Ageing limits stemness and tumorigenesis by reprogramming iron homeostasis.

Ageing is associated with a decline in the number and fitness of adult stem cells. Ageing-associated loss of stemness is posited to suppress tumorigenesis, but this hypothesis has not been tested in vivo. Here we use physiologically aged autochthonous genetically engineered mouse models and primary cells to demonstrate that ageing suppresses lung cancer initiation and progression by degrading the stemness of the alveolar cell of origin.

Multiplex digital profiling of DNA methylation heterogeneity for sensitive and cost-effective cancer detection in low-volume liquid biopsies.

Molecular alterations in cancerous tissues exhibit intercellular genetic and epigenetic heterogeneity, complicating the performance of diagnostic assays, particularly for early cancer detection. Conventional liquid biopsy methods have limited sensitivity and/or ability to assess epigenetic heterogeneity of rare epiallelic variants cost-effectively. We report an approach, named REM-DREAMing (Ratiometric-Encoded Multiplex Discrimination of Rare EpiAlleles by Melt), which leverages a digital microfluidic platform that incorporates a ratiometric fluorescence multiplex detection scheme and precise digital high-resolution melt analysis to enable low-cost, parallelized analysis of heterogeneous methylation patterns on a molecule-by-molecule basis for the detection of cancer in liquid biopsies.

Streamlined miRNA loading of surface protein-specific extracellular vesicle subpopulations through electroporation.

Background: Extracellular vesicles (EVs) have emerged as an exciting tool for targeted delivery of therapeutics for a wide range of diseases. As nano-scale membrane-bound particles derived from living cells, EVs possess inherent capabilities as carriers of biomolecules. However, the translation of EVs into viable therapeutic delivery vehicles is challenged by lengthy and inefficient processes for cargo loading and pre- and post-loading purification of EVs, resulting in limited quantity and consistency of engineered EVs.

Prognostic utility of circulating tumor DNA methylation analysis in stage IV colorectal cancer.

Background And Objectives: Our aim in this study was to investigate the usefulness of circulating tumor (ct) DNA methylation analysis for predicting long-term outcomes after resection in Stage IV colorectal cancer (CRC).

Methods: Methylation analyses were performed on 95 plasma samples from patients with CRC who underwent surgery. The methylation status (relative methylation value: RMV) of CpG within the promoter region of three genes (CHFR, SOX11, and CDO1) was assessed to quantitative methylation-specific PCR (qMSP) analysis.

Single-molecule epiallelic profiling of DNA derived from routinely collected Pap specimens for noninvasive detection of ovarian cancer.

Recent advances in molecular analyses of ovarian cancer have revealed a wealth of promising tumour-specific biomarkers, including protein, DNA mutations and methylation; however, reliably detecting such alterations at satisfactorily high sensitivity and specificity through low-cost methods remains challenging, especially in early-stage diseases. Here we present PapDREAM, a new approach that enables detection of rare, ovarian-cancer-specific aberrations of DNA methylation from routinely-collected cervical Pap specimens. The PapDREAM approach employs a microfluidic platform that performs highly parallelized digital high-resolution melt to analyze locus-specific DNA methylation patterns on a molecule-by-molecule basis at or near single CpG-site resolution at a fraction (< 1/10th) of the cost of next-generation sequencing techniques.

Aging limits stemness and tumorigenesis in the lung by reprogramming iron homeostasis.

Aging is associated with a decline in the number and fitness of adult stem cells . Aging-associated loss of stemness is posited to suppress tumorigenesis , but this hypothesis has not been tested . Here, using physiologically aged autochthonous genetically engineered mouse models and primary cells , we demonstrate aging suppresses lung cancer initiation and progression by degrading stemness of the alveolar cell of origin.

The intricate interplay between cancer stem cells and cell-of-origin of cancer: implications for therapeutic strategies.

Background: Cancer stem cells (CSCs) have emerged as pivotal players in tumorigenesis, disease progression, and resistance to therapies.

Objective: This comprehensive review delves into the intricate relationship between CSCs and the cell-of-origin in diverse cancer types.

Design: Comprehensive review of thematically-relevant literature.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition.

Current and Emerging Methods for Ovarian Cancer Screening and Diagnostics: A Comprehensive Review.

With a 5-year survival rate of less than 50%, ovarian high-grade serous carcinoma (HGSC) is one of the most highly aggressive gynecological malignancies affecting women today. The high mortality rate of HGSC is largely attributable to delays in diagnosis, as most patients remain undiagnosed until the late stages of -disease. There are currently no recommended screening tests for ovarian cancer and there thus remains an urgent need for new diagnostic methods, particularly those that can detect the disease at early stages when clinical intervention remains effective.

Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR.

Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations.

High-throughput sample processing for methylation analysis in an automated, enclosed environment.

Variation in methylcytosine is perhaps the most well-studied epigenetic mechanism of gene regulation. Methods that have been developed and implemented for assessing DNA methylation require sample DNA to be extracted, purified and chemically-processed through bisulfite conversion before downstream analysis. While some automated solutions exist for each of these individual process steps, a fully integrated solution for accomplishing the entire process in a high-throughput manner has yet to be demonstrated.

High performance methylated DNA markers for detection of colon adenocarcinoma.

Background: Colon cancer (CC) is treatable if detected in its early stages. Improved CC detection assays that are highly sensitive, specific, and available at point of care are needed. In this study, we systematically selected and tested methylated markers that demonstrate high sensitivity and specificity for detection of CC in tissue and circulating cell-free DNA.

Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing.

Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity.

Mutation and methylation profiles of ectopic and eutopic endometrial tissues.

Adenomyosis and peritoneal endometriosis are common gynecologic lesions; they are characterized by aberrant locations of normal-appearing endometrium in myometrium and peritoneal surface, respectively. Both ectopic lesions are speculated to originate from uterine eutopic endometrium, which is composed of epithelium and stroma, but how these two different tissue types co-evolve in ectopic locations remains unclear. Here, we analyzed exome-wide mutations and global methylation in microdissected epithelium and stroma separately in paired adenomyosis, peritoneal endometriosis, and endometrium to investigate their relationship.

A phase 2 trial of gemcitabine and docetaxel in patients with metastatic colorectal adenocarcinoma with methylated checkpoint with forkhead and ring finger domain promoter and/or microsatellite instability phenotype.

We previously reported CHFR methylation in a subset of colorectal cancer (CRC; ∼30%) with high concordance with microsatellite instability (MSI). We also showed that CHFR methylation predicted for sensitivity to docetaxel, whereas the MSI-high phenotypes were sensitive to gemcitabine. We hypothesized that this subset of patients with CRC would be selectively sensitive to gemcitabine and docetaxel.

Ligation-Enabled Fluorescence-Coding PCR for High-Dimensional Fluorescence-Based Nucleic Acid Detection.

Polymerase chain reaction (PCR) is by far the most commonly used method of nucleic acid amplification and has likewise been employed for a plethora of diagnostic purposes. Nonetheless, multiplexed PCR-based detection schemes have hitherto been largely limited by technical challenges associated with nonspecific interactions and other limitations inherent to traditional fluorescence-based assays. Here, we describe a novel strategy for multiplexed PCR-based analysis called Ligation-eNabled fluorescence-Coding PCR (LiNC PCR) that exponentially enhances the multiplexing capability of standard fluorescence-based PCR assays.

Epigenomic Reprogramming toward Mesenchymal-Epithelial Transition in Ovarian-Cancer-Associated Mesenchymal Stem Cells Drives Metastasis.

A role for cancer cell epithelial-to-mesenchymal transition (EMT) in cancer is well established. Here, we show that, in addition to cancer cell EMT, ovarian cancer cell metastasis relies on an epigenomic mesenchymal-to-epithelial transition (MET) in host mesenchymal stem cells (MSCs). These reprogrammed MSCs, termed carcinoma-associated MSCs (CA-MSCs), acquire pro-tumorigenic functions and directly bind cancer cells to serve as a metastatic driver/chaperone.

Methylomic Landscapes of Ovarian Cancer Precursor Lesions.

Purpose: The current paradigm in the development of high-grade serous ovarian carcinoma (HGSC) proposes that the majority of HGSCs arise from precursor serous tubal intraepithelial carcinoma (STIC) lesions of the fallopian tube. Here we survey genome-wide methylation in HGSC precursor lesions to identify genomic regions that exhibit high-specificity differential hypermethylation for potential use as biomarkers for detecting STIC and HGSC at stages when curative intervention likely remains feasible.

Experimental Design: We first identified quality control criteria for performing reliable methylomic analysis of DNA-limited tubal precursor lesions with the Illumina Infinium MethylationEPIC array.

Promoter methylation of ADAMTS1 and BNC1 as potential biomarkers for early detection of pancreatic cancer in blood.

Background: Despite improvements in cancer management, most pancreatic cancers are still diagnosed at an advanced stage. We have recently identified promoter DNA methylation of the genes ADAMTS1 and BNC1 as potential blood biomarkers of pancreas cancer. In this study, we validate this biomarker panel in peripheral cell-free tumor DNA of patients with pancreatic cancer.

Multilayer microfluidic array for highly efficient sample loading and digital melt analysis of DNA methylation.

Liquid biopsies contain a treasure of genetic and epigenetic biomarkers that contain information for the detection and monitoring of human disease. DNA methylation is an epigenetic modification that is critical to determining cellular phenotype and often becomes altered in many disease states. In cancer, aberrant DNA methylation contributes to carcinogenesis and can profoundly affect tumor evolution, metastatic potential, and resistance to therapeutic intervention.

Long Interspersed Nuclear Element 1 Retrotransposons Become Deregulated during the Development of Ovarian Cancer Precursor Lesions.

There is growing evidence that most high-grade serous ovarian carcinomas likely arise from local dissemination of precursor lesions of the fallopian tube. Evolution of these lesions from early p53 signatures to latter-stage, serous tubal intraepithelial carcinomas (STICs) is characterized by cytologic atypia, accumulation of somatic mutations, and genomic instability, the etiologies of which remain unclear. Long interspersed element 1 (LINE-1) retrotransposon is expressed in many carcinomas, including high-grade serous ovarian carcinoma, where it contributes to genomic instability; however, the timing of LINE-1 activation during this evolution has yet to be elucidated.

Facile profiling of molecular heterogeneity by microfluidic digital melt.

This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes.

Methylomic Analysis of Ovarian Cancers Identifies Tumor-Specific Alterations Readily Detectable in Early Precursor Lesions.

Purpose: High-grade serous ovarian carcinoma (HGSOC) typically remains undiagnosed until advanced stages when peritoneal dissemination has already occurred. Here, we sought to identify HGSOC-specific alterations in DNA methylation and assess their potential to provide sensitive and specific detection of HGSOC at its earliest stages.

Experimental Design: MethylationEPIC genome-wide methylation analysis was performed on a discovery cohort comprising 23 HGSOC, 37 non-HGSOC malignant, and 36 histologically unremarkable gynecologic tissue samples.

Defining, distinguishing and detecting the contribution of heterogeneous methylation to cancer heterogeneity.

DNA methylation is a fundamental means of epigenetic gene regulation that occurs in virtually all cell types. In many higher organisms, including humans, it plays vital roles in cell differentiation and homeostatic maintenance of cell phenotype. The control of DNA methylation has traditionally been attributed to a highly coordinated, linear process, whose dysregulation has been associated with numerous pathologies including cancer, where it occurs early in, and even prior to, the development of neoplastic tissues.