Biology subjects

Shirure, V. S.

Publications and source records attributed to Shirure, V. S..

7 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↗

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↗

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↗

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↗

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↗