Isolation and Characterization of Urinary Extracellular Vesicles from Healthy Donors and Patients with Castration-Resistant Prostate Cancer

Prostate cancer (PCa) is the most commonly diagnosed malignancy among men in developed countries. The five-year survival rate for men diagnosed with early-stage PCa is approximately 100%, while it is less than 30% for castration-resistant PCa (CRPC). Currently, the detection of prostate-specific antigens as biomarkers for the prognosis of CRPC is criticized because of its low accuracy, high invasiveness, and high false-positive rate. Therefore, it is important to identify new biomarkers for prediction of CRPC progression. Extracellular vesicles (EVs) derived from tumors have been highlighted as potential markers for cancer diagnosis and prognosis. Specifically, urinary EVs directly reflect changes in the pathophysiological conditions of the urogenital system because it is exposed to prostatic secretions. Thus, detecting biomarkers in urinary EVs provides a promising approach for performing an accurate and non-invasive liquid biopsy for CPRC. In this study, we effectively isolated urinary EVs with low protein impurities using size-exclusion chromatography combined with ultrafiltration. After EV isolation and characterization, we evaluated the miRNAs in urinary EVs from healthy donors and patients with CRPC. The results indicated that miRNAs (miR-21-5p, miR-574-3p, and miR-6880-5p) could be used as potential biomarkers for the prognosis of CRPC. This analysis of urinary EVs contributes to the fast and convenient prognosis of diseases, including CRPC, in the clinical setting.     

Critical Roles of Circular RNA in Tumor Metastasis via Acting as a Sponge of miRNA/isomiR

Circular RNAs (circRNAs), a class of new endogenous non-coding RNAs (ncRNAs), are closely related to the carcinogenic process and play a critical role in tumor metastasis. CircRNAs can lay the foundation for tumor metastasis via promoting tumor angiogenesis, make tumor cells gain the ability of migration and invasion by regulating epithelial-mesenchymal transition (EMT), interact with immune cells, cytokines, chemokines, and other non-cellular components in the tumor microenvironment, damage the normal immune function or escape the immunosuppressive network, and further promote cell survival and metastasis. Herein, based on the characteristics and biological functions of circRNA, we elaborated on the effect of circRNA via circRNA-associated competing endogenous RNA (ceRNA) network by acting as miRNA/isomiR sponges on tumor angiogenesis, cancer cell migration and invasion, and interaction with the tumor microenvironment (TME), then explored the potential interactions across different RNAs, and finally discussed the potential clinical value and application as a promising biomarker. These results provide a theoretical basis for the further application of metastasis-related circRNAs in cancer treatment. In summary, we briefly summarize the diverse roles of a circRNA-associated ceRNA network in cancer metastasis and the potential clinical application, especially the interaction of circRNA and miRNA/isomiR, which may complicate the RNA regulatory network and which will contribute to a novel insight into circRNA in the future.     

Hypercholesterolemia in Cancer and in Anorexia Nervosa: A Hypothesis for a Crosstalk

The relationship between cholesterol and cancer has been widely demonstrated. Clinical studies have shown changes in blood cholesterol levels in cancer patients. In parallel, basic research studies have shown that cholesterol is involved in the mechanisms of onset and progression of the disease. On the other hand, anorexic patients have high cholesterol levels and a high susceptibility to cancer. In this review, we first present a brief background on the relations among nutrition, eating disorders and cancer. Using several notable examples, we then illustrate the changes in cholesterol in cancer and in anorexia nervosa, providing evidence for their important relationship. Finally, we show a new possible link between cholesterol disorder in cancer and in anorexia nervosa.     

Limited Accuracy of Pan-Trk Immunohistochemistry Screening for NTRK Rearrangements in Follicular-Derived Thyroid Carcinoma

Patients with advanced thyroid cancer harboring NTRK rearrangements can be treated with highly effective selective inhibitors. Immunohistochemistry (IHC) analysis, to detect Trk protein expression, represents an appealing screening strategy for NTRK rearrangements, but its efficacy has been poorly explored in thyroid cancer. The aim of this study is to investigate the diagnostic utility of Trk IHC in the identification of NTRK rearrangements. A series of 26 follicular-derived thyroid tumors, positive for NTRK rearrangements, and 28 NTRK fusion-negative controls were retrospectively analyzed by IHC using the pan-Trk monoclonal antibody (clone {"type":"entrez-protein","attrs":{"text":"EPR17341","term_id":"523383444","term_text":"EPR17341"}}EPR17341) on the Ventana system. Area under the curve (AUC), sensitivity and specificity were calculated by ROC analysis. Trk expression was detected in 25 samples, including 22 out of the 26 NTRK-rearranged (84.6%) and three out of 28 NTRK-negative samples (10.7%). Four out of twenty-six NTRK-rearranged thyroid tumors were negative for Trk expression (15.4%), all carrying the ETV6/NTRK3 fusion. The AUC, sensitivity and specificity were 0.87, 0.85 and 0.89, respectively. A screening based on IHC analysis showed limited sensitivity and specificity in the identification of NTRK-rearranged tumors. Since falsely negative results could preclude the administration of effective targeted drugs, alternative detection strategies should be considered for thyroid cancer.     

Synthesis,In Vitro,and Computational Studies of PTP1B Phosphatase Inhibitors Based on Oxovanadium(IV) and Dioxovanadium(V) Complexes

One of the main goals of recent bioinorganic chemistry studies has been to design and synthesize novel substances to treat human diseases. The promising compounds are metal-based and metal ion binding components such as vanadium-based compounds. The potential anticancer action of vanadium-based compounds is one of area of investigation in this field. In this study, we present five oxovanadium(IV) and dioxovanadium(V) complexes as potential PTP1B inhibitors with anticancer activity against the MCF-7 breast cancer cell line, the triple negative MDA-MB-231 breast cancer cell line, and the human keratinocyte HaCaT cell line. We observed that all tested compounds were effective inhibitors of PTP1B, which correlates with anticancer activity. [VO(dipic)(dmbipy)]·2 H₂O (Compound 4) and [VOO(dipic)](2-phepyH)·H₂O (Compound 5) possessed the greatest inhibitory effect, with IC₅₀ 185.4 ± 9.8 and 167.2 ± 8.0 nM, respectively. To obtain a better understanding of the relationship between the structure of the examined compounds and their activity, we performed a computer simulation of their binding inside the active site of PTP1B. We observed a stronger binding of complexes containing dipicolinic acid with PTP1B. Based on our simulations, we suggested that the studied complexes exert their activity by stabilizing the WPD-loop in an open position and limiting access to the P-loop.     

Identification of Spliceogenic Variants beyond Canonical GT-AG Splice Sites in Hereditary Cancer Genes

Pathogenic/likely pathogenic variants in susceptibility genes that interrupt RNA splicing are a well-documented mechanism of hereditary cancer syndromes development. However, if RNA studies are not performed, most of the variants beyond the canonical GT-AG splice site are characterized as variants of uncertain significance (VUS). To decrease the VUS burden, we have bioinformatically evaluated all novel VUS detected in 732 consecutive patients tested in the routine genetic counseling process. Twelve VUS that were predicted to cause splicing defects were selected for mRNA analysis. Here, we report a functional characterization of 12 variants located beyond the first two intronic nucleotides using RNAseq in APC, ATM, FH, LZTR1, MSH6, PALB2, RAD51C, and TP53 genes. Based on the analysis of mRNA, we have successfully reclassified 50% of investigated variants. 25% of variants were downgraded to likely benign, whereas 25% were upgraded to likely pathogenic leading to improved clinical management of the patient and the family members.     

PVC低发泡实心板挤出成型技术要点

在PVC低发泡实心板挤出成型中,控制聚合物熔体的流动和发泡是保证产品质量的关键,重点介绍了生产工艺和原理,并分析了生产中的技术要点,提出了解决的办法。     

A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping

We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures. Notably, earlier studies with fixed co-cultures were limited by the absence of a quantitative method for identifying sub-populations of co-cultured cells, or for quantitating transfer relative to control populations of SAOS-2 or fibroblasts cultured alone. We now overcome that limitation by a novel Cartesian plot analysis that identifies individual co-cultured cells as belonging to one of five distinct cell populations, and also gives numerical measure of similarity to control cell populations. We verified the utility of the method by first confirming the previously established relationship between SAOS-2 morphology and uptake of fibroblast contents, and also demonstrated similar effects in other cancer cell lines including from melanomas, and cancers of the ovary and colon. The method was extended to examine global DNA methylation, and while there was no clear effect on SAOS-2 DNA methylation, co-cultured fibroblasts had greatly reduced DNA methylation, similar to cancer associated fibroblasts.     

Tumor-Localized Administration of α-GalCer to Recruit Invariant Natural Killer T Cells and Enhance Their Antitumor Activity against Solid Tumors

Invariant natural killer T (iNKT) cells have the capacity to mount potent anti-tumor reactivity and have therefore become a focus in the development of cell-based immunotherapy. iNKT cells attack tumor cells using multiple mechanisms with a high efficacy; however, their clinical application has been limited because of their low numbers in cancer patients and difficulties in infiltrating solid tumors. In this study, we aimed to overcome these critical limitations by using α-GalCer, a synthetic glycolipid ligand specifically activating iNKT cells, to recruit iNKT to solid tumors. By adoptively transferring human iNKT cells into tumor-bearing humanized NSG mice and administering a single dose of tumor-localized α-GalCer, we demonstrated the rapid recruitment of human iNKT cells into solid tumors in as little as one day and a significantly enhanced tumor killing ability. Using firefly luciferase-labeled iNKT cells, we monitored the tissue biodistribution and pharmacokinetics/pharmacodynamics (PK/PD) of human iNKT cells in tumor-bearing NSG mice. Collectively, these preclinical studies demonstrate the promise of an αGC-driven iNKT cell-based immunotherapy to target solid tumors with higher efficacy and precision.     

Technical Validation and Clinical Implications of Ultrasensitive PCR Approaches for EGFR-Thr790Met Mutation Detection in Pretreatment FFPE Samples and in Liquid Biopsies from Non-Small Cell Lung Cancer Patients

In pretreatment tumor samples of EGFR-mutated non-small cell lung cancer (NSCLC) patients, EGFR-Thr790Met mutation has been detected in a variable prevalence by different ultrasensitive assays with controversial prognostic value. Furthermore, its detection in liquid biopsy (LB) samples remains challenging, being hampered by the shortage of circulating tumor DNA (ctDNA). Here, we describe the technical validation and clinical implications of a real-time PCR with peptide nucleic acid (PNA-Clamp) and digital droplet PCR (ddPCR) for EGFR-Thr790Met detection in diagnosis FFPE samples and in LB. Limit of blank (LOB) and limit of detection (LOD) were established by analyzing negative and low variant allele frequency (VAF) FFPE and LB specimens. In a cohort of 78 FFPE samples, both techniques showed an overall agreement (OA) of 94.20%. EGFR-Thr790Met was detected in 26.47% of cases and was associated with better progression-free survival (PFS) (16.83 ± 7.76 vs. 11.47 ± 1.83 months; p = 0.047). In LB, ddPCR was implemented in routine diagnostics under UNE-EN ISO 15189:2013 accreditation, increasing the detection rate of 32.43% by conventional methods up to 45.95%. During follow-up, ddPCR detected EGFR-Thr790Met up to 7 months before radiological progression. Extensively validated ultrasensitive assays might decipher the utility of pretreatment EGFR-Thr790Met and improve its detection rate in LB studies, even anticipating radiological progression.