RESUMEN
Neutrophil infiltration into tissues is essential for host defense and pathogen clearance. Although many of the signaling pathways involved in the transendothelial migration of neutrophils are known, the role of the endothelium in regulating neutrophil behavior in response to infection within interstitial tissues remains unclear. Here we developed a microscale 3-dimensional (3D) model that incorporates an endothelial lumen, a 3D extracellular matrix, and an intact bacterial source to model the host microenvironment. Using this system, we show that an endothelial lumen significantly increased neutrophil migration toward a source of Pseudomonas aeruginosa Surprisingly, we found neutrophils, which were thought to be short-lived cells in vitro, migrate for up to 24 hours in 3D in the presence of an endothelial lumen and bacteria. In addition, we found that endothelial cells secrete inflammatory mediators induced by the presence of P aeruginosa, including granulocyte-macrophage colony-stimulating factor (GM-CSF), a known promoter of neutrophil survival, and interleukin (IL)-6, a proinflammatory cytokine. We found that pretreatment of neutrophils with a blocking antibody against the IL-6 receptor significantly reduced neutrophil migration to P aeruginosa but did not alter neutrophil lifetime, indicating that secreted IL-6 is an important signal between endothelial cells and neutrophils that mediates migration. Taken together, these findings demonstrate an important role for endothelial paracrine signaling in neutrophil migration and survival.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Interleucina-6/biosíntesis , Neutrófilos/metabolismo , Células Endoteliales/inmunología , Endotelio Vascular/inmunología , Humanos , Interleucina-6/inmunología , Neutrófilos/inmunología , Comunicación Paracrina/fisiología , Pseudomonas aeruginosa , Migración Transendotelial y Transepitelial/fisiologíaRESUMEN
Whether human cancer follows a hierarchical or stochastic model of differentiation is controversial. Furthermore, the factors that regulate cancer stem-like cell (CSC) differentiation potential are largely unknown. We used a novel microfluidic single-cell culture method to directly observe the differentiation capacity of four heterogeneous ovarian cancer cell populations defined by the expression of the CSC markers aldehyde dehydrogenase (ALDH) and CD133. We evaluated 3,692 progeny from 2,833 cells. We found that only ALDH(+)CD133(+) cells could generate all four ALDH(+/-)CD133(+/-) cell populations and identified a clear branched differentiation hierarchy. We also observed a single putative stochastic event. Within the hierarchy of cells, bone morphologenetic protein 2 (BMP2) is preferentially expressed in ALDH(-)CD133(-) cells. BMP2 promotes ALDH(+)CD133(+) cell expansion while suppressing the proliferation of ALDH(-)CD133(-) cells. As such, BMP2 suppressed bulk cancer cell growth in vitro but increased tumor initiation rates, tumor growth, and chemotherapy resistance in vivo whereas BMP2 knockdown reduced CSC numbers, in vivo growth, and chemoresistance. These data suggest a hierarchical differentiation pattern in which BMP2 acts as a feedback mechanism promoting ovarian CSC expansion and suppressing progenitor proliferation. These results explain why BMP2 suppresses growth in vitro and promotes growth in vivo. Together, our results support BMP2 as a therapeutic target in ovarian cancer.
Asunto(s)
Proteína Morfogenética Ósea 2/fisiología , Neoplasias Ováricas/patología , Antígeno AC133 , Aldehído Deshidrogenasa/metabolismo , Antígenos CD/metabolismo , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/metabolismo , Proteína Morfogenética Ósea 2/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Técnicas de Silenciamiento del Gen , Glicoproteínas/metabolismo , Humanos , Microfluídica , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Péptidos/metabolismoRESUMEN
Innate immune cell infiltration into neoplastic tissue is the first line of defense against cancer and can play a deterministic role in tumor progression. Here, we describe a series of assays, using a reconfigurable microscale assay platform (i.e. Stacks), which allows the study of immune cell infiltration in vitro with spatiotemporal manipulations. We assembled Stacks assays to investigate tumor-monocyte interactions, re-education of activated macrophages, and neutrophil infiltration. For the first time in vitro, the Stacks infiltration assays reveal that primary tumor-associated fibroblasts from specific patients differ from that associated with the benign region of the prostate in their ability to limit neutrophil infiltration as well as facilitate monocyte adhesion and anti-inflammatory monocyte polarization. These results show that fibroblasts play a regulatory role in immune cell infiltration and that Stacks has the potential to predict individual patients' cancer-immune response.
Asunto(s)
Fibroblastos Asociados al Cáncer , Neoplasias , Línea Celular Tumoral , Humanos , Macrófagos , Masculino , Monocitos , Microambiente TumoralRESUMEN
The study of intercellular signalling networks requires organotypic microscale systems that facilitate the culture, conditioning and manipulation of cells. Here, we describe a reconfigurable microfluidic cell-culture system that facilitates the assembly of three-dimensional tissue models by stacking layers that contain preconditioned microenvironments. By using principles of open and suspended microfluidics, the Stacks system is easily assembled or disassembled to provide spatial and temporal manoeuvrability in two-dimensional and three-dimensional assays of multiple cell types, enabling the modelling of sequential paracrine-signalling events, such as tumour-cell-mediated differentiation of macrophages and macrophage-facilitated angiogenesis. We used Stacks to recapitulate the in vivo observation that different prostate cancer tissues polarize macrophages with distinct gene-expression profiles of pro-inflammatory and anti-inflammatory cytokines. Stacks also enabled us to show that these two types of macrophages signal distinctly to endothelial cells, leading to blood vessels with different morphologies. Our proof-of-concept experiments exemplify how Stacks can efficiently model multicellular interactions and highlight the importance of spatiotemporal specificity in intercellular signalling.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Transducción de Señal , Análisis Espacio-Temporal , Citocinas/metabolismo , Células Endoteliales , Humanos , Macrófagos , Técnicas Analíticas Microfluídicas/instrumentación , Microfluídica/instrumentación , Transcriptoma , Microambiente TumoralRESUMEN
While organotypic approaches promise increased relevance through the inclusion of increased complexity (e.g., 3D extracellular microenvironment, structure/function relationships, presence of multiple cell types), cell source is often overlooked. Induced pluripotent stem cell (iPSC)-derived cells are potentially more physiologically relevant than cell lines, while also being less variable than primary cells, and recent advances have made them commercially available at costs similar to cell lines. Here, the use of induced pluripotent stem cell-derived endothelium for the generation of a functional microvessel model is demonstrated. High precision structural and microenvironmental control afforded by the design approach synergizes with the advantages of iPSC to produce microvessels for modeling endothelial biology in vitro. iPSC microvessels show endothelial characteristics, exhibit barrier function, secrete angiogenic and inflammatory mediators, and respond to changes in the extracellular microenvironment by altering vessel phenotype. Importantly, when deployed in the investigation of neutrophils during innate immune recruitment, the presence of the iPSC endothelial vessel facilitates neutrophil extravasation and migration toward a chemotactic source. Relevant cell sources, such as iPSC, combine with organotypic models to open the way for improved and increasingly accessible in vitro tissue, disease, and patient-specific models.
Asunto(s)
Endotelio/citología , Células Madre Pluripotentes Inducidas/citología , Animales , Células Endoteliales/citología , Humanos , Dispositivos Laboratorio en un Chip , Microvasos/citología , Neutrófilos/citologíaRESUMEN
Considerable evidence suggests that self-renewal and differentiation of cancer stem-like cells, a key cell population in tumorgenesis, can determine the outcome of disease. Though the development of microfluidics has enhanced the study of cellular lineage, it remains challenging to retrieve sister cells separately inside enclosed microfluidics for further analyses. In this work, we developed a photomechanical method to selectively detach and reliably retrieve target cells from enclosed microfluidic chambers. Cells cultured on carbon nanotube-polydimethylsiloxane composite surfaces can be detached using shear force induced through irradiation of a nanosecond-pulsed laser. This retrieval process has been verified to preserve cell viability, membrane proteins, and mRNA expression levels. Using the presented method, we have successfully performed 96-plex single-cell transcriptome analysis on sister cells in order to identify the genes altered during self-renewal and differentiation, demonstrating phenomenal resolution in the study of cellular lineage.
Asunto(s)
Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Diferenciación Celular , Línea Celular Tumoral , Linaje de la Célula , Supervivencia Celular , Humanos , Dispositivos Laboratorio en un Chip , Análisis de la Célula Individual/instrumentación , Análisis de la Célula Individual/métodosRESUMEN
Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (<100 cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced by tumor-stromal interactions.
Asunto(s)
Neoplasias de la Mama/patología , Comunicación Celular , Regulación Neoplásica de la Expresión Génica , Dispositivos Laboratorio en un Chip , Células Madre Neoplásicas/patología , Células del Estroma/patología , Microambiente Tumoral , Animales , Neoplasias de la Mama/metabolismo , Adhesión Celular , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo/instrumentación , Diseño de Equipo , Estudios de Factibilidad , Femenino , Humanos , Ratones , Células Madre Neoplásicas/metabolismo , Polihidroxietil Metacrilato/química , Prueba de Estudio Conceptual , Análisis de la Célula Individual , Células del Estroma/metabolismo , Propiedades de SuperficieRESUMEN
Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.
Asunto(s)
Microfluídica/métodos , Células Madre Neoplásicas/citología , Esferoides Celulares/citología , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Hidrogeles/química , Dispositivos Laboratorio en un Chip , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones SCID , Microfluídica/instrumentación , Microscopía Electrónica de Rastreo , Células Madre Neoplásicas/metabolismo , Polihidroxietil Metacrilato/química , Receptores Notch/genética , Receptores Notch/metabolismo , Análisis de la Célula Individual/métodos , Esferoides Celulares/metabolismo , Trasplante HeterólogoRESUMEN
Tumor cell migration toward and intravasation into capillaries is an early and key event in cancer metastasis, yet not all cancer cells are imbued with the same capability to do so. This heterogeneity within a tumor is a fundamental property of cancer. Tools to help us understand what molecular characteristics allow a certain subpopulation of cells to spread from the primary tumor are thus critical for overcoming metastasis. Conventional in vitro migration platforms treat populations in aggregate, which leads to a masking of intrinsic differences among cells. Some migration assays reported recently have single-cell resolution, but these platforms do not provide for selective retrieval of the distinct migrating and non-migrating cell populations for further analysis. Thus, to study the intrinsic differences in cells responsible for chemotactic heterogeneity, we developed a single-cell migration platform so that individual cells' migration behavior can be studied and the heterogeneous population sorted based upon chemotactic phenotype. Furthermore, after migration, the highly chemotactic and non-chemotactic cells were retrieved and proved viable for later molecular analysis of their differences. Moreover, we modified the migration channel to resemble lymphatic capillaries to better understand how certain cancer cells are able to move through geometrically confining spaces.
Asunto(s)
Quimiotaxis , Técnicas Analíticas Microfluídicas , Análisis de la Célula Individual/métodos , Línea Celular Tumoral , Movimiento Celular , Humanos , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Análisis de la Célula Individual/instrumentaciónRESUMEN
Cancer heterogeneity has received considerable attention for its role in tumor initiation and progression, and its implication for diagnostics and therapeutics in the clinic. To facilitate a cellular heterogeneity study in a low cost and highly efficient manner, we present a microfluidic platform that allows traceable clonal culture and characterization. The platform captures single cells into a microwell array and cultures them for clonal expansion, subsequently allowing on-chip characterization of clonal phenotype and response against drug treatments. Using a heterogeneous prostate cancer model, the PC3 cell line, we verified our prototype, identifying three different sub-phenotypes and correlating their clonal drug responsiveness to cell phenotype.
RESUMEN
Cancer-stromal cell interactions are a critical process in tumorigenesis. Conventional dish-based assays, which simply mix two cell types, have limitations in three aspects: 1) limited control of the cell microenvironment; 2) inability to study cell behavior in a single-cell manner; and 3) have difficulties in characterizing single cell behavior within a highly heterogeneous cell population (e.g. tumor). An innovative use of microfluidic technology is for improving the spatial resolution for single cell assays. However, it is challenging to isolate the paired interacting cells while maintaining nutrient renewal. In this work, two-phase flow was used as a simple isolation method, separating the microenvironment of each individual chamber. As nutrients in an isolated chamber are consumed by cells, media exchange is required. To connect the cell culture chamber to the media exchange layer, we demonstrated a 3D microsystem integration technique using vertical connections fabricated by deep reactive-ion etching (DRIE). Compared to previous approaches, the presented process allows area reduction of vertical connections by an order of magnitude, enabling compact 3D integration. A semi-permeable membrane was sandwiched between the cell culture layer and the media exchange layer. The selectivity of the semi-permeable membrane results in the retention of the signaling proteins within the chamber while allowing free diffusion of nutrients (e.g., glucose and amino acids). Thus, paracrine signals are accumulated inside the chamber without cross-talk between cells in other chambers. Utilizing these innovations, we co-cultured UM-SCC-1 (head and neck squamous cell carcinoma) cells and endothelial cells to simulate tumor proliferation enhancement in the vascular endothelial niche.