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).

1. Design and fabrication
<ol>
	<li>Device design
	<ol>
		<li>For microfluidic device fabrication, create an SU-8 mold using a 200 &#181;m layer of SU-8 spin-coated onto a Si-wafer (RCA-1 cleaned and 2% hydrogen fluoride (HF) treated), followed by a single mask photolithography step as described previously8,9.</li>
		<li>Cast a 4 mm thick polydimethylsiloxane (PDMS) replica from the SU-8 mold to generate a durable polyurethane mold for downstre...

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

<ol>
	<li>Siegel, R. L., Miller, K. D., Wagle, N. S., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2023.">Cancer statistics, 2023.</a> CA Cancer J Clin. 73 (1), 17-48 (2023).</li><li>Hachey, S. J., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+tumor+chip+technology.">Applications of tumor chip technology.</a> Lab Chip. 18 (19), 2893-2912 (2018).</li><li>Ewald, M. L., Chen, Y. H., Lee, A. P., Hughes, C. C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+niche+in+next+generation+microphysiological+systems.">The vascular niche in next generation microphysiological systems.</a> Lab Chip. 21 (17), 3615-3616 (2021).</li><li>Osaki, T., Sivathanu, V., Kamm, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularized+microfluidic+organ-chips+for+drug+screening,+disease+models+and+tissue+engineering.">Vascularized microfluidic organ-chips for drug screening, disease models and tissue engineering.</a> Curr Opin Biotechnol. 52, 116-123 (2018).</li><li>Shirure, V. S., Hughes, C. C. W., George, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+vascularized+organoid-on-a-chip+models.">Engineering vascularized organoid-on-a-chip models.</a> Annu Rev Biomed Eng. 23, 141-167 (2021).</li><li>Del Piccolo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-on-chip+modeling+of+organ-specific+cancer+and+metastasis.">Tumor-on-chip modeling of organ-specific cancer and metastasis.</a> Adv Drug Deliv Rev. 175, 113798 (2021).</li><li>Sontheimer-Phelps, A., Hassell, B. A., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+cancer+in+microfluidic+human+organs-on-chips.">Modelling cancer in microfluidic human organs-on-chips.</a> Nat Rev Cancer. 19 (2), 65-81 (2019).</li><li>Sobrino, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+microtumors+in+vitro+supported+by+perfused+vascular+networks.">3D microtumors in vitro supported by perfused vascular networks.</a> Sci Rep. 6, 31589 (2016).</li><li>Phan, D. T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vascularized+and+perfused+organ-on-a-chip+platform+for+large-scale+drug+screening+applications.">A vascularized and perfused organ-on-a-chip platform for large-scale drug screening applications.</a> Lab Chip. 17 (3), 511-520 (2017).</li><li>Hachey, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+vascularized+micro-tumor+model+of+human+colorectal+cancer+recapitulates+in+vivo+responses+to+standard-of-care+therapy.">An in vitro vascularized micro-tumor model of human colorectal cancer recapitulates in vivo responses to standard-of-care therapy.</a> Lab Chip. 21 (7), 1333-1351 (2021).</li><li>Hachey, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Human+Vascularized+Micro-Tumor+Model+of+Patient-Derived+Colorectal+Cancer+Recapitulates+Clinical+Disease.">A Human Vascularized Micro-Tumor Model of Patient-Derived Colorectal Cancer Recapitulates Clinical Disease.</a> Transl Res. 255, 97-108 (2023).</li><li>Liu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+in+vitro+vascularized+micro-organ+and+micro-tumor+models+are+reproducible+organ-on-a-chip+platforms+for+studies+of+anticancer+drugs.">Human in vitro vascularized micro-organ and micro-tumor models are reproducible organ-on-a-chip platforms for studies of anticancer drugs.</a> Toxicology. 445, 152601 (2020).</li><li>Jahid, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-based+Design+of+CDC42+Effector+Interaction+Inhibitors+for+the+Treatment+of+Cancer.">Structure-based Design of CDC42 Effector Interaction Inhibitors for the Treatment of Cancer.</a> Cell Rep. 39 (4), 110760 (2022).</li><li>Hsu, Y. H., Moya, M. L., Hughes, C. C. W., George, S. C., Lee, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+platform+for+generating+large-scale+nearly+identical+human+microphysiological+vascularized+tissue+arrays.">A microfluidic platform for generating large-scale nearly identical human microphysiological vascularized tissue arrays.</a> Lab Chip. 13 (15), 2990-2998 (2013).</li><li>Moya, M. L., Hsu, Y. H., Lee, A. P., Christopher, C. W. H., George, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+perfused+human+capillary+networks.">In vitro perfused human capillary networks.</a> Tissue Eng - Part C: Methods. 19 (9), 730-737 (2013).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+on-chip+microfluidic+pressure+regulator+that+facilitates+reproducible+loading+of+cells+and+hydrogels+into+microphysiological+system+platforms.">An on-chip microfluidic pressure regulator that facilitates reproducible loading of cells and hydrogels into microphysiological system platforms.</a> Lab Chip. 16 (5), 868-876 (2016).</li><li>Phan, D. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier-on-a-chip:+Microphysiological+systems+that+capture+the+complexity+of+the+blood-central+nervous+system+interface.">Blood-brain barrier-on-a-chip: Microphysiological systems that capture the complexity of the blood-central nervous system interface.</a> Exp Biol Med. 242 (17), 1669-1678 (2017).</li><li>Kurokawa, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cell-derived+endothelial+cells+for+three-dimensional+microphysiological+systems.">Human induced pluripotent stem cell-derived endothelial cells for three-dimensional microphysiological systems.</a> Tissue Eng Part C: Methods. 23 (8), 474-484 (2017).</li><li>Romero-L&#243;pez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recapitulating+the+human+tumor+microenvironment:+Colon+tumor-derived+extracellular+matrix+promotes+angiogenesis+and+tumor+cell+growth.">Recapitulating the human tumor microenvironment: Colon tumor-derived extracellular matrix promotes angiogenesis and tumor cell growth.</a> Biomaterials. 116, 118-129 (2017).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Carpenter, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+Image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: Image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7 (10), R100 (2006).</li><li>Zudaire, E., Gambardella, L., Kurcz, C., Vermeren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+tool+for+quantitative+analysis+of+vascular+networks.">A computational tool for quantitative analysis of vascular networks.</a> PLoS one. 6 (11), e27385 (2011).</li><li>Corliss, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REAVER:+A+program+for+improved+analysis+of+high-resolution+vascular+network+images.">REAVER: A program for improved analysis of high-resolution vascular network images.</a> Microcirculation. 27 (5), e12618 (2020).</li><li>Urban, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+learning+for+drug+discovery+and+cancer+research:+Automated+analysis+of+vascularization+images.">Deep learning for drug discovery and cancer research: Automated analysis of vascularization images.</a> IEEE/ACM Trans Comput Biol Bioinform. 16 (3), 1029-1035 (2019).</li></ol>

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, vascula...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Fabrication</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>(3-Mercaptopropyl)trimethoxysilane, 95%&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>175617-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Greiner Bio-One&#160;&#956;Clear Bottom 96-well Polystyrene Microplates</td>
			<td>Greiner Bio-One</td>
			<td>655096</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol &#8805;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>Cell culture/Loading</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioTek Lionheart FX Automated Microscope</td>
			<td>Agilent&#160;</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 Clostridium histolyticum (type 4)</td>
			<td>Sigma-Aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Hank&#8217;s Balanced Salt Solution, 1X without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-021-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning&#160;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&#8211;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 &#8211; 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&#160;</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.

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

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

Our tumor-on-a-chip platform was developed to physiologically model the growth of human vascularized micro tumors, and allows us to answer both basic and translational research questions. Questions that focus on basic tumor biology, drug efficacy, tumor responsiveness, underlying immunologic processes, potential immunotherapeutic intervention, and personalized therapy approaches. Effectively translating preclinical discoveries into potent cancer treatments hinges on faithfully replicating the disease state and model systems.This protocol outlines the establishment of the vascularized micro tumor, or VMT, to mimic the tumor microenvironment in vitro. The VMT faithfully reproduces key aspects of a human tumor, yielding clinically relevant findings. Our model allows us to perform physiologically relevant, translational human cancer research.Specifically, our protocol, in contrast to other models, incorporates dynamic flow conditions, enables the integration of immune cells and immunotherapy, and further allows us for the incorporation of personalized medicine approaches. We are employing the VMT model to propel personalized medicine initiatives forward in both preclinical and clinical arenas. This model deepens our understanding of microenvironmental factors in cell-cell interactions, influencing therapeutic resistance and cancer progression.Moreover, the VMT shows promise in discovering customized patient-specific treatments for enhanced clinical care.

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 (<str...

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

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.

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

establishing-physiologic-human-vascularized-micro-tumor-model-for

Research

JoVE Journal

Cancer Research

1.2K ビュー.  University of California, Irvine. 当社の腫瘍オンチッププラットフォームは、ヒト血管新生微小腫瘍の増殖を生理学的にモデル化するために開発され、基礎研究とトランスレーショナル研究の両方の疑問に答えることができます。基本的な腫瘍生物学、薬効、腫瘍反応性、根底にある免疫学的プロセス、潜在的な免疫療法的介入、および個別化治療アプローチに焦点を当てた質問。前臨床の発見を強力?...

ビデオ: 著者スポットライト:トランスレーショナルがん研究のモデルとしてのヒト血管新生微小腫瘍の作成 

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.

https://cloudfront.jove.com/CDNSource/teasers/65865_b.jpg

orthotopic transplantation of breast tumors as preclinical models for breast cancer

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug...

working with human tissues for translational cancer research

Working with Human Tissues for Translational Cancer Research

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited...

preparing a vascularized micro-tumor system using human cells for studying tumors and drug responses 

Preparing a Vascularized Micro-Tumor System Using Human Cells for Studying Tumors and Drug Responses   

creating anatomically accurate and reproducible intracranial xenografts of human brain tumors

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

a modified heterotopic swine hind limb transplant model for translational vascularized composite allotransplantation (vca) research

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation VCA Research

Vascularized Composite Allotransplantations (VCA) have become a clinical reality. However, broad clinical application of VCA is limited by chronic multi-drug immunosuppression. The authors present a reliable and reproducible large animal model to translate novel immunomodulatory strategies that can minimize or potentially eliminate the need of immunosuppression in...

A Modified Heterotopic Swine Hind Limb Transplant Model for Translational Vascularized Composite Allotransplantation (VCA) Research

modified heterotopic hindlimb osteomyocutaneous flap model in the rat for translational vascularized composite allotransplantation research

Modified Heterotopic Hindlimb Osteomyocutaneous Flap Model in the Rat for Translational Vascularized Composite Allotransplantation Research

Vascularized composite allograft offers life-altering benefits to transplant recipients, but the biological causes of graft rejection and vasculopathy remain poorly understood. The rodent surgical model presented here offers a reproducible, clinically relevant model of transplantation, allowing researchers to evaluate rejection events and potential therapeutic strategies to prevent their...

venous thrombosis assay in a mouse model of cancer

Author Spotlight: Advancing Cancer Associated Thrombosis Research in Rodent Models 

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous...

Optimized Venous Thrombosis Assay in a Mouse Cancer Model Using Vascular Clips

https://cloudfront.jove.com/CDNSource/teasers/65518_b.jpg

orienteering as a tool for cognitive research: an implementation guide

Author Spotlight: Utilizing Orienteering in Research to Improve Brain Health and Spatial Memory

The sport of orienteering is emerging as an effective way to train both the body and brain as it combines physical activity with spatial navigation. The current manuscript offers a guide on how to implement the sport of orienteering in...

Implementing Orienteering for Body and Brain Training 

https://cloudfront.jove.com/CDNSource/teasers/66833_b.jpg

establishment of patient-derived xenograft mouse model with human osteosarcoma tissues

Author Spotlight: Replicating Human Osteosarcoma Progression in Immunodeficient Mice for Cancer Study

The present protocol describes the method for establishing a patient-derived xenograft (PDX) mouse model using human osteosarcoma...

Protocol for Osteosarcoma PDX Mouse Models: Studying Growth and Drug Resistance

https://cloudfront.jove.com/CDNSource/teasers/66491_b.jpg

testing cancer immunotherapeutics in a humanized mouse model bearing human tumors

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the...

engineering of human blood-induced microglia-like cells for reverse-translational brain research

Author Spotlight: Induced Microglia-Like Cell Technology to Shed Light on the Role of Microglial Dysfunction in Neuropsychiatric Disorders

This study delineates a novel approach for the establishment of human monocyte-derived microglia-like (iMG) cells that enable the indirect assessment of brain inflammation. This presents a cellular model that may be beneficial to research focusing on potential inflammation of the brain and associated neuropsychiatric...

Engineering of Human Blood-Induced Microglia-like Cells for Reverse-Translational Brain Research

generation of comprehensive thoracic oncology database - tool for translational research

Generation of Comprehensive Thoracic Oncology Database - Tool for Translational Research

A thoracic oncology database was developed to serve as a comprehensive repository for clinical and laboratory data for the purposes of translational research. The database will serve translational cancer researchers within the Thoracic Oncology Research Program. This database is adaptable to other cancer models, as well as other human...

a human peripheral blood mononuclear cell (pbmc) engrafted humanized xenograft model for translational immuno-oncology (i-o) research

A Human Peripheral Blood Mononuclear Cell PBMC Engrafted Humanized Xenograft Model for Translational Immuno-oncology I-O Research

We describe a human peripheral blood mononuclear cell (PBMC) &#8212; based humanized xenograft mouse model for translational immuno-oncology research. This protocol could serve as a general guideline for establishing and characterizing similar models for I-O therapy...

A Human Peripheral Blood Mononuclear Cell (PBMC) Engrafted Humanized Xenograft Model for Translational Immuno-oncology (I-O) Research

translational orthotopic models of glioblastoma multiforme

Translational Orthotopic Models of Glioblastoma Multiforme

Here, we describe a preclinical orthotopic mouse model for GBM, established by intracranial injection of cells derived from genetically engineered mouse model tumors. This model displays the disease hallmarks of human GBM. For translational studies, the mouse brain tumor is tracked by in vivo MRI and histopathology.

creating matched in vivo/in vitro patient-derived model pairs of pdx and pdx-derived organoids for cancer pharmacology research

Creating Matched In vivo/In vitro Patient-Derived Model Pairs of PDX and PDX-Derived Organoids for Cancer Pharmacology Research

A method is described to create organoids using patient-derived xenografts (PDX) for in vitro screening, resulting in matched pairs of in vivo/in vitro models. PDX tumors were harvested/processed into small pieces mechanically or enzymatically, followed by the Clevers&#8217; method to grow tumor organoids that were passaged, cryopreserved and characterized against the original...

circulating tumor cell lines: an innovative tool for fundamental and translational research

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

robust detection of gene amplification in formalin-fixed paraffin-embedded samples by fluorescence in situ hybridization

Author Spotlight: FISH as a Tool for Precise Gene Amplification Assessment in Cancer Specimens

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

Enhancing FISH Analysis for Oncogenic Drivers in Cancer Tissue Samples

https://cloudfront.jove.com/CDNSource/teasers/66978_b.jpg

a hepatocellular cancer patient&#45;derived organoid xenograft model to investigate impact of liver regeneration on tumor growth

Author Spotlight: Hepatocellular Carcinoma Research Through Patient&amp;#45;Derived Organoid Engraftment in Liver Regeneration Models

Organoids generated from patient tumors are orthotopically injected into the mouse liver. The resection of non-tumorous liver tissue leads to a regenerative environment in the liver tissue where the tumor is...

Patient&amp;#45;Derived Hepatocarcinoma Cell Organoids for Liver Regeneration Study

https://cloudfront.jove.com/CDNSource/teasers/66245_b.jpg

To begin, place the HBSS cell dissociation reagent EGM-2 and DMEM in a 37 degree Celsius water bath for 10 to 15 minutes. Thaw thrombin and laminin overnight at 4 degrees Celsius and fibrinogen at room temperature. Add 1.5 microliters of thrombin into a 500 microliter microcentrifuge tube.Place a UV sterilized high-throughput plates into a desiccator for 30 minutes to remove air trapped in the microfluidics. Place the T75 flask under the microscope at 4x magnification to confirm cell confluency and transduction efficiency. Wash the cells twice with five milliliters of HBSS.Then, add one milliliter of dissociation reagent to the flask and incubate at 37 degrees Celsius with 5%carbon dioxide for one to two minutes. Gently tap the flask using the palm and observe under the microscope that all cells have been lifted. Wash the cells with nine milliliters of appropriate media and collect suspension to a 15-milliliter conical tube.Immediately remove a small aliquot and count the cells. Centrifuge the cell suspension at 300G and four degrees Celsius for three to five minutes. Then, remove the supernatant and resuspend the pellet in appropriate media on ice.Using the given equation, calculate the number of cells needed. Resuspend the cells at a concentration of 1 times 10 to the 6 cells per milliliter, and calculate the volume of cells needed using the following equation. Centrifuge the required volume of cell suspension as demonstrated.Then, in a calculated volume of fibrinogen, resuspend the pellet on ice.

Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System

To begin, place the UV-sterilized high-throughput plates and thrombin aliquots into the tissue culture hood. Place the prepared cell fibrin mix on ice to slow clotting. Using a P20 pipette, draw six microliters of the cell fibrin mix.Then gently place the pipette tip into the thrombin aliquot and mix twice without introducing air bubbles. Lift the high-throughput plate at an angle and quickly insert the pipette tip into one of the loading ports. Push the pipette plunger to the first stop in a smooth and fluid motion and inject the cell fibrin mix into the tissue chamber.Ensure the gel crosses entirely through the chamber. Gently place the plate flat without removing the pipette tip or displacing the pipette. Remove the pipette tip using a hand from the P20 and leave it in the loading port hole.After two minutes, remove the pipette tips with gentle twisting motions from the loading ports and place the lid on the plate. Incubate the plate for 15 to 20 minutes at 37 degrees Celsius for gel polymerization. Place the microfluidic device on the microscope stage to observe the even distribution of cells throughout the chamber without any air bubbles.Insert the P20 pipette tip into M1 or M3 and expel four microliters of laminin slowly to coat the entire top panel. Remove the pipette tip as demonstrated previously and incubate the plate at 37 degrees Celsius in 5%carbon dioxide for 10 minutes. Add 275 microliters of EGM-2 complete media to the uncoupled reservoirs of wells located in rows A and B.Insert the P200 pipette tip into the media inlet hole of the wells containing 275 microliters of EGM-2 and slowly expel 75 microliters of medium, watching the media travel through the channel and bubble up on the other side.After removing the pipette tip, push the remaining media from the tip into the media reservoir. Add 50 microliters of media to entirely cover the low side wells in rows G and H.Incubate the plates for one to two hours as demonstrated. Under a microscope, identify any bubbles in the media channels and remove them by reintroducing 75 microliters of media into the channels.Check medium inlets or outlets for air bubbles using the naked eye. Then insert the P200 pipette tip into the hole and pull the bubble out by lifting the plunger. In vascularized micro-organs or VMOs, endothelial cells initially distributed evenly within the tissue chamber but by day two, they began to stretch out and luminize.By day four, the endothelial cells anastomosed with the outer microfluidic channels, forming a continuous vascular network. The tight vascular barrier function was confirmed by perfusing FITC-dextran through the microvascular network with minimal leakage. Perfusing MDA-MB-231 cancer cells into the VMOs resulted in their adherence to the endothelial lining and subsequent extravasation into the extracellular space, forming multiple micrometastases within 24 hours post-perfusion.Through time-lapse microscopic imaging, the extravasation of T-cells into the extracellular space of the VMOs was observed over 45 minutes. In vascularized micro-tumors with fully-formed vessels, T-cells rapidly adhered to the vascular wall upon perfusion.