Cancer is characterized by hypomethylation-associated silencing of large chromatin domains, whose contribution to tumorigenesis is uncertain. Through high-resolution genome-wide single-cell DNA methylation sequencing, we identify 40 core domains that are uniformly hypomethylated from the earliest detectable stages of prostate malignancy through metastatic circulating tumor cells (CTCs). Nested among these repressive domains are smaller loci with preserved methylation that escape silencing and are enriched for cell proliferation genes. Transcriptionally silenced genes within the core hypomethylated domains are enriched for immune-related genes; prominent among these is a single gene cluster harboring all five CD1 genes that present lipid antigens to NKT cells and four IFI16-related interferon-inducible genes implicated in innate immunity. The re-expression of CD1 or IFI16 murine orthologs in immuno-competent mice abrogates tumorigenesis, accompanied by the activation of anti-tumor immunity. Thus, early epigenetic changes may shape tumorigenesis, targeting co-located genes within defined chromosomal loci. Hypomethylation domains are detectable in blood specimens enriched for CTCs.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

When evaluating EpCAM-based enrichment technologies for circulating tumour cells (CTCs), the cell lines used should closely resemble real CTCs, meaning the EpCAM expression of CTCs needs to be known, but also the EpCAM expression of cell lines at different institutions and times is important. As the number of CTCs in the blood is low, we enriched CTCs through the depletion of leukocytes from diagnostic leukapheresis products of 13 prostate cancer patients and measured EpCAM expression using quantitative flow cytometry. Antigen expression was compared between multiple institutions by measuring cultures from each institution. Capture efficiency was also measured for one of the used cell lines. Results show CTCs derived from castration-sensitive prostate cancer patients have varying but relatively low EpCAM expression, with median expression per patient ranging from 35 to 89,534 (mean 24,993) molecules per cell. A large variation in the antigen expression of identical cell lines cultured at different institutions was found, resulting in recoveries when using the CellSearch system ranging from 12 up to 83% for the same cell line. We conclude that large differences in capture efficiency can occur while using the same cell line. To closely resemble real CTCs from castration-sensitive prostate cancer patients, a cell line with a relatively low EpCAM expression should be used, and its expression should be monitored frequently.
© 2023. The Author(s).

The rise and fall of estrogen and progesterone across menstrual cycles and during pregnancy regulates breast development and modifies cancer risk. How these hormones impact each cell type in the breast remains poorly understood because they act indirectly through paracrine networks. Using single-cell analysis of premenopausal breast tissue, we reveal a network of coordinated transcriptional programs representing the tissue-level response to changing hormone levels. Our computational approach, DECIPHER-seq, leverages person-to-person variability in breast composition and cell state to uncover programs that co-vary across individuals. We use differences in cell-type proportions to infer a subset of programs that arise from direct cell-cell interactions regulated by hormones. Further, we demonstrate that prior pregnancy and obesity modify hormone responsiveness through distinct mechanisms: obesity reduces the proportion of hormone-responsive cells, whereas pregnancy dampens the direct response of these cells to hormones. Together, these results provide a comprehensive map of the cycling human breast.
Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Long non-coding RNAs (lncRNAs) are critical for regulating HOX genes, aberration of which is a dominant mechanism for leukemic transformation. How HOX gene-associated lncRNAs regulate hematopoietic stem cell (HSC) function and contribute to leukemogenesis remains elusive. We found that HOTTIP is aberrantly activated in acute myeloid leukemia (AML) to alter HOXA-driven topologically associated domain (TAD) and gene expression. HOTTIP loss attenuates leukemogenesis of transplanted mice, while reactivation of HOTTIP restores leukemic TADs, transcription, and leukemogenesis in the CTCF-boundary-attenuated AML cells. Hottip aberration in mice abnormally promotes HSC self-renewal leading to AML-like disease by altering the homeotic/hematopoietic gene-associated chromatin signature and transcription program. Hottip aberration acts as an oncogenic event to perturb HSC function by reprogramming leukemic-associated chromatin and gene transcription.
Copyright © 2019 Elsevier Inc. All rights reserved.

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.