Biology subjects

George, S. C.

Publications and source records attributed to George, S. C..

18 recordsLinked to original sources

bioRxiv2026

Hydrodynamic shear enables enrichment of functional tumor antigen-reactive T cells

Adoptive T-cell therapies rely on the identification and expansion of rare tumor-reactive T cells, yet current enrichment strategies are limited by the low abundance of these cells and complexity of their functional enrichment. Here, we present a microfluidic platform that exploits hydrodynamic shear as a controllable parameter for enriching antigen-specific T cells through peptide-major histocompatibility complex (pMHC)-mediated capture. An eight-channel microfluidic device was engineered to simultaneously interrogate a range of wall shear stresses while maintaining uniform cell delivery, enabling systematic identification of shear conditions that maximize antigen-specific enrichment. Using engineered MART-1-specific Jurkat cells, we demonstrate that T-cell capture is jointly regulated by wall shear stress and pMHC density, with intermediate shear preferentially enriching antigen-specific cells over nonspecific binders. Translation of the optimal operating condition to a high-throughput single-shear device enabled approximately 35-fold enrichment of antigen-specific T cells from peripheral blood mononuclear cells containing only 0.05% target cells. We further show that peptide-MHC complexes isolated directly from melanoma whole-cell lysates support shear-dependent enrichment comparable to recombinant pMHCs. Finally, primary MART-1-specific CD8 T cells enriched using tumor-derived pMHCs retained the ability to recognize melanoma cells and upregulated the activation marker CD137 following antigen-specific stimulation. Together, these findings establish hydrodynamic shear as an orthogonal parameter for antigen-specific T-cell enrichment and provide a framework for integrating force-based selection with tumor-derived pMHCs to isolate functional antigen-specific T cells using tumor-derived pMHCs.

bioengineering↗

bioRxiv2025

A physiological microfluidic blood-brain-barrier model for in vitro study of nanoparticle trafficking and accumulation

Although the blood-brain barrier (BBB) restricts passage of most molecules, various naturally occurring and synthetic nanoparticles are nonetheless found within the brain parenchyma. To study the mechanisms underlying this phenomenon, we developed a microfluidic BBB model (mBBB) using human cerebral microvascular endothelial cells (HCMECs) in direct contact with primary human astrocytes and pericytes within a physiologically relevant extracellular matrix. The horizontal architecture enables high-resolution imaging across the full barrier interface and allows direct assessment of nanoparticle transport and accumulation. This in vitro platform recapitulates key features of the BBB, including selective permeability, junctional protein expression, and receptor-mediated uptake pathways. Using this system, the trafficking and accumulation of structurally distinct nanoparticles, including liposomes, nanoplastics, and extracellular vesicles (EVs), were compared. Among these, heterologous EVs exhibit the highest transport efficiency. Analysis of nanoparticle properties suggest that ligand presentation and membrane composition, rather than size or stiffness, primarily govern BBB penetration. The mBBB platform provides a high-throughput, imaging-based framework to systematically interrogate nanoparticle trafficking across the BBB and offers a translational tool for both drug delivery and neurotoxicity screening.

bioengineering↗

bioRxiv2025

Targeting Neutrophil Extracellular Traps to inhibit Colon Cancer Tumor Necrosis and Metastasis

Necrosis, conventionally thought of as a passive consequence of aggressive tumor growth, is associated with poor prognosis in colorectal cancer (CRC). We recently discovered that necrosis can be caused by neutrophils and neutrophil extracellular traps (NETs) aggregates driving vascular occlusion within the tumor vasculature in models of breast cancer. Here, we evaluated the role of NETs in inducing necrosis and metastasis in CRC. We found that the numbers of neutrophils primed to form NETs were elevated in the circulation of patients with CRC as compared to controls. CD177Low neutrophils were also elevated, and they showed reduced extravasation capacity with intact ability to form NETs. The extent of necrosis correlated with metastasis (stage IV disease), independent of tumor size, in our human cohort. In both human and murine CRC tumors, necrotic regions were characterized by neutrophil infiltration and NET accumulation, and NET aggregates were observed in the vasculature next to the necrotic regions. Single cell RNA sequencing and spatial transcriptomic analysis of human CRC and liver metastases revealed that necrotic tumors activate pathways associated with increased metastatic potential, including epithelial-to-mesenchymal-transition. Using a mouse model of DNA mismatch repair proficient CRC, we found neutrophil infiltration and NETs increased with tumor progression. Genetic or pharmacological inhibition of NET formation decreased necrosis and metastasis, and importantly enhanced chemotherapy efficacy. Altogether, our findings show that NET formation in human CRC is a key feature of tumor necrosis, that it is associated with metastasis, and further suggest that preventing NET formation may offer clinical benefits to CRC patients.

cancer biology↗

bioRxiv2024

A nucleotide code governs Lis1's ability to relieve dynein autoinhibition

Dynein-1 is a microtubule motor responsible for the transport of cytoplasmic cargoes. Activation of motility requires it first overcome an autoinhibited state prior to its assembly with dynactin and a cargo adaptor. Studies suggest that Lis1 may relieve dyneins autoinhibited state. However, evidence for this mechanism is lacking. We first set out to determine the rules governing dynein-Lis1 binding, which reveals that their binding affinity is regulated by the nucleotide-bound states of each of three nucleotide-binding pockets within the dynein motor domain. We also find that distinct nucleotide codes coordinate dynein-Lis1 binding stoichiometry by impacting binding affinity at two different sites within the dynein motor domain. Electron microscopy reveals that a 1 Lis1:1 dynein complex directly promotes an open, uninhibited conformational state of dynein, whereas a 2:1 complex resembles the autoinhibited state. Cryo-EM analysis reveals the structural basis for Lis1 opening dynein relies on interactions with the linker domain.

biochemistry↗

bioRxiv2023

A microfluidic strategy to capture antigen-specific high affinity B cells

Assessing B cell affinity to pathogen-specific antigens prior to or following exposure could facilitate the assessment of immune status. Current standard tools to assess antigen-specific B cell responses focus on equilibrium binding of the secreted antibody in serum. These methods are costly, time-consuming, and assess antibody affinity under zero-force. Recent findings indicate that force may influence BCR-antigen binding interactions and thus immune status. Here, we designed a simple laminar flow microfluidic chamber in which the antigen (hemagglutinin of influenza A) is bound to the chamber surface to assess antigen-specific BCR binding affinity of five hemagglutinin-specific hybridomas under 65- to 650-pN force range. Our results demonstrate that both increasing shear force and bound lifetime can be used to enrich antigen-specific high affinity B cells. The affinity of the membrane-bound BCR in the flow chamber correlates well with the affinity of the matched antibodies measured in solution. These findings demonstrate that a microfluidic strategy can rapidly assess BCR-antigen binding properties and identify antigen-specific high affinity B cells. This strategy has the potential to both assess functional immune status from peripheral B cells and be a cost-effective way of identifying individual B cells as antibody sources for a range of clinical applications.

bioengineering↗

bioRxiv2023

Krumholzibacteriota and Deltaproteobacteria contain rare genetic potential to liberate carbon from monoaromatic compounds in subsurface coal seams

Biogenic methane in subsurface coal seam environments is produced by diverse consortia of microbes. Although this methane is useful for global energy security, it remains unclear which microbes can liberate carbon from the coal. Most of this carbon is relatively resistant to biodegradation, as it is contained within aromatic rings. Thus, to explore for coal-degrading taxa in the subsurface, this study used coal seam metagenomes to reconstruct important metagenome-assembled genomes (MAGs) using a key genomic marker for the anaerobic degradation of monoaromatic compounds as a guide: the benzoyl-CoA reductase gene (bcrABCD). Three taxa were identified with this genetic potential. The first was a novel taxon from the Krumholzibacteriota phylum, which this study is the first to describe. This Krumholzibacteriota sp. contained a full set of genes for benzoyl-CoA dearomatisation, in addition to other genes for anaerobic catabolism of monoaromatics. Analysis of Krumholzibacteriota MAGs from other environments revealed that this genetic potential may be common within this phylum, and thus they may be important organisms for the liberation of recalcitrant carbon on a global scale. Further, two taxa from the Deltaproteobacteria class were also implicated in monoaromatic degradation; two geographically unrelated Syntrophorhabdus aromaticivorans MAGs, and a Syntrophaceae sp. MAG. Each of these three taxa are potential rate-limiting organisms for subsurface coal-to-methane biodegradation. Their description here provides an understanding of their function within the coal seam microbiome, and will help inform future efforts in coal bed methane stimulation, anoxic bioremediation of organic pollutants, and assessments of anoxic carbon cycling and emissions. ImportanceSubsurface coal seams are highly anoxic and oligotrophic environments, where the main source of carbon is "locked away" within aromatic rings. Despite these challenges, biogenic methane accumulates within many of these coal seams, which implies that the coal seam microbiome can "unlock" this carbon source in situ. For over two decades, researchers have been working to understand which organisms are responsible for these processes. This study provides the first descriptions of these organisms. Here, we report metagenomic insights into the liberation of carbon from aromatic molecules typically found within coal, the degradation pathways involved, and descriptions of the Krumholzibacteriota sp., Syntrophorhabdus aromaticivorans, and Syntrophaceae sp. that contain this genetic potential. Additionally, this is the first time that the Krumholzibacteriota phylum has been implicated in anaerobic dearomatisation of aromatic hydrocarbons. This potential is identified here in numerous taxa within the phylum from other subsurface environments, implicating Krumholzibacteriota in global-scale carbon-cycling processes.

microbiology↗

bioRxiv2022

Engineered vasculature induces functional maturation of pluripotent stem cell-derived islet organoids

Blood vessels play a critical role in pancreatic islet health and function, yet current culture methods to generate islet organoids from human pluripotent stem cells (SC-islets) lack a vascular component. Here, we engineered 3D vascularized SC-islet organoids by assembling SC-islet cells, human primary endothelial cells (ECs) and fibroblasts both in a non-perfused model and a microfluidic device with perfused vessels. Vasculature improved stimulus-dependent Ca2+ influx into SC-{beta}-cells, a hallmark of {beta}-cell function that is blunted in non-vascularized SC-islets. We show that an islet-like basement membrane is formed by vasculature and contributes to the functional improvement of SC-{beta}-cells. Furthermore, cell-cell communication networks based on scRNA-seq data predicted BMP2/4-BMPR2 signaling from ECs to SC-{beta}-cells. Correspondingly, BMP4 augmented the SC-{beta}-cell Ca2+ response and insulin secretion. These vascularized SC-islet models will enable further studies of crosstalk between {beta}-cells and ECs and can serve as in vivo-mimicking platforms for disease modeling and therapeutic testing.

cell biology↗

bioRxiv2022

Convection and extracellular matrix binding control interstitial transport of extracellular vesicles

Extracellular vesicles (EVs) influence a host of normal and pathophysiological processes in vivo. Compared to soluble mediators, EVs are relatively large (~30-150 nm) and can traffic a wide range of proteins on their surface including extracellular matrix (ECM) binding proteins. We isolated EVs from the MCF10 series - a model human cell line of breast cancer progression - and demonstrated increasing presence of laminin-binding integrins 3{beta}1 and 6{beta}1 on the EVs as the malignant potential of the MCF10 cells increased. Transport of the EVs within a microfluidic device under controlled physiological interstitial flow (0.15-0.75 m/s) demonstrated that convection was the dominant mechanism of transport. Binding of the EVs to the ECM enhanced the spatial concentration and gradient, which was partially mitigated by blocking integrins 3{beta}1 and 6{beta}1. Our studies demonstrate that convection and ECM binding are the dominant mechanisms controlling EV interstitial transport and should be leveraged in the design of nanotherapeutics.

bioengineering↗

bioRxiv2022

Automated protein-protein structure prediction of the T cell receptor-peptide major histocompatibility complex

T Cell Receptor (TCR) recognition of a peptide-major histocompatibility complex (pMHC) is a crucial component of the adaptive immune response. The identification of therapeutically relevant TCR-pMHC pairs is a significant bottleneck in the implementation of TCR-based immunotherapies but may be augmented by computational methodologies. The ability to computationally design TCRs to target a specific pMHC will require an automated integration of next-generation sequencing, protein-protein structure prediction, molecular dynamics (MD), and TCR ranking. We present a generic pipeline to evaluate patient-specific, sequence-based TCRs to a target pMHC. Using the three most frequently expressed TCRs from 16 colorectal cancer patients, we predicted the protein-protein structure of the TCRs to the target CEA peptide-MHC using Modeller and ColabFold. Then, these TCR-pMHC structures were compared by performing an automated molecular dynamics equilibration. ColabFold generates starting configurations that require, on average, a ~2.5X reduction in simulation time to equilibrate TCR-pMHC structures compared to Modeller. In addition, the structural differences between Modeller and ColabFold are demonstrated by an increase in root mean square deviation (~0.20 nm) between clusters of equilibrated configurations, which can impact the number of hydrogen bonds and Lennard-Jones contacts between the TCR and pMHC. Finally, we identify a TCR ranking criteria that may be used to prioritize TCRs for evaluation of in vitro immunogenicity.

biophysics↗

bioRxiv2021

A vascularized 3D model of the human pancreatic islet for ex vivo study of immune cell-islet interaction

Insulin is an essential regulator of blood glucose homeostasis that is produced exclusively by {beta} cells within the pancreatic islets of healthy individuals. In those affected by diabetes, immune inflammation, damage, and destruction of islet {beta} cells leads to insulin deficiency and hyperglycemia. Current efforts to understand the mechanisms underlying {beta} cell damage in diabetes rely on in vitro-cultured cadaveric islets. However, isolation of these islets involves removal of crucial matrix and vasculature that supports islets in the intact pancreas. Unsurprisingly, these islets demonstrate reduced functionality over time in standard culture conditions, thereby limiting their value for understanding native islet biology. Leveraging a novel, vascularized micro-organ (VMO) approach, we have recapitulated elements of the native pancreas by incorporating isolated human islets within a three-dimensional matrix nourished by living, perfusable blood vessels. Importantly, these islets show long-term viability and maintain robust glucose-stimulated insulin responses. Furthermore, vessel-mediated delivery of immune cells to these tissues provides a model to assess islet-immune cell interactions and subsequent islet killing -- key steps in type 1 diabetes pathogenesis. Together, these results establish the islet-VMO as a novel, ex vivo platform for studying human islet biology in both health and disease.

cell biology↗

bioRxiv2021

T Cell Receptor Non-Equilibrium Kinetics

An atomic-scale mechanism describing the role of mechanosensing in T Cell Receptor (TCR) recognition of peptides in the binding groove of the peptide-major histocompatibility complex (pMHC) may inform the design of novel TCRs for immunotherapies. Using steered molecular dynamic simulations, our study demonstrates that mutations to peptides in the binding groove of the pMHC - which are known to discretely alter the T cell response to an antigen - influence MHC conformation and thus the overall strength of the TCR-pMHC bond including duration and length under constant load. Moreover, physiochemical features of the TCR-pMHC dynamic bond strength, such as hydrogen bonds and Lennard-Jones contacts, correlate with the immunogenic response elicited by the specific peptide in the MHC groove. Thus, formation of transient TCR-pMHC bonds is a characteristic of immunogenic peptides and is mediated by stabilized interactions.x

biophysics↗

bioRxiv2021

The Atomic-Level Physiochemical Determinants of T Cell Receptor Dissociation Kinetics

The rational design of T Cell Receptors (TCRs) for immunotherapy has stagnated due to a limited understanding of the dynamic physiochemical features of the TCR that elicit an immunogenic response. The physiochemical features of the TCR-peptide major histocompatibility complex (pMHC) bond dictate bond lifetime which, in turn, correlates with immunogenicity. Here, we: i) characterize the force-dependent dissociation kinetics of the bond between a TCR and a set of pMHC ligands using Steered Molecular Dynamics (SMD); and ii) implement a machine learning algorithm to identify which physiochemical features of the TCR govern dissociation kinetics. Our results demonstrate that the total number of hydrogen bonds between the CDR2{beta}-MHC({beta}), CDR1-Peptide, and CDR3{beta}-Peptide are critical features that determine bond lifetime. We propose that amino acid substitutions to these hypervariable regions of the TCR can efficiently manipulate immunogenicity and thus be used in the rational design of TCRs for immunotherapy.

immunology↗

bioRxiv2021

Exploiting synergistic interactions of Medicago sativa L. and Paraburkholderia tropica for enhanced biodegradation of diesel fuel hydrocarbons

The biotechnological application of microorganisms for rhizoremediation of contaminated sites requires the development of plant-microbe symbionts capable of plant growth promotion and hydrocarbon degradation. Studies focusing on microbial consortia are often difficult to reproduce, thereby necessitating the need for culturable single bacterial species for biotechnological applications. Through genomic analyses and plant growth experiments, we examined the synergistic interactions of Medicago sativa L. and Paraburkholderia tropica for enhanced remediation of diesel fuel-contaminated soils. Comparative genomics revealed strong potential of P. tropica for plant growth-promotion, chemotaxis and motility, root nodulation and colonization, and diesel fuel degradation. Plant growth experiments confirmed that P. tropica thrived in the contaminated soils and effectively enhanced M. sativa growth. Geochemical analysis showed that the M. sativa + P. tropica treatment resulted in an efficient degradation of diesel fuel hydrocarbons within two months, offering great prospects for enhanced biodegradation of organic pollutants.

microbiology↗

bioRxiv2021

Metagenomic insight into the plant growth-promoting potential of a diesel-degrading bacterial consortium for enhanced rhizoremediation application

The slow rate of natural attenuation of organic pollutants, together with unwanted environmental impacts of traditional remediation strategies, has necessitated the exploration of plant-microbe systems for enhanced bioremediation applications. The identification of microorganisms capable of promoting both plant growth and hydrocarbon degradation is crucial to the success of plant-based remediation techniques. Through successive enrichments of a soil sample from a historic oil-contaminated site in Wietze, Germany, we isolated a plant growth-promoting and hydrocarbon-degrading bacterial consortium. Metagenome analysis of the consortium led to the identification of genes and taxa putatively associated with these processes. The majority of the coding DNA sequences involved in these reactions were affiliated to Acidocella aminolytica and Acidobacterium capsulatum. In microcosm experiments performed in association with Medicago sativa L., the consortium achieved 91% rhizodegradation of diesel fuel hydrocarbons within 60 days, indicating its potential for biotechnological applications in the remediation of sites contaminated by organic pollutants.

microbiology↗

bioRxiv2021

Diversity and metagenome analysis of a hydrocarbon-degrading bacterial consortium from asphalt lakes located in Wietze, Germany

The pollution of terrestrial and aquatic environments by petroleum contaminants, especially diesel fuel, is a persistent environmental threat requiring cost-effective and environmentally sensitive remediation approaches. Bioremediation is one such approach, but is dependent on the availability of microorganisms with the necessary metabolic abilities and environmental adaptability. The aim of this study was to examine the microbial community in a petroleum contaminated site, and isolate organisms potentially able to degrade hydrocarbons. Through successive enrichment of soil microorganisms from samples of an historic petroleum contaminated site in Wietze, Germany, we isolated a bacterial consortium using diesel fuel hydrocarbons as sole carbon and energy source. The 16S rRNA gene analysis revealed the dominance of Alphaproteobacteria. We further reconstructed a total of 18 genomes from both the original soil sample and the isolated consortium. The analysis of both the metagenome of the consortium and the reconstructed metagenome-assembled genomes show that the most abundant bacterial genus in the consortium, Acidocella, possess many of the genes required for the degradation of diesel fuel aromatic hydrocarbons, which are often the most toxic component. This can explain why this genus proliferated in all the enrichment cultures. Therefore, this study reveals that the microbial consortium isolated in this study and its dominant genus, Acidocella, could potentially serve as an effective inoculum for the bioremediation of sites polluted with diesel fuel or other organic contaminants.

microbiology↗

bioRxiv2020

Tumor-on-a-chip platform to interrogate the role of macrophages in tumor progression

Tumor-infiltrating leukocytes, in particular macrophages, play an important role in tumor behavior and clinical outcome. The spectrum of macrophage subtypes ranges from antitumor "M1"-type to protumor "M2"-type macrophages. Tumor-associated macrophages (TAMs) typically display phenotypic features of both M1 and M2, and the population distribution is thought to be dynamic and evolve as the tumor progresses. However, our understanding of how TAMs impact the tumor microenvironment remains limited by the lack of appropriate 3D in vitro models that can capture cell to cell dynamics at high spatial and temporal resolution. Using our recently developed micro-physiological "tumor-on-a-chip" (TOC) device, we present here our findings on the impact of defined macrophage subsets on tumor behavior. The TOC device design contains three adjacent and connected chambers in which both the upper and lower chambers are loaded with tumor cells while the central chamber contains a dynamic, perfused, living microvascular network. Introduction of human pancreatic or colorectal cancer cells together with M1-polorized macrophages significantly inhibited tumor growth and tumor-induced angiogenesis. Protein analysis and antibody-based neutralization studies confirmed that these effects were mediated through production of chemokines CXCL9, CXCL10, and CXCL11. By contrast, M2-macrophages mediated increased tumor cell migration into the vascularized chamber and did not inhibit tumor growth or angiogenesis. In fact, single-cell RNA-sequencing showed that M2 macrophages further segregated endothelial cells into two distinct subsets, corresponding to static cells in vessels versus active cells involved in angiogenesis. The impact of M2 macrophages was mediated mostly by production of MMP7 and ANGPT2. In summary, our data demonstrate the utility of the TOC device to mechanistically probe biological questions in a 3D in vitro microenvironment. Insight BoxMacrophages in the tumor microenvironment are key determinants of tumor behavior and clinical outcome. The macrophage subset composition and its functional impact change as tumors progress or during treatment, but adequate models to study this are lacking. We developed a tumor-on-a-chip model of perfused 3D tumor growth to probe the impact of defined macrophage subsets. Our data is consistent with previously described macrophage activity in the tumor microenvironment, and provides potential new molecular targets. Herein, we demonstrate feasibility of probing immuno-oncology questions in a 3D in vitro microenvironment and at a spatiotemporal resolution.

cancer biology↗

bioRxiv2020

Organ-on-a-chip model of vascularized human bone marrow niches

Animal models of bone marrow have limited spatial and temporal resolution to observe biological events (intravasation and cellular egress) and are inadequate to dissect dynamic events at the niche level (100 microns). Utilizing microfluidic and stem cell technology, we present a 3D in vitro model of human bone marrow that contains perivascular and endosteal niches complete with dynamic, perfusable vascular networks. We demonstrate that our model can perform in vivo functions including maintenance and differentiation of CD34+ hematopoietic stem/progenitor cells (HSPC) for up to fourteen days, egress of myeloid progenitors, and expression of markers consistent with in vivo human bone marrow. The platform design enables the addition of tissue niches at a later timepoint to probe mechanisms such as tumor cell migration. Overall, we present a novel organ-on-a-chip platform that is capable of recapitulating the human bone marrow microenvironment to observe hematopoietic phenomena at high spatial and temporal resolution.

bioengineering↗

bioRxiv2020

High-resolution transcriptional and morphogenetic profiling of cells from micropatterned human embryonic stem cell gastruloid cultures

During mammalian gastrulation, germ layers arise and are shaped into the body plan while extraembryonic layers sustain the embryo. Human embryonic stem cells, cultured with BMP4 on extracellular matrix micro-discs, reproducibly differentiate into gastruloids, expressing markers of germ layers and extraembryonic cells with radial organization. Single-cell RNA sequencing and cross-species comparisons with mouse and cynomolgus monkey gastrulae suggest that gastruloids contain seven major cell types, including epiblast, ectoderm, mesoderm, endoderm, and extraembryonic trophoblast- and amnion-like cells. Upon gastruloid dissociation, single cells reseeded onto micro-discs were motile and aggregated with the same but segregated from distinct cell types. Ectodermal cells segregated from endodermal and extraembryonic cells but mixed with mesodermal cells. Our work demonstrates that the gastruloid system supports primate-specific features of embryogenesis, and that gastruloid cells exhibit evolutionarily conserved sorting behaviors. This work generates a resource for transcriptomes of human extraembryonic and embryonic germ layers differentiated in a stereotyped arrangement.

developmental biology↗