Biology subjects

Roberts, L. M.

Publications and source records attributed to Roberts, L. M..

8 recordsLinked to original sources

bioRxiv2025

Engineered lymphatic stroma model applications in central nervous system leukemia

Central nervous system (CNS) involvement frequently occurs in acute lymphoblastic leukemia (ALL), but many questions remain about how leukemia cells access, persist in, and exploit the CNS. The CNS is protected by the meninges, which are fluid filled membranes that surround the brain. Within the meninges, meningeal lymphatic vessels drain cerebrospinal fluid to cervical lymph nodes for immunosurveillance, a potential pathway for leukemia cell migration into or egress from the CNS. Here, we utilize tissue engineered models of the meningeal lymphatics and lymph node stroma to probe how leukemia cells interact with these key tissues. We first demonstrate that standard-of-care chemotherapeutics can damage meningeal lymphatic barriers. Next, we showed that soluble factors from the meningeal lymphatics can support leukemia cell growth while soluble factors from the lymph node model under flow can promote leukemia cell migration. Finally, we show that leukemia cells migrate through the lymph node model under both static and flow conditions. Overall, we have demonstrated the feasibility of using engineered lymphatic models to study leukemia cell behavior in the CNS with the goal of expanding the available experimental platforms for understanding CNS metastasis and relapse. Insight BoxLeukemia commonly infiltrates the central nervous system (CNS), requiring intensive CNS-directed therapies that are often ineffective and cause both acute and long-term toxicities, especially in pediatric patients. The meningeal lymphatics and the deep cervical lymph nodes constitute a pivotal axis in CNS immunity, facilitating drainage of fluid and waste and enabling peripheral immune surveillance in response to CNS-derived signals. Here, we employ in vitro models of the meningeal lymphatics and lymph node stroma to demonstrate their crosstalk in influencing leukemia cell growth and migration. These engineered platforms serve as valuable tools for uncovering mechanistic insights into the meningeal-lymph node axis in the context of CNS-leukemia relapse.

bioengineering↗

bioRxiv2024

Demonstration of chemotherapeutic mediated lymphatic changes in meningeal lymphatics in vitro, ex vivo, and in vivo

Systemic chemotherapeutics target cancer cells but are also known to impact other cells away from the tumor. Questions remain whether systemic chemotherapy crosses the blood-brain barrier and causes inflammation in the periphery that impacts the central nervous system (CNS) downstream. The meningeal lymphatics are a critical component that drain cerebrospinal fluid from the CNS to the cervical lymph nodes for immunosurveillence. To develop new tools for understanding chemotherapy-mediated effects on the meningeal lymphatics, we present two novel models that examine cellular and tissue level changes. Our in vitro tissue engineered model of a meningeal lymphatic vessel lumen, using a simple tissue culture insert system with both lymphatic endothelial and meningeal cells, examines cell disruption. Our ex vivo model culturing mouse meningeal layers probes structural changes and remodeling, correlating to an explant tissue level. To gain a holistic understanding, we compare our in vitro and ex vivo models to in vivo studies for validation and a three-tier methodology for examining the chemotherapeutic response of the meningeal lymphatics. We have demonstrated that the meningeal lymphatics can be disrupted by systemic chemotherapy but show differential responses to platinum and taxane chemotherapies, emphasizing the need for further study of off-target impacts in the CNS.

bioengineering↗

bioRxiv2022

Hyaluronic acid biosynthesis promotes an invasive, stem-like cancer cell phenotype by broadly altering metabolism

Metastasis is the leading cause of breast cancer-related deaths and often driven by invasion and cancer-stem like cells (CSCs). Both the CSC phenotype and invasion have been associated with increased hyaluronic acid (HA) production. How these independent observations are connected, and which role metabolism plays in this process remains unclear due in part to the lack of convergent approaches that integrate engineered model systems, computational tools, and cancer biology. Using microfluidic invasion models, metabolomics, computational flux balance analysis (FBA), and bioinformatic analysis of patient data we investigated the functional links between the stem-like, invasive, and metabolic phenotype of breast cancer cells as a function of HA biosynthesis. Our results suggest that CSCs are more invasive than non-CSCs and that broad metabolic changes caused by overproduction of HA play a role in this process. Accordingly, overexpression of hyaluronic acid synthases (HAS) 2 or 3 induced a metabolic phenotype that promoted breast cancer cell stemness and invasion in vitro and upregulated a transcriptomic signature that was predictive of increased invasion and worse survival in patients. Collectively, this study suggests that HA overproduction leads to metabolic adaptations that help satisfy the energy demands necessary for 3D invasion of breast cancer stem cells further highlighting the importance of engineered model systems and multidisciplinary approaches in cancer research.

cancer biology↗

bioRxiv2021

Fusobacterium nucleatum infection induces pancreatic cancer cell proliferation and migration through regulation of host cytokine signaling

Pancreatic ductal adenocarcinoma (PDAC) harbors a complex tumor microbiome that has been implicated in cancer progression and resistance to chemotherapy. Recent clinical investigations uncovered a correlation between high loads of intratumor Fusobacterium nucleatum and decreased patient survival. Here we show that healthy and cancerous pancreatic cell lines harboring intracellular F. nucleatum secrete increased levels of cancer-associated cytokines including GM-CSF, CXCL1, IL-8, and MIP-3. We report that GM-CSF (granulocyte-macrophage colony stimulating factor) secretion directly increases the proliferation and migration of pancreatic cancer cells via an autocrine mechanism, notably in the absence of immune cell participation. Furthermore, we show that non-cancerous pancreatic epithelial cells do not exhibit increased proliferation or migration in response to these cytokines, but nevertheless, their secreted cytokines stimulate these responses in cancerous cell lines through paracrine signaling. Our results provide evidence that intratumor F. nucleatum in the pancreas elicits an infection-specific cytokine secretion profile from both normal and cancerous cells that adversely contributes to cancer progression through autocrine and paracrine mechanisms. Therefore, these results support the importance of investigating the contributions of both microbiome and host driven processes in pancreatic cancer to guide future therapeutic interventions.

cancer biology↗

bioRxiv2021

Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, particularly with respect to immune responses, we utilized the rhesus macaque model of SARS-CoV-2 infection. Two cohorts of eight older (16-23 years) and eight younger (3-5 years) rhesus macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at pre-defined timepoints in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication dynamics were limited between age cohorts. Transcriptional signatures of inflammation-associated genes in cells isolated from bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both younger and older animals. These findings suggested that age did not substantially skew major facets of acute disease in this model. However, age-specific divergence of immune responses emerged during the post-acute phase of infection (7-21 dpi). Older animals exhibited sustained local inflammatory innate responses while local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, in contrast to persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling in SARS-CoV-2-infected rhesus macaques suggests that age may delay or impair the induction of anti-viral cellular immune responses and delay efficient return to immune homeostasis following acute infection.

immunology↗

bioRxiv2020

Validation and Application of a Bench Top Cell Sorter in a BSL-3 Containment Setting

Rigorous assessment of the cellular and molecular changes during infection typically requires isolation of specific immune cell subsets for downstream application. While there are numerous options for enrichment/isolation of cells from tissues, fluorescent activated cell sorting (FACS) is accepted as a method that results in superior purification of a wide variety of cell types. Flow cytometry requires extensive fluidics and aerosol droplets can be generated during collection of target cells. Pathogens such as Francisella tularensis, Mycobacterium tuberculosis, Yersinia pestis, and SARS-CoV-2 require manipulation at biosafety level-3 (BSL-3). Due to the concern of potential aerosolization of these pathogens, use of flow cytometric-based cell sorting in these laboratory settings requires placement of the equipment in dedicated biosafety cabinets within the BSL-3. For many researchers, this is often not possible due to expense, space, or expertise available. Here we describe the safety validation and utility of a completely closed cell sorter that results in gentle, rapid, high purity, and safe sorting of cells on the benchtop at BSL-3. We also provide data demonstrating the need for cell sorting versus bead purification and the applicability of this technology for BSL-3 and potentially BSL-4 related infectious disease projects. Adoption of this technology will significantly expand our ability to uncover important features of the most dangerous infectious diseases leading to faster development of novel vaccines and therapeutics.

immunology↗

bioRxiv2020

The Genetic Architecture of Leaf Stable Carbon Isotope Composition in Zea mays and the Effect of Transpiration Efficiency on Elemental Accumulation

With increased demand on freshwater resources for agriculture, it is imperative that more water-use efficient crops are developed. Leaf stable carbon isotope composition, {delta}13C, is a proxy for transpiration efficiency and a possible tool for breeders, but the underlying mechanisms effecting {delta}13C in C4 plants are not known. It has been suggested that differences in specific leaf area, which potentially reflects variation in internal CO2 diffusion, can impact leaf {delta}13C. However, at this point the relationship has not been tested in maize. Furthermore, although it is known that water movement is important for elemental uptake, it is not clear how manipulation of transpiration for increased water-use efficiency may impact nutrient accumulation. Here we characterize the underlying genetic architecture of leaf {delta}13C and test its relationship to specific leaf area and the ionome in four biparental populations of maize. Five significant QTL for leaf {delta}13C were identified, including both novel QTL as well as some that were identified previously in maize kernels. One of the QTL regions contains an Erecta-like gene, the ortholog of which has been shown to regulate transpiration efficiency and leaf {delta}13C in Arabidopsis. Our data does not support a relationship between {delta}13C and specific leaf area, and of the 19 elements analyzed, only a weak correlation between molybdenum and {delta}13C was detected. Together these data begin to build a genetic understanding of leaf {delta}13C in maize and suggest the potential to improve plant water use without significantly influencing elemental homeostasis. Article SummaryQuantitative genetics approaches were used to investigate the genetic architecture of leaf stable carbon isotope discrimination ({delta}13C) in maize. Developing a better understanding of leaf {delta}13C could facilitate its use in breeding for reduced transpirational water loss. Several genomic regions were identified that contribute to the variation observed in leaf {delta}13C. Furthermore, contrary to what has been observed in other species, leaf {delta}13C was not correlated with specific leaf area. Finally, a leaf ionomic analysis indicates that a reduction in transpiration, and thus mass flow, would not result in a decrease in nutrient accumulation.

plant biology↗

bioRxiv2018

Stable Carbon Isotopes in Zea mays

The increasing demand for food production and predicted climate change scenarios highlight the need for improvements in crop sustainability. The efficient use of water will become increasingly important for rainfed agricultural crops even in fertile regions that have historically received ample precipitation. Improvements in water-use efficiency in Zea mays have been limited, and warrants a renewed effort aided by molecular breeding approaches. Progress has been constrained by the difficulty of measuring water-use in a field environment. The stable carbon isotope composition ({delta}13C) of the leaf has been proposed as an integrated signature of carbon fixation with a link to stomatal conductance. However, additional factors affecting leaf {delta}13C exist, and a limited number of studies have explored this trait in Z. mays. Here we present an extensive characterization of leaf {delta}13C in Z. mays. Significant variation in leaf {delta}13C exists across diverse lines of Z. mays, which we show to be heritable across several environments.\n\nFurthermore, we examine temporal and spatial variation in leaf {delta}13C to determine the optimum sampling time to maximize the use of leaf {delta}13C as a trait. Finally, our results demonstrate the relationship between transpiration and leaf {delta}13C in the field and the greenhouse. Decreasing transpiration and soil moisture are associated with decreasing leaf {delta}13C. Taken together these results outline a strategy for using leaf {delta}13C and reveal its usefulness as a measure of transpiration efficiency under well-watered conditions rather than a predictor of performance under drought.\n\nSignificance StatementThis study identifies sources of variation in stable carbon isotopes of maize leaves and establishes the framework for connecting leaf {delta}13C and transpiration efficiency.

plant biology↗