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The aryl hydrocarbon receptor (AhR) is a sensor of products of tryptophan metabolism and a potent modulator of immunity. Here, we examined the impact of AhR in tumor-associated macrophage (TAM) function in pancreatic ductal adenocarcinoma (PDAC). TAMs exhibited high AhR activity and Ahr-deficient macrophages developed an inflammatory phenotype. Deletion of Ahr in myeloid cells or pharmacologic inhibition of AhR reduced PDAC growth, improved efficacy of immune checkpoint blockade, and increased intra-tumoral frequencies of IFNγ+CD8+ T cells. Macrophage tryptophan metabolism was not required for this effect. Rather, macrophage AhR activity was dependent on Lactobacillus metabolization of dietary tryptophan to indoles. Removal of dietary tryptophan reduced TAM AhR activity and promoted intra-tumoral accumulation of TNFα+IFNγ+CD8+ T cells; provision of dietary indoles blocked this effect. In patients with PDAC, high AHR expression associated with rapid disease progression and mortality, as well as with an immune-suppressive TAM phenotype, suggesting conservation of this regulatory axis in human disease.
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Tolerância Imunológica/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Triptofano/imunologia , Macrófagos Associados a Tumor/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Humanos , Indóis/imunologia , Indóis/metabolismo , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Microbiota/imunologia , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Triptofano/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/metabolismoRESUMO
Mass cytometry permits the high dimensional analysis of complex biological samples; however, some techniques are not yet integrated into the mass cytometry workflow due to reagent availability. The use of self-labeling protein systems, such as HaloTag, are one such application. Here, we describe the design and implementation of the first mass cytometry ligands for use with HaloTag. "Click"-amenable HaloTag warheads were first conjugated onto poly(l-lysine) or poly(acrylic acid) polymers that were then functionalized with diethylenetriaminepentaacetic acid (DTPA) lutetium metal chelates. Kinetic analysis of the HaloTag labeling rates demonstrated that the structure appended to the 1-chlorohexyl warhead was key to success. A construct with a diethylene glycol spacer appended to a benzamide gave similar rates (kobs â¼ 102 M-1 s-1), regardless of the nature of the polymer. Comparison of the polymer with a small molecule chelate having rapid HaloTag labeling kinetics (kobs â¼ 104 M-1 s-1) suggests the polymers significantly reduced the HaloTag labeling rate. HEK293T cells expressing surface-exposed GFP-HaloTag fusions were labeled with the polymeric constructs and 175Lu content measured by cytometry by time-of-flight (CyTOF). Robust labeling was observed; however, significant nonspecific binding of the constructs to cells was also present. Heavily pegylated polymers demonstrated that nonspecific binding could be reduced to allow cells bearing the HaloTag protein to be distinguished from nonexpressing cells.
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Hidrolases , Polímeros , Proteínas , Humanos , Ligantes , Cinética , Células HEK293RESUMO
Aldehydes are attractive bioorthogonal coupling partners. The ease of manipulation of aldehydes and their orthogonality to other classes of bioorthogonal reactions have inspired the exploration of chemistries, which generate irreversible conjugates. Similarly, nitrones have been shown to be potent 1,3-dipoles in bioorthogonal reactions when paired with strained alkynes. Here, we combine the reactivity of nitrones with the simplicity of aldehydes using an N-allylglyoxylamide, in a cascade reaction with an N-alkylhydroxylamine to produce a bicyclic isoxazolidine. The reaction is found to be catalyzed by 5-methoxyanthranilic acid and proceeds at pH 7 with favorable kinetics. Using the HaloTag7 protein bearing an N-alkylhydroxylamine, we show the reaction to be bioorthogonal in a complex cell lysate and to proceed well at the surface of a HEK293 cell. Furthermore, the reaction is compatible with a typical strain-promoted alkyne-azide click reaction. The characteristics of this reaction suggest it will be a useful addition to the pallet of bioorthogonal reactions that have revolutionized chemical biology.
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Óxidos de Nitrogênio , Proteínas , Humanos , Células HEK293 , Proteínas/química , Óxidos de Nitrogênio/química , Alcinos/química , Aldeídos , Azidas/química , Reação de CicloadiçãoRESUMO
Interactions between tumor cells and the extracellular matrix (ECM) are an important factor contributing to therapy failure in cancer patients. Current in vitro breast cancer spheroid models examining the role of mechanical properties on spheroid response to chemotherapy are limited by the use of two-dimensional cell culture, as well as simultaneous variation in hydrogel matrix stiffness and other properties, e.g., hydrogel composition, pore size, and cell adhesion ligand density. In addition, currently used hydrogel matrices do not replicate the filamentous ECM architecture in a breast tumor microenvironment. Here, we report a collagen-alginate hydrogel with a filamentous architecture and a 20-fold variation in stiffness, achieved independently of other properties, used for the evaluation of estrogen receptor-positive breast cancer spheroid response to doxorubicin. The variation in hydrogel mechanical properties was achieved by altering the degree of cross-linking of alginate molecules. We show that soft hydrogels promote the growth of larger MCF-7 tumor spheroids with a lower fraction of proliferating cells and enhance spheroid resistance to doxorubicin. Notably, the stiffness-dependent chemotherapeutic response of the spheroids was temporally mediated: it became apparent at sufficiently long cell culture times, when the matrix stiffness has influenced the spheroid growth. These findings highlight the significance of decoupling matrix stiffness from other characteristics in studies of chemotherapeutic resistance of tumor spheroids and in development of drug screening platforms.
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Neoplasias da Mama , Esferoides Celulares , Neoplasias da Mama/tratamento farmacológico , Técnicas de Cultura de Células , Matriz Extracelular , Feminino , Humanos , Hidrogéis , Microambiente TumoralRESUMO
The tumour microenvironment (TME) determines vital aspects of tumour development, such as tumour growth, metastases and response to therapy. Cancer-associated fibroblasts (CAFs) are abundant and extremely influential in this process and interact with cellular and matrix TME constituents such as endothelial and immune cells and collagens, fibronectin and elastin, respectively. However, CAFs are also the recipients of signals-both chemical and physical-that are generated by the TME, and their phenotype effectively evolves alongside the tumour mass during tumour progression. Amid a rising clinical interest in CAFs as a crucial force for disease progression, this review aims to contextualise the CAF phenotype using the chronological framework of the CAF life cycle within the evolving tumour stroma, ranging from quiescent fibroblasts to highly proliferative and secretory CAFs. The emergence, properties and clinical implications of CAF activation are discussed, as well as research strategies used to characterise CAFs and current clinical efforts to alter CAF function as a therapeutic strategy.
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Fibroblastos Associados a Câncer/patologia , Neoplasias/fisiopatologia , Progressão da Doença , Humanos , Microambiente Tumoral/genéticaRESUMO
There is a clinical need for new, more effective treatments for chronic wounds in diabetic patients. Lack of epithelial cell migration is a hallmark of nonhealing wounds, and diabetes often involves endothelial dysfunction. Therefore, targeting re-epithelialization, which mainly involves keratinocytes, may improve therapeutic outcomes of current treatments. In this study, we present an integrin-binding prosurvival peptide derived from angiopoietin-1, QHREDGS (glutamine-histidine-arginine-glutamic acid-aspartic acid-glycine-serine), as a therapeutic candidate for diabetic wound treatments by demonstrating its efficacy in promoting the attachment, survival, and collective migration of human primary keratinocytes and the activation of protein kinase B Akt and MAPKp42/44 The QHREDGS peptide, both as a soluble supplement and when immobilized in a substrate, protected keratinocytes against hydrogen peroxide stress in a dose-dependent manner. Collective migration of both normal and diabetic human keratinocytes was promoted on chitosan-collagen films with the immobilized QHREDGS peptide. The clinical relevance was demonstrated further by assessing the chitosan-collagen hydrogel with immobilized QHREDGS in full-thickness excisional wounds in a db/db diabetic mouse model; QHREDGS showed significantly accelerated and enhanced wound closure compared with a clinically approved collagen wound dressing, peptide-free hydrogel, or blank wound controls. The accelerated wound closure resulted primarily from faster re-epithelialization and increased formation of granulation tissue. There were no observable differences in blood vessel density or size within the wound; however, the total number of blood vessels was greater in the peptide-hydrogel-treated wounds. Together, these findings indicate that QHREDGS is a promising candidate for wound-healing interventions that enhance re-epithelialization and the formation of granulation tissue.
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Diabetes Mellitus Experimental/patologia , Hidrogéis/farmacologia , Peptídeos/farmacologia , Reepitelização , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/farmacologia , Colágeno/farmacologia , Humanos , Peróxido de Hidrogênio/toxicidade , Proteínas Imobilizadas/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Peptídeos/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reepitelização/efeitos dos fármacosRESUMO
The profound metabolic reprogramming that occurs in cancer cells has been investigated primarily in two-dimensional cell cultures, which fail to recapitulate spatial aspects of cell-to-cell interactions as well as tissue gradients present in three-dimensional tumours. Here, we describe an engineered model to assemble three-dimensional tumours by rolling a scaffold-tumour composite strip. By unrolling the strip, the model can be rapidly disassembled for snapshot analysis, allowing spatial mapping of cell metabolism in concert with cell phenotype. We also show that the establishment of oxygen gradients within samples that are shaped by oxygen-dependent signalling pathways, as well as the consequential variations in cell growth, response to hypoxic gradients extending from normoxia to severe hypoxia, and therapy responsiveness, are consistent with those of tumours in vivo. Moreover, by using liquid chromatography tandem mass spectrometry, we mapped cellular metabolism and identified spatially defined metabolic signatures of cancer cells to reveal both known and novel metabolic responses to hypoxia.
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Neoplasias/metabolismo , Oxigênio/metabolismo , Engenharia Tecidual , Alicerces Teciduais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Transdução de SinaisRESUMO
Recapitulating the architecture of native tissue remains a significant challenge, impeding the progress of engineering tissues. Imposing appropriate organization is especially challenging in tissues that contain multiple cellular components in complex structural units. One solution is to mimic developmental processes in embryos. In an embryo, cells are organized by tissue patterning, whereby induction of fate-determining genes is spatially controlled to generate patterns of cell differentiation and maturation. Following patterning, the imposed cell organization is further reinforced by implementation of compartment boundaries, which prevent intermingling of cells from distinct phenotypic domains, thereby ensuring preservation of proper cell organization in growing and reorganizing tissues. Both morphogenic processes utilize a conserved set of fundamental principles, the implementation of which leads to highly regulated cell organization. In this article, we review these patterning principles in vivo and reflect on the progress made by tissue engineers in mimicking tissue patterning ex vivo.
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Biomimética/métodos , Padronização Corporal/fisiologia , Desenvolvimento Embrionário/fisiologia , Técnicas de Cultura de Órgãos/métodos , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Técnicas de Cultura Celular por Lotes/instrumentação , Técnicas de Cultura Celular por Lotes/métodos , Biomimética/instrumentação , Humanos , Técnicas de Cultura de Órgãos/instrumentação , Engenharia Tecidual/instrumentaçãoRESUMO
Directed migration of groups of cells is a critical aspect of tissue morphogenesis that ensures proper tissue organization and, consequently, function. Cells moving in groups, unlike single cells, must coordinate their migratory behavior to maintain tissue integrity. During directed migration, cells are guided by a combination of mechanical and chemical cues presented by neighboring cells and the surrounding extracellular matrix. One important class of signals that guide cell migration includes topographic cues. Although the contact guidance response of individual cells to topographic cues has been extensively characterized, little is known about the response of groups of cells to topographic cues, the impact of such cues on cell-cell coordination within groups, and the transmission of nonautonomous contact guidance information between neighboring cells. Here, we explore these phenomena by quantifying the migratory response of confluent monolayers of epithelial and fibroblast cells to contact guidance cues provided by grooved topography. We show that, in both sparse clusters and confluent sheets, individual cells are contact-guided by grooves and show more coordinated behavior on grooved versus flat substrates. Furthermore, we demonstrate both in vitro and in silico that the guidance signal provided by a groove can propagate between neighboring cells in a confluent monolayer, and that the distance over which signal propagation occurs is not significantly influenced by the strength of cell-cell junctions but is an emergent property, similar to cellular streaming, triggered by mechanical exclusion interactions within the collective system.
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Comunicação Celular , Movimento Celular , Células Epiteliais/citologia , Fibroblastos/citologia , Transdução de Sinais , Contagem de Células , Linhagem Celular , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Junções Intercelulares/metabolismo , Masculino , Modelos BiológicosRESUMO
Grooved substrates are commonly used to guide cell alignment and produce in vitro tissues that mimic certain aspects of in vivo cellular organization. These more sophisticated tissues provide valuable in vitro models for testing drugs and for dissecting out molecular mechanisms that direct tissue organization. To increase the accessibility of these tissue models we describe a simple and yet reproducible strategy to produce 1 µm-spaced grooved well plates suitable for conducting automated analysis of cellular responses. We characterize the alignment of four human cell types: retinal epithelial cells, umbilical vein endothelial cells, foreskin fibroblasts, and human pluripotent stem-cell-derived cardiac cells on grooves. We find all cells align along the grooves to differing extents at both sparse and confluent densities. To increase the sophistication of in vitro tissue organization possible, we also created hybrid substrates with controlled patterns of microgrooved and flat regions that can be identified in real-time using optical microscopy. Using our hybrid patterned surfaces we explore: (i) the ability of neighboring cells to provide a template to organize surrounding cells that are not directly exposed to grooved topographic cues, and (ii) the distance over which this template effect can operate in confluent cell sheets. We find that in fibroblast sheets, but not epithelial sheets, cells aligned on grooves can direct alignment of neighboring cells in flat regions over a limited distance of approximately 200 µm. Our hybrid surface plate provides a novel tool for studying the collective response of groups of cells exposed to differential topographical cues.
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Comunicação Celular/fisiologia , Ensaios de Triagem em Larga Escala/instrumentação , Engenharia Tecidual/instrumentação , Linhagem Celular , Células Cultivadas , Desenho de Equipamento , Humanos , Propriedades de SuperfícieRESUMO
Rapid advances in tissue engineering have resulted in more complex and physiologically relevant 3D in vitro tissue models with applications in fundamental biology and therapeutic development. However, the complexity provided by these models is often not leveraged fully due to the reductionist methods used to analyze them. Computational and mathematical models developed in the field of systems biology can address this issue. Yet, traditional systems biology has been mostly applied to simpler in vitro models with little physiological relevance and limited cellular complexity. Therefore, integrating these two inherently interdisciplinary fields can result in new insights and move both disciplines forward. In this review, we provide a systematic overview of how systems biology has been integrated with 3D in vitro tissue models and discuss key application areas where the synergies between both fields have led to important advances with potential translational impact. We then outline key directions for future research and discuss a framework for further integration between fields.
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Understanding the highly complex tumor-immune landscape is an important goal for developing novel immune therapies for solid cancers. To this end, 3D cancer-immune models have emerged as patient-relevant in vitro tools for modeling the tumor-immune landscape and the cellular interactions within it. In this review, we provide an overview of the components and applications of 3D cancer-immune models and discuss their evolution from 2015 to 2023. Specifically, we observe trends in primary cell-sourced, T cell-based complex models used for therapy evaluation and biological discovery. Finally, we describe the challenges of implementing 3D cancer-immune models and the opportunities for maximizing their potential for deciphering the complex tumor-immune microenvironment and identifying novel, clinically relevant drug targets.
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Background: Functional evaluation of molecules that are predicted to promote stem cell mediated endogenous repair often requires in vivo transplant studies that are low throughput and hinder the rate of discovery. To offer greater throughput for functional validation studies, we miniaturized, simplified and expanded the functionality of a previously developed muscle endogenous repair (MEndR) in vitro assay that was shown to capture significant events of in vivo muscle endogenous repair. Methods: The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with human myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), enriched from digested mouse skeletal muscle tissue using an improved magnetic-activated cell sorting protocol, are engrafted within the engineered template. Murine MuSCs are fluorescently labeled, enabling co-evaluation of human and mouse Pax7+ cell responses to drug treatments. A regenerative milieu is introduced by injuring the muscle tissue with a myotoxin to initiate endogenous repair "in a dish". Phenotypic data is collected at endpoints with a high-content imaging system and is analyzed using ImageJ-based image analysis pipelines. Results: The miniaturized format and modified manufacturing protocol cuts reagent costs in half and hands-on seeding time ~ threefold, while the image analysis pipelines save 40 h of labour. By evaluating multiple commercially available human primary myoblast lines in 2D and 3D culture, we establish quality assurance metrics for cell line selection that standardizes myotube template quality. In vivo outcomes (enhanced muscle production and Pax7+ cell expansion) to a known modulator of MuSC mediated repair (p38/ß MAPK inhibition) are recapitulated in the miniaturized culture assay, but only in the presence of stem cells and the regenerative milieu. Discussion: The miniaturized predictive assay offers a simple, scaled platform to co-investigate human and mouse skeletal muscle endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline. Supplementary Information: The online version contains supplementary material available at 10.1186/s44330-024-00005-4.
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Modeling the heterogeneity of the tumor microenvironment (TME) in vitro is essential to investigating fundamental cancer biology and developing novel treatment strategies that holistically address the factors affecting tumor progression and therapeutic response. Thus, the development of new tools for both in vitro modeling, such as patient-derived organoids (PDOs) and complex 3D in vitro models, and single cell omics analysis, such as single-cell RNA-sequencing, represents a new frontier for investigating tumor heterogeneity. Specifically, the integration of PDO-based 3D in vitro models and single cell analysis offers a unique opportunity to explore the intersecting effects of interpatient, microenvironmental, and tumor cell heterogeneity on cell phenotypes in the TME. In this review, the current use of PDOs in complex 3D in vitro models of the TME is discussed and the emerging directions in the development of these models are highlighted. Next, work that has successfully applied single cell analysis to PDO-based models is examined and important experimental considerations are identified for this approach. Finally, open questions are highlighted that may be amenable to exploration using the integration of PDO-based models and single cell analysis. Ultimately, such investigations may facilitate the identification of novel therapeutic targets for cancer that address the significant influence of tumor-TME interactions.
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Neoplasias , Humanos , Biologia , Organoides , Fenótipo , Análise de Célula Única , Microambiente TumoralRESUMO
Patient-derived organoids have emerged as a useful tool to model tumour heterogeneity. Scaling these complex culture models while enabling stratified analysis of different cellular sub-populations, however, remains a challenge. One strategy to enable higher throughput organoid cultures is the scaffold-supported platform for organoid-based tissues (SPOT). SPOT allows the generation of flat, thin, and dimensionally-defined microtissues in both 96- and 384-well plate footprints that are compatible with longitudinal image-based readouts. SPOT is currently manufactured manually, however, limiting scalability. In this study, an automation approach to engineer tumour-mimetic 3D microtissues in SPOT using a liquid handler is optimized and comparable within- and between-sample variation to standard manual manufacturing is shown. Further, a liquid handler-supported cell extraction protocol to support single-cell-based end-point analysis using high-throughput flow cytometry and multiplexed cytometry by time of flight is developed. As a proof-of-value demonstration, 3D complex tissues containing different proportions of tumour and stromal cells are generated to probe the reciprocal impact of co-culture. It is also demonstrated that primary patient-derived organoids can be incorporated into the pipeline to capture patient-level tumour heterogeneity. It is envisioned that this automated 96/384-SPOT workflow will provide opportunities for future applications in high-throughput screening for novel personalized therapeutic targets.
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Neoplasias , Humanos , Fluxo de Trabalho , Técnicas de Cocultura , Neoplasias/patologia , Ensaios de Triagem em Larga Escala/métodos , Automação , OrganoidesRESUMO
The spatial configuration of cells in the tumor microenvironment (TME) affects both cancer and fibroblast cell phenotypes contributing to the clinical challenge of tumor heterogeneity and therapeutic resistance. This is a particular challenge in stroma-rich pancreatic ductal adenocarcinoma (PDAC). Here, a versatile system is described to study the impact of tissue architecture on cell phenotype using PDAC as a model system. This fully human system encompassing both primary pancreatic stellate cells and primary organoid cells using the TRACER platform to allow the creation of user-defined TME architectures that have been inferred from clinical PDAC samples and are analyzed by CyTOF to characterize cells extracted from the system. High dimensional characterization using CyTOF demonstrates that tissue architecture leads to distinct hypoxia and proliferation gradients. Furthermore, phenotypic markers for both cell types are also graded in ways that cannot be explained by either hypoxia or coculture alone. This demonstrates the importance of using complex models encompassing cancer cells, stromal cells, and allowing control over architecture to explore the impact of tissue architecture on cell phenotype. It is anticipated that this model will help decipher how tissue architecture and cell interactions regulate cell phenotype and hence cellular and tissue heterogeneity.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Técnicas de Cocultura , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/patologia , Fenótipo , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Protein purification is a ubiquitous procedure in biochemistry and the life sciences, and represents a key step in the protein production pipeline. The need for scalable and parallel protein purification systems is driven by the demands for increasing the throughput of recombinant protein characterization. Therefore, automating the process to simultaneously handle multiple samples with minimal human intervention is highly desirable, yet there are only a handful of such systems that have been developed, all of which are closed source and expensive. To address this challenge, we present REVOLVER, a 3D-printed programmable protein purification system based on gravity-column workflows and controlled by Arduino boards that can be built for under $130 USD. REVOLVER takes a cell lysate sample and completes a full protein purification process with almost no human intervention and yields results indistinguishable from those obtained by an experienced biochemist when purifying a real-world protein sample. We further present and describe MULTI-VOLVER, a scalable version of the REVOLVER that allows for parallel purification of up to six samples and can be built for under $250 USD. Both systems can help accelerate protein purification and ultimately link them to bio-foundries for protein characterization and engineering.
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Topographical cues are known to influence cell organization both in native tissues and in vitro. In the trachea, the matrix beneath the epithelial lining is composed of collagen fibres that run along the long axis of the airway. Previous studies have shown that grooved topography can induce morphological and cytoskeletal alignment in epithelial cell lines. In the present work we assessed the impact of substrate topography on the organization of primary human tracheal epithelial cells (HTECs) and human induced pluripotent stem cell (hiPSC)-derived airway progenitors and the resulting alignment of cilia after maturation of the airway cells under Air-Liquid-Interface (ALI) culture. Grooves with optimized dimensions were imprinted into collagen vitrigel membranes (CVM) to produce gel inserts for ALI culture. Grooved CVM substrates induced cell alignment in HTECs and hiPSC airway progenitors in submerged culture. Further, both cell types were able to terminally differentiate into a multi-ciliated epithelium on both flat and groove CVM substrates. When exposed to ALI conditions, HTECs lost alignment after 14 days. Meanwhile, hiPSC-derived airway progenitors maintained their alignment throughout 31 days of ALI culture. Interestingly, neither initial alignment on the grooves, nor maintained alignment on the grooves induced alignment of cilia basal bodies, an indication of the direction of ciliary beating direction in the airway cells. Planar organization of airway cells during or prior to ciliogenesis therefore does not appear to be a feasible strategy to control cilia organization and subsequent airway epithelial function and additional cues are likely necessary to produce cilia alignment.
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Hidrogéis , Células-Tronco Pluripotentes Induzidas , Células Cultivadas , Cílios , Células Epiteliais , Epitélio , HumanosRESUMO
Tumors contain heterogeneous and dynamic populations of cells that do not all display the fast-proliferating properties that traditional chemotherapies target. There is a need therefore, to develop novel treatment strategies that target diverse tumor cell properties. Identifying therapy combinations is challenging however. Current approaches have relied on cell lines cultured in monolayers with treatment response being assessed using endpoint metabolic assays, which although enable large-scale throughput, do not capture tumor heterogeneity. Here, a 3D in vitro tumor model using micro-molded hydrogels (microgels), the Gels for Live Analysis of Compartmentalized Environments (GLAnCE) platform, is adapted into a 96-well plate format (96-GLAnCE) that integrates patient-derived organoids (PDOs) and is combined with longitudinal automated imaging to address these limitations. Using 96-GLAnCE, two measures of tumor aggressiveness are quantified, tumor cell growth and in situ regrowth after drug treatment, in both cell lines and PDOs. The use of longitudinal image-based readouts enables the identification of tumor cell phenotypes with cell population and subpopulation resolution that cannot be detected by standard bulk-soluble assays. 96-GLAnCE is a versatile and robust platform that combines 3D-ECM based models, PDOs, and real-time assay readouts, to provide an additional tool for pre-clinical anti-cancer drug discovery for the identification of novel targets with translatable clinical significance.