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...

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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 ...