Analysis of Carcinogenic Involvement of MicroRNA Pattern in Peripheral Non-Cancerous Tissues and Chronic Viral Liver Injury.

Risk factors for hepatocarcinogenesis include chronic inflammation due to viral infection, liver fibrosis, and aging. In this study, we separated carcinogenic and non-carcinogenic cases due to hepatitis C virus (HCV) infection, aiming to comprehensively analyze miRNA expression in liver tissues by age, and identify factors that contribute to carcinogenesis. Total RNA was extracted from 360 chronic hepatitis C (CH), 43 HCV infected hepatocellular carcinoma (HCC), and surrounding non-tumor (SNT) tissues. MicroRNA (miRNA) expression patterns were analyzed using microarray. Using machine learning, we extracted characteristic miRNA expression patterns for each disease and age. There were no age-dependent changes in miRNA expression in the disease-specific comparisons; however, miRNA expression differed among the age groups of 50, 60, and 70 years of age between CH and SNT. The expression of miRNA was different between SNT and HCC only in patients in their 70s. Of the 55 miRNAs with significant differences in expression between CH and SNT, 34 miRNAs showed significant differences in expression even in the degree of liver fibrosis. The observation that miRNAs involved in hepatocarcinogenesis differ at different ages suggests that the mechanisms of carcinogenesis differ by age group as well. We also found that many miRNAs whose expression did not affect liver fibrosis were involved in carcinogenesis. These findings are expected to define biomarkers for detection of HCC at early stage, and develop novel therapeutic targets for HCC.

Exploring extracellular matrix and prostaglandin pathway alterations across varying resection margin distances of right-sided colonic adenocarcinoma.

BACKGROUND: Surgical resection followed by indicated adjuvant therapy offers potential curative treatment in colonic adenocarcinoma. Beyond the well-established seed and soil theory of colon cancer progression, the 'normal-appearing' tissues near the tumor are not genuinely normal and remain as remnants in patients following surgery. Our objective was to elucidate the alteration of gene expression and pathways across various distances of resection margins in right-sided colonic adenocarcinoma. METHODS: Twenty-seven fresh samples of primary cancer and 56 matched non-tumor tissues adjacent to the tumor (NAT) were collected from patients with resectable right-sided colon cancer. NAT were systematically obtained at varying distances (1, 5, and 10 cm) on both proximal and distal sides. Comprehensive gene expression analysis was performed using 770-gene PanCancer Progression Panel, delineating distinctive pathways and functional predictions for each region. RESULTS: Distinctive gene signatures and pathways exhibited by normal-appearing tissues were discovered at varying distances from cancer. Notably, SFRP2, PTGDS, COL1A1, IL1B, THBS2, PTGIS, COL1A2, NPR1, and BGN were upregulated, while ENPEP, MMP1, and NRCAM were downregulated significantly in 1-cm tissue compared to farther distances. Substantial alterations in the extracellular matrix (ECM) and prostaglandin/thromboxane synthesis were significantly evident at the 1-cm distance. Functional analysis indicated enhanced cell viability and survival, alongside reduced cellular death and apoptosis. CONCLUSIONS: Different distances exerted a significant impact on gene alteration within the normal-looking mucosa surrounding primary cancer, influenced by various mechanisms. These findings may highlight potential therapeutic targets related to the ECM and prostaglandin/thromboxane pathways for treatment strategies.

Bioimaging and Sensing Thiols In Vivo and in Tumor Tissues Based on a Near-Infrared Fluorescent Probe with Large Stokes Shift.

The well-known small-molecule biothiols have been used to maintain the normal metabolism of peroxy radicals, forming protein structures, resisting cell apoptosis, regulating metabolism, and protecting the homeostasis of cells in the organism. A large amount of research has found that abnormal levels of the above biothiols can cause some adverse diseases, such as changes in hair pigmentation, a slower growth rate, delayed response, excessive sleep and skin diseases. In order to further investigate the exact intracellular molecular mechanism of biothiols, it is imperative to explore effective strategies for real-time biothiol detection in living systems. In this work, a new near-infrared (NIR) emission fluorescence probe (probe 1) for sensitive and selective detection of biothiols was devised by combining dicyanoisophorone derivatives with the dinitrobenzenesulfonyl (DNBS) group. As expected, probe 1 could specifically detect biothiols (Cys, Hcy and GSH) through the dinitrobenzenesulfonyl group to form dye 2, which works as a signaling molecule for sensing biothiols in real samples. Surprisingly, probe 1 showed superior sensing characteristics and low-limit detection towards biothiols (36.0 nM for Cys, 39.0 nM for Hcy and 48.0 nM for GSH) with a large Stokes shift (134 nm). Additionally, the function of probe 1 as a platform for detecting biothiols was confirmed by confocal fluorescence imaging of biothiols in MCF-7 cells and zebrafish. More importantly, the capability of probe 1 in vivo has been further evaluated by imaging the overexpressed biothiols in tumor tissue. It is reasonable to believe that probe 1 can provide a valuable method to explore the relationship between biothiols and the genesis of tumor.

A benzothiazole-based fluorescence probe for imaging of peroxynitrite during ferroptosis and diagnosis of tumor tissues.

Ferroptosis, as a new regulated mode of cell death, is mainly characterized by iron-dependent accumulation of lipid peroxides related to reactive oxygen species (ROS). However, the changes of peroxynitrite (ONOO-) during ferroptosis are not very clear due to the limited reports. On the other hand, ONOO- has become an endogenous toxicant leading to cell apoptosis and necrosis. Thus, it is urgent to develop molecular tools with high selectivity and sensitivity to monitor intracellular ONOO-. Herein, we presented a fluorescent probe, BTMO-PN, by introducing a phenylboronic acid ester as the ONOO- recognition site into benzothiazolyl derivative. BTMO-PN exhibited a rapid and marked fluorescence enhancement signal toward ONOO-, owing to the ONOO--triggered the cleavage of phenylboronic acid ester to release strongly fluorescent BTMO. Moreover, BTMO-PN could image endogenous and exogenous ONOO- changes in live cells. Importantly, using BTMO-PN, we demonstrated the up-generation of ONOO- levels in cancer cells during ferroptosis. Furthermore, BTMO-PN has successfully been applied for distinguishing tumor tissues from normal tissues with excellent contrast, making it great potential for cancer diagnosis by detecting the ONOO- changes.

A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a 'green' xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

Investigation of the Distribution of Magnetic Nanoparticles in Tumor Tissues by the Method of Scanning Magnetic Force Nanotomography.

The development of effective biomedical technologies using magnetic nanoparticles (MNPs) for the tasks of oncotherapy and nanodiagnostics requires the development and implementation of new methods for the analysis of micro- and nanoscale distributions of MNPs in the volume of cells and tissues. The paper presents a new approach to three-dimensional analysis of MNP distributions - scanning magnetic force nanotomography as applied to the study of tumor tissues. Correlative reconstruction of MNP distributions and nanostructure features of the studied tissues made it possible to quantitatively estimate the parameters of three-dimensional distributions of composite nanoparticles based on silicon and iron oxide obtained by femtosecond laser ablation and injected intravenously and intratumorally into tumor tissue samples of B16/F1 mouse melanoma. The developed technology based on the principles of scanning probe nanotomography is applicable for studying the features of three-dimensional micro- and nanoscale distributions of magnetic nanoparticles in biomaterials, cells and tissues of various types.

1 H NMR-based metabolomics reveal overlapping discriminatory metabolites and metabolic pathway disturbances between colorectal tumor tissues and fecal samples.

Previous studies have compared fecal metabolites from healthy and colorectal cancer (CRC) patients to predict the pro-CRC signatures. However, the systemic mechanistic link between feces and colonic tissues of CRC patients is still limited. The current study was a paralleled investigation of colonic tumor tissues and their normal adjacent tissues alongside patient-matched feces by using 1 H nuclear magnetic resonance spectroscopy combined with pattern recognition to investigate how fecal metabolic phenotypes are linked to the changes in colorectal tumor profiles. A set of overlapping discriminatory metabolites across feces and tumor tissues of CRC were identified, including elevated levels of lactate, glutamate, alanine, succinate and reduced amounts of butyrate. These changes could indicate the networks for metabolic pathway perturbations in CRC potentially involved in the disruptions of glucose and glycolytic metabolism, TCA cycle, glutaminolysis, and short chain fatty acids metabolism. Furthermore, changes in fecal acetate were positively correlated with alterations of glucose and myo-inositol in colorectal tumor tissues, implying enhanced energy production for rapid cell proliferation. Compared to other fecal metabolites, acetate demonstrated the highest diagnostic performance for diagnosing CRC, with an AUC of 0.843 in the training set, and a good predictive ability in the validation set. Overall, these associations provide evidence of distinct metabolic signatures and metabolic pathway disturbances between the colonic tissues and feces within the same individual, and changes of fecal metabolic signature could reflect the CRC tissue microenvironment, highlighting the significance of the distinct fecal metabolic profiles as potential novel and noninvasive relevant indicators for CRC detection.

Screening and identification of non-inflammatory specific protein markers in Wilms' tumor tissues.

Wilms' tumor is one of the most common malignancies in children, and early diagnosis is critical for its subsequent treatment and prognosis. Our previous study employed proteomics to investigate protein markers in the serum of Wilms' tumor children. The present study aimed to identify specific protein markers in Wilms' tumor. Proteomic comparison of Wilms' tumor with normal kidney tissues and the sera of systemic inflammatory response syndrome (SIRS) controls was performed. Surface-enhanced laser desorption ionization time-of-flight (SELDI-TOF-MS) identified a protein with m/z 8350 as specific to Wilms' tumor. The target protein was purified using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified as profilin-1 by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF). Its expression was validated using real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Our data identify profilin-1 as a potential protein marker for Wilms' tumor and demonstrate the feasibility of the above procedures for screening and identification of tumor-specific protein markers.

Establishing a correlation between RIN and A260/280 along with the multivariate evaluation of factors affecting the quality of RNA in cryopreserved cancer bio-specimen.

Biomarkers are biological characteristic to measure and evaluate normal and pathological states. To define expression-based molecular biomarkers, high-quality tissue samples are a prerequisite for the preparation of standard RNA. It is already known that RIN number defines the RNA quality, however its relation with A260/280 ratio and Ct value is not defined clearly; therefore, understanding an association will provide a reliable method for describing RNA quality. Multiple cryopreserved human tumor tissue types from ACTREC Biorepository and TMH-INTTR were analyzed for the effect of storage time on RNA quality. The RNA from tumor samples were isolated and analyzed by RIN, A260/280 ratio, and Ct value to establish inter-relationships. Around 50% samples had a RIN of ≥ 6.9 and A260/280 ≤ 2.04; 27% had a RIN ≥ 5 and A260/280 ≤ 2.08, and remaining 23% displayed RIN < 5 and A260/280 > 2.08. However, the RNA quality has no association with the storage period of tissue samples. Moreover, all samples which had A260/280 ≤ 2.08 showed acceptable Ct values of 17-24. The data clearly suggests that the A260/280 ratio is able to predict RNA quality. To the best of our knowledge, this is the first Indian report analyzing the labile nucleic acid-RNA quality from different cancer tissue types cryopreserved for diverse time periods. In conclusion, RIN and A260/280 ratio can help in predicting the quality of RNA independently; however, both together provide better assurance for further downstream processing.

HILIC/ESI-MS determination of gangliosides and other polar lipid classes in renal cell carcinoma and surrounding normal tissues.

Negative-ion hydrophilic liquid chromatography-electrospray ionization mass spectrometry (HILIC/ESI-MS) method has been optimized for the quantitative analysis of ganglioside (GM3) and other polar lipid classes, such as sulfohexosylceramides (SulfoHexCer), sulfodihexosylceramides (SulfoHex2Cer), phosphatidylglycerols (PG), phosphatidylinositols (PI), lysophosphatidylinositols (LPI), and phosphatidylserines (PS). The method is fully validated for the quantitation of the studied lipids in kidney normal and tumor tissues of renal cell carcinoma (RCC) patients based on the lipid class separation and the coelution of lipid class internal standard with the species from the same lipid class. The raw data are semi-automatically processed using our software LipidQuant and statistically evaluated using multivariate data analysis (MDA) methods, which allows the complete differentiation of both groups with 100% specificity and sensitivity. In total, 21 GM3, 28 SulfoHexCer, 26 SulfoHex2Cer, 10 PG, 19 PI, 4 LPI, and 7 PS are determined in the aqueous phase of lipidomic extracts from kidney tumor tissue samples and surrounding normal tissue samples of 20 RCC patients. S-plots allow the identification of most upregulated (PI 40:5, PI 40:4, GM3 34:1, and GM3 42:2) and most downregulated (PI 32:0, PI 34:0, PS 36:4, and LPI 16:0) lipids, which are primarily responsible for the differentiation of tumor and normal groups. Another confirmation of most dysregulated lipids is performed by the calculation of fold changes together with T and p values to highlight their statistical significance. The comparison of HILIC/ESI-MS data and matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) data confirms that lipid dysregulation patterns are similar for both methods. Graphical abstract ᅟ.

Evaluation of genome-wide genotyping concordance between tumor tissues and peripheral blood.

Tumor tissues were potential resources in cancer susceptibility studies. To assess the genotyping concordance between tumor tissues and peripheral blood, we conducted this study in a large sample size and genome-wide scale. Genome-wide genotypes of human colon adenocarcinoma (COAD) retrieved from The Cancer Genome Atlas (TCGA) was analyzed. A total of 387 pairs of matched fresh frozen tumor tissues and peripheral blood samples passed the quality control processes. High concordant rate (94.85% with no-calls and 97.89% without no-calls) was found between tumor tissues and peripheral blood. The discordant rate raised with the increase of heterozygote rate, and the tendency was statistically significant. The total missing rate was 3.10%. We also verified 14 susceptibility SNPs and the average genotyping concordant rate was 97.42%. These findings suggest that majority of SNPs could be accurately genotyped using DNA isolated from tumor tissues.

Quality Assessments of Long-Term Quantitative Proteomic Analysis of Breast Cancer Xenograft Tissues.

Clinical proteomics requires large-scale analysis of human specimens to achieve statistical significance. We evaluated the long-term reproducibility of an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomics strategy using one channel for reference across all samples in different iTRAQ sets. A total of 148 liquid chromatography tandem mass spectrometric (LC-MS/MS) analyses were completed, generating six 2D LC-MS/MS data sets for human-in-mouse breast cancer xenograft tissues representative of basal and luminal subtypes. Such large-scale studies require the implementation of robust metrics to assess the contributions of technical and biological variability in the qualitative and quantitative data. Accordingly, we derived a quantification confidence score based on the quality of each peptide-spectrum match to remove quantification outliers from each analysis. After combining confidence score filtering and statistical analysis, reproducible protein identification and quantitative results were achieved from LC-MS/MS data sets collected over a 7-month period. This study provides the first quality assessment on long-term stability and technical considerations for study design of a large-scale clinical proteomics project.

MicroRNA-101 inhibits human hepatocellular carcinoma progression through EZH2 downregulation and increased cytostatic drug sensitivity.

BACKGROUND & AIMS: Oncogene polycomb group protein enhancer of zeste homolog 2 (EZH2) has been proposed to be a target gene of putative tumor suppressor microRNA-101 (miR-101). The aim of our study was to investigate the functional role of both miR-101 and EZH2 in human hepatocellular carcinoma (HCC). METHODS: MiR-101 and EZH2 expressions were evaluated in tumor tissues of 99 HCC patients and 7 liver cancer cell lines by real-time PCR. Luciferase reporter assay was employed to validate whether EZH2 represents a target gene of miR-101. The effect of miR-101 on HCC growth as well as programmed cell death was studied in vitro and in vivo. RESULTS: MiR-101 expression was significantly downregulated in most of HCC tissues and all cell lines, whereas EZH2 was significantly overexpressed in most of HCC tissues and all cell lines. There was a negative correlation between expression levels of miR-101 and EZH2. Luciferase assay results confirmed EZH2 as a direct target gene of miR-101, which negatively regulates EZH2 expression in HCC. Ectopic overexpression of miR-101 dramatically repressed proliferation, invasion, colony formation as well as cell cycle progression in vitro and suppressed tumorigenicity in vivo. Furthermore, miR-101 inhibited autophagy and synergized with either doxorubicin or fluorouracil to induce apoptosis in tumor cells. CONCLUSION: Tumor suppressor miR-101 represses HCC progression through directly targeting EZH2 oncogene and sensitizes liver cancer cells to chemotherapeutic treatment. Our findings provide significant insights into molecular mechanisms of hepatocarcinogenesis and may have clinical relevance for the development of novel targeted therapies for HCC.

Stage dependent microbial dynamics in hepatocellular carcinoma and adjacent normal liver tissues.

The interactive pathway of the gut-liver axis underscores the significance of microbiome modulation in the pathogenesis and progression of various liver diseases, including hepatocellular carcinoma (HCC). This study aims to investigate the disparities in the composition and functionality of the hepatic microbiota between tumor tissues and adjacent normal liver tissues, and their implications in the etiology of HCC. We conducted a comparative analysis of the hepatic microbiome between adjacent normal liver tissues and tumor tissues from HCC patients. Samples were categorized according to the modified Union for International Cancer Control (mUICC) staging system into Non-tumor, mUICC stage I, mUICC stage II, and mUICC stage III groups. Microbial richness and community composition were analyzed, and phylogenetic profiles were examined to identify significantly altered microbial taxa among the groups. Predicted metabolic pathways were analyzed using PICRUSt2. Our analysis did not reveal significant differences in microbial richness and community composition with the development of HCC. However, phylogenetic profiling identified significantly altered microbial taxa among the groups. Sphingobium, known for degrading polychlorinated biphenyls (PCBs), exhibited a significantly negative correlation with clinical indices in HCC patients. Conversely, Sphingomonas, a gut bacterium associated with various liver diseases, showed a positive correlation. Predicted metabolic pathways suggested a correlation between atrazine degradation and valine, leucine, and isoleucine biosynthesis with mUICC stage and tumor size. Our results underscore the critical link between hepatic microbial composition and function and the HCC tumor stage, suggesting a potentially pivotal role in the development of HCC. These findings highlight the importance of targeting the hepatic microbiome for therapeutic strategies in HCC.

Bioinformatics analysis and validation of CSRNP1 as a key prognostic gene in non-small cell lung cancer.

Cysteine and serine-rich nuclear protein 1 (CSRNP1) has shown prognostic significance in various cancers, but its role in non-small cell lung cancer (NSCLC) remains elusive. We investigated CSRNP1 expression in NSCLC cases using bioinformatics tools from the GEO public repository and validated our findings through RT-qPCR in tumor and adjacent normal tissues. KEGG and GO enrichment analyses were employed to unveil the significant deregulation in signaling pathways. Additionally, clinical significance of CSRNP1 in NSCLC was determined through receiver operating curve (ROC) analysis, and its impact on survival was assessed using Kaplan-Meier analysis. To explore the functional impact of CSRNP1, we silenced its expression in NSCLC cells and assessed the effects on cell viability, migration, and invasion using MTT, Transwell, and wound-healing assays, respectively. Additionally, we investigated the influence of CSRNP1 silencing on the phosphorylation patterns of critical signaling proteins such as p53, p-Akt, and p-MDM2. Our results demonstrated significantly lower CSRNP1 expression in NSCLC tumor tissues (P < 0.01). ROC analysis indicated that NSCLC patients with high CSRNP1 expression exhibited extended overall survival and disease-free survival. Furthermore, CSRNP1 silencing promoted NSCLC cells viability, migration, and invasion (P < 0.05). Mechanistically, CSRNP1 silencing led to increased phosphorylation of AKT and MDM2, along with a concurrent reduction in p53 protein expression, suggesting its impact on NSCLC through deregulated cell cycle processes. In conclusion, our study underscores the significance of CSRNP1 in NSCLC pathogenesis, offering insights for targeted therapeutic interventions of NSCLC.

Intelligent tumor tissue classification for Hybrid Health Care Units.

Introduction: In the evolving healthcare landscape, we aim to integrate hyperspectral imaging into Hybrid Health Care Units to advance the diagnosis of medical diseases through the effective fusion of cutting-edge technology. The scarcity of medical hyperspectral data limits the use of hyperspectral imaging in disease classification. Methods: Our study innovatively integrates hyperspectral imaging to characterize tumor tissues across diverse body locations, employing the Sharpened Cosine Similarity framework for tumor classification and subsequent healthcare recommendation. The efficiency of the proposed model is evaluated using Cohen's kappa, overall accuracy, and f1-score metrics. Results: The proposed model demonstrates remarkable efficiency, with kappa of 91.76%, an overall accuracy of 95.60%, and an f1-score of 96%. These metrics indicate superior performance of our proposed model over existing state-of-the-art methods, even in limited training data. Conclusion: This study marks a milestone in hybrid healthcare informatics, improving personalized care and advancing disease classification and recommendations.

Differential Expression of lincRNA-ROR Spliced Transcript Variants in Breast Cancer.

Background: We investigated the expression pattern of a human stem cell-specific, large intergenic noncoding RNA (lincRNA) regulator of reprogramming (lincRNA-ROR) and its spliced transcript variants in breast tumors. Breast cancer is the leading cause of cancer mortality in women; therefore, finding a reliable diagnostic tumor marker, based on the molecular profile of tumor cells, is warranted. Methods: qRT-PCR was used to investigate the expression alteration of a specific stem cell-related lincRNA and its spliced transcript variants in breast tumors which provided by the Iran National Tumor Bank (2014-2016). Suitability of lincRNA-ROR and expression alterations of its spliced transcript variants as breast tumor biomarkers were examined by ROC curve analysis. Results: Expression was significantly upregulated in lincRNA-ROR variants 1 (NR-048536) and 4 (AB844432) and downregulated in variant 3 (AB844431), with expression levels failing to distinguish between breast tumor types, grades, and malignancy stages. Whereas ROC curve analysis gave good scores to the expressions of variants 1 (AUC=0.7675, P=0.003) and 3 (AUC=0.9383, P=0.00173), suggesting their suitability as potential breast tumor biomarkers, it gave an AUC score of 0.6033 for lincRNA-ROR spliced variant 4 (P=0.4118), denoting its unsuitability as a breast cancer biomarker. Conclusion: Aberrant expressions of lincRNA-ROR spliced transcript variants could serve as reliable biomarkers with potential usefulness in breast cancer diagnosis. However, further research should elucidate the role and tissue expression of lincRNA-ROR spliced transcript variants in various cancers.

Banking of Tumor Tissues: Effect of Preanalytical Variables in the Phase of Pre- and Postacquisition on RNA Integrity.

Background: RNA integrity of tumor tissues from 12 common organs was measured, and tumor tissues from liver were found to have the best RNA integrity in our previous study. The effects of preanalytical variables in the phase of pre- and postacquisition on RNA integrity were further assessed in hepatocellular carcinoma (HCC) tissues in this study. Methods: RNA integrity number (RIN) was measured in tissues from 146 HCC patients. First, 42 fresh HCC tumor tissues were newly collected to assess the effect of various preanalytical variables in the phase of preacquisition on RNA integrity. Second, eight paired HCC tumor and normal tissues were newly collected and used in the gradient course study of ex vivo ischemia time and freeze-thaw cycles on RNA integrity. Finally, 96 stock-frozen tumor tissues with various years of frozen storage were used to assess the effect of cryopreservation time. Results: RNA integrity was found to be independent of patient age, sex, clinical stage, tumor location, HBV infection status, tumor diameter, and surgical approach, but affected by tumor grade. Tumor tissues with a greater tumor grade had lower RIN. With the prolongation of ex vivo ischemia time, freeze-thaw cycles, and cryopreservation time, the RIN of HCC tissues showed decreasing trends. Significant decreases in RIN of the tumor and normal tissues were observed at 6 and 2 hours of ex vivo ischemia time, respectively, and significantly decreased RIN of tumor tissues was observed after six freeze-thaw cycles and 6 years of cryopreservation. Conclusions: Preanalytical variables in the phase of preacquisition such as tumor grade, and in the postacquisition phase such as ex vivo ischemia time, freeze-thaw times, and freeze-storage time both have effects on RNA integrity of HCC tissues. Tissue-based translational research should pay attention to preanalytical variables when collecting and utilizing tumor tissues.

Patient-derived organoids in translational oncology and drug screening.

Patient-derived organoids (PDO) are a new biomedical research model that can reconstruct phenotypic and genetic characteristics of the original tissue and are useful for research on pathogenesis and drug screening. To introduce the progression in this field, we review the key factors of constructing organoids derived from epithelial tissues and cancers, covering culture medium and matrix, morphological characteristics, genetic profiles, high-throughput drug screening, and application potential. We also discuss the co-culture system of cancer organoids with tumor microenvironment (TME) associated cells. The co-culture system is widely used in evaluating crosstalk of cancer cells with TME components, such as fibroblasts, endothelial cells, immune cells, and microorganisms. The article provides a prospective for standardized cultivation mode, automatic morphological evaluation, and drug sensitivity screening using high-throughput methods.

Imaging of a novel ratio γ-glutamyl transpeptidase fluorescent probe in living cells and biopsies.

Herein, a novel fluorescent probe, GTP, was developed for monitoring the GGT (γ-glutamyl transpeptidase) level in living cells and biopsies. It consisted of the typical recognition group γ-Glu (γ-Glutamylcysteine) and the fluorophore (E)-4-(4-aminostyryl)-1-methylpyridin-1-ium iodide. With a ratio response between the signal intensity at 560 nm and 500 nm (RI560/I500), it could be important complement for the turn-on ones. With the linear range of 0-50 U/L, the limit of detection was calculated as 0.23 µM. The detection system showed the strongest response near pH 7.4, and exhibited steady fluorescence signals for at least 48 h. With high selectivity, good anti-interference and low cytotoxicity, GTP was suitable for physiological applications. By monitoring the GGT level with the ratio values in the green and blue channels, the probe GTP could distinguish cancer cells from normal cells. Furthermore, in the mouse tissues and humanization tissue samples, the probe GTP could also recognize the tumor tissues from the normal ones.