Biology subjects

Knapp, D. J.

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

11 recordsLinked to original sources

bioRxiv2026

Tumour-stroma signaling drives a reversible program with neural-like features in cancer-associated fibroblasts

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, in part due to a dense and highly plastic tumor microenvironment dominated by cancer-associated fibroblasts (CAFs). In previous work, we identified a CAF population expressing a neural-associated NES+SOX4+NOTCH1+ program that increased with disease progression. The signals driving this state and the mechanisms regulating it remained unclear. Here, we investigated how tumor-derived signals activate this program and whether it represents a stable fibroblast identity or a reversible state. Using spatial transcriptomic data spanning normal pancreas to invasive carcinoma, we identified EGF, FGF2 and BDNF as candidate tumor-to-fibroblast signals associated with disease progression. Functional experiments in patient-derived CAFs and human fibroblast cell lines showed that EGF and FGF2 are sufficient to induce the NES+SOX4+NOTCH1+ program. Growth factor withdrawal reversed the NES+SOX4+NOTCH1+ program at both the protein and transcriptional levels, indicating that this state is dynamic and environment dependent. Single-cell transcriptomic analysis further revealed a multi-phase response involving early loss of fibroblast-associated features, transient activation of neural-associated genes, and a later stress-associated state. Regulatory analyses identified ETS-family transcription factors as key regulators of the program, while phosphoproteomic profiling and inhibitor studies showed that MAPK/ERK signaling plays a central role in its transcriptional activation. Finally, pan-cancer single-cell analysis showed that this state is enriched in CAFs and increases from normal to tumor tissue across multiple cancer types. Together, these findings define a reversible, growth factor-driven CAF program and identify signaling mechanisms that may provide opportunities to selectively modulate tumor-supportive fibroblast states.

cancer biology↗

bioRxiv2026

Spatial multi-omics resolve epithelium-fibroblast gradients and highlight NESTIN-NOTCH1-expressing subepithelial fibroblasts during human pancreatic tumorigenesis

Intraductal papillary mucinous neoplasms (IPMNs) are cystic precursors of pancreatic ductal adenocarcinoma undergoing dynamic epithelial and stromal remodeling during progression. In this study, we integrated computational pathology, cyclic immunofluorescence, and Xenium spatial transcriptomics to define the spatial organization of the IPMN microenvironment. We queried cell-based histopathological features and identified stromal patterns strongly associated with epithelium proximity. Cyclic immunofluorescence and Xenium revealed a subepithelial gradient extending from myofibroblasts to inflammatory fibroblasts. Xenium further identified a distinct subepithelial NOTCH1-NESTIN-expressing fibroblast subset in myofibroblasts that expands with tumor progression. By recapitulating the spatial remodeling event using human pancreatic tumor cells and primary myofibroblast co-culture models, we demonstrated the capacity of pancreatic tumor cells to induce a NESTIN-expressing state in neighboring myofibroblasts. These findings reveal new dynamic epithelial-stromal interactions through subepithelial NESTIN+ fibroblasts associated with pancreatic tumorigenesis and immediate microenvironment remodeling by active tumor cells, highlighting the power of computational pathology integrated with spatial transcriptomics.

cancer biology↗

bioRxiv2025

Cell State Chaos Underpins the Evolution of SMARCA4-Deficient Dedifferentiated Endometrial Cancer

Dedifferentiated endometrial carcinoma (DDEC) is a histologically unique cancer type, wherein well-differentiated regions lie adjacent to morphologically distinct, high-grade lesions that are histologically undifferentiated. Previous studies have determined that in nearly half of the cases dedifferentiation is associated with the genomic inactivation of SMARCA4, a catalytic subunit belonging to the SWI/SNF chromatin remodelling complex (SWI/SNF CRC), suggesting that SMARCA4 loss causes dedifferentiation. Herein, using gene editing, we reveal that when serially passaged in mice, SMARCA4-deficient endometrial cancer cells repeatably and predictably generate heterogeneous admixtures of differentiated and undifferentiated cells, resembling human DDEC. Surprisingly, despite this metamorphosis, SMARCA4 loss does not induce lineage plasticity nor reprogramming to a less differentiated fate. Rather, single-cell sequencing combined with barcoding demonstrated that SMARCA4 loss induces a dysregulated epigenome that allows cells to randomly move through cellular states that are otherwise shared with SMARCA4-expressing well-differentiated cancer cells. This finding was validated using a cohort of patient samples, such that epithelial fate markers (E-CADHERIN) can be detected in morphologically undifferentiated cells. Collectively, this work constitutes the first repeatable model of human dedifferentiated cancer and suggests that histological dedifferentiation is not due to the acquisition of a stem cell-like fate. Rather, undifferentiated tissue emerges due to epigenomic dysfunction concomitant with the arbitrary movement of cancer cells between cellular states.

cancer biology↗

bioRxiv2025

A Rare Multipotent Peg-like Epithelial Cell is a Candidate Cell-of-Origin for High-Grade Serous Ovarian Cancer

To illuminate the origins of high-grade serous ovarian cancer (HGSOC), the most lethal and common form of ovarian cancer, we have created a comprehensive living organoid biobank of human fallopian tube tissue, which is thought to be the origin of this cancer. Through optimized culture protocols and integrated multi-omic profiling--including single-cell RNA sequencing, chromatin accessibility (ATAC) analysis, proteomics, and secretomics--we assembled the largest molecular atlas of the fallopian tube epithelium to date. This resource revealed diverse epithelial lineages and regulatory networks, including a rare, multipotent epithelial subpopulation with hybrid epithelial-mesenchymal features. Spatially localized to the basal epithelium and resembling mesonephric developmental precursors, these cells exhibit transcriptomic and proteomic similarities to the mesenchyme-like subtype of HGSOC, implicating them as potential cells-of-origin. Their molecular identity is preserved in organoid models, enabling future mechanistic and translational studies. This resource, which advances fundamental understanding of epithelial hierarchy and cancer susceptibility, provides a platform to inform early detection and prevention strategies for aggressive forms of ovarian cancer. HighlightsO_LIEstablishment of a clinically annotated fallopian tube organoid biobank enables delineation of epithelial lineage hierarchies and differentiation capacity. C_LIO_LIMulti-omics integration defines robust, lineage-specific transcriptional and regulatory networks in the fallopian tube epithelium. C_LIO_LIA rare basal epithelial subpopulation with mesenchymal features aligns with a mesenchyme-like subtype of high-grade serous ovarian cancer. C_LIO_LIRare basal peg cells exhibit fetal mesonephric developmental transcriptional programs and are maintained ex-vivo in fallopian tube organoids. C_LI

cancer biology↗

bioRxiv2025

Receptor Tyrosine Kinase Profiling Identifies Chronic Constitutive Floodgate Oxidative Signaling in Glutathione-Independent Human Mammary Luminal Progenitor Cells

The human mammary epithelium contains a subset of luminal progenitor (LP) cells that are distinct from basal cells in both lineage potential and redox biology. LPs are uniquely equipped to tolerate oxidative stress through glutathione-independent mechanisms and have been implicated as candidate cells of origin in basal-like breast cancers. In this study, we identify the receptor tyrosine kinase (RTK) cKIT (CD117), as a defining feature of LPs and a key mediator of their expansion. cKIT is developmentally restricted to the LP compartment via Polycomb-mediated epigenetic repression in basal and luminal-committed cells. It is expressed in scattered epithelial cells within both ductal and alveolar regions of resting human mammary glands. Using RTK-engineered MCF10A models, we demonstrate that cKIT ligand/stem cell factor (SCF)-activated wildtype cKIT signaling is sufficient to drive proliferation in the absence of epidermal growth factor (EGF) and that cKIT is responsive not only to canonical ligands but also to hydrogen peroxide (H2O2). In primary human LPs, cKIT is rapidly phosphorylated upon exposure to SCF and H2O2, with concomitant AKT activation. These responses are enhanced when cKIT and EGFR signaling are co-engaged, suggesting a cooperative mitogenic program. In mammary gland, phosphorylation of the antioxidant enzyme PRDX1 is selectively detected in LPs, consistent with a floodgate model of redox signaling in which transient oxidative inactivation of peroxiredoxins (PRDXs) facilitates RTK signaling under elevated intracellular reactive oxygen species conditions. Clinically, elevated cKIT expression is associated with shorter progression-free survival in certain basal-like breast cancer, supporting a link between LP-like redox signaling states and aggressive tumor behavior. Together, these findings define a redox-integrated RTK signaling axis centered on cKIT that drives LP expansion and is associated with poor outcomes in a subset of basal breast cancers. This work establishes a mechanistic framework for targeting redox-responsive progenitor populations in both regenerative and oncologic context.

cancer biology↗

bioRxiv2024

A Digital Microfluidic Platform for the Microscale Production of Functional Immune Cell Therapies

Genetically engineering human immune cells has been shown to be an effective approach for developing novel cellular therapies to treat a wide range of diseases. To expand the scope of these cellular therapies while solving persistent challenges, extensive research and development is still required. Electroporation has recently emerged as one of the most popular techniques for inserting biological payloads into human immune cells to perform genetic engineering. However, several recent studies have reported that electroporation can negatively impact cell functionality. Additionally, the requirement to use large amounts of cells and expensive payloads to achieve efficient delivery can drive up the costs of development efforts. Here we use a digital microfluidic enabled electroporation system (referred to as triDrop) and compare them against two state-of-the-art commercially available systems for the engineering of human T cells. We describe the ability to use triDrop for highly viable, highly efficient transfection while using substantially fewer cells and payload. Subsequently, we perform transcriptomic analysis on cells engineered with each of the three systems and show that electroporation with triDrop lead to less dysregulation of several functionally relevant pathways. Finally, in a direct comparison of immunotherapeutic functionality, we show that T cells engineered with triDrop have an improved ability to mount an immune response when presented with tumor cells. These results show that the triDrop platform is uniquely suited to produce functionally engineered immune cells while also reducing the costs of cell engineering compared to other commercially available systems.

bioengineering↗

bioRxiv2024

Puberty Blocker and Aging Impact on Testicular Cell States and Function

Spermatogonial stem cell (SSC) acquisition of meiotogenetic state during puberty to produce genetically diverse gametes is blocked by drugs collectively referred as puberty blocker (PB). Investigating the impact of PB on juvenile SSC state and function is challenging due to limited tissue access and clinical data. Herein, we report largest clinically annotated juvenile testicular biorepository with all children with gender dysphoria on chronic PB treatment highlighting shift in pediatric patient demography in US. At the tissue level, we report mild-to-severe sex gland atrophy in PB treated children. We developed most extensive integrated single-cell RNA dataset to date (>100K single cells; 25 patients), merging both public and novel (52 month PB-treated) datasets, alongside innovative computational approach tailed for germ cells and evaluated the impact of PB and aging on SSC. We report novel constitutional ranges for each testicular cell type across the entire age spectrum, distinct effects of treatments on prepubertal vs adult SSC, presence of spermatogenic epithelial cells exhibiting post-meiotic-state, irrespective of age, puberty status, or PB treatment. Further, we defined distinct effects of PB and aging on testicular cell lineage composition, and SSC meiotogenetic state and function. Using single cell data from prepubertal and young adult, we were able to accurately predict sexual maturity based both on overall cell type proportions, as well as on gene expression patterns within each major cell type. Applying these models to a PB-treated patient that they appeared pre-pubertal across the entire tissue. This combined with the noted gland atrophy and abnormalities from the histology data raise a potential concern regarding the complete reversibility and reproductive fitness of SSC. The biorepository, data, and research approach presented in this study provide unique opportunity to explore the impact of PB on testicular reproductive health.

developmental biology↗

bioRxiv2023

Time- and lineage-resolved transcriptional profiling uncovers gene expression programs and clonal relationships that underlie human T lineage specification

T cells develop from multi-potent hematopoietic progenitors in the thymus and provide adaptive protection against pathogens and cancer. However, the emergence of human T cell-competent blood progenitors, and their subsequent specification to the T lineage, has been challenging to capture in real time. Here, we leveraged a pluripotent stem cell differentiation system to understand the transcriptional dynamics and cell fate restriction events that underlie this critical developmental process. Time-resolved single cell RNA sequencing revealed that cell-cycle exit, downregulation of the multipotent hematopoietic program, and upregulation of >90 lineage-associated transcription factors all occur within a highly co-ordinated and narrow developmental window. Computational gene-regulatory network inference elucidated the transcriptional logic of T lineage specification, uncovering an important role for YBX1. We mapped the differentiation cell fate hierarchy using transcribed lineage barcoding and mathematical trajectory inference and discovered that mast and myeloid potential bifurcate from each other early in haematopoiesis, upstream of T lineage restriction. Collectively, our analyses provide a quantitative, time-resolved model of human T cell specification with relevance for regenerative medicine and developmental immunology.

immunology↗

bioRxiv2023

Near-perfect precise on-target editing of human hematopoietic stem and progenitor cells

Precision gene editing in primary hematopoietic stem and progenitor cells (HSPCs) would facilitate both curative treatments for monogenic disorders as well as disease modelling. Precise efficiencies even with the CRISPR/Cas system, however, remain limited. Through an optimization of guide RNA delivery, donor design, and additives, we have now obtained mean precise editing efficiencies >90% on primary cord blood HSCPs with minimal toxicity and without observed off-target editing. The main protocol modifications needed to achieve such high efficiencies were the addition of the DNA-PK inhibitor AZD7648, and the inclusion of spacer-breaking silent mutations in the donor in addition to mutations disrupting the PAM sequence. Critically, editing was even across the progenitor hierarchy, did not substantially distort the hierarchy or affect lineage outputs in colony-forming cell assays or the frequency of high self-renewal potential long-term culture initiating cells. As modelling of many diseases requires heterozygosity, we also demonstrated that the overall editing and zygosity can be tuned by adding in defined mixtures of mutant and wild-type donor. With these optimizations, editing at near-perfect efficiency can now be accomplished directly in human HSPCs. This will open new avenues in both therapeutic strategies and disease modelling.

bioengineering↗

bioRxiv2018

Precise tuning of gene expression output levels in mammalian cells

Precise, analogue regulation of gene expression is critical for development, homeostasis and regeneration in mammals. In contrast, widely employed experimental and therapeutic approaches such as knock-in/out strategies are more suitable for binary control of gene activity, while RNA interference (RNAi) can lead to pervasive off-target effects and unpredictable levels of repression. Here we report on a method for the precise control of gene expression levels in mammalian cells based on engineered, synthetic microRNA response elements (MREs). To develop this system, we established a high-throughput sequencing approach for measuring the efficacy of thousands of miR-17 MRE variants. This allowed us to create a library of microRNA silencing-mediated fine-tuners (miSFITs) of varying strength that can be employed to control the expression of user specified genes. To demonstrate the value of this technology, we used a panel of miSFITs to tune the expression of a peptide antigen in a mouse melanoma model. This analysis revealed that antigen expression level is a key determinant of the anti-tumour immune response in vitro and in vivo. miSFITs are a powerful tool for modulating gene expression output levels with applications in research and cellular engineering.

synthetic biology↗

bioRxiv2018

Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

Spatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here we developed a variant screening platform to identify differential sequence determinants of human tRNA promoter and processing activities. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enabled highly specific guide RNA production from a Pol-II promoter.

bioengineering↗