Reconfigurable open microfluidics for studying the spatiotemporal dynamics of paracrine signalling

Yu, Jiaquan; Berthier, Jean; Craig, Alexandria; Groot, Theodorus E. de; Sparks, Sidney; Ingram, Patrick; Jarrard, David F.; Huang, Wei; Beebe, David J.; Theberge, Ashleigh B.

doi:10.1038/s41551-019-0421-4

Cited by 77 publications

(110 citation statements)

References 43 publications

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“…The Stacks system was combined with metabolic autofluorescence imaging to monitor tumor-mediated changes in macrophage metabolism and migration ( Fig. 1A) 27 . Microscale 3D co-cultures of tumor cells and macrophages were established in the Stacks 3D system using primary human cancer cells, and cell lines from mouse and human origin ( Fig 1A).…”

Section: Resultsmentioning

confidence: 99%

“…The Stacks system was previously characterized for diffusion gradients and cell viability, survival, and recruitment 27 . To determine non-specific changes in metabolism within the Stacks system, metabolic activity of macrophages 1 hour after seeding was assessed for RAW264.7 macrophage monocultures and RAW264.7+PyVMT mouse breast carcinoma co-cultures.…”

Section: Metabolic Imaging Validation: Macrophage Stimulation In 2d Imentioning

confidence: 99%

“…Combined tumor and immune cell culture within an extracellular matrix (ECM) further enables paracrine signaling between cell layers and protein-protein interactions necessary for cell motility 25,26 . These microscale platforms also provide a simple approach to recapitulate in vivo tumor-associated environmental conditions (e.g., hypoxia, nutrient availability) that regulate both macrophage phenotype and function [23][24][25]27 . These representative 3D tumor models provide attractive systems to evaluate macrophage heterogeneity in response to spatiotemporal changes in the microenvironment.…”

Section: Introductionmentioning

confidence: 99%

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Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Heaster

Humayun

et al. 2020

Preprint

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Macrophages within the tumor microenvironment (TME) exhibit a spectrum of pro-tumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate twophoton autofluorescence imaging of NAD(P)H and FAD to non-destructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours post-stimulation for mouse macrophages (RAW 264.7) stimulated with IFN-γ or IL-4 plus IL-13 in 2D culture, validating that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse PyVMT or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor co-cultures exhibited significantly different FAD mean lifetimes and greater migration than mono-cultures at 24, 48, and 72 hours post-seeding. In co-cultures with primary human cancer cells, actively-migrating monocyte-derived macrophages had greater redox ratios (NAD(P)H/FAD intensity) compared to passively-migrating monocytes at 24 and 48 hours post-seeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumorimmune crosstalk within the 3D TME.

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Section: Resultsmentioning

confidence: 99%

Section: Metabolic Imaging Validation: Macrophage Stimulation In 2d Imentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Heaster

Humayun

et al. 2020

Preprint

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“…Building upon the foundation created by previous in vitro models, we present the creation of a novel microscale in vitro granuloma model that can be adapted to study the soluble factor signaling between granulomas and their surrounding microenvironment immediately following infection. Using a recently developed modular microfluidic coculture platform, known as “Stacks”(Yu et al 2019), we demonstrate a multi-layered coculture that can be spatially and temporally manipulated to mimic different microenvironments and timepoints. The Stacks platform utilizes suspended cultures, wherein a droplet is contained in a well consisting of walls but lacking a ceiling or floor (Casavant et al, n.d.; Humayun, Chow, and Young 2018; Berthier et al 2019), thereby enabling users to vertically stack layers containing different cell types and place them in signaling contact.…”

Section: Introductionmentioning

confidence: 99%

“…The Stacks platform utilizes suspended cultures, wherein a droplet is contained in a well consisting of walls but lacking a ceiling or floor (Casavant et al, n.d.; Humayun, Chow, and Young 2018; Berthier et al 2019), thereby enabling users to vertically stack layers containing different cell types and place them in signaling contact. (Yu et al 2019) The modular component of the Stacks, as well as of other microfluidic platforms, offers a notable advantage as users can optimize model conditions individually and connect each component to create different complex systems. (Yu et al 2019; Ong et al 2019) As a proof of concept, we use a model mycobacterial strain known to induce granuloma formation in vitro(Puissegur et al 2004; Seitzer and Gerdes 2003), Mycobacterium bovis Bacillus Calmette-Guerin (BCG), with human blood-derived immune cells and validate its ability to form an in vitro granuloma model on the microscale (4 μL culture volume) in a layer of the Stacks platform.…”

Section: Introductionmentioning

confidence: 99%

A modular microscale granuloma model for immune-microenvironment signaling studiesin vitro

Berry

Gower

et al. 2020

Preprint

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Tuberculosis (TB) is one of the most potent infectious diseases in the world, causing more deaths than any other single infectious agent. TB infection is caused by inhalation of Mycobacterium tuberculosis (Mtb) and subsequent phagocytosis and migration into the lung tissue by innate immune cells (e.g., alveolar macrophages, neutrophils, dendritic cells), resulting in the formation of a fused mass of immune cells known as the granuloma. Considered the pathological hallmark of TB, the granuloma is a complex microenvironment that is crucial for pathogen containment as well as pathogen survival. Disruption of the delicate granuloma microenvironment via numerous stimuli, such as variations in cytokine secretions, nutrient availability, and the makeup of immune cell population, can lead to an active infection. Herein, we present a novel in vitro model to examine the soluble factor signaling between a mycobacterial infection and its surrounding environment. Adapting a newly developed suspended microfluidic platform, known as Stacks, we established a modular microscale infection model containing human immune cells and a model mycobacterial strain that can easily integrate with different microenvironmental cues through simple spatial and temporal “stacking” of each module of the platform. We validate the establishment of suspended microscale (4 μL) infection cultures that secrete increased levels of proinflammatory factors IL-6, VEGF, and TNFα upon infection and form 3D aggregates (granuloma model) encapsulating the mycobacteria. As a proof of concept to demonstrate the capability of our platform to examine soluble factor signaling, we cocultured an in vitro angiogenesis model with the granuloma model and quantified morphology changes in endothelial structures as a result of culture conditions (P < 0.05 when comparing infected vs. uninfected coculture systems). We envision our modular in vitro granuloma model can be further expanded and adapted for studies focusing on the complex interplay between granulomatous structures and their surrounding microenvironment, as well as a complementary tool to augment in vivo signaling and mechanistic studies.

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Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media

Plou

Garcı́a

Charconnet

et al. 2020

Adv Funct Materials

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The composition and intercellular interactions of tumor cells in the tissues dictate the biochemical and metabolic properties of the tumor microenvironment. The metabolic rewiring has a profound impact on the properties of the microenvironment, to an extent that monitoring such perturbations could harbor diagnostic and therapeutic relevance. A growing interest in these phenomena has inspired the development of novel technologies with sufficient sensitivity and resolution to monitor metabolic alterations in the tumor microenvironment. In this context, surface-enhanced Raman scattering (SERS) can be used for the label-free detection and imaging of diverse molecules of interest among extracellular components. Herein, the application of nanostructured plasmonic substrates comprising Au nanoparticles, self-assembled as ordered superlattices, to the precise SERS detection of selected tumor metabolites, is presented. The potential of this technology is first demonstrated through the analysis of kynurenine, a secreted immunomodulatory derivative of the tumor metabolism and the related molecules tryptophan and purine derivatives. SERS facilitates the unambiguous identification of trace metabolites and allows the multiplex detection of their characteristic fingerprints under different conditions. Finally, the effective plasmonic SERS substrate is combined with a hydrogel-based three-dimensional cancer model, which recreates the tumor microenvironment, for the real-time imaging of metabolite alterations and cytotoxic effects on tumor cells.

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Reconfigurable open microfluidics for studying the spatiotemporal dynamics of paracrine signalling

Cited by 77 publications

References 43 publications

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

A modular microscale granuloma model for immune-microenvironment signaling studiesin vitro

Multiplex SERS Detection of Metabolic Alterations in Tumor Extracellular Media

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