Exploring the impact of insufficient thermal ablation on hepatocellular carcinoma: NDST2 overexpression mechanism and its role in facilitating growth and invasion of residual cancer cells.

OBJECTIVE: Incomplete thermal ablation (ITA) fosters the malignancy of residual cells in Hepatocellular carcinoma (HCC) with unclear mechanisms now. This study aims to investigate the expression changes of NDST2 following ITA of HCC and its impact on residual cancer cells. METHODS: An in vitro model of heat stress-induced liver cancer was constructed to measure the expression of NDST2 using Quantitative Real-Time PCR and Western blotting experiments. The sequencing data from nude mice were used for validation. The clinical significance of NDST2 in HCC was evaluated by integrating datasets. Gene ontology and pathway analysis were conducted to explore the potential signaling pathways regulated by NDST2. Additionally, NDST2 was knocked down in heat stress-induced HCC cells, and the effects of NDST2 on these cells were verified using Cell Counting Kit-8 assays, scratch assays, and Transwell assays. RESULTS: NDST2 expression levels are elevated in HCC, leading to a decrease in overall survival rates of HCC patients. Upregulation of immune checkpoint levels in high NDST2-expressing HCC may contribute to immune evasion by liver cancer cells. Additionally, the low mutation rate of NDST2 in HCC suggests a relatively stable expression of NDST2 in this disease. Importantly, animal and cell models treated with ITA demonstrate upregulated expression of NDST2. Knockdown of NDST2 in heat stress-induced liver cancer cells results in growth inhibition associated with gene downregulation. CONCLUSION: The upregulation of NDST2 can accelerate the progression of residual HCC after ITA, suggesting a potential role for NDST2 in the therapeutic efficacy and prognosis of residual HCC.

Overexpression of PLK1 Molecule Following Incomplete Thermal Ablation Promotes the Proliferation and Invasion of Residual Hepatocellular Carcinoma.

TAT, a widely used treatment for HCC, can exacerbate the progression of residual HCC. The present study investigated the mechanism of action of PLK1 following ITA of HCC. The PLK1 levels in HCC were determined using qRT-PCR from clinical patient samples, IHC from tissue microarray, and data from globally high-throughput data and microarrays. The PLK1 levels and their effect on the biological phenotype of heat-stress HCC cells were evaluated through in vitro experiments. We detected PLK1 abnormal expression in HCC models of nude mice subjected to ITA. We detected the effects of different PLK1 expression levels on EMT pathway proteins. PLK1 exhibited an overexpression in HCC tissues with an SMD of 1.19 (3414 HCC and 3036 non-HCC tissues were included), distinguishing HCC from non-HCC effectively (AUC = 0.9). The qRT-PCR data from clinical HCC patient samples and IHC from HCC tissue microarray results also indicated an overexpressed level. In the incomplete ablation models, an increased PLK1 expression was found in both heat-stress cells and subcutaneous tumors. The upregulation of PLK1 following ITA was found to enhance the malignancy of HCC and exacerbate the proliferation, migration, and invasion of residual HCC cells, whereas PLK1 knockdown suppressed the biological malignancy of HCC cells. Meanwhile, PLK1 has different regulatory effects on various EMT pathway proteins. PLK1 promotes the progression of residual HCC by activating EMT pathway after ITA, which might provide a novel idea for the treatment and prognosis of residual HCC.

Intra-individual comparison of Sonazoid contrast-enhanced ultrasound and SonoVue contrast-enhanced ultrasound in diagnosing hepatocellular carcinoma.

PURPOSE: To assess whether the diagnostic performance of Sonazoid contrast-enhanced ultrasound (SZUS) is non-inferior to that of SonoVue contrast-enhanced ultrasound (SVUS) in diagnosing hepatocellular carcinoma (HCC) in individuals with high risk. MATERIALS AND METHODS: This prospective study was conducted from October 2020 to May 2022 and included participants with a high risk of HCC who underwent SZUS and SVUS. All lesions were confirmed by clinical or pathological diagnosis. Each nodule was classified according to the Contrast-Enhanced Ultrasound Liver Imaging Reporting and Data System version 2017 (CEUS LI-RADS v2017) for SVUS and SZUS and the modified CEUS LI-RADS (using Kupffer phase defect instead of late and mild washout) for SZUS. The diagnostic performance of both two modalities for all observations was compared. Analysis of the vascular phase and Kupffer phase imaging characteristics of CEUS was performed. RESULTS: One hundred and fifteen focal liver lesions from 113 patients (94 HCCs, 12 non-HCC malignancies, and 9 benign lesions) were analysed. According to CEUS LI-RADS (v2017), SVUS and SZUS showed similar sensitivity (71.3% vs. 72.3%) and specificity (85.7% vs. 81.0%) in HCC diagnosis. However, the modified CEUS LI-RADS did not significantly improve the diagnostic efficacy of Sonazoid compared to CEUS LI-RADS v2017, having equivalent sensitivity (73.4% vs. 72.3%) and specificity (81.0% vs. 81.0%). The agreement between SVUS and SZUS for all observations was 0.610 (95% CI 0.475, 0.745), while for HCCs it was 0.452 (95% CI 0.257, 0.647). CONCLUSION: Using LI-RADS v2017, SZUS and SVUS showed non-inferior efficacy in evaluating HCC lesions. In addition, adding Kupffer phase defects to SZUS does not notably improve its diagnostic efficacy.

RAD21: A Key Transcriptional Regulator in the Development of Residual Liver Cancer.

Objective: Thermal ablation is a commonly used therapy for hepatocellular carcinoma (HCC). Nevertheless, inadequate ablation can lead to the survival of residual HCC, potentially causing rapid progression. The underlying mechanisms for this remain unclear. This study explores the molecular mechanism responsible for the rapid progression of residual HCC. Methods: We established an animal model of inadequate ablation in BALB/c nude mice and identified a key transcriptional regulator through high-throughput sequencing. Subsequently, we conducted further investigations on RAD21. We evaluated the expression and clinical significance of RAD21 in HCC and studied its impact on HCC cell function through various assays, including CCK-8, wound healing, Transwell migration and invasion. In vitro experiments established an incomplete ablation model verifying RAD21 expression and function. Using ChIP-seq, we determined potential molecules regulated by RAD21 and investigated how RAD21 influences residual tumor development. Results: High RAD21 expression in HCC was confirmed and correlated with low tumor cell differentiation, tumor growth, and portal vein thrombosis. Silencing RAD21 inhibited the migration, invasion, and proliferation significantly in liver cancer cells. Patients with high RAD21 levels showed elevated multiple inhibitory immune checkpoint levels and a lower response rate to immune drugs. Heat treatment intensified the malignant behavior of liver cancer cells, resulting in increased migration, invasion, and proliferation. After subjecting it to heat treatment, the results indicated elevated RAD21 levels in HCC. Differentially expressed molecules regulated by RAD21 following incomplete ablation were primarily associated with the VEGF signaling pathway, focal adhesion, angiogenesis, and hepatocyte growth factor receptor signaling pathway etc. Conclusion: The upregulation of RAD21 expression after incomplete ablation may play a crucial role in the rapid development of residual tumors and could serve as a novel therapeutic target.

Glioma-derived small extracellular vesicles induce pericyte-phenotype transition of glioma stem cells under hypoxic conditions.

BACKGROUND: Glioblastoma (GBM) is the most common and lethal primary brain tumor characterized by extensive vascularization. Anti-angiogenic therapy for this cancer offers the possibility of universal efficacy. However, preclinical and clinical studies suggest that anti-VEGF drugs, such as Bevacizumab, actively promote tumor invasion, which ultimately leads to a therapy-resistant and recurrent phenotype of GBMs. Whether Bevacizumab can improve survival over chemotherapy alone remains debated. Herein, we emphasize the importance of small extracellular vesicles (sEVs) internalization by glioma stem cells (GSCs) in giving rise to the failure of anti-angiogenic therapy in the treatment of GBMs and discover a specific therapeutic target for this damaging disease. METHODS: To experimentally prove that hypoxia conditions promote the release of GBM cells-derived sEVs, which could be taken up by the surrounding GSCs, we used an ultracentrifugation strategy to isolate GBM-derived sEVs under hypoxic or normoxic conditions, performed bioinformatics analysis and multidimensional molecular biology experiments, and established a xenograft mouse model. RESULTS: The internalization of sEVs by GSCs was proven to promote tumor growth and angiogenesis through the pericyte-phenotype transition. Hypoxia-derived sEVs could efficiently deliver TGF-ß1 to GSCs, thus resulting in the activation of the TGF-ß signaling pathway and the consequent pericyte-phenotype transition. Specifically targeting GSC-derived pericytes using Ibrutinib can reverse the effects of GBM-derived sEVs and enhance the tumor-eradicating effects when combined with Bevacizumab. CONCLUSION: This present study provides a new interpretation of the failure of anti-angiogenic therapy in the non-operative treatment of GBMs and discovers a promising therapeutic target for this intractable disease.

Integrative radiomics and transcriptomics analyses reveal subtype characterization of non-small cell lung cancer.

OBJECTIVES: To assess whether integrative radiomics and transcriptomics analyses could provide novel insights for radiomic features' molecular annotation and effective risk stratification in non-small cell lung cancer (NSCLC). METHODS: A total of 627 NSCLC patients from three datasets were included. Radiomics features were extracted from segmented 3-dimensional tumour volumes and were z-score normalized for further analysis. In transcriptomics level, 186 pathways and 28 types of immune cells were assessed by using the Gene Set Variation Analysis (GSVA) algorithm. NSCLC patients were categorized into subgroups based on their radiomic features and pathways enrichment scores using consensus clustering. Subgroup-specific radiomics features were used to validate clustering performance and prognostic value. Kaplan-Meier survival analysis with the log-rank test and univariable and multivariable Cox analyses were conducted to explore survival differences among the subgroups. RESULTS: Three radiotranscriptomics subtypes (RTSs) were identified based on the radiomics and pathways enrichment profiles. The three RTSs were characterized as having specific molecular hallmarks: RTS1 (proliferation subtype), RTS2 (metabolism subtype), and RTS3 (immune activation subtype). RTS3 showed increased infiltration of most immune cells. The RTS stratification strategy was validated in a validation cohort and showed significant prognostic value. Survival analysis demonstrated that the RTS strategy could stratify NSCLC patients according to prognosis (p = 0.009), and the RTS strategy remained an independent prognostic indicator after adjusting for other clinical parameters. CONCLUSIONS: This radiotranscriptomics study provides a stratification strategy for NSCLC that could provide information for radiomics feature molecular annotation and prognostic prediction. KEY POINTS: • Radiotranscriptomics subtypes (RTSs) could be used to stratify molecularly heterogeneous patients. • RTSs showed relationships between molecular phenotypes and radiomics features. • The RTS algorithm could be used to identify patients with poor prognosis.

Molecular hallmarks of breast multiparametric magnetic resonance imaging during neoadjuvant chemotherapy.

PURPOSE: To identify molecular basis of four parameters obtained from dynamic contrast-enhanced magnetic resonance imaging, including functional tumor volume (FTV), longest diameter (LD), sphericity, and contralateral background parenchymal enhancement (BPE). MATERIAL AND METHODS: Pretreatment-available gene expression profiling and different treatment timepoints MRI features were integrated for Spearman correlation analysis. MRI feature-related genes were submitted to hypergeometric distribution-based gene functional enrichment analysis to identify related Kyoto Encyclopedia of Genes and Genomes annotation. Gene set variation analysis was utilized to assess the infiltration of distinct immune cells, which were used to determine relationships between immune phenotypes and medical imaging phenotypes. The clinical significance of MRI and relevant molecular features were analyzed to identify their prediction performance of neoadjuvant chemotherapy (NAC) and prognostic impact. RESULTS: Three hundred and eighty-three patients were included for integrative analysis of MRI features and molecular information. FTV, LD, and sphericity measurements were most positively significantly correlated with proliferation-, signal transmission-, and immune-related pathways, respectively. However, BPE did not show marked correlation relationships with gene expression alteration status. FTV, LD and sphericity all showed significant positively or negatively correlated with some immune-related processes and immune cell infiltration levels. Sphericity decreased at 3 cycles after treatment initiation was also markedly negatively related to baseline sphericity measurements and immune signatures. Its decreased status could act as a predictor for prediction of response to NAC. CONCLUSION: Different MRI features capture different tumor molecular characteristics that could explain their corresponding clinical significance.

METTL3/IGF2BP3 axis inhibits tumor immune surveillance by upregulating N6-methyladenosine modification of PD-L1 mRNA in breast cancer.

BACKGROUND: Continual expression of PD-L1 in tumor cells is critical for tumor immune escape and host T cell exhaustion, however, knowledge on its clinical benefits through inhibition is limited in breast cancer. N6-methyladenosine (m6A) plays a crucial role in multiple biological activities. Our study aimed to investigate the regulatory role of the m6A modification in PD-L1 expression and immune surveillance in breast cancer. METHODS: MeRIP-seq and epitranscriptomic microarray identified that PD-L1 is the downstream target of METTL3. MeRIP-qPCR, absolute quantification of m6A modification assay, and RIP-qPCR were used to examine the molecular mechanism underlying METTL3/m6A/IGF2BP3 signaling axis in PD-L1 expression. B-NDG and BALB/c mice were used to construct xenograft tumor models to verify the phenotypes upon METTL3 and IGF2BP3 silencing. In addition, breast cancer tissue microarray was used to analyze the correlation between PD-L1 and METTL3 or IGF2BP3 expression. RESULTS: We identified that PD-L1 was a downstream target of METTL3-mediated m6A modification in breast cancer cells. METTL3 knockdown significantly abolished m6A modification and reduced stabilization of PD-L1 mRNA. Additionally, METTL3-mediated PD-L1 mRNA activation was m6A-IGF2BP3-dependent. Moreover, inhibition of METTL3 or IGF2BP3 enhanced anti-tumor immunity through PD-L1-mediated T cell activation, exhaustion, and infiltration both in vitro and in vivo. PD-L1 expression was also positively correlated with METTL3 and IGF2BP3 expression in breast cancer tissues. CONCLUSION: Our study suggested that METTL3 could post-transcriptionally upregulate PD-L1 expression in an m6A-IGF2BP3-dependent manner to further promote stabilization of PD-L1 mRNA, which may have important implications for new and efficient therapeutic strategies in the tumor immunotherapy.

The LXR-623-induced long non-coding RNA LINC01125 suppresses the proliferation of breast cancer cells via PTEN/AKT/p53 signaling pathway.

LXR-623 (WAY-252623), a liver X receptor agonist, reduces atherosclerotic plaque progression and remarkably inhibits the proliferation of glioblastoma cells, owing to its brain-penetrant ability. However, the role of LXR-623 against the proliferation of other cancer cells and the underlying mechanism remain unknown. Long non-coding RNAs (lncRNAs) serve as novel and crucial regulators that participate in cancer tumorigenesis and diverse biological processes. Here, we report a previously uncharacterized mechanism underlying lncRNA-mediated exocytosis of LXR-623 via the phosphatase and tensin homolog (PTEN)/protein kinase B (AKT)/p53 axis to suppress the proliferation of cancer cells in vitro. We found that LXR-623 significantly inhibited the proliferation and induced apoptosis and cell cycle arrest at S phase in breast cancer cells in a concentration- and time-dependent manner. Experiments using a xenograft mouse model revealed the inhibitory effects of LXR-623 on tumor growth. We used lncRNA microarray to investigate the potential genes regulated by LXR-623. As a result, LINC01125 was found to be significantly upregulated in the cells treated with LXR-623. Gain- and loss-of-function assays were conducted to investigate the anti-proliferation role of LINC01125. LINC01125 knockdown resulted in the inhibition of the cytotoxic effect of LXR-623; in contrast, LINC01125 overexpression significantly enhanced the effect of LXR-623. LXR-623 and LINC01125-mediated anti-growth regulation is, at least in part, associated with the participation of the PTEN/AKT/mouse double minute 2 homolog (MDM2)/p53 pathway. In addition, SF1670, a specific PTEN inhibitor with prolonged intracellular retention, may strongly block the anti-proliferation effect induced by LXR-623 and LINC01125 overexpression. Chromatin immunoprecipitation (ChIP) assay results suggest that p53 binds to the promoter of LINC01125 to strengthen the expression of the PTEN/AKT pathway. Taken together, our findings suggest that LXR-623 possesses significant antitumor activity in breast cancer cells that is partly mediated through the upregulation in LINC01125 expression and enhancement in apoptosis via the PTEN/AKT/MDM2/p53 pathway.

Resveratrol provides neuroprotection by regulating the JAK2/STAT3/PI3K/AKT/mTOR pathway after stroke in rats.

Ischemic stroke is a common disease with high mortality and morbidity worldwide. One of the important pathophysiological effects of ischemic stroke is apoptosis. A neuroprotective effect is defined as the inhibition of neuronal apoptosis to rescue or delay the infarction in the surviving ischemic penumbra. Resveratrol is a natural polyphenol that reportedly prevents cerebral ischemia injury by regulating the expression of PI3K/AKT/mTOR. Therefore, this study aimed to elucidate the neuroprotective effect of resveratrol on cerebral ischemia/reperfusion injury and to investigate the signaling pathways and mechanisms through which resveratrol regulates apoptosis in the ischemic penumbra. Rats were subjected to middle cerebral artery occlusion for 2 h followed by 24 h reperfusion. Cerebral infarct volume was measured using 2% TTC staining. TUNEL staining was conducted to evaluate neuronal apoptosis. Western blotting and immunohistochemistry were used to detect the proteins involved in the JAK2/STAT3/PI3K/AKT/mTOR pathway. The results suggested that resveratrol significantly improved neurological function, reduced cerebral infarct volume, decreased neuronal damage, and markedly attenuated neuronal apoptosis; these effects were attenuated by the inhibition of PI3K/AKT with LY294002 and JAK2/STAT3 with AG490. We also found that resveratrol significantly upregulated the expression of p-JAK2, p-STAT3, p-AKT, p-mTOR, and BCL-2 and downregulated expression of cleaved caspase-3 and BAX, which was partially reversed by LY294002 and AG490. These results suggested that resveratrol provides a neuroprotective effect against cerebral ischemia/reperfusion injury, which is partially mediated by the activation of JAK2/STAT3 and PI3K/AKT/mTOR. Resveratrol may indirectly upregulate the PI3K/AKT/mTOR pathway by activating JAK2/STAT3.