Biology subjects

Wirtz, D.

Publications and source records attributed to Wirtz, D..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

Precision-engineered biomimetics: the human fallopian tube

The fallopian tube has an essential role in several physiological and pathological processes from pregnancy to ovarian cancer. However, there are no biologically relevant models to study its pathophysiology. The state-of-the-art organoid model has been compared to two-dimensional tissue sections and molecularly assessed providing only cursory analyses of the models accuracy. We developed a novel multi-compartment organoid model of the human fallopian tube that was meticulously tuned to reflect the compartmentalization and heterogeneity of the tissues composition. We validated this organoids molecular expression patterns, cilia-driven transport function, and structural accuracy through a highly iterative platform wherein organoids are compared to a three-dimensional, single-cell resolution reference map of a healthy, transplantation-quality human fallopian tube. This organoid model was precision-engineered to match the human microanatomy. One sentence summaryTunable organoid modeling and CODA architectural quantification in tandem help design a tissue-validated organoid model.

bioengineering↗

bioRxiv2023

Serine synthesis pathway upregulated by E-cadherin is essential for the proliferation and metastasis of breast cancers

The loss of E-cadherin (E-cad), an epithelial cell adhesion molecule, has been implicated in the epithelial-mesenchymal transition (EMT), promoting invasion and migration of cancer cells and, consequently, metastasis. However, recent studies have demonstrated that E-cad supports the survival and proliferation of metastatic cancer cells, suggesting that our understanding of E-cad in metastasis is far from comprehensive. Here, we report that E-cad upregulates the de novo serine synthesis pathway (SSP) in breast cancer cells. The SSP provides metabolic precursors for biosynthesis and resistance to oxidative stress, critically beneficial for E-cad-positive breast cancer cells to achieve faster tumor growth and more metastases. Inhibition of PHGDH, a rate-limiting enzyme in the SSP, significantly and specifically hampered the proliferation of E-cad-positive breast cancer cells and rendered them vulnerable to oxidative stress, inhibiting their metastatic potential. Our findings reveal that E-cad adhesion molecule significantly reprograms cellular metabolism, promoting tumor growth and metastasis of breast cancers.

cancer biology↗

bioRxiv2023

Age-related increases in fibroblasts-secreted IGFBP2 increase melanoma cell invasion and lipid synthesis.

Aged melanoma patients (>65 years old) have more aggressive disease relative to young patients (<55 years old) for reasons that are not completely understood. Analysis of the young and aged secretome from human dermal fibroblasts identified >5-fold levels of insulin-like growth factor binding protein 2 (IGFBP2) in the aged fibroblast secretome. IGFBP2 functionally triggers upregulation of the PI3K-dependent fatty acid biosynthesis program in melanoma cells through increases in FASN. Melanoma cells co-cultured with aged dermal fibroblasts have higher levels of lipids relative to young dermal fibroblasts, which can be lowered by silencing IGFBP2 expression in fibroblasts, prior to treating with conditioned media. Conversely, ectopically treating melanoma cells with recombinant IGFBP2 in the presence of conditioned media from young fibroblasts, promoted lipid synthesis and accumulation in the melanoma cells. Neutralizing IGFBP2 in vitro reduces migration and invasion in melanoma cells, and in vivo studies demonstrate that neutralizing IGFBP2 in syngeneic aged mice, ablates tumor growth as well as metastasis. Conversely, ectopic treatment of young mice with IGFBP2 in young mice increases tumor growth and metastasis. Our data reveal that aged dermal fibroblasts increase melanoma cell aggressiveness through increased secretion of IGFBP2, stressing the importance of considering age when designing studies and treatment. SignificanceThe aged microenvironment drives metastasis in melanoma cells. This study reports that IGFBP2 secretion by aged fibroblasts induces FASN in melanoma cells and drives metastasis. Neutralizing IGFBP2 decreases melanoma tumor growth and metastasis.

cancer biology↗

bioRxiv2023

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

cancer biology↗

bioRxiv2022

Tumor proliferation and invasion are coupled through cell-extracellular matrix friction

Cell proliferation and invasion are two key drivers of tumor progression and are traditionally considered two independent cellular processes regulated by distinct pathways. Through in vitro and in silico methods, we provide evidence that these two processes are intrinsically coupled through matrix-adhesion friction. Using novel tumor spheroids, we show that both tumor cell proliferation and invasion are limited by a volumetric carrying capacity of the system, i.e. maximum spatial cell concentration supported by the systems total cell count, nutrient consumption rate, and collagen gel mechanical properties. To manipulate these phenotypes in breast cancer cells, we modulate the expression of E-cadherin and its associated role in adhesion, invasion, and proliferation. We integrate these results into a mixed-constitutive formulation to computationally delineate the contributions of cellular and extracellular adhesion, stiffness, and mechanical properties of the extracellular matrix (ECM) to the proliferative and invasive fates of breast cancer tumor spheroids. Both approaches conclude that the dominant drivers of tumor fate are system properties modulating cell-ECM friction, such as E-cadherin dependent cell-ECM adhesion and matrix pore size.

biophysics↗

bioRxiv2022

MLL1 regulates cytokine-driven cell migration and metastasis

Cell migration is a critical requirement for cancer metastasis. Cytokine production and its role in cancer cell migration have been traditionally associated with immune cells in the tumor microenvironment. MLL1 is a histone methyltransferase that controls 3D cell migration via the secretion of cytokines, IL-6 and TGF-{beta}1, by the cancer cells themselves. In vivo, MLL1 depletion reduced metastatic burden and prolonged survival. MLL1 exerts its effects with its scaffold protein, Menin. Mechanistically, the MLL1-Menin interaction controls actin filament assembly via the IL-6/pSTAT3/Arp3 axis and acto-myosin contractility via the TGF-{beta}1/Gli2/ROCK1/2/pMLC2 axis, which regulate dynamic protrusion generation and 3D cell migration. MLL1 also regulates cell proliferation via mitosis-based and cell cycle-related pathways. Combining an MLL1-Menin inhibitor with Paclitaxel, a standard chemotherapeutic, abrogated tumor growth and metastasis in a syngeneic model. These results highlight the potential of targeting the MLL1 in metastasis prevention and its potential to be combined with currently administered chemotherapeutics. Statement of SignificanceWe identify MLL1 as being vital to metastasis, which causes the vast majority of cancer-related deaths. MLL1 controls cell migration, a requirement for metastasis, by regulating the secretion of cytokines. MLL1 inhibition lowers metastatic burden independent of its impact on primary tumor growth, highlighting its anti-metastatic potential in TNBC.

cancer biology↗

bioRxiv2022

Spatial transcriptomics of FFPE pancreatic intraepithelial neoplasias reveals cellular and molecular alterations of progression to pancreatic ductal carcinoma

Spatial transcriptomics (ST) is a powerful new approach to characterize the cellular and molecular architecture of the tumor microenvironment. Previous single-cell RNA-sequencing (scRNA-seq) studies of pancreatic ductal adenocarcinoma (PDAC) have revealed a complex immunosuppressive environment characterized by numerous cancer associated fibroblasts (CAFs) subtypes that contributes to poor outcomes. Nonetheless, the evolutionary processes yielding that microenvironment remain unknown. Pancreatic intraepithelial neoplasia (PanIN) is a premalignant lesion with potential to develop into PDAC, but the formalin-fixed and paraffin-embedded (FFPE) specimens required for PanIN diagnosis preclude scRNA-seq profiling. We developed a new experimental pipeline for FFPE ST analysis of PanINs that preserves clinical specimens for diagnosis. We further developed novel multi-omics analysis methods for threefold integration of imaging, ST, and scRNA-seq data to analyze the premalignant microenvironment. The integration of ST and imaging enables automated cell type annotation of ST spots at a single-cell resolution, enabling spot selection and deconvolution for unique cellular components of the tumor microenvironment (TME). Overall, this approach demonstrates that PanINs are surrounded by the same subtypes of CAFs present in invasive PDACs, and that the PanIN lesions are predominantly of the classical PDAC subtype. Moreover, this new experimental and computational protocol for ST analysis suggests a biological model in which CAF-PanIN interactions promote inflammatory signaling in neoplastic cells which transitions to proliferative signaling as PanINs progress to PDAC. SummaryPancreatic intraepithelial neoplasia (PanINs) are pre-malignant lesions that progress into pancreatic ductal adenocarcinoma (PDAC). Recent advances in single-cell technologies have allowed for detailed insights into the molecular and cellular processes of PDAC. However, human PanINs are stored as formalin-fixed and paraffin-embedded (FFPE) specimens limiting similar profiling of human carcinogenesis. Here, we describe a new analysis protocol that enables spatial transcriptomics (ST) analysis of PanINs while preserving the FFPE blocks required for clinical assessment. The matched H&E imaging for the ST data enables novel machine learning approaches to automate cell type annotations at a single-cell resolution and isolate neoplastic regions on the tissue. Transcriptional profiles of these annotated cells enable further refinement of imaging-based cellular annotations, showing that PanINs are predominatly of the classical subtype and surrounded by PDAC cancer associated fibroblast (CAF) subtypes. Applying transfer learning to integrate ST PanIN data with PDAC scRNA-seq data enables the analysis of cellular and molecular progression from PanINs to PDAC. This analysis identified a transition between inflammatory signaling induced by CAFs and proliferative signaling in PanIN cells as they become invasive cancers. Altogether, this integration of imaging, ST, and scRNA-seq data provides an experimental and computational approach for the analysis of cancer development and progression.

cancer biology↗

bioRxiv2022

Mechanotransductive feedback control of endothelial cell motility and vascular morphogenesis

Vascular morphogenesis requires persistent endothelial cell motility that is responsive to diverse and dynamic mechanical stimuli. Here, we interrogated the mechanotransductive feedback dynamics that govern endothelial cell motility and vascular morphogenesis. We show that the transcriptional regulators, YAP and TAZ, are activated by mechanical cues to transcriptionally limit cytoskeletal and focal adhesion maturation, forming a conserved mechanotransductive feedback loop that mediates human endothelial cell motility in vitro and zebrafish intersegmental vessel (ISV) morphogenesis in vivo. This feedback loop closes in 4 hours, achieving cytoskeletal equilibrium in 8 hours. Feedback loop inhibition arrested endothelial cell migration in vitro and ISV morphogenesis in vivo. Inhibitor washout at 3 hrs, prior to feedback loop closure, restored vessel growth, but washout at 8 hours, longer than the feedback timescale, did not, establishing lower and upper bounds for feedback kinetics in vivo. Mechanistically, YAP and TAZ induced transcriptional suppression of RhoA signaling to maintain dynamic cytoskeletal equilibria. Together, these data establish the mechanoresponsive dynamics of a transcriptional feedback loop necessary for persistent endothelial cell migration and vascular morphogenesis.

cell biology↗

bioRxiv2021

Senescent stroma induces nuclear deformations in cancer cells via the inhibition of RhoA/ROCK/myosin II-based cytoskeletal tension

The presence of senescent cells within tissues has been functionally linked to malignant transformations. Here, using tension-gauge tethers technology, particle-tracking microrheology, and quantitative microscopy, we demonstrate that senescent associated secretory phenotype (SASP) derived from senescent fibroblasts impose nuclear lobulations and volume shrinkage on malignant cells, which stems from the loss of RhoA/ROCK/myosin II-based cortical tension. This loss in cytoskeletal tension induces decreased cellular contractility, adhesion, and increased mechanical compliance. These SASP-induced morphological changes are in part mediated by lamin A/C. These findings suggest that SASP induces a defective outside-in mechanotransduction, from actomyosin fibers in the cytoplasm to the nuclear lamina, thereby triggering a cascade of biophysical and biomolecular changes in cells that associate with malignant transformations.

cancer biology↗

bioRxiv2020

Deep Learning Identification of Stiffness Markers in Breast Cancer

While essential to our understanding of solid tumor progression, the study of cell and tissue mechanics has yet to find traction in the clinic. Determining tissue stiffness, a mechanical property known to promote a malignant phenotype in vitro and in vivo, is not part of the standard algorithm for the diagnosis and treatment of breast cancer. Instead, clinicians routinely use mammograms to identify malignant lesions and radiographically dense breast tissue is associated with an increased risk of developing cancer. Whether breast density is related to tumor tissue stiffness, and what cellular and non-cellular components of the tumor contribute the most to its stiffness are not well understood. Through training of a deep learning network and mechanical measurements of fresh patient tissue, we create a bridge in understanding between clinical and mechanical markers. The automatic identification of cellular and extracellular features from hematoxylin and eosin (H&E)-stained slides reveals that global and local breast tissue stiffness best correlate with the percentage of straight collagen. Global breast tissue mechanics correlate weakly with the percentage of blood vessels and fibrotic tissue, and non-significantly with the percentage of fat, ducts, tumor cells, and wavy collagen in tissue. Importantly, the percentage of dense breast tissue does not directly correlate with tissue stiffness or straight collagen content.

cancer biology↗

bioRxiv2020

In situ characterization of the 3D microanatomy of the pancreas and pancreatic cancer at single cell resolution

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer. Accumulating evidence indicates the tumor microenvironment is highly associated with tumorigenesis through regulation of cellular physiology, signaling systems, and gene expression profiles of cancer cells. Yet the mechanisms by which the microenvironment evolves from normal pancreas architecture to precursor lesions and invasive cancer is poorly understood. Obtaining high-content and high-resolution information from a complex tumor microenvironment in large volumetric landscapes represents a key challenge in the field of cancer biology. To address this challenge, we established a novel method to reconstruct three-dimensional (3D) centimeter-scale tissues containing billions of cells from serially sectioned histological samples, utilizing deep learning approaches to recognize eight distinct tissue subtypes from hematoxylin and eosin stained sections at micrometer and single-cell resolution. Using samples from a range of normal, precancerous, and invasive pancreatic cancer tissue, we map in 3D modes of cancer invasion in the tumor microenvironment, and emphasize the need for further 3D quantification of biological systems.

cancer biology↗

bioRxiv2020

Multi-compartment tumor organoids

Organoid cultures are widely used because they preserve many features of cancer cells in vivo. Here, we developed high-throughput oil-in-water droplet microtechnology to generate highly uniform, small-volume, multi-compartment organoids. Each organoid culture features a microenvironmental architecture that mimics both the basement membrane and stromal barriers. This matrix architecture, which allows accessing both proliferative and invasive features of cancer cells in a single platform, has profound effect on observed drug responsiveness and tumor progression that correlate well with in vivo and clinical outcomes. The method was tested on multiple types of cancer cells including primary cells and immortalized cell lines, and we determined our platform is suitable even for cells of poor organoid-forming ability. These new organoids also allow for direct orthotopic mouse implantation of cancer cells with unprecedented success.

cancer biology↗

bioRxiv2020

E-cadherin promotes cell hyper-proliferation in breast cancer

The loss of the intercellular adhesion molecule E-cadherin is a hallmark of the epithelial- mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Accordingly, E-cadherin has long been considered a tumor suppressor gene. Using novel multi-compartment spheroids and multiple in vivo models, we show that E-cadherin promotes a hyper-proliferative phenotype in breast cancer cells via interaction with the transmembrane receptor EGFR. This interaction results in the activation of the MEK/ERK signaling pathway, leading to a significant increase in proliferation via the activation of transcription factors including c-Fos. Pharmacological inhibition of MEK activity in E-cadherin positive breast cancer cells significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor growth and identifies a potential new target to treat hyper-proliferative E-cadherin-positive breast tumors.

cancer biology↗

bioRxiv2020

Fractional re-distribution among cell motility states during ageing

Ageing in humans is associated with a decreased capacity to regulate cell physiology. Cellular properties, such as cell morphology and cell mechanics, encode ageing information and as a result can be used as robust ageing biomarkers. Using a panel of dermal fibroblasts derived from healthy donors spanning a wide age range, we observe an age-related reduction in average cell motility, which we show is not due to the decreased motility of all cells, but results from fractional re-distribution among motility states. By taking advantage of the single-cell nature of our motility data, we show that cells can be classified based on spatial and activity patterns that define age-dependent motility states. These findings highlight an important feature of ageing cells shown by the decrease in the heterogeneity of cell movement in older adults, that potentially offer new mechanistic insights into the ageing process and avenues for novel biomarker development.

bioengineering↗

bioRxiv2019

Confronting p-hacking: addressing p-value dependence on sample size

Biomedical research has come to rely on p-values as a deterministic measure for data-driven decision making. In the largely extended null-hypothesis significance testing (NHST) for identifying statistically significant differences among groups of observations, a single p-value computed from sample data is routinely compared with a threshold, commonly set to 0.05, to assess the evidence against the hypothesis of having non-significant differences among groups, or the null hypothesis. Because the estimated p-value tends to decrease when the sample size is increased, applying this methodology to large datasets results in the rejection of the null hypothesis, making it not directly applicable in this specific situation. Herein, we propose a systematic and easy-to-follow method to detect differences based on the dependence of the p-value on the sample size. The proposed method introduces new descriptive parameters that overcome the effect of the size in the p-value interpretation in the framework of large datasets, reducing the uncertainty in the decision about the existence of biological/clinical differences between the compared experiments. This methodology enables both the graphical and quantitative characterization of the differences between the compared experiments guiding the researchers in the decision process. An in-depth study of the proposed methodology is carried out using both simulated and experimentally obtained data. Simulations show that under controlled data, our assumptions on the p-value dependence on the sample size holds. The results of our analysis in the experimental datasets reflect the large scope of this approach and its interpretability in terms of common decision-making and data characterization tasks. For both simulated and real data, the obtained results are robust to sampling variations within the dataset.

bioinformatics↗

bioRxiv2019

The role of integrin αv and CD44 in GBM migration in human brain

Glioblastoma (GBM) is the most common primary adult malignant brain tumor. Recurrence is driven invading tumor cells that escape surgical resection and demonstrate resistance to standard-of-care chemotherapy and radiotherapy. A large body of research has been conducted on tumor cell motility. However, typical in vitro models make use of polystyrene culture dishes, which exhibit significantly different physical parameters than brain tissue. Here we report on the use of human organotypic brain slices as an ex vivo approach for the dynamic study of GBM cell motility. Temporal lobectomy tissue from epilepsy patients was obtained and cut into 350{micro}m thick slices. After the tissue slices had a weeks incubation for recovery, fluorescently labeled tumor cells were seeded. We then tracked individual tumor cells using time-lapse fluorescent confocal microscopy. Quantification of motility characteristics, including mean squared displacement, total path length, and consistency, allowed for comparison of different conditions, including knockdown of cell surface proteins integrin v (ITGAV) and CD44. Human organotypics demonstrated minimal variability across specimen in terms of motility parameters, including total path length, averaged instantaneous velocity, and consistency. Knockdown of the traditional motility protein ITGAV showed little effect on overall motility while knockdown of CD44 resulted in a significant reduction in both averaged instantaneous velocity and total path length. When the same parameters were examined using Matrigel, ITGAV and CD44 both showed decreased motility, highlighting the impact of the physical environment on cell behavior. Finally, cell motility in mouse organotypic slices was decreased when compared to human organotypic slices. Here we demonstrate the use of human organotypic brain slices in the study of GBM cell invasion. This model system offers a physiologically-relevant environment in which to examine the dynamic process of cell motility.

cancer biology↗

bioRxiv2019

YAP/TAZ as a Novel Regulator of cell volume

How mammalian cells regulate their physical size is currently poorly understood, in part due to the difficulty of accurately quantifying cell volume in a high throughput manner. Here, using the fluorescence exclusion method, we demonstrate that the mechanosensitive transcriptional regulators YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are novel regulators of single cell volume. We report that the role of YAP/TAZ in cell volume regulation must go beyond its influence on total cell cycle duration or the cell shape to explain the observed changes in volume. Moreover, for our experimental conditions, volume regulation by YAP/TAZ is independent of mTOR. Instead, we find YAP/TAZ directly impacts the cell division volume. Based on the idea that YAP/TAZ is a mechanosensor, we find that inhibiting the assembly of myosin and cell tension slows cell cycle progression from G1 to S. These results suggest that YAP/TAZ and the Hippo pathway may be modulating cell volume in combination with cytoskeletal tension during cell cycle progression.

bioengineering↗

bioRxiv2017

Computational discovery of tissue morphology biomarkers in very long-term survivors with pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer, with an average 5-year survival rate of only 8%. Within PDAC patients, however, there is a small subset of patients who survive >10 years. Deciphering underlying reasons behind prolonged survival could potentially provide new opportunities to treat PDAC; however, no genomic, transcriptomic, proteomic, or clinical signatures have been found to robustly separate this subset of patients. Digital pathology, in combination with machine learning, provides an opportunity to computationally search for tissue morphology patterns associated with disease outcomes. Here, we developed a computational framework to analyze whole-slide images (WSI) of PDAC patient tissue and identify tissue-morphology signatures for very long term surviving patients. Our results indicate that less tissue morphology heterogeneity is significantly linked to better patient survival and that the extra-tumoral space encodes prognostic information for survival. Based on information from morphological heterogeneity in the tumor and its adjacent area, we established a machine learning model with an AUC of 0.94. Our analysis workflow highlighted a quantitative visual-based tissue phenotype analysis that also allows direct interaction with pathology. This study demonstrates a pathway to accelerate the discovery of undetermined tissue morphology associated with pathogenesis states and prognosis and diagnosis of patients by utilizing new computational approaches.

bioinformatics↗