3D Microfluidic Model Research Articles

Development and validation of a controlled heating apparatus for long-term MRI of 3D microfluidic tumor models.

Conventional testing of novel contrast agents for magnetic resonance imaging (MRI) involves cell and animal studies. However, 2D cultures lack dynamic flow and in vivo MRI is limited by regulatory approval of long-term anesthesia use. Microfluidic tumor models (MTMs) offer a cost-effective, reproducible, and high throughput platform for bridging cell and animal models. Yet, MRI of microfluidic devices is challenging, due to small fluid volumes generating low sensitivity. For the first time, an MRI of MTMs was performed at low field strength (1 T) using conventional imaging equipment without microcoils. To enable longitudinal MRI, we developed (1) CHAMP-3 (controlled heating apparatus for microfluidics and portability) which heats MTMs during MRI scans and (2) an MRI-compatible temperature monitoring system. CHAMP-3 maintained chip surface temperature at ~37°C and the media inside at ~35.5°C. Enhanced T1-weighted MRI contrast was achieved in 3D MTMs with free manganese (Mn2+) solutions and Mn2+ labeled tumor cells.

Dysfunctional mechanotransduction regulates the progression of PIK3CA-driven vascular malformations.

Somatic activating mutations in PIK3CA are common drivers of vascular and lymphatic malformations. Despite common biophysical signatures of tissues susceptible to lesion formation, including compliant extracellular matrix and low rates of perfusion, lesions vary in clinical presentation from localized cystic dilatation to diffuse and infiltrative vascular dysplasia. The mechanisms driving the differences in disease severity and variability in clinical presentation and the role of the biophysical microenvironment in potentiating progression are poorly understood. Here, we investigate the role of hemodynamic forces and the biophysical microenvironment in the pathophysiology of vascular malformations, and we identify hemodynamic shear stress and defective endothelial cell mechanotransduction as key regulators of lesion progression. We found that constitutive PI3K activation impaired flow-mediated endothelial cell alignment and barrier function. We show that defective shear stress sensing in PIK3CA E542K endothelial cells is associated with reduced myosin light chain phosphorylation, junctional instability, and defective recruitment of vinculin to cell-cell junctions. Using 3D microfluidic models of the vasculature, we demonstrate that PIK3CA E542K microvessels apply reduced traction forces and are unaffected by flow interruption. We further found that draining transmural flow resulted in increased sprouting and invasion responses in PIK3CA E542K microvessels. Mechanistically, constitutive PI3K activation decreased cellular and nuclear elasticity resulting in defective cellular tensional homeostasis in endothelial cells which may underlie vascular dilation, tissue hyperplasia, and hypersprouting in PIK3CA-driven venous and lymphatic malformations. Together, these results suggest that defective nuclear mechanics, impaired cellular mechanotransduction, and maladaptive hemodynamic responses contribute to the development and progression of PIK3CA-driven vascular malformations.

Paracrine Ovarian Cancer Cell-Derived CSF1 Signaling Regulates Macrophage Migration Dynamics in a 3D Microfluidic Model that Recapitulates In Vivo Infiltration Patterns in Patient-Derived Xenografts.

A high density of macrophages in the ovarian cancer microenvironment is associated with disease progression and poor outcomes. Understanding cancer-macrophage interaction mechanisms that establish this pro-tumorigenic microenvironment is critical for developing macrophage-targeted therapies. Here, 3D microfluidic assays and patient-derived xenografts are utilized to define the role of cancer-derived colony stimulating factor 1 (CSF1) on macrophage infiltration dynamics toward ovarian cancer cells. It is demonstrated that multiple ovarian cancer models promote the infiltration of macrophages into a 3D extracellular matrix in vitro in a cell density-dependent manner. Macrophages exhibit directional migration and increased migration speed under both direct interactions with cancer cells embedded within the matrix and paracrine crosstalk with cancer cells seeded in an independent microchannel. It is also found that platinum-based chemotherapy increases macrophage recruitment and the levels of cancer cell-derived CSF1. Targeting CSF1 signaling under baseline or chemotherapy-treatment conditions reduces the number of infiltrated macrophages. It is further shown that results obtained with the 3D microfluidic model reflect the recruitment profiles of macrophages in patient-derived xenografts in vivo. These findings highlight the role of CSF1 signaling in establishing macrophage-rich ovarian cancer microenvironments, as well as the utility of microfluidic models in recapitulating 3D tumor ecosystems and dissecting cancer-macrophage signaling.

Microphysiologically Engineered Vessel-Tumor Model to Investigate Vascular Transport Dynamics of Immune Cells.

Cancer immunotherapy has emerged as a promising therapeutic strategy to combat cancer effectively. However, it is hard to observe and quantify how this in vivo process happens. Three-dimensional (3D) microfluidic vessel-tumor models offer valuable capability to study how immune cells transport during cancer progression. We presented an advanced 3D vessel-supported tumor model consisting of the endothelial lumen and vessel network for the study of T cells' transportation. The process of T cell transport through the vessel network and interaction with tumor spheroids was represented and monitored in vitro. Specifically, we demonstrate that the endothelial glycocalyx serving in the T cells' transport can influence the endothelium-immune interaction. Furthermore, after vascular transport, how programmed cell death protein 1 (PD-1) immune checkpoint inhibition influences the delivered activated-T cells on tumor killing was evaluated. Our in vitro vessel-tumor model provides a microphysiologically engineered platform to represent T cell vascular transportation during tumor immunotherapy. The reported innovative vessel-tumor platform is believed to have the potential to explore the tumor-induced immune response mechanism and preclinically evaluate immunotherapy's effectiveness.

In vitro 3D microfluidic peritoneal metastatic colorectal cancer model for testing different oxaliplatin-based HIPEC regimens.

Treatment of colorectal peritoneal metastases with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (HIPEC) is still evolving. Conducting a randomized trial is challenging due to the high heterogeneity in the presentation of peritoneal disease and various surgical approaches. Biological research may facilitate more rapid translation of information into clinical practice. There is an emerging need for a preclinical model to improve HIPEC treatment protocols in terms of drug doses and treatment durations. The aim of the study is to design a tool that serves as an invitro three-dimensional (3D) microfluidic peritoneal metastatic colorectal cancer model to test the efficacy of different HIPEC treatments. We determined the effects of current therapy options using a 3D static disease model on human colon carcinoma cell lines (HCT 116) and transforming growth factor-β1 induced epithelial-to-mesenchymal transition (EMT) HCT 116 lines at 37 °C and 42 °C for 30, 60, and 120 min. We determined oxaliplatin's half maximal inhibitory concentrations in a 3D static culture by using viability assay. Clinical practices of HIPEC were applied in the developed model. EMT-induced HCT 116 cells were less sensitive to oxaliplatin treatment compared to non-induced cells. We observed increased cytotoxicity when increasing the temperature from 37 °C to 42 °C and extending the treatment duration from 30 to 120 min. We found that 200 mg/m2 oxaliplatin administered for 120 min is the most effective HIPEC treatment option within the framework of clinic applications. The tool map provide insights into creating more realistic pre-clinical tools that could be used for a patient-based drug screening.

Exploring the Interplay of RUNX2 and CXCR4 in Melanoma Progression.

Overexpression of the Runt-related transcription factor 2 (RUNX2) has been reported in several cancer types, and the C-X-C motif chemokine receptor 4 (CXCR4) has an important role in tumour progression. However, the interplay between CXCR4 and RUNX2 in melanoma cells remains poorly understood. In the present study, we used melanoma cells and a RUNX2 knockout (RUNX2-KO) in vitro model to assess the influence of RUNX2 on CXCR4 protein levels along with its effects on markers associated with cell invasion and autophagy. Osteotropism was assessed using a 3D microfluidic model. Moreover, we assessed the impact of CXCR4 on the cellular levels of key cellular signalling proteins involved in autophagy. We observed that melanoma cells express both RUNX2 and CXCR4. Restored RUNX2 expression in RUNX2 KO cells increased the expression levels of CXCR4 and proteins associated with the metastatic process. The protein markers of autophagy LC3 and beclin were upregulated in response to increased CXCR4 levels. The CXCR4 inhibitor WZ811 reduced osteotropism and activated the mTOR and p70-S6 cell signalling proteins. Our data indicate that the RUNX2 transcription factor promotes the expression of the CXCR4 chemokine receptor on melanoma cells, which in turn promotes autophagy, cell invasiveness, and osteotropism, through the inhibition of the mTOR signalling pathway. Our data suggest that RUNX2 promotes melanoma progression by upregulating CXCR4, and we identify the latter as a key player in melanoma-related osteotropism.

Hyaluronic acid turnover controls the severity of cerebral cavernous malformations in bioengineered human micro-vessels.

Cerebral cavernous malformations (CCMs) are vascular lesions that predominantly form in blood vessels of the central nervous system upon loss of the CCM multimeric protein complex. The endothelial cells within CCM lesions are characterized by overactive MEKK3 kinase and KLF2/4 transcription factor signaling, leading to pathological changes such as increased endothelial cell spreading and reduced junctional integrity. Concomitant to aberrant endothelial cell signaling, non-autonomous signals from the extracellular matrix (ECM) have also been implicated in CCM lesion growth and these factors might explain why CCM lesions mainly develop in the central nervous system. Here, we adapted a three-dimensional microfluidic system to examine CCM1 deficient human micro-vessels in distinctive extracellular matrices. We validate that pathological hallmarks are maintained in this model. We further show that key genes responsible for homeostasis of hyaluronic acid, a major extracellular matrix component of the central nervous system, are dysregulated in CCM. Supplementing the matrix in our model with distinct forms of hyaluronic acid inhibits pathological cell spreading and rescues barrier function. Hyaluronic acid acts by dampening cell-matrix adhesion signaling in CCM, either downstream or in parallel of KLF2/4. This study provides a proof-of-principle that ECM embedded 3D microfluidic models are ideally suited to identify how changes in ECM structure and signaling impact vascular malformations.

Relatively Rare Populations of Invasive Cells Drive Progression of Heterogeneous Tumors.

Breast tumors often display an astonishing degree of spatial and temporal heterogeneity, which are associated with cancer progression, drug resistance, and relapse. Triple-negative breast cancer (TNBC) is a particularly aggressive and heterogeneous subtype for which targeted therapies are scarce. Consequently, patients with TNBC have a poorer overall prognosis compared to other breast cancer patients. Within heterogeneous tumors, individual clonal subpopulations may exhibit differences in their rates of growth and degrees of invasiveness. We hypothesized that such phenotypic heterogeneity at the single-cell level may accelerate tumor progression by enhancing the overall growth and invasion of the entire tumor. To test this hypothesis, we isolated and characterized clonal subpopulations with distinct morphologies and biomarker expression from the inherently heterogeneous 4T1 mouse mammary carcinoma cell line. We then leveraged a 3D microfluidic tumor model to reverse-engineer intratumoral heterogeneity and thus investigate how interactions between phenotypically distinct subpopulations affect tumor growth and invasion. We found that the growth and invasion of multiclonal tumors were largely dictated by the presence of cells with epithelial and mesenchymal traits, respectively. The latter accelerated overall tumor invasion, even when these cells comprised less than 1% of the initial population. Consistently, tumor progression was delayed by selectively targeting the mesenchymal subpopulation. This work reveals that highly invasive cells can dominate tumor phenotype and that specifically targeting these cells can slow the progression of heterogeneous tumors, which may help inform therapeutic approaches. The online version contains supplementary material available at 10.1007/s12195-023-00792-w.

Impact of brain organoid-derived sEVs on metastatic adaptation and invasion of breast carcinoma cells through a microphysiological system.

Brain metastases are common in triple-negative breast cancer (TNBC), suggesting a complex process of cancer spread. The mechanisms enabling TNBC cell adaptation and proliferation in the brain remain unclear. Small extracellular vesicles (sEVs) play a crucial role in communication between breast carcinoma cells and the brain. However, the lack of relevant models hinders understanding of sEV-mediated communication. The present study assesses the impact of brain organoid-derived sEVs (BO-sEVs) on various behaviours of the MDA-MB-231 cell line, chosen as a representative of TNBC in a 3D microfluidic model. Our results demonstrate that 150-200 nm sEVs expressing CD63, CD9, and CD81 from brain organoid media decrease MDA-MB-231 cell proliferation, enhance their wound-healing capacity, alter their morphology into more mesenchymal mode, and increase their stemness. BO-sEVs led to heightened PD-L1, CD49f, and vimentin levels of expression in MDA-MB-231 cells, suggesting an amplified immunosuppressive, stem-like, and mesenchymal phenotype. Furthermore, these sEVs also induced the expression of neural markers such as GFAP in carcinoma cells. The cytokine antibody profiling array also showed that BO-sEVs enhanced the secretion of MCP-1, IL-6, and IL-8 by MDA-MB-231 cells. Moreover, sEVs significantly enhance the migration and invasion of carcinoma cells toward brain organoids in a 3D organoid-on-a-chip system. Our findings emphasize the potential significance of metastatic site-derived sEVs as pivotal mediators in carcinoma progression and adaptation to the brain microenvironment, thereby unveiling novel therapeutic avenues.

Osteogenesis and osteoclastogenesis on a chip: Engineering a self-assembling 3D coculture

Healthy bone is maintained by the process of bone remodeling. An unbalance in this process can lead to pathologies such as osteoporosis which are often studied with animal models. However, data from animals have limited power in predicting the results that will be obtained in human clinical trials. In search for alternatives to animal models, human in vitro models are emerging as they address the principle of reduction, refinement, and replacement of animal experiments (3Rs). At the moment, no complete in vitro model for bone-remodeling exists. Microfluidic chips offer great possibilities, particularly because of the dynamic culture options, which are crucial for in vitro bone formation. In this study, a scaffold free, fully human, 3D microfluidic coculture model of bone remodeling is presented. A bone-on-a-chip coculture system was developed in which human mesenchymal stromal cells differentiated into the osteoblastic lineage and self-assembled into scaffold free bone-like tissues with the shape and dimensions of human trabeculae. Human monocytes were able to attach to these tissues and to fuse into multinucleated osteoclast-like cells, establishing the coculture. Computational modeling was used to determine the fluid flow induced shear stress and strain in the formed tissue. Furthermore, a set-up was developed allowing for long-term (35 days) on-chip cell culture with benefits including continuous fluid-flow, low bubble formation risk, easy culture medium exchange inside the incubator and live cell imaging options. This on-chip coculture is a crucial advance towards developing in vitro bone remodeling models to facilitate drug testing.

Blood–brain barrier microfluidic chips and their applications

As a natural dynamic barrier separating blood from brain parenchyma, the blood–brain barrier (BBB) is mainly composed of brain microvascular endothelial cells (BMECs), pericytes, astrocytes, and a variety of neurons. The BBB regulates the highly selective transport of various substances between the brain and blood and maintains the stability of the central nervous system (CNS). Owing to this tight control, the BBB represents a formidable challenge for the delivery of drugs and other exogenous compounds into the CNS, which has bottlenecked the development of many drugs for CNS diseases. Therefore, efficient and precise in vitro models of the BBB are needed to assess the efficacy and toxicity of drugs targeting the CNS to inform drug design and to improve the success rate of agents that enter clinical evaluation. In vitro BBB models have rapidly advanced from the early two-dimensional (2D) static models to the current three-dimensional (3D) dynamic microfluidic chips. Although the commonly used, static, in vitro BBB models are simple to construct and TEER values are convenient to detect, the static models do not provide an ideal (i.e., accurate) BBB environment, since they lack the correct physiological size/scale and hemodynamic shear stress, both of which play substantial roles in promoting and maintaining EC differentiation into a specific BBB phenotype. Compared with traditional static models, 3D microfluidic models thus enable cells to react in a manner more closely resembling in vivo behavior by simulating a microenvironment with more natural signal transduction. As a result, the dynamic 3D BBB model can more accurately recapitulate the structure and function of the human BBB. Here we summarize the recent progress in in vitro microfluidic BBB chips and their research applications as well as discuss the prospects and challenges for where the technology is headed.

1035 Understanding dynamic immune responses within a 3D microfluidic model of human skin

1035 Understanding dynamic immune responses within a 3D microfluidic model of human skin

Abstract 4613: Inhibition of myosin II in triple-negative breast cancer cells limits macrophage recruitment in a 3D environment

Abstract Among all breast cancer subtypes, triple-negative breast cancer (TNBC) exhibits the least favorable outcomes with a complex tumor microenvironment that facilitates metastatic progression. Remodeling of the extracellular matrix at the tumor invasive front is associated with increased cancer cell motility and poor outcomes. Myosin II, a key cytoskeletal regulator, plays a central role in cancer cell metastatic potential. In the clinic, high expression of myosin II in breast tumors has been associated with distant metastasis and lower survival rates. Interestingly, breast tumors overexpressing myosin II exhibited a higher density of tumor-associated macrophages and an immunosuppressive microenvironment. However, the relationship between myosin II activation in TNBC cancer cells and macrophage recruitment remains poorly understood. Here we employed a novel 3D microfluidic model of tumor-macrophage co-culture to study macrophage infiltration toward cancer cells with high spatiotemporal resolution. Using a panel of TNBC cell lines we found that high myosin II expression in cancer cells correlated with higher macrophage recruitment in a 3D matrix. Pharmacologic or genetic inhibition of myosin II reduced macrophage recruitment. Furthermore, using our microfluidic devices, we controlled the spatial distribution of cancer cells in a 3D matrix and separated the role of cancer cell-secreted paracrine factors from cancer cell-mediated matrix remodeling on macrophage recruitment. Analysis of matrix remodeling over time revealed that targeting myosin II in cancer cells limited the establishment of migratory tracks that facilitated macrophage recruitment. In sum, our studies revealed the critical role of cancer cell-driven matrix remodeling in promoting macrophage infiltration that can be therapeutically targeted to reverse the establishment of an immunosuppressive microenvironment. Citation Format: Youngbin Cho, Ruxuan Li, Ioannis Zervantonakis. Inhibition of myosin II in triple-negative breast cancer cells limits macrophage recruitment in a 3D environment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4613.

Multiplexed Detection of Molecular Interactions with DNA Origami Engineered Cells in 3D Collagen Matrices.

The interactions of cells with signaling molecules present in their local microenvironment maintain cell proliferation, differentiation, and spatial organization and mediate progression of diseases such as metabolic disorders and cancer. Real-time monitoring of the interactions between cells and their extracellular ligands in a three-dimensional (3D) microenvironment can inform detection and understanding of cell processes and the development of effective therapeutic agents. DNA origami technology allows for the design and fabrication of biocompatible and 3D functional nanodevices via molecular self-assembly for various applications including molecular sensing. Here, we report a robust method to monitor live cell interactions with molecules in their surrounding environment in a 3D tissue model using a microfluidic device. We used a DNA origami cell sensing platform (CSP) to detect two specific nucleic acid sequences on the membrane of B cells and dendritic cells. We further demonstrated real-time detection of biomolecules with the DNA sensing platform on the surface of dendritic cells in a 3D microfluidic tissue model. Our results establish the integration of live cells with membranes engineered with DNA nanodevices into microfluidic chips as a highly capable biosensor approach to investigate subcellular interactions in physiologically relevant 3D environments under controlled biomolecular transport.

Shining a Light on Cancer-Photonics in Microfluidic Tumor Modeling and Biosensing.

Microfluidic platforms represent a powerful approach to miniaturizing important characteristics of cancers, improving in vitro testing by increasing physiological relevance. Different tools can manipulate cells and materials at the microscale, but few offer the efficiency and versatility of light and optical technologies. Moreover, light-driven technologies englobe a broad toolbox for quantifying critical biological phenomena. Herein, the role of photonics in microfluidic 3D cancer modeling and biosensing from three major perspectives is reviewed. First, optical-driven technologies are looked upon, as these allow biomaterials and living cells to be manipulated with microsized precision and present opportunities to advance 3D microfluidic models by engineering cancer microenvironments' hallmarks, such as their architecture, cellular complexity, and vascularization. Second, the growing field of optofluidics is discussed, exploring how optical tools can directly interface microfluidic chips, enabling the extraction of relevant biological data, from single fluorescent signals to the complete 3D imaging of diseased cells within microchannels. Third, advances in optical cancer biosensing are reviewed, focusing on how light-matter interactions can detect biomarkers, rare circulating tumor cells, and cell-derived structures such as exosomes. Photonic technologies' current challenges and caveats in microfluidic 3D cancer models are overviewed, outlining future research avenues that may catapult the field.

A simplified three-dimensional numerical simulation approach for surface acoustic wave tweezers

Standing surface acoustic waves (SSAWs) have been extensively used as acoustic tweezers to manipulate, transport, and separate microparticles and biological cells in a microscale fluidic environment, with great potentials for biomedical sensing, genetic analysis, and therapeutics applications. Currently, there lacks an accurate, reliable, and efficient three-dimensional (3D) modeling platform to simulate behaviors of micron-size particles/cells in acoustofluidics, which is crucial to provide the guidance for the experimental studies. The major challenge for achieving this is the computational complexity of 3D modeling. Herein, a simplified but effective 3D SSAW microfluidic model was developed to investigate the separation and manipulation of particles. This model incorporates propagation attenuation of the surface waves to increase the modeling accuracy, while simplifies the modeling of piezoelectric substrates and the wall of microchannel by determining the effective propagation region of the substrate. We have simulated the SSAWs microfluidics device, and systematically analyzed effects of voltage, tilt angle, and flow rate on the separation of the particles under the SSAWs. The obtained simulation results are compared with those obtained from the experimental studies, showing good agreements. This simplified modeling platform could become a convenient tool for acoustofluidic research.

The Release of Inflammatory Mediators from Acid-Stimulated Mesenchymal Stromal Cells Favours Tumour Invasiveness and Metastasis in Osteosarcoma.

Simple SummaryWe aimed to validate the correlation between tumour glycolysis/acidosis and inflammation in osteosarcoma-associated mesenchymal stromal cells and investigate the role of acidity-induced inflammation in the development of metastasis in this very aggressive cancer. We confirmed the presence of an acidic microenvironment in osteosarcoma xenografts, both subcutaneous and orthotopic, using state-of-the-art imaging technologies; corroborated the correlation between tumour glycolysis, acidosis, and inflammatory markers in human patients; and finally, explored the use of anti-IL6 antibody to target these pathogenic pathways, using advanced 3D microfluidic models. In the future, advanced imaging systems for the measurement of tumour glycolysis and/or pH may help identify osteosarcoma patients who would benefit from anti-IL6 therapies to complement conventional therapy.Osteosarcoma is the most frequent primary malignant bone tumour with an impressive tendency to metastasise. Highly proliferative tumour cells release a remarkable amount of protons into the extracellular space that activates the NF-kB inflammatory pathway in adjacent stromal cells. In this study, we further validated the correlation between tumour glycolysis/acidosis and its role in metastases. In patients, at diagnosis, we found high circulating levels of inflammatory mediators (IL6, IL8 and miR-136-5p-containing extracellular vesicles). IL6 serum levels significantly correlated with disease-free survival and 18F-FDG PET/CT uptake, an indirect measurement of tumour glycolysis and, hence, of acidosis. In vivo subcutaneous and orthotopic models, co-injected with mesenchymal stromal (MSC) and osteosarcoma cells, formed an acidic tumour microenvironment (mean pH 6.86, as assessed by in vivo MRI-CEST pH imaging). In these xenografts, we enlightened the expression of both IL6 and the NF-kB complex subunit in stromal cells infiltrating the tumour acidic area. The co-injection with MSC also significantly increased lung metastases. Finally, by using 3D microfluidic models, we directly showed the promotion of osteosarcoma invasiveness by acidosis via IL6 and MSC. In conclusion, osteosarcoma-associated MSC react to intratumoural acidosis by triggering an inflammatory response that, in turn, promotes tumour invasiveness at the primary site toward metastasis development.

3D microfluidic tumor models for biomimetic engineering of glioma niche and detection of cell morphology, migration and phenotype change

In this work, an integrated 3-dimensional microfluidic device was developed for simulation of the immune microenvironment of glioma niche through the co-culture of three kinds of related cells. Glioma cells, endothelial cells and macrophages were co-cultured together in the microfluidic device, spatially separated by the design of a coffer structure and the use of hydrogel. This platform enabled separate monitoring of the morphology change and migration of cells, as well as molecular interactions between different kinds of cells. Tumor cells were found to exhibit EMT like shape change to become thinner, and sensitive perception and taxis toward macrophages. The influence of tumor cells and the microenvironment, macrophages would be re-educated and the phenotype could be changed from M1 (tumor-suppressive) to M2 (tumor-supportive), which could be validated through cytokines analysis. This 3D microfluidic tumor model provides a powerful tool for studying the biological properties of glioma niche.

Implementation and characterization of a physiologically relevant flow waveform in a 3D microfluidic model of the blood–brain barrier

Previous in vitro studies interrogating the endothelial response to physiologically relevant flow regimes require specialized pumps to deliver time-dependent waveforms that imitate in vivo blood flow. The aim of this study is to create a low-cost and broadly adaptable approach to mimic physiological flow, and then use this system to characterize the effect of flow separation on velocity and shear stress profiles in a three-dimensional (3D) topology. The flow apparatus incorporates a programmable linear actuator that superposes oscillations on a constant mean flow driven by a peristaltic pump to emulate flow in the carotid artery. The flow is perfused through a 3D in vitro model of the blood-brain barrier designed to induce separated flow. Experimental flow patterns measured by microparticle image velocimetry and modeled by computational fluid dynamics reveal periodic changes in the instantaneous shear stress along the channel wall. Moreover, the time-dependent flow causes periodic flow separation zones, resulting in variable reattachment points during the cycle. The effects of these complex flow regimes are assessed by evaluating the integrity of the in vitro blood-brain barrier model. Permeability assays and immunostaining for proteins associated with tight junctions reveal barrier breakdown in the region of disturbed flow. In conclusion, the flow system described here creates complex, physiologically relevant flow profiles that provide deeper insight into the fluid dynamics of separated flow and pave the way for future studies interrogating the cellular response to complex flow regimes.

The effects of biofilms on tumor progression in a 3D cancer-biofilm microfluidic model

Components within the tumor microenvironment, such as intratumoral bacteria (IB; within tumors), affect tumor progression. However, current experimental models have not explored the effects of extratumoral bacteria (EB; outside tumors) on cancer progression. Here, we developed a microfluidic platform to analyze the influence of bacterial distribution on bladder cancer progression under defined conditions, using uropathogenic Escherichia coli. This was achieved by establishing coating (CT) and colonizing (CL) models to simulate the different invasion and colonization modes of IB and EB in tumor tissues. We demonstrated that both EB and IB induced closer cell-cell contacts within the tumor cluster, but cancer cell viability was reduced only in the presence of IB. Interestingly, cancer stem cell counts increased significantly in the presence of EB. These outcomes were due to the formation of extracellular DNA-based biofilms by EB. Triple therapy of DNase (anti-biofilm agent), ciprofloxacin (antibiotic), and doxorubicin (anti-cancer drug) could effectively eradicate biofilms and tumors simultaneously. Our preclinical proof-of-concept provides insights on how bacteria can influence tumor progression and facilitate future research on anti-biofilm cancer management therapies.