Biology subjects

Knapp, D. J. H. F.

Publications and source records attributed to Knapp, D. J. H. F..

4 recordsLinked to original sources

bioRxiv2026

The Human Male Mammary Gland has Similar Epithelial Populations to Female but Distinct Composition and Transcriptional Properties

The normal adult male breast has not been characterized at single-cell resolution, leaving the cellular basis of male breast cancer (MBC) biology undefined. Here we present an integrated single-cell RNA sequencing atlas of the adult human breast comprising 174,471 cells from 17 donors (3 male, 14 female), including 18,117 male-derived cells. This revealed that the male breast retains all three epithelial populations, basal (BC), luminal progenitor (LP), and luminal committed cells (LC), but with an increase in LC at the expense of BC and LP across all three male donors. Male LC were distinguished from female by elevated ESR1 and PGR mRNA, enrichment of RNA processing and ribosome biogenesis programs, reduced inflammatory cytokine and growth factor signaling, elevated estradiol gene set enrichment scores, and higher inferred activity of developmental patterning transcription factors. This pattern was observed across differential expression, gene ontology, ligand profiling, and regulon-based analyses, and was not restricted to sex chromosome-linked gene expression. This is consistent with the near-universal estrogen receptor (ER) positivity that characterizes MBC clinically. This atlas provides the first cellular and transcriptional reference for the normal male breast and a resource for investigating sex differences in mammary biology, germline susceptibility variant interpretation, and modeling breast malignancies.

cancer biology↗

bioRxiv2021

DLL4 and VCAM1 enhance the emergence of T cell-competent hematopoietic progenitors from human pluripotent stem cells

T cells are key mediators of the adaptive immune response and show tremendous efficacy as cellular therapeutics. However, obtaining primary T cells from human donors is expensive and variable. Pluripotent stem cells (PSCs) have the potential to serve as a consistent and renewable source of T cells, but differentiating PSCs into hematopoietic progenitors with T cell potential remains a significant challenge. Here, we developed an efficient serum- and feeder-free protocol for differentiating human PSCs into hematopoietic progenitors and T cells. This defined method allowed us to study the impact of individual recombinant proteins on blood emergence and lineage potential. We demonstrate that the presence of DLL4 and VCAM1 during the endothelial-to-hematopoietic transition (EHT) enhances downstream progenitor T cell output by >80-fold. Using single cell transcriptomics, we showed that these two proteins synergise to drive strong notch signalling in nascent hematopoietic stem and progenitor cells and that VCAM1 additionally drives a pro-inflammatory transcriptional program. Finally, we applied this differentiation method to study the impact of cytokine concentration dynamics on T cell maturation. We established optimised media formulations that enabled efficient and chemically defined differentiation of CD8{beta}+, CD4-, CD3+, TCR{beta}+ T cells from PSCs.

bioengineering↗

bioRxiv2021

Barcoded Competitive Clone-Initiating Cell (BC-CIC) Analysis Reveals Differences in Ovarian Cancer Cell Genotype and Niche Specific Clonal Fitness During Growth and Metastasis In Vivo

During oncogenesis, pathogenic clones develop which contain cells capable of spreading throughout the body, ultimately compromising vital organ functions and physiology. Understanding how metastatic clones develop and spread is critical for improving cancer treatments. However, our understanding of these processes has been hampered by a paucity of quantitative methodologies to comprehensively map, track and characterize such clones. To address this shortcoming, we have developed a DNA barcoding and next-generation sequencing based system-wide clonal tracking technology integrated with a computational data analysis pipeline called Clone-Initiating Cell (CIC) Calculator. The CIC Calculator interfaces with the CIC Morbus Mandala (CIC-MM) plot, a novel tool to visually comprehend and detect four distinct categories that explains their complex relationships with various tissues/organ sites. Further, we describe machine learning approaches to study CIC number, frequency, and estimate clone size and distribution demonstrating distinct growth patterns, and their inter-relationships and their routes of metastatic spread at clonal resolution. We demonstrate these methodologies, using our novel multifunctional lentiviral barcode libraries, and specifically barcoded tubal-ovarian metastatic OVCAR5 cell lines (engineered to express varying levels of metastasis promoting LRRC15 gene) and co-injected cells in a competitive CIC assay into tubal or ovarian sites in highly immunodeficient NSG mice. DNA was isolated from primary tumors, omental/bowel metastasis and system-wide anatomical site/organs. Amplicon sequencing libraries were constructed with spike-in-control barcodes (serving as internal calibration controls) to estimate absolute clone sizes. The computational pipeline CIC Calculator was then used to deconvolute and filter the data, set stringent thresholds, and generate high-quality information on CIC numbers and frequencies, clone sizes, linkages across sites and classify clones based on their extent of metastatic activity. Using of CIC-MM plot, statistical models and machine learning approaches, we generated high-resolution clonal maps of metastasis for each animal. The information generated included clone types and system-wide metastasis, similar and dissimilar clonal patterns of dominance at heterotopic sites and their routes of metastases. The data revealed previously unknown influences of cellular genotype and their implanted sites on selecting certain clones with specific system-wide clonal patterns, and identified rare LRRC15 expressor clones (classified as CIC.Toti) predisposed to exploit all sites, albeit at varying degrees of dominance. The genomic technology and computational methodology described here are tissue-agnostic. They enable rapid adoption for an investigation into various stages of system-wide metastasis and growth of transplantable malignant cells at the highest clonal resolution.

cancer biology↗

bioRxiv2019

Variable Retention of Differentiation-specific DNA Replication Timing in Human Pediatric Leukemia

Human B-lineage precursor acute lymphoid leukemias (BCP-ALLs) comprise a group of genetically and clinically distinct disease entities with features of differentiation arrest at known stages of normal B-lineage differentiation. We previously showed BCP-ALL cells display unique and clonally heritable DNA-replication timing (RT) programs; i.e., programs describing the variable order of replication of megabase-scale chromosomal units of DNA in different cell types. To determine the extent to which BCP-ALL RT programs mirror or deviate from specific stages of normal human B-cell differentiation, we transplanted immunodeficient mice with quiescent normal human CD34+ cord blood cells and obtained RT signatures of the regenerating B-lineage populations. We then compared these with RT signatures for leukemic cells from a large cohort of BCP-ALL patients. The results identify BCP-ALL subtype-specific features that resemble specific stages of B-cell differentiation and features that appear associated with relapse. These results suggest the genesis of BCP-ALL involves alterations in RT that reflect clinically relevant leukemia-specific genetic and/or epigenetic changes. SUMMARYGenome-wide DNA replication timing profiles of >100 pediatric leukemic samples and normally differentiating human B-lineage cells isolated from xenografted immunodeficient mice were generated. Comparison of these identified potentially clinically relevant features that both match and deviate from the normal profiles.

cancer biology↗