Extracellular Vesicles Released After Cranial Radiation: An Insight into an Early Mechanism of Brain Injury

Cranial radiation is important for treating both primary brain tumors and brain metastases. A potential delayed side effect of cranial radiation is neurocognitive function decline. Early detection of CNS injury might prevent further neuronal damage. Extracellular vesicles (EVs) have emerged as a potential diagnostic tool because of their unique membranous characteristics and cargos. We investigated whether EVs can be an early indicator of CNS injury by giving C57BJ/6 mice 10 Gy cranial IR. EVs were isolated from sera to quantify: 1) number of EVs using nanoparticle tracking analysis (NTA); 2) Glial fibrillary acidic protein (GFAP), an astrocyte marker; and 3) protein-bound 4-hydroxy-2-nonenal (HNE) adducts, an oxidative damage marker. Brain tissues were prepared for immunohistochemistry staining and protein immunoblotting. The results demonstrate: 1) increased GFAP levels (p \u3c 0.05) in EVs, but not brain tissue, in the IR group; and 2) increased HNE-bound protein adduction levels (p \u3c 0.05). The results support using EVs as an early indicator of cancer therapy-induced neuronal injury

Enrichment of cancer stem cells via β-catenin contributing to the tumorigenesis of hepatocellular carcinoma

Abstract Background Hepatocellular carcinoma (HCC) is among the deadliest cancers due to its heterogeneity, contributing to chemoresistance and recurrence. Cancer stem-like cells (CSCs) are suggested to play an important role in HCC tumorigenesis. This study investigates the role of Wnt/β-catenin pathway in CSC enrichment and the capabilities of these CSCs in tumor initiation in orthotopic immunocompetent mouse model. Methods HCC-CSCs were enriched using established serum-free culture method. Wnt/β-catenin pathway activation and its components were analyzed by western blot and qRT-PCR. The role of β-catenin in enrichment of CSC spheroids was confirmed using siRNA interference. Tumorigenic capabilities were confirmed using orthotopic immunocompetent mouse model by injecting 2 × 106 Hepa1–6 CSC spheroids or control cells in upper left liver lobe. Results The serum-free cultured Hepa1–6 cells demonstrated self-renewal, spheroid formation, higher EpCAM expression, increased Hoechst-33342 efflux, and upregulated Wnt/β-catenin signaling. Wnt/β-catenin pathway upregulation was implicated with the downstream targets, i.e., c-MYC, Cyclin-D1, and LEF1. Also, we found that GSK-3β serine-9 phosphorylation increased in Hepa1–6 spheroids. Silencing β-catenin by siRNA reversed spheroid formation phenotype. Mice injected with Hepa1–6 CSC spheroids showed aggressive tumor initiation and growth compared with mice injected with control cells. Conclusions Successfully induced Hepa1–6 spheroids were identified with CSC-like properties. Aberrant β-catenin upregulation mediated by GSK-3β was observed in the Hepa1–6 spheroids. The β-catenin mediated CSC enrichment in the induced spheroids possesses the capability of tumor initiation in immunocompetent mice. Our study suggests plausible cell dedifferentiation mediated by β-catenin contributes to CSC-initiated HCC tumor growth in vivo

Serological biomarkers associate ultrasound characteristics of steatohepatitis in mice with liver cancer

Abstract Background Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of lesions ranging from steatosis to a complex pattern, nonalcoholic steatohepatitis (NASH). Ultrasonography provides important information on hepatic architecture for steatosis. NASH patients have an increased risk of hepatocellular carcinoma (HCC). Early detection of NASH is critical for clinicians to advise on necessary treatments to prevent the onset of HCC. Methods We established a NASH-HCC mouse model using diethylnitrosamine as a carcinogen to induce HCC and a high-fat diet to induce metabolic disorders. Characteristics of ultrasound imaging and potential serum biomarkers were investigated for detection of steatohepatitis and HCC in mice. Results The data suggested that ultrasound imaging of hyperechoic masses was potentially linked to the gross finding of HCC nodules, which was further confirmed by the histology. Positive correlation between serum fibroblast growth factor 15 and acoustic attenuation coefficient was found in mice with steatohepatitis. Combined with the serum markers, the increased acoustic attenuation coefficient could be a useful diagnostic parameter of ultrasound imaging for NASH detection. Conclusions This study demonstrates that a combination of serum fibroblast growth factor 15 and acoustic attenuation coefficient could be a sensitive marker for steatohepatitis and to predict carcinogenic initiation and progression of HCC in mice. These results might help for the design of ultrasound and surrogate markers in screening NASH patients who could be at risk of HCC

Additional file 1: of Enrichment of cancer stem cells via β-catenin contributing to the tumorigenesis of hepatocellular carcinoma

Supplementary experiments and routine methods. Figure S1. Spheroid formation capability of HCC cell lines. Figure S2. β-catenin expression in HepG2 cell line. Figure S3. Knockdown efficiency of siRNA transfection. Routine Methods: Immunocytochemistry (ICC) Staining, Flow-Cytometry Analysis, Protein Extraction and Western Blot. Table S1. Primary antibodies (catalog info and dilution). Table S2. HRP conjugated secondary antibodies (catalog info and dilution). Table S3. Important reagents (catalog info). Table S4. Sequence details for RNA interference experiments. (DOCX 573 kb

Additional file 1: of Up-regulation of FGF15/19 signaling promotes hepatocellular carcinoma in the background of fatty liver

Tables S1-S3, list of antibodies, promers and siRNAs. Method for ultrasound image acquisition. Figure legends for Figures S1-S4. (DOCX 18 kb

Additional file 4: of Up-regulation of FGF15/19 signaling promotes hepatocellular carcinoma in the background of fatty liver

Figure S3. Representative images of EpCAM and β-Catenin in liver parenchyma in all 4 experimental groups at month 10 and in cultured cells treated with FFA and BAS. Fluorescent staining was performed using FITC tagged anti-EpCAM and anti-β-Catenin antibodies on the frozen tissue sections of mice. Fluorescent staining for HCC cells was carried out on the 8 well chamber slide seeded Hepal-6 cells in response to FFA treatment. DAPI (4′,6-diamidino-2-phenylindole) fluorescent stain was performed to detect nucleus as counter staining. UT: untreated; CD: control diet; HFD: high fat diet; DEN: N-nitrosodiethylamine; FFA: free fatty acid; BSA: bovine serum albumin. (JPG 4260 kb

Additional file 2: of Up-regulation of FGF15/19 signaling promotes hepatocellular carcinoma in the background of fatty liver

Figure S1. Representative gross anatomy and ultrasound images from all 4 experimental groups at month 2 month 6, and month 10. On visual pattern, tumor showed as HCC nodule, while the ultrasound appearance of HCC showed either to be hyperechoic or hypoechoic. M: month; UT: untreated; CD: control diet; HFD: high fat diet; DEN: N-nitrosodiethylamine. Black arrow head: HCC nodules on liver; White arrow head: HCC nodules on ultrasound images. (JPG 715 kb

Additional file 5: of Up-regulation of FGF15/19 signaling promotes hepatocellular carcinoma in the background of fatty liver

Figure S4. Upper: Representative Western blot for β-Klotho proteins detection (AFP, FASN, FGFR4 and β-Catenin) of 3 paired tissues (HCC tissue and adjacent benign tissue) from HCC patients. Lower: quantification of AFP, FASN, FGFR4 and β-Catenin by Western blot analysis in tissues (HCC tissue and adjacent benign tissue) from 33 HCC patients. T: HCC tissue; A: adjacent benign tissue. *: P < 0.05 vs adjacent benign tissue. (JPG 608 kb

Additional file 3: of Up-regulation of FGF15/19 signaling promotes hepatocellular carcinoma in the background of fatty liver

Figure S2. The body weights, liver weights, serum and tissue triglyceride levels, alpha fetoprotein (AFP) and alanine transaminase (ALT) levels in all 4 experimental groups at month 2 month 6, and month 10. Glucose tolerance test (GTT) and insulin tolerance test (ITT) were recorded in all 4 experimental groups at month 10. UT: untreated; CD: control diet; HFD: high fat diet; DEN: N-nitrosodiethylamine. *: P < 0.05 vs UT + CD. (JPG 1775 kb