cancer tissues was verified. An optical fluorescence probe (Po-12) containing a NIRF dye and TIGIT peptide was synthesized for evaluating the modulatory effect of 125I seed brachytherapy. Lymphocytes uptake by Po-12 were detected by FCM and confocal microscopy. The distribution and accumulation of Po-12 in vivo were explored by NIRF imaging in subcutaneous and orthotopic tumors. IHC and IF staining were used to verify the expression of TIGIT in the tumors. Results: TIGIT was highly expressed in HCC and increased with tumor differentiation. The dye-labeled peptide (Po-12) retained a stable binding affinity for the TIGIT protein in vitro. Accumulation of fluorescence intensity (FI) increased with time extended in subcutaneous H22 tumors, and the optimal point is 1 h. TIGIT was highly expressed on lymphocytes infiltrated in tumors and could be suppressed by 125I seed brachytherapy. Accumulation of Po-12-Cy5 was increased in tumor-bearing groups while declined in 125I radiation group.

Sarcopenia increases the risk of early biliary infection after percutaneous transhepatic biliary stent placement.

Purpose: To assess the association between sarcopenia and the risk of early biliary infection (EBI) after percutaneous transhepatic biliary stent (PTBS) placement in patients with inoperable biliary tract cancer (BTC). Patients and methods: In this single center, retrospective observational study, patients diagnosed with inoperable BTC undergoing PTBS placement between January 2013 and July 2021 were enrolled. Preoperative sarcopenia was defined based on skeletal muscle mass measured by computed tomography images on the level of third lumbar vertebra within one month before PTBS placement. Patients were divided into two groups in accordance with the status of sarcopenia. Univariate and further multivariate logistic analyses were performed to determine predictors for EBI. Stratified and interactive analyses were conducted to investigate the stability of results. Further receiver operating characteristic curve was performed to determine the predictive value of sarcopenia on EBI after PTBS placement. Results: Totally, 134 patients were included in this retrospective study, with 45 (33.6%) patients characterized as sarcopenia. The incidence rate of EBI was 26.9% (36/134). Multivariate analyses demonstrated that sarcopenia [Odds ratio (OR), 2.75; 95%CI: 1.11-6.77; P=0.028], obstruction length (OR, 1.04; 95%CI: 1.00-1.08; P=0.030) and diabetes (OR, 2.46; 95%CI: 1.01-5.96; P=0.047) were significant predictors of EBI. There were no significant interactions in different subgroups (P for interaction > 0.05). Moreover, the areas under the curves (AUC) revealed that the combined index containing sarcopenia, obstruction length, and diabetes showed the better predictive value (AUC= 0.723) than either one alone. Conclusion: Sarcopenia increased the risk of EBI in patients with inoperable BTC after PTBS placement. Preoperative assessment of sarcopenia may aid in risk stratification. Patients with sarcopenia should be given intensive monitoring.

Tumor oxygenation nanoliposome synergistic hypoxia-inducible-factor-1 inhibitor enhanced Iodine-125 seed brachytherapy for esophageal cancer.

Iodine-125 (125I) brachytherapy has become one of the most effective palliative treatment options for advanced esophageal cancer. However, resistance toward 125I brachytherapy caused by pre-existing tumor hypoxia and hypoxia-inducible factor 1 (HIF-1) signaling pathway activation represents a significant limitation in esophageal cancer treatment. To circumvent these problems, herein, we proposed an innovative strategy to alleviate radioresistance of brachytherapy by co-encapsulating catalase (CAT) and HIF-1 inhibitor-acriflavine (ACF) into the hydrophilic cavities of liposome, termed as "ACF-CAT@Lipo". Under overexpressed H2O2 stimulation in the tumor region, the fabricated ACF-CAT@Lipo can generate an amount of O2 and alleviate tumor hypoxia in vitro and in vivo. Furthermore, cooperating with ACF, the expression of hypoxia-related protein (e.g. HIF-1α, VEGF, MMP-2) are obviously decreased. Importantly, the copious oxygenation and the significant inhibition expression of HIF-1α can further improve the radiosensitivity of 125I brachytherapy and finally realize the eradication of esophageal cancer in vivo. The oxygen enrichment and HIF-1 inhibition function of ACF-CAT@Lipo provides a new strategy to overcome the brachytherapy resistance of esophageal cancer therapy.

Microorganism-enabled photosynthetic oxygeneration and ferroptosis induction reshape tumor microenvironment for augmented nanodynamic therapy.

Nanodynamic therapy (NDT) based on reactive oxygen species (ROS) generation has been envisioned as a distinct modality for efficient cancer treatment. However, insufficient ROS generation and partial ROS consumption frequently limit the theraputic effect and outcome of NDT owing to the low oxygen (O2) tension and high glutathione (GSH) level in tumor microenvironment (TME). To circumvent these critical issues, we herein proposed and engineered the biodegradable GSH-depletion Mn(III)-riched manganese oxide nanospikes (MnOx NSs) with the photosynthetic bacterial cyanobacteria (Cyan) as a high-efficient and synergistic platform to reshape TME by simultaneously increasing oxygen content and decreasing GSH level. Specifically, under the trigger of acidity, MnOx NSs reacted with photosynthetic oxygen can generate toxic singlet oxygen (1O2). Moreover, MnOx NSs significantly reduced intracellular GSH, resulting in decreased GPX4 activity, which induced tumor cell non-apoptotic ferroptosis. Consequently, this combined strategy based on coadministration with Cyan and MnOx NSs demonstrated the superior antitumor efficacy via amplification of oxidative stress in 4T1 tumor-bearing mice for the synergetic oxygen-augmented nanodynamic/ferroptosis therapy. This work highlights a facile synergistic micro-/nano-system with the specific capability of reshaping TME to augment the sensitivity and therapeutic efficacy of NDT in solid hypoxic tumor therapy.

The Prognostic Value of Sarcopenia and Myosteatosis in Biliary Tract Cancer After Palliative Treatment With Radiation-Emitting Metallic Stent.

Background: Radiation-emitting metallic stent (REMS) placement is increasingly used for malignant biliary obstruction (MBO) caused by unresectable biliary tract carcinoma (UBTC) in clinical practice. The study is aimed to evaluate the prognostic value of sarcopenia, myosteatosis, and their combination on overall survival (OS) in patients treated with REMS for UBTC. Methods: Patients diagnosed with UBTC who underwent REMS placement between January 2013 and May 2021 were included consecutively in this retrospective study. Sarcopenia and myosteatosis were defined based on skeletal muscle index (SMI) and skeletal muscle attenuation (SMA), respectively, which were measured by computer tomography (CT) images on the level of the third lumbar vertebral body before REMS placement. Patients were categorized into two groups by sex-specific cutoff value for sarcopenia and myosteatosis, and OS rates were compared between the groups. Univariate and multivariate cox regression analyses were used to assess factors associated with OS. Results: Data of 135 patients included were retrospectively reviewed and analyzed. Median OS was 7.17 months in total cohort. Patients in the sarcopenia group had significant poorer OS than those in the non-sarcopenia group (median: 3.23 vs. 11.60 months, p < 0.001). OS was shorter in patients with myosteatosis than those without myosteatosis (median: 4.40 vs. 9.17 months, p < 0.001). Sarcopenia (odds ratio [OR] = 9.61; 95% CI = 5.41-17.09; p < 0.001) and myosteatosis (OR = 1.70; 95% CI = 1.13-2.57; p = 0.012) were significantly associated with OS. Combining sarcopenia and myosteatosis (CSM) showed a better predictive accuracy in OS than either one (area under curves: CSM vs. sarcopenia = 0.760 vs. 0.698, p = 0.049; CSM vs. myosteatosis = 0.760 vs. 0.671, p = 0.006). Conclusion: Sarcopenia and myosteatosis are negative predictors of survival in patients who underwent REMS placement for UBTC. CSM seemed to show a better prognostic value than either sarcopenia or myosteatosis alone. They can be used preoperatively for risk evaluation.

Interventional Treatment for Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is the second most common type of primary liver malignancy. The latest classification includes intrahepatic cholangiocarcinoma and extrahepatic cholangiocarcinoma, with the latter one further categorized into perihilar and distal cholangiocarcinoma. Although surgical resection is the preferred treatment for CCA, less than half of the patients are actually eligible for radical surgical resection. Interventional treatment, such as intra-arterial therapies, ablation, and brachytherapy (iodine-125 seed implantation), has become an acceptable palliative treatment for patients with unresectable CCA. For these patients, interventional treatment is helpful for locoregional control, symptom relief, and improving quality of life. Herein, in a timely and topical manner, we will review these advances and highlight future directions of research in this article.

CDCA2 promotes tumorigenesis and induces radioresistance in oesophageal squamous cell carcinoma cells.

Cell division cycle­associated 2 (CDCA2) overexpression has been demonstrated to serve a significant role in tumorigenesis in certain types of cancer. Nevertheless, its role in tumour proliferation and radioresistance in oesophageal squamous cell carcinoma (ESCC) remains to be elucidated. Thus, the present study aimed to elucidate these roles. Data were downloaded from The Cancer Genome Atlas (TCGA) to compare the gene expression profiles. The expression of CDCA2 was higher in ESCC tissues compared with normal tissues. Gene set enrichment analysis was performed based on the ESCC cohorts in TCGA database. This demonstrated that higher expression of CDCA2 was significantly associated with the expression of related components of the cell cycle phase transition and G2/M phase transition pathways. Collectively, the results revealed that CDCA2 could serve as an underlying target to regulate tumour growth and radioresistance among patients with ESCC.

Hypoxic tumour cell-derived exosomal miR-340-5p promotes radioresistance of oesophageal squamous cell carcinoma via KLF10.

BACKGROUND: Radiotherapy resistance is a major obstacle in the treatment of oesophageal squamous cell carcinoma (OSCC). Hypoxia is a critical cause of radioresistance. However, the communication between hypoxic cells and aerobic cells via exosomes during the transfer of radiation resistance remains unclear. METHODS: Exo-miR-340-5p levels were analysed by RNA-seq and qRT-PCR. We co-cultured OSCC cells with isolated normoxic and hypoxic exosomes to study their impact on radiosensitivity. We used a specific exo-miR-340-5p mimic and knock-down retrovirus to explore the role of this miRNA in the transfer of radioresistance from hypoxic to normoxic cells. Dual-luciferase reporter and RIP assays were used to verify KLF10 as a putative target of miR-340-5p. Several in vitro assays were conducted and xenograft models were established to investigate the effect of exo-miR-340-5p on OSCC radiosensitivity. The plasma exo-miR-340-5p levels in OSCC patients were analysed to study the clinical value of this parameter. RESULTS: Hypoxic exosomes alleviated radiation-induced apoptosis and accelerated DNA damage repair. miR-340-5p was highly expressed in hypoxic exosomes and was transferred into normoxic cells, where it induced radioresistance. Overexpression of miR-340-5p in normoxic OSCC cells mimicked the radioresistance of cells co-cultured with hypoxic exosomes. Knockdown of miR-340-5p in hypoxic exosomes reversed the radioresistance effect, indicating that exo-miR-340-5p is critical for hypoxic EV-transferred radioresistance. KLF10 was identified as the direct target of miR-340-5p. Moreover, metformin was found to increase the expression of KLF10 and enhance the radiosensitivity of OSCC. Higher levels of miR-340-5p in the plasma exosomes from OSCC patients are related to a poorer radiotherapy response and prognosis. CONCLUSIONS: Hypoxic tumour cell-derived exosomal miR-340-5p confers radioresistance in OSCC by targeting KLF10/UVRAG, suggesting that miR-340-5p could be a potential biomarker and therapeutic target for the enhancement of radiosensitivity in OSCC. Metformin can increase KLF10 expression, which ameliorates the radioresistance induced by exo-miR-340-5p transfer. Therefore, metformin could be further investigated as a therapeutic option for the treatment of OSCC.

DNA methylation data-based prognosis-subtype distinctions in patients with esophageal carcinoma by bioinformatic studies.

Esophageal carcinoma (ESCA) is caused by the accumulation of genetic and epigenetic alterations in esophageal mucosa. Of note, the earliest and the most frequent molecular behavior in the complicated pathogenesis of ESCA is DNA methylation. In the present study, we downloaded data of 178 samples from The Cancer Genome Atlas (TCGA) database to explore specific DNA methylation sites that affect prognosis in ESCA patients. Consequently, we identified 1,098 CpGs that were significantly associated with patient prognosis. Hence, these CpGs were used for consensus clustering of the 178 samples into seven clusters. Specifically, the samples in each group were different in terms of age, gender, tumor stage, histological type, metastatic status, and patient prognosis. We further analyzed 1,224 genes in the corresponding promoter regions of the 1,098 methylation sites, and enriched these genes in biological pathways with close correlation to cellular metabolism, enzymatic synthesis, and mitochondrial autophagy. In addition, nine representative specific methylation sites were screened using the weighted gene coexpression network analysis. Finally, a prognostic prediction model for ESCA patients was built in both training and validation cohorts. In summary, our study revealed that classification based on specific DNA methylation sites could reflect ESCA heterogeneity and contribute to the improvement of individualized treatment and precise prognostic prediction.

The ATPase subunit of ATP6V1C1 inhibits autophagy and enhances radiotherapy resistance in esophageal squamous cell carcinoma.

Radiotherapy is one of the primary therapeutic modalities for patients diagnosed esophageal squamous cell carcinoma(ESCC). Previous studies have shown that chemotherapy resistance could be linked with the overexpression vascular ATPases(V-ATPase) subunits genes. However, it is unknown whether V-ATPase subunits genes play a role in radiotherapy resistance. The aim of this study was to investigate the effect of the ATP6V1C1 in radiotherapy resistance. siRNA and plasmids were used to transfect low expression of ATP6V1C1 in TE13 (human ESCC cell) and high expressed in ECA109 (human ESCC cell), respectively. To observe proliferation, radiosensitivity, apoptosis and DNA-damage response, colony formation assays, EDU assays, flow cytometry and γH2AX assay were used with or without radiation exposure, separately. The quantities of the autophagosomes and autolysosomes by immunofluorescence were calculated. Autophagic microstructure were discovered by transmission electron microscopy, and the study also repeated in vivo by nude mice. Western blot assay was applied to prove changes in relative proteins. We found that suppressing ATP6V1C1 increased the sensitivity of ESCC cells after RT. Silencing ATP6V1C1 with IR suppressed the tumor growth and promoted autophagy. Besides, the underlying mechanism of ATP6V1C1, which is not fatally disrupted, is that ATP6V1C1 with ionizing radiation (IR)decreased apoptosis and inhibited autophagy may by activating mTOR signaling to suppress radiosensitivity for ESCC cells. Thus, we first reported that the ATP6V1C1 may represent a potential radiotherapeutic target by effect on radiation sensitivity for ESCC.

MiR-450a-5p inhibits autophagy and enhances radiosensitivity by targeting dual-specificity phosphatase 10 in esophageal squamous cell carcinoma.

Radioresistance reduces the success of therapy for patients with ESCC. Enhancing our understanding of the cardinal principles of radioresistance may improve the response of patients to irradiation. MicroRNAs perform a key role in posttranscriptional regulation, which is linked with the response of tumors to irradiation. Here, we successfully constructed a radioresistant cell line model, ECA109R, from parental esophageal cancer cell line ECA109. We used RNA-Seq analysis and qRT-PCR to compare the miRNA expression profiles of the ECA109 and ECA109R cell lines. The results revealed that miR-450a-5p was downregulated in the radioresistant cells. Functional analysis indicated that miR-450a-5p increases cellular radiosensitivity and suppresses autophagy in ESCC cells. We utilized a luciferase reporter assay to identify the target gene, DUSP10, as an indispensable regulator of the p38 and SAPK/JNK signaling pathways. Upregulation or downregulation of DUSP10 expression could reverse the effects of miR-450a-5p overexpression or inhibition. Tumor xenograft experiments verified that miR-450a-5p overexpression could increase sensitivity to radiation therapy in vivo. In general, our findings indicate that miR-450a-5p is a latent radiosensitizer and may represent a potential novel therapeutic target for radioresistance in ESCC.

Hypoxia induced changes in miRNAs and their target mRNAs in extracellular vesicles of esophageal squamous cancer cells.

BACKGROUND: Extracellular vesicles (EVs) are endogenous membrane vesicles with a diameter of 30-200 nm. It has been reported that hypoxic cancer cells can release numerous EVs to mediate multiple regional and systemic effects in the tumor microenvironment. METHODS: In this study, we used ultracentrifugation to extract EVs secreted by TE-13, an esophageal squamous carcinoma (ESCC) cell line during normoxia and hypoxia and performed high-throughput sequencing to detect exosomal miRNAs. Gene ontology (GO) and KEGG pathway analyses were used to reveal pathways potentially regulated by the miRNAs. RESULTS: A total of 10 810 miRNAs were detected; 50 were significantly upregulated and 34 were significantly downregulated under hypoxic environment. GO analysis identified enrichment of protein binding, regulation of transcription (DNA-templated), and membrane as molecular function, biological process, and cellular component, respectively. KEGG pathway analysis revealed cancer-associated pathways, phospholipase D signaling pathway, autophagy, focal adhesion and AGE-RAGE signaling as the key pathways. Further verification experiment from qRT-PCR indicated that miR-128-3p, miR-140-3p, miR-340-5p, miR-452-5p, miR-769-5p and miR-1304-p5 were significantly upregulated in EVs from hypoxia TE-13 cells while miR-340-5p was significantly upregulated in two other ESCC cells, ECA109 and TE-1. CONCLUSION: This study, for the first time reveals changes in the expression of exosomal miRNAs in hypoxic ESCC cells and these findings will act as a resource to study the hypoxic tumor microenvironment and ESCC EVs.

Synthesis and Preliminary Evaluation of a Novel 18F-Labeled 2-Nitroimidazole Derivative for Hypoxia Imaging.

OBJECTIVE: Hypoxia is prevalent in tumors and plays a pivotal role in resistance to chemoradiotherapy. 18F-MISO (18F-labeled fluoromisonidazole) is currently the preferred choice of PET hypoxia tracers in clinical practice, but has severe disadvantages involving complex labeling methods and low efficient imaging due to lipophilicity. We aimed to design a novel nitroimidazole derivative labeled by 18F via a chelation technique to detect hypoxic regions and provide a basis for planning radiotherapy. MATERIALS AND METHODS: First, we synthesized a 2-nitroimidazole precursor, 2-[4-(carboxymethyl)-7-[2-(2-(2-nitro-1H-imidazol-1-yl)acetamido)ethyl]-1,4,7-triazanonan-1-yl]acetic acid (NOTA-NI). For 18F-labeling, a 18F solution was reacted with a mixture of AlCl3 and NOTA-NI at pH 3.5 and 100°C for 20 min, and the radiochemical purity and stability were evaluated. Biological behaviors of Al18F-NOTA-NI were analyzed by an uptake study in ECA109 normoxic and hypoxic cells, and a biodistribution study and microPET imaging in ECA109 xenografted mice. RESULTS: Al18F-NOTA-NI required a straightforward and efficient labeling procedure compared with 18F-MISO. The uptake values were distinctly higher in hypoxic tumor cells. Animal studies revealed that the imaging agent was principally excreted via the kidneys. Due to hydrophilicity, the radioactivities in blood and muscle were decreased, and we could clearly distinguish xenografted tumors from para-carcinoma tissue by PET imaging. CONCLUSIONS: The nitroimidazole tracer Al18F-NOTA-NI steadily accumulated in hypoxic areas in tumors and was rapidly eliminated from normal tissue. It appears to be a promising candidate for hypoxia imaging with high sensitivity and resolution.