A Microfluidic System for the Investigation of Tumor Cell Extravasation (original) (raw)
Related papers
In Vitro Model of Tumor Cell Extravasation
PLoS ONE, 2013
Tumor cells that disseminate from the primary tumor and survive the vascular system can eventually extravasate across the endothelium to metastasize at a secondary site. In this study, we developed a microfluidic system to mimic tumor cell extravasation where cancer cells can transmigrate across an endothelial monolayer into a hydrogel that models the extracellular space. The experimental protocol is optimized to ensure the formation of an intact endothelium prior to the introduction of tumor cells and also to observe tumor cell extravasation by having a suitable tumor seeding density. Extravasation is observed for 38.8% of the tumor cells in contact with the endothelium within 1 day after their introduction. Permeability of the EC monolayer as measured by the diffusion of fluorescently-labeled dextran across the monolayer increased 3.8 fold 24 hours after introducing tumor cells, suggesting that the presence of tumor cells increases endothelial permeability. The percent of tumor cells extravasated remained nearly constant from1 to 3 days after tumor seeding, indicating extravasation in our system generally occurs within the first 24 hours of tumor cell contact with the endothelium.
Microfluidic device for studying tumor cell extravasation in cancer metastasis
2010 Biomedical Sciences and Engineering Conference, 2010
Metastasis is the process by which cancer spreads to form secondary tumors at downstream locations throughout the body. This uncontrolled spreading is the leading cause of death in patients with epithelial cancers and is the main reason that suppressing and targeting cancer has proven to be so challenging. Tumor cell extravasation is one of the key steps in cancer's progression towards a metastatic state. This occurs when circulating tumor cells found within the blood stream are able to transmigrate through the endothelium lining and basement membrane of the vasculature to form metastatic tumors at secondary sites within the body. Predicting the likelihood of this occurrence in patients, or being able to determine specific markers involved in this process could lead to preventative measures targeting these types of cancer; moreover, this may lead to the discovery of novel anti-metastatic drugs. We have developed a microfluidic device that has shown the extravasation of fluorescently labeled tumor cells across an endothelial cell lined membrane coated with matrigel followed by the formation of colonies. This device provides the advantages of combining a controlled environment, mimicking that found within the body, with real-time monitoring capabilities allowing for the study of these biomarkers and cellular interactions along with other potential mechanisms involved in the process of extravasation.
Mechanisms of tumor cell extravasation in an in vitro microvascular network platform
Integrative biology : quantitative biosciences from nano to macro, 2013
A deeper understanding of the mechanisms of tumor cell extravasation is essential in creating therapies that target this crucial step in cancer metastasis. Here, we use a microfluidic platform to study tumor cell extravasation from in vitro microvascular networks formed via vasculogenesis. We demonstrate tight endothelial cell-cell junctions, basement membrane deposition and physiological values of vessel permeability. Employing our assay, we demonstrate impaired endothelial barrier function and increased extravasation efficiency with inflammatory cytokine stimulation, as well as positive correlations between the metastatic potentials of MDA-MB-231, HT-1080, MCF-10A and their extravasation capabilities. High-resolution time-lapse microscopy reveals the highly dynamic nature of extravasation events, beginning with thin tumor cell protrusions across the endothelium followed by extrusion of the remainder of the cell body through the formation of small (~1 μm) openings in the endothelia...
Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function
Proceedings of the National Academy of Sciences, 2012
Entry of tumor cells into the blood stream is a critical step in cancer metastasis. Although significant progress has been made in visualizing tumor cell motility in vivo, the underlying mechanism of cancer cell intravasation remains largely unknown. We developed a microfluidic-based assay to recreate the tumor-vascular interface in three-dimensions, allowing for high resolution, real-time imaging, and precise quantification of endothelial barrier function. Studies are aimed at testing the hypothesis that carcinoma cell intravasation is regulated by biochemical factors from the interacting cells and cellular interactions with macrophages. We developed a method to measure spatially resolved endothelial permeability and show that signaling with macrophages via secretion of tumor necrosis factor alpha results in endothelial barrier impairment. Under these conditions intravasation rates were increased as validated with live imaging. To further investigate tumor-endothelial (TC-EC) signaling, we used highly invasive fibrosarcoma cells and quantified tumor cell migration dynamics and TC-EC interactions under control and perturbed (with tumor necrosis factor alpha) barrier conditions. We found that endothelial barrier impairment was associated with a higher number and faster dynamics of TC-EC interactions, in agreement with our carcinoma intravasation results. Taken together our results provide evidence that the endothelium poses a barrier to tumor cell intravasation that can be regulated by factors present in the tumor microenvironment.
The Use of Microfluidic Platforms to Probe the Mechanism of Cancer Cell Extravasation
Advanced Healthcare Materials
laboratory tools and model systems have not yet translated into improved patient outcomes as attrition rates of clinical trials on cancer interventions remain high. [3] The two primary causes of trial failures are attributed to poor efficacy and safety. [4] There is growing sentiment that these failures arise from the inability of preclinical models to recapitulate the molecular complexity of human disease. [5] Some recent notable and well-documented failed clinical trials illuminate the major differences in underlying physiology between humans and animals. For example, the drug TGN1412, an antibody to CD28, was used in primate models that were specifically selected for expressing 100% homology with the binding site for TGN1412 on human CD28. [6-8] The drug was well tolerated in several animal studies where only moderate increases in select chemokines were measured over several days. The phase 1 study of safety in humans was catastrophic. [7] Within hours of receiving a single dose 500 times smaller than safely tolerated in animal studies, all six healthy volunteers became critically ill through a massive systemic inflammatory response. [9] Further, of the new cancer therapeutics with proven safety, an exceedingly small fraction demonstrate efficacy in humans. [4] These limitations, in addition to growing ethical and financial consideration associated with animal models in research studies, have led to the proposal of eliminating animal models in preclinical efforts entirely. [10] In their place, there is an emerging need for novel approaches that more directly recapitulate human physiology in studies of cancer progression. The void of experimental tools is now being filled with microfluidic systems engineered to investigate the process of cancer progression. [11,12] The main advantages of microfabricated devices in studies of cancer progression are fourfold. They allow precise control over the cellular constituents, extracellular components, and physical environment, and they are amenable high magnification visualization. Importantly, cellular and noncellular constituents that are incorporated in microfluidic devices can be derived from human sources. Recreating microvascular geometry and incorporating human-based constituents in a microfluidic platform provides an alternative to animal models that may recapitulate cancer metastasis in an in vitro model system that is favorable to detailed experimental investigation. The key steps of the metastatic process have been detailed elsewhere. [13-15] The most difficult step in the metastatic process to study may be extravasation since it is a rare event that occurs well hidden within intact organs. Despite the difficulty in studying this process, the molecular mechanisms underlying extravasation Powerful experimental tools have contributed a wealth of novel insight into cancer etiology from the organ to the subcellular levels. However, these advances in understanding have outpaced improvements in clinical outcomes. One possible reason for this shortcoming is the reliance on animal models that do not fully replicate human physiology. An alternative in vitro approach that has recently emerged features engineered microfluidic platforms to investigate cancer progression. These devices allow precise control over cellular components, extracellular constituents, and physical forces, while facilitating detailed microscopic analysis of the metastatic process. This review focuses on the recent use of microfluidic platforms to investigate the mechanism of cancer cell extravasation.
bioRxiv (Cold Spring Harbor Laboratory), 2020
Throughout the process of metastatic dissemination, tumor cells are continuously subjected to mechanical forces resulting from complex fluid flows due to changes in pressures in their local microenvironments. While these forces have been associated with invasive phenotypes in 3D matrices, their role in key steps of the metastatic cascade, namely extravasation and subsequent interstitial migration, remains poorly understood. In this study, an in vitro model of the human microvasculature was employed to subject tumor cells to physiological luminal, trans-endothelial, and interstitial flows to evaluate their effects on those key steps of metastasis. Luminal flow promoted the extravasation potential of tumor cells, possibly as a result of their increased intravascular migration speed. Trans-endothelial flow increased the speed with which tumor cells transmigrated across the endothelium as well as their migration speed in the matrix following extravasation. In addition, tumor cells possessed a greater propensity to migrate in close proximity to the endothelium when subjected to physiological flows, which may promote the successful formation of metastatic foci. These results show important roles of fluid flow during extravasation and invasion, which could determine the local metastatic potential of tumor cells.
In vitro models of the metastatic cascade: from local invasion to extravasation
Drug Discovery Today, 2013
A crucial event in the metastatic cascade is the extravasation of circulating cancer cells from blood capillaries to the surrounding tissues. The past 5 years have been characterized by a significant evolution in the development of in vitro extravasation models, which moved from traditional transmigration chambers to more sophisticated microfluidic devices, enabling the study of complex cell-cell and cell-matrix interactions in multicellular, controlled environments. These advanced assays could be applied to screen easily and rapidly a broad spectrum of molecules inhibiting cancer cell endothelial adhesion and extravasation, thus contributing to the design of more focused in vivo tests. Keywords extravasation; cancer; in vitro; microfluidics; drug The past four decades were characterized by promising successes in cancer treatment and detection, through the development of devices reducing surgical invasiveness or enabling early diagnosis, and the discovery of drugs blocking primary tumor progression, thus reducing cancer mortality and improving life quality for patients with terminal disease [1].
Nature Protocols, 2017
Distant metastasis, which results in >90% of cancer related deaths, is enabled by hematogenous dissemination of tumor cells via the circulation. This requires the completion of a sequence of complex steps including transit, initial arrest, extravasation, survival and proliferation. Increased understanding of the cellular and molecular players enabling each of these steps is key in uncovering new opportunities for therapeutic intervention during early metastatic dissemination. Here, we describe an in vitro model of the human microcirculation with the potential to recapitulate discrete steps of early metastatic seeding, including arrest, transendothelial migration and early micrometastases formation. The microdevice features self-organized human microvascular networks formed over 4-5 days, after which tumor can be perfused and extravasation events easily tracked over 72 hours, via standard confocal microscopy. Contrary to most in vivo and in vitro extravasation assays, robust and rapid scoring of extravascular cells combined with high-resolution imaging can be easily achieved due to the confinement of the vascular network to one plane close to the surface of the device. This renders extravascular cells clearly distinct and allows tumor cells of interest to be identified quickly compared to those in thick tissues. The ability to generate large numbers of devices (~36) per experiment coupled with fast quantitation further allows for highly parametric studies, which is required when testing multiple genetic or pharmacological perturbations. This is coupled with the capability for live tracking of single cell extravasation events allowing both tumor and endothelial morphological
Procoagulant tumor microvesicles attach to endothelial cells on biochips under microfluidic flow
Biomicrofluidics, 2019
Tumour patients are at a high risk of venous thromboembolism (VTE) and the mechanism by which this occurs may involve tumour derived microvesicles (MV). Previously, it has been shown that tumour MV become attached to endothelial cells in static conditions. To investigate whether this process occurs under physiologically relevant flow rates, tumour MV were perfused across a microfluidic device coated with growing human umbilical endothelial cells (HUVECs). Cell lines were screened for their ability to form tumour spheroids and two cell lines, ES-2 and U87 were selected, formed spheroids were transferred to a microfluidic chip and a second endothelial cell biochip was coated with HUVECs and the two chips linked. Media was flowed through the spheroid chip to the endothelial chip and procoagulant activity (PCA) of the tumour media was determined by one-stage prothrombin time assay. Tumour MV were also quantified by flow cytometry before and after interaction with HUVECs. Confocal images showed HUVECs acquired fluorescence from MV attachment. Labelled MV were proportionally lost from MV rich media with time when flowed over HUVECs and was not observed on a control chip. The loss of MV was accompanied by a proportional reduction in PCA. Flow cytometry, confocal microscopy and live flow imagery captured under pulsatile flow confirmed an associated between tumour MV and HUVECs. Tumour MV attached to endothelial cells under physiological flow rates which may be relevant to the VTE pathways in cancer patients.
2019
Tumour patients are at a high risk of venous thromboembolism (VTE) and the mechanism by which this occurs may involve tumour derived microvesicles (MV). Previously, it has been shown that tumour MV become attached to endothelial cells in static conditions. To investigate whether this process occurs under physiologically relevant flow rates, tumour MV were perfused across a microfluidic device coated with growing human umbilical endothelial cells (HUVECs). Cell lines were screened for their ability to form tumour spheroids and two cell lines, ES-2 and U87 were selected, formed spheroids were transferred to a microfluidic chip and a second endothelial cell biochip was coated with HUVECs and the two chips linked. Media was flowed through the spheroid chip to the endothelial chip and procoagulant activity (PCA) of the tumour media was determined by one-stage prothrombin time assay. Tumour MV were also quantified by flow cytometry before and after interaction with HUVECs. Confocal images showed HUVECs acquired fluorescence from MV attachment. Labelled MV were proportionally lost from MV rich media with time when flowed over HUVECs and was not observed on a control chip. The loss of MV was accompanied by a proportional reduction in PCA. Flow cytometry, confocal microscopy and live flow imagery captured under pulsatile flow confirmed an associated between tumour MV and HUVECs. Tumour MV attached to endothelial cells under physiological flow rates which may be relevant to the VTE pathways in cancer patients.