Organoid Modeling of the Tumor Immune Microenvironment.
2018; 175 (7): 1972
Invitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor Tcell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.
View details for PubMedID 30550791
Alternative TSSs are co-regulated in single cells in the mouse brain.
Molecular systems biology
2017; 13 (5): 930-?
Alternative transcription start sites (TSSs) have been extensively studied genome-wide for many cell types and have been shown to be important during development and to regulate transcript abundance between cell types. Likewise, single-cell gene expression has been extensively studied for many cell types. However, how single cells use TSSs has not yet been examined. In particular, it is unknown whether alternative TSSs are independently expressed, or whether they are co-activated or even mutually exclusive in single cells. Here, we use a previously published single-cell RNA-seq dataset, comprising thousands of cells, to study alternative TSS usage. We find that alternative TSS usage is a regulated process, and the correlation between two TSSs expressed in single cells of the same cell type is surprisingly high. Our findings indicate that TSSs generally are regulated by common factors rather than being independently regulated or stochastically expressed.
View details for DOI 10.15252/msb.20167374
View details for PubMedID 28495919
Cancer-specific changes in DNA methylation reveal aberrant silencing and activation of enhancers in leukemia.
2017; 129 (7): e13-e25
Acute myeloid leukemia (AML) is characterized by an impaired differentiation process leading to an accumulation of immature blasts in the blood. One feature of cytogenetically normal AML is alterations to the DNA methylome. We analyzed 57 AML patients with normal karyotype by using Illumina's 450k array and showed that aberrant DNA methylation is significantly altered at enhancer regions and that the methylation levels at specific enhancers predict overall survival of AML patients. The majority of sites that become differentially methylated in AML occur in regulatory elements of the human genome. Hypermethylation associates with enhancer silencing. In addition, chromatin immunoprecipitation sequencing analyses showed that a subset of hypomethylated sites correlate with enhancer activation, indicated by increased H3K27 acetylation. DNA hypomethylation is therefore not sufficient for enhancer activation. Some sites of hypomethylation occur at weak/poised enhancers marked with H3K4 monomethylation in hematopoietic progenitor cells. Other hypomethylated regions occur at sites inactive in progenitors and reflect the de novo acquisition of AML-specific enhancers. Altered enhancer dynamics are reflected in the gene expression of enhancer target genes, including genes involved in oncogenesis and blood cell development. This study demonstrates that histone variants and different histone modifications interact with aberrant DNA methylation and cause perturbed enhancer activity in cytogenetically normal AML that contributes to a leukemic transcriptome.
View details for DOI 10.1182/blood-2016-07-726877
View details for PubMedID 28003272
Single-cell mRNA isoform diversity in the mouse brain.
2017; 18 (1): 126-?
Alternative mRNA isoform usage is an important source of protein diversity in mammalian cells. This phenomenon has been extensively studied in bulk tissues, however, it remains unclear how this diversity is reflected in single cells.Here we use long-read sequencing technology combined with unique molecular identifiers (UMIs) to reveal patterns of alternative full-length isoform expression in single cells from the mouse brain. We found a surprising amount of isoform diversity, even after applying a conservative definition of what constitutes an isoform. Genes tend to have one or a few isoforms highly expressed and a larger number of isoforms expressed at a low level. However, for many genes, nearly every sequenced mRNA molecule was unique, and many events affected coding regions suggesting previously unknown protein diversity in single cells. Exon junctions in coding regions were less prone to splicing errors than those in non-coding regions, indicating purifying selection on splice donor and acceptor efficiency.Our findings indicate that mRNA isoform diversity is an important source of biological variability also in single cells.
View details for DOI 10.1186/s12864-017-3528-6
View details for PubMedID 28158971
View details for PubMedCentralID PMC5291953
Amplification-free sequencing of cell-free DNA for prenatal non-invasive diagnosis of chromosomal aberrations
2015; 105 (3): 150-158
Cell-free DNA has been used for fetal rhesus factor and sex determination, fetal aneuploidy screening, cancer diagnostics and monitoring, and other applications. However current methods of using cell free DNA require amplification, which leads to allelic dropout and bias especially when starting with small amounts of DNA. Here we describe an amplification-free method for sequencing of cell-free DNA, even from low levels of starting material. We evaluated this method in the context of prenatal diagnosis of fetal aneuploidy and compared it with a PCR-based library preparation method as well as a recently described method using unique molecular identifiers (UMI). All methods performed well, however coverage was increased by the amplification-free method and GC-induced bias was reduced by both the amplification-free method and the UMI method. Future diagnostic applications including whole genome sequencing of cell-free DNA will benefit from amplification-free sequencing.
View details for DOI 10.1016/j.ygeno.2014.12.005
View details for Web of Science ID 000349782700004
View details for PubMedID 25543032
- Counting absolute numbers of molecules using unique molecular identifiers NATURE METHODS 2012; 9 (1): 72-U183