ABSTRACT
The isolation and enrichment of specific extracellular vesicle (EV) subpopulations are essential in the context of precision medicine. However, the current methods predominantly rely on a single-positive marker and are susceptible to interference from soluble proteins or impurities. This limitation represents a significant obstacle to the widespread application of EVs in biological research. Herein, a novel approach that utilizes proximity ligation assay (PLA) and DNA-RNA hybridization are proposed to facilitate the binding of two proteins on the EV membrane in advance enabling the isolation and enrichment of intact EVs with double-positive membrane proteins followed by using functionalized magnetic beads for capture and enzymatic cleavage for isolated EVs release. The isolated subpopulations of EVs can be further utilized for cellular uptake studies, high-throughput small RNA sequencing, and breast cancer diagnosis. Hence, developing and implementing a specialized system for isolating and enriching a specific subpopulation of EVs can enhance basic and clinical research in this field.
Subject(s)
Breast Neoplasms , Extracellular Vesicles , Humans , Female , Membrane Proteins/metabolism , Breast Neoplasms/metabolism , Extracellular Vesicles/metabolism , RNA , Immunomagnetic SeparationABSTRACT
To gain mechanistic insights into the functions and developmental dynamics of tumor-infiltrated immune cells, especially B-lymphocytes, here we combine single-cell RNA-sequencing and antigen receptor lineage analysis to characterize a large number of triple-negative breast cancer infiltrated immune cells and report a comprehensive atlas of tumor-infiltrated B-lymphocytes. The single-cell transcriptional profiles reveal significant heterogeneity in tumor-infiltrated B-cell subgroups. The single-cell antigen receptor analyses demonstrate that compared with those in peripheral blood, tumor-infiltrated B-cells have more mature and memory B-cell characteristics, higher clonality, more class switching recombination and somatic hypermutations. Combined analyses suggest local differentiation of infiltrated memory B-cells within breast tumors. The B-cell signatures based on the single-cell RNA-sequencing results are significantly associated with improved survival in breast tumor patients. Functional analyses of tumor-infiltrated B-cell populations suggest that mechanistically, B-cell subgroups may contribute to immunosurveillance through various pathways. Further dissection of tumor-infiltrated B-cell populations will provide valuable clues for tumor immunotherapy.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/immunology , Gene Expression Profiling , Lymphocytes, Tumor-Infiltrating/immunology , Receptors, Antigen/metabolism , Sequence Analysis, RNA , Single-Cell Analysis , Aged , Breast Neoplasms/blood , Female , Gene Expression Regulation, Neoplastic , Humans , Immunoglobulin Heavy Chains/genetics , Kaplan-Meier Estimate , Middle Aged , Transcriptome/geneticsABSTRACT
OBJECTIVE: To detect potential variant in an ethical Han Chinese pedigree affected with breast cancer. METHODS: The proband and her relatives were subjected to next-generation sequencing using a target capture sequencing kit containing 121 cancer-related genes. Candidate variants were selected by analysis of their type, frequency in population, and segregation with the phenotype. Candidate variant was verified by Sanger sequencing and TA cloning. RESULTS: A c.2013_2014ins GT variant was detected in the BRCA1 gene among all breast cancer patients from this pedigree but not among healthy females. The variant was not recorded in the 1000 Genome Project database or the Exome Aggregation Consortium (ExAC) database. The frameshifting insertion was predicted to form an premature stop codon in gene transcript and can give rise to a truncated protein. CONCLUSION: The BRCA1 c.2013_2014ins GT variant probably underlies the pathogenesis of breast cancer in this Chinese pedigree.
Subject(s)
BRCA1 Protein/genetics , Breast Neoplasms/genetics , Asian People , Exome , Female , High-Throughput Nucleotide Sequencing , Humans , Pedigree , PhenotypeSubject(s)
CRISPR-Cas Systems , Gene Editing , Genome , Animals , Cell Line , Gene Targeting , Humans , Mice , RNA, Guide, KinetoplastidaABSTRACT
CRISPR/dCas9 is a versatile tool that can be used to recruit various effectors and fluorescent molecules to defined genome regions where it can modulate genetic and epigenetic markers, or track the chromatin dynamics in live cells. In vivo applications of CRISPR/dCas9 in animals have been challenged by delivery issues. We generate and characterize a mouse strain with dCas9-EGFP ubiquitously expressed in various tissues. Studying telomere dynamics in these animals reveals surprising results different from those observed in cultured cell lines. The CRISPR/dCas9 knock-in mice provide an important and versatile tool to mechanistically study genome functions in live animals.
