We thank members of Dr. Christopher Hughes&#39; lab for their valued input into the procedures described, as well as our collaborators in Dr. Abraham Lee&#39;s lab for their assistance with platform design and fabrication. This work was supported by the following grants: UG3/UH3 TR002137, R61/R33 HL154307, 1R01CA244571, 1R01 HL149748, U54 CA217378 (CCWH) and TL1 TR001415 and W81XWH2110393 (SJH).

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

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CCWH has an equity interest in Aracari Biosciences, Inc., which is commercializing a version of the technology described in this paper. The terms of this arrangement have been reviewed and approved by the University of California, Irvine, in accordance with its conflict-of-interest policies. There are no other conflicts of interest.

Nearly every tissue in the body receives nutrients and oxygen through the vasculature, making it a critical component for realistic disease modeling and drug screening in vitro. Moreover, several malignancies and disease states are defined by vascular endothelial dysfunction and hyperpermeability3. Notably, in cancer, tumor-associated vasculature is often ill-perfused, disrupted, and leaky, thus acting as a barrier to therapeutic and immune cell delivery to the tumor. Furthermore, vasculature serves as a conduit through which cancer cells can metastasize to seed distant tissues and facilitates cell-cell communications that dampen the immune response while further promoting cancer cell growth and dissemination. These phenomena highlight the crucial role the vascular niche plays in therapeutic resistance and cancer progression and the need to accurately model the tumor microenvironment during preclinical study. Yet standard in vitro model systems fail to include appropriate stromal and vascular components or incorporate dynamic flow conditions. To address these shortcomings in current model systems, methods to establish a well-characterized microphysiological system that supports the formation of a living, perfused human micro-tumor (VMT) for physiologic oncology research were presented. Importantly, the VMT models key characteristics of aberrant tumor-associated vessels and tumor-stromal interactions, making it ideal for biomimetic disease modeling and therapeutic efficacy testing10.
For ease of use, the platform does not require any external pumps or valves and, owing to the 96-well plate format can be adapted to standard culture equipment and workflows. Further, different device iterations to address distinct biological questions and established tissue- and patient-specific compartments have been validated8,9,10,11,12,13,14,15,16,17. While the platform can be adapted to virtually any organ- or tissue-specific use by integrating various cell types, cells must be tested first for growth and vasculogenic capacity within the VMO/VMT at varying cell concentrations to determine the optimal seeding density and co-culture conditions. To establish the vasculature, human endothelial colony-forming cell-derived endothelial cells (ECFC-EC) can be purchased commercially or freshly isolated from cord blood by selecting CD31+ cells. Human umbilical vein endothelial cells (HUVEC) can also be used to establish vasculature within the VMO/VMT and can either be purchased commercially or freshly isolated from umbilical cords. Additionally, induced pluripotent stem cell-derived endothelial cells (iPSC-EC) have been successfully tested in the platform, opening the possibility of a completely autologous system18. Commercially derived fibroblasts (standard, normal human lung fibroblasts for their vasculogenic potential) work well in the VMO/VMT, and some primary-derived stromal cell populations can be incorporated or substituted as well. Primary-derived tumors can be introduced into the VMT as single cells, spheroids, organoids, or tumor chunks. Matrix composition can be modified according to experimental needs, including spiking with collagens, laminin, fibronectin, or even decellularized tissue matrices19.
The protocol includes several critical steps where special care is essential to avoid common issues (Figure 5). During loading, ensure homogeneous mixing of the cell/fibrin slurry by careful pipetting and smooth introduction into the tissue chamber (Figure 5A). Apply proper pressure to expel the gel completely into the chamber to prevent partial loading (Figure 5B). Visualizing the cell/fibrin slurry transversing the entire tissue chamber is necessary to ensure complete chamber filling and can be facilitated by placing a gloved finger behind the device unit. Avoid pressing too hard on the micropipette plunger to prevent bursting the cell/fibrin mix into the microfluidic channels (Figure 5C). Care must be taken not to introduce air bubbles during pipetting to prevent interference with tissue development and downstream applications (Figure 5D). Proper mixing and loading speed are crucial to avoid areas of inconsistent clotting (Figure 5E) while removing the pipette tip prematurely may also disrupt the gel in the chamber (Figure 5F). Practice loadings are recommended to familiarize users with the timed element of the procedure and the loading step. Further, properly introducing laminin into the microfluidic channels is crucial for EC migration, anastomosis with outer channels, and the formation of a continuous, perfusable network for nutrient delivery. Incomplete or absent channel lining will lead to poor perfusion results and unusable VMO/VMT.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65865/65865fig05v2.jpg" /> 
Figure 5. Common mistakes with loading. (A) Microfluidic device unit properly loaded without defects. (B) Cell/fibrin mix was not introduced completely into the chamber, resulting in partial loading. (C) Too much pressure applied during loading, resulting in gel bursting into the microfluidic channel, blocking flow. (D)&#160;Air bubbles introduced into the cell/fibrin mix within the chamber while pipetting. (E) Improper mixing of the cell/fibrin mix or slow loading causing inconsistencies in clotting. (F) Removing the pipette tip from the loading port before the gel has sufficiently set will result in disruption of the cell/fibrin mix in the tissue chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65865/65865fig05largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Robust and standardized workflows for analysis are crucial in VMO/VMT studies, as they generate substantial amounts of imaging data. Image processing and analytical methods for VMO/VMT have been described previously8,9,10,11,12,13. For tumor quantitative analysis, the fluorescent intensity in the color channel representing the tumor cells is measured using open-source software like ImageJ/Fiji (National Institute of Health)20 or CellProfiler (Broad Institute)21. The threshold of tumor micrographic images is set to select the fluorescent tumor region, and the mean fluorescence intensity is measured within that region. The tumor&#39;s total fluorescent intensity is calculated as the product of the fluorescent area and its mean intensity, normalized to baseline values (pre-treatment) to obtain the fold change in tumor growth per device over the experimental period. Regarding vessel quantitative analysis, AngioTool (National Cancer Institute)22, ImageJ/Fiji macro scripts, or MATLAB software, such as REAVER23, can be used to quantify total vessel length, number of endpoints, number of junctions, average vessel length, vessel diameter, mean lacunarity, and vessel percentage area. Machine learning algorithms can be integrated into workflows for automatic analysis of vascular images to identify compounds that effectively disrupt the vasculature24. Perfusion images are analyzed by measuring the change in fluorescence intensity within regions of the extracellular space and calculating the permeability coefficient10. Finite element simulations of intraluminal flow inside a microvascular network can be performed using COMSOL Multiphysics25.&#160;Implementation of standardized analytical methods is critical for extracting meaningful insights from the vast amount of data generated in VMT studies.
The protocol outlined here will allow the user to leverage the VMO/VMT platform to study many aspects of tumor biology, including tumor growth/progression, tumor metastasis, intra-tumor T cell dynamics, and tumor response to chemotherapy and anti-angiogenic treatment. To enable physiologically relevant immuno-oncology studies, it was demonstrated how freshly isolated T cells perfuse through the microvasculature, extravasate across the endothelial cell barrier, and migrate into the tissue construct. Time-lapse microscopic confocal imaging was presented as a tool to view spatially random, temporally rapid events, including T cell extravasation, that are not readily visualized with other model systems. In addition, we have previously tested multiple types of antineoplastic drugs in the VMT, including chemotherapeutics, small molecule/tyrosine kinase inhibitors, monoclonal antibodies (such as anti-PD1 and bevacizumab), anti-angiogenic compounds, and vascular stabilizing agents, underscoring how the platform can be used to test different classes of drugs targeting both the tumor and the associated stroma8,9,10,11,12,13. Effluent can be collected from the platform and analyzed for various cytokines as well as exosomes. In future studies, the VMT platform can be used to assess the sensitivity of tumor cells to T-cell-mediated attack at the individual patient level. In conclusion, the VMT is a flexible and powerful platform and one that is ideal for studying tumor biology, where remodeling of the vascular and stromal components is key to tumor progression.

Cancer remains a major health concern worldwide and is the second leading cause of death in the United States. For the year 2023 alone, the National Center for Health Statistics anticipates more than 1.9 million new cancer cases and over 600,000 cancer deaths occurring in the US1, highlighting the urgent need for effective treatment approaches. However, currently, only 5.1% of anti-cancer therapeutics entering clinical trials ultimately gain FDA approval. Failure of promising candidates to successfully progress through clinical trials can be partially attributed to the use of non-physiological model systems, such as 2D and spheroid cultures, during preclinical drug development2. These classical cancer models lack essential components of the tumor microenvironment, such as a stromal niche, associated immune cells, and perfused vasculature, which are key determinants of therapeutic resistance and disease progression. Thus, a new model system that better mimics the human in vivo tumor microenvironment is necessary to improve the clinical translation of preclinical findings.
The field of tissue engineering is rapidly advancing, providing improved methods for studying human diseases in laboratory settings. One significant development is the emergence of microphysiological systems (MPS), also known as organ chips or tissue chips, which are functional, miniaturized human organs capable of replicating healthy or diseased conditions3,4,5. Within this context, tumor chips, which are three-dimensional microfluidic-based in vitro human tumor models, have been developed for oncology research2,3,4,5,6,7,8,9,10,11,12,13. These advanced models incorporate biochemical and biophysical cues within a dynamic tumor microenvironment, enabling researchers to study tumor behavior and responses to treatments in a more physiologically relevant context. However, despite these advancements, few groups have successfully incorporated a living, functional vasculature, particularly one that self-patterns in response to physiologic flow3,4,5,6. The inclusion of a functional vascular network is crucial as it allows for modeling physical barriers that affect drug or cell delivery, cell homing to distinct microenvironments, and transendothelial migration of tumor, stromal, and immune cells. By including this feature, the tumor chip can better represent the complexities observed in the in vivo tumor microenvironment.
To address this unmet need, we have developed a novel drug-screening platform that enables micro-vessel networks to form within a microfluidic device8,9,10,11,12,13,14,15,16. This base organ chip platform, termed the vascularized micro-organ (VMO), can be adapted to virtually any organ system to replicate original tissue physiology for disease modeling, drug screening, and personalized medicine applications. VMOs are established by co-culturing endothelial colony-forming cell-derived endothelial cells (ECFC-EC), HUVEC or iPSC-EC (hereafter EC), and multiple stromal cells in the chamber, including normal human lung fibroblasts (NHLF), which remodel the matrix, and pericytes that wrap and stabilize the vessels. The VMO can also be established as a cancer model system by co-culturing tumor cells with the associated stroma to create a vascularized micro-tumor (VMT)8,9,10,11,12,13, or tumor chip, model. Through the co-culture of multiple cell types in a dynamic flow environment, perfused microvascular networks form de novo in the tissue chambers of the device, where vasculogenesis is closely regulated by interstitial flow rates14,15. Medium is driven through the microfluidic channels of the device by a hydrostatic pressure head that supplies the surrounding cells of the tissue chamber with nutrients exclusively through the micro-vessels, with a permeability coefficient of 1.2 x 10-7 cm/s, similar to what is seen for capillaries in vivo8.
The incorporation of self-organizing micro-vessels into the VMT model represents a significant breakthrough because it: 1) mimics the structure and function of vascularized tumor masses in vivo; 2) can model key steps of metastasis, including tumor-endothelial and stromal cell interactions; 3) establishes physiologically selective barriers for nutrient and drug delivery, improving pharmaceutical screening; and 4) allows direct assessment of drugs with anti-angiogenic and anti-metastatic capabilities. By replicating the in vivo delivery of nutrients, drugs, and immune cells in a complex 3D microenvironment, the VMO/VMT platform is a physiologically relevant model that can be used to perform drug screening and study cancer, vascular or organ-specific biology. Importantly, the VMT supports the growth of various types of tumors, including colon cancer, melanoma, breast cancer, glioblastoma, lung cancer, peritoneal carcinomatosis, ovarian cancer, and pancreatic cancer8,9,10,11,12,13. In addition to being low-cost, easily established, and arrayed for high throughput experiments, the microfluidic platform is fully optically compatible for real-time image analysis of tumor-stromal interactions and response to stimuli or therapeutics. Each cell type in the system is labeled with a different fluorescent marker to allow direct visualization and tracking of cell behavior throughout the entire experiment, creating a window into the dynamic tumor microenvironment. We have previously shown that the VMT more faithfully models in vivo tumor growth, architecture, heterogeneity, gene expression signatures, and drug responses than standard culture modalities10. Importantly, the VMT supports the growth and study of patient-derived cells, including cancer cells, which better models the pathology of the parent tumors than standard spheroid cultures and further advances personalized medicine efforts11. This manuscript outlines the methods for establishing the VMT, showcasing its utility for studying human cancers.

<table><tbody><tr><td>&lt;strong&gt;Fabrication&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(3-Mercaptopropyl)trimethoxysilane, 95%&amp;nbsp;</td><td>Sigma-Aldrich</td><td>175617-100G</td><td></td></tr><tr><td>Greiner Bio-One&amp;nbsp;&amp;mu;Clear Bottom 96-well Polystyrene Microplates</td><td>Greiner Bio-One</td><td>655096</td><td></td></tr><tr><td>Methanol &amp;ge;99.8% ACS</td><td>VWR Chemicals BDH</td><td>BDH1135-1LP</td><td></td></tr><tr><td>MILTEX Sterile Disposable Biopsy Punch with Plunger, 1mm diameter,</td><td>Integra Miltex</td><td>33-31AA-P/25</td><td></td></tr><tr><td>PDMS membrane</td><td>PAX Industries</td><td>HT-6240</td><td></td></tr><tr><td>Plasma Cleaner PDC-001</td><td>Harrick Plasma</td><td>N/A</td><td></td></tr><tr><td>Smooth-Cast 385</td><td>Smooth-On</td><td>N/A</td><td></td></tr><tr><td>SP Bel-Art Lab Companion Clear Polycarbonate Cabinet Style Vacuum Desiccator</td><td>Bel-Art</td><td>F42400-4031</td><td></td></tr><tr><td>Standard Lids with Condensation Rings, 96-well plate</td><td>VWR</td><td>82050-827</td><td></td></tr><tr><td>SYLGARD 184 Silicone Elastomer Kit (PDMS)</td><td>Dow</td><td>4019862</td><td></td></tr><tr><td>&lt;strong&gt;Cell culture/Loading&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BioTek Lionheart FX Automated Microscope</td><td>Agilent&amp;nbsp;</td><td>CYT5MFAW</td><td></td></tr><tr><td>CELLvo Human Endothelial Progenitor Cells</td><td>StemBioSys</td><td>N/A</td><td></td></tr><tr><td>Collagen I, rat tail</td><td>Enzo Life Sciences</td><td></td><td></td></tr><tr><td>Collagenase from &lt;em&gt;Clostridium histolyticum&lt;/em&gt; (type 4)</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>Corning Hank&amp;rsquo;s Balanced Salt Solution, 1X without calcium and magnesium</td><td>Corning</td><td>21-021-CV</td><td></td></tr><tr><td>Corning&amp;nbsp;DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate</td><td>Corning</td><td>10013CV</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>EGM-2 Endothelial Cell Growth Medium-2 BulletKit</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>Fibrinogen from bovine plasma</td><td>Neta Scientific</td><td>SIAL-341573</td><td></td></tr><tr><td>Fibronectin human plasma</td><td>Sigma-Aldrich</td><td>F0895</td><td></td></tr><tr><td>Fluorescein isothiocyanate&amp;ndash;dextran (70kDa)</td><td>Sigma-Aldrich</td><td>FD70S-1G</td><td></td></tr><tr><td>Gelatin from porcine skin</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>Hyaluronidase from sheep testes (type 4)</td><td>Sigma-Aldrich</td><td>H6254</td><td></td></tr><tr><td>Laminin Mouse Protein</td><td>Gibco</td><td>23017015</td><td></td></tr><tr><td>Leica TCS SP8</td><td>Leica</td><td>N/A</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC</td><td>HTB-26</td><td></td></tr><tr><td>NHLF &amp;ndash; Normal Human Lung Fibroblasts</td><td>Lonza</td><td>CC-2512</td><td></td></tr><tr><td>Nikon Eclipse Ti</td><td>Nikon</td><td>N/A</td><td></td></tr><tr><td>Paraformaldehyde 4% in 0.1M Phosphate BufferSaline, pH 7.4</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>15735-90-1L</td><td></td></tr><tr><td>PBMCs - Peripheral blood mononuclear cells</td><td>Lonza</td><td>CC-2702</td><td></td></tr><tr><td>PBS, pH 7.4</td><td>Gibco</td><td>10010049</td><td></td></tr><tr><td>Premium Grade Fetal Bovine Serum (FBS), Heat Inactivated</td><td>Avantor Seradigm</td><td>97068-091</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Invitrogen</td><td>P10144</td><td></td></tr><tr><td>Quick-RNA Microprep Kit</td><td>Zymo Research</td><td>R1051</td><td></td></tr><tr><td>Thrombin from bovine plasma</td><td>Sigma-Aldrich</td><td>T4648</td><td></td></tr><tr><td>Triton X-100 (Electrophoresis),</td><td>Fisher BioReagents</td><td>BP151-100</td><td></td></tr><tr><td>TrypLE Express Enzyme (1X), phenol red</td><td>Gibco</td><td>12605028</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco</td><td>25300062</td><td></td></tr><tr><td>Vasculife</td><td>Lifeline Cell Technology</td><td>LL-0003</td><td></td></tr></tbody></table>

establishing a physiologic human vascularized micro-tumor model for cancer research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Following the protocols outlined here, VMOs and VMTs were established using commercially purchased EC, NHLF, and, for VMT, the triple-negative breast cancer cell line MDA-MB-231. Established VMOs were also perfused with cancer cells to mimic metastasis. In each model, by day 5 of co-culture, a vascular network self-assembles in response to gravity-driven flow across the tissue chamber, serving as a conduit for in vivo like delivery of nutrients, therapeutics, and cancer or immune cells to the stromal niche (Figure 4). VMOs were first established by introducing mCherry-labeled EC into the tissue chamber, as shown in Figure 4A (day 0 of culture), with an even distribution of cells. On day 2 of VMO culture, EC begin to stretch out and lumenize (Figure 4B), and by day 4, EC have anastomosed with the outer microfluidic channels and form a continuous vascular network (Figure 4C). After the vasculature formed anastomoses and lined the outer channels, VMO tissue was perfused with 70 kD FITC-dextran to confirm vascular patency (Figure 4D). FITC-dextran was introduced into the media reservoir with the highest hydrostatic pressure and allowed to perfuse across the tissue chamber via micro-vessels from the high-pressure side to the low-pressure side, as indicated by the arrows. In the VMO, FITC-dextran fully perfused the microvascular network within 15 min with minimal vascular leak, confirming tight vascular barrier function (Figure 4E). MDA-MB-231 cells were then perfused into VMO, where cells adhered to the endothelial lining (Figure 4F) and extravasated into the extra-vascular space within 24 h post-perfusion, forming multiple micro-metastases within the tissue chamber (Figure 4G). Time-lapse microscopic fluorescent images were taken every 50 ms with 4x and 10x air objectives on an inverted confocal microscope to observe cancer cells perfusing through the microvasculature in real-time (Supplementary Video 1, Supplementary Video 2).
In the VMO, T cells can be seen extravasating into the extracellular space over the course of 45 min (Figure 4H-I). Time-lapse fluorescent micrographs were taken on a confocal microscope to acquire a z-stack of 150 &#181;m depth every 15 min to observe T cell extravasation in real time (Supplementary Video 3). As shown in Figure 4J, MDA-MB-231 VMT with fully formed, non-leaky vessels were perfused with T cells (yellow), many of which rapidly adhered to the vascular wall (arrowheads; Figure 4K, Supplementary Video 4, Supplementary Video 5). These results, in addition to prior studies8,9,10,11,12,13,14,15, demonstrate the utility of the VMO and VMT platforms for immunology and immune-oncology research, respectively.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65865/65865fig04.jpg" /> 
Figure 4. Representative results for MDA-MB-231 VMT and VMO. (A)&#160;VMO on day 0 immediately after loading cells into the tissue chamber. EC are shown in red. Scale bar = 500 &#181;m. (B) By day 2 of VMO culture, EC begin to stretch in response to flow. (C)&#160;VMO day 4 shows that the vascular network is anastomosed with the outer microfluidic channels, and vessels are nearly mature. (D)&#160;VMO networks are fully perfused and patent on day 5 of culture. Vessels shown in red, 70 kD FITC-dextran green. The direction of flow is indicated by arrows. Scale bar = 500 &#181;m. (E) Zoom view of perfused VMO. Scale bar = 100 &#181;m. (F) MDA-MB-231 (cyan) is perfused through the same VMO network shown in E, and at time 0, cancer cells have adhered to the endothelial vessel lining (arrowheads). Scale bar = 100 &#181;m. (G) By 24 h, MDA-MB-231 cells have extravasated into the extracellular space, establishing multiple micro-metastases within the vascular niche. Scale bar = 100 &#181;m. (H) Time-lapse confocal fluorescent microscopy reveals T cell extravasation through a micro-vessel within the VMO (I) over the course of 45 min. Arrowheads denote areas of extravasation. Scale bar = 50 &#181;m. (J) Triple-negative breast cancer cell line MDA-MB-231 is established in the VMT and perfused on day 5. Scale bar = 500 &#181;m. The vascular network shows minimal leak at 15 min post-perfusion (inset, scale bar = 100 &#181;m). (K) MDA-MB-231 VMT (same as in B) is perfused with T cells (yellow), with multiple areas of T cell adherence to the vascular wall (arrowheads). Scale bar = 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65865/65865fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1. Perfusion of ovarian cancer cells in the VMO. Time-lapse fluorescent microscopy of COV362&#160;cells (cyan) perfused through a vascular network (red) and imaged at 4x objective every 50 ms for 1 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65865/supplemental_video_1.zip" target="_blank">Please click here to download of this Video.</a>
Supplementary Video 2. Perfusion of triple-negative breast cancer cells in the VMO. Time-lapse fluorescent microscopy of MDA-MB-231 cells (cyan) perfused through a vascular network (red) and imaged at 10x objective every 50 ms for 30 s. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65865/supplemental_video_2.zip" target="_blank">Please click here to download of this Video.</a>
Supplementary Video 3. T cell perfusion of VMO. Time-lapse confocal fluorescent microscopy captured the process of T cell extravasation through a micro-vessel within the VMO over a 45 min duration. Z-stack images were obtained every 15 min, with a step size of 2 &#181;m and a depth of 150 &#181;m. The vessel is red, T cells are yellow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65865/supplemental_video_3.zip" target="_blank">Please click here to download of this Video.</a>
Supplementary Video 4. T cell perfusion of VMT. Time-lapse fluorescent&#160;microscopy of T cells perfused through MDA-MB-231 VMT at 4x objective. Images were acquired every 50 ms for 30 s. T cells are shown in yellow, MDA-MB-231 in cyan, and vessels/EC in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65865/supplemental_video_4.zip" target="_blank">Please click here to download of this Video.</a>
Supplementary Video 5. Zoom view of T cell-VMT perfusion. Magnified 10x view of MDA-MB-231 VMT perfused with T cells (from Supplementary Video 4). Images were acquired every 50 ms for 30 s. T cells are shown in yellow, MDA-MB-231 in cyan, and vessels/EC in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65865/supplemental_video_5.zip" target="_blank">Please click here to download of this Video.</a>

Watch this Scientific Journal Video about Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research at JoVE.com

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

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Nanyang Technological University

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JoVE Journal

Pharmacokinetics and Pharmacodynamics

Cancer Research

Unit 1

Pharmacokinetics: Drug Biotransformation

SCIENTISTS IN ACTION

1.5K Views.  University of California, Irvine. This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures. 

Video: Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Bioengineering

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional veins. The purpose of this article is to carefully describe the establishment of a murine xenograft model that mimics human VM and is able to reflect patient heterogeneity. Hyper-activating non-inherited (somatic) TEK (TIE2) and PIK3CA mutations in endothelial cells (EC) have been identified as the main drivers of pathological vessel enlargement in VM. The following protocol describes the isolation, purification and expansion of patient-derived EC expressing mutant TIE2 and/or PIK3CA. These EC are injected subcutaneously into the back of immunodeficient athymic mice to generate ectatic vascular channels. Lesions generated with TIE2 or PIK3CA-mutant EC are visibly vascularized within 7&#8210;9 days of injection and recapitulate histopathological features of VM patient tissue. This VM xenograft model provides a reliable platform to investigate the cellular and molecular mechanisms driving VM formation and expansion. In addition, this model will be instrumental for translational studies testing the efficacy of novel drug candidates in preventing the abnormal vessel enlargement seen in human VM.

We present a detailed protocol to generate a murine xenograft model of venous malformation. This model is based on the subcutaneous injection of patient-derived endothelial cells containing hyper-activating TIE2 and/or PIK3CA gene mutations. Xenograft lesions closely recapitulate the histopathological features of VM patient tissue.

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

Developmental Biology

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Medicine

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

Summary

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Chapters in this video

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Tissue Engineering of a Human 3D in vitro Tumor Test System

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A Patient-Derived Xenograft Model for Venous Malformation

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Bioengineering Human Microvascular Networks in Immunodeficient Mice