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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

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

Published: September 15th, 2023

DOI:

10.3791/65865

1Molecular Biology and Biochemistry, University of California, Irvine, 2Biomedical Engineering, 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.

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. 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.

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, du....

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1. Design and fabrication

  1. Device design
    1. For microfluidic device fabrication, create an SU-8 mold using a 200 µm 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.
    2. Cast a 4 mm thick polydimethylsiloxane (PDMS) replica from the SU-8 mold to generate a durable polyurethane mold for downstre.......

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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 (

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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.......

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We thank members of Dr. Christopher Hughes' lab for their valued input into the procedures described, as well as our collaborators in Dr. Abraham Lee'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).

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NameCompanyCatalog NumberComments
Fabrication
(3-Mercaptopropyl)trimethoxysilane, 95% Sigma-Aldrich175617-100G
Greiner Bio-One μClear Bottom 96-well Polystyrene MicroplatesGreiner Bio-One655096
Methanol ≥99.8% ACSVWR Chemicals BDHBDH1135-1LP
MILTEX Sterile Disposable Biopsy Punch with Plunger, 1mm diameter,Integra Miltex33-31AA-P/25
PDMS membranePAX IndustriesHT-6240
Plasma Cleaner PDC-001Harrick PlasmaN/A
Smooth-Cast 385Smooth-OnN/A
SP Bel-Art Lab Companion Clear Polycarbonate Cabinet Style Vacuum DesiccatorBel-ArtF42400-4031
Standard Lids with Condensation Rings, 96-well plateVWR82050-827
SYLGARD 184 Silicone Elastomer Kit (PDMS)Dow4019862
Cell culture/Loading
BioTek Lionheart FX Automated MicroscopeAgilent CYT5MFAW
CELLvo Human Endothelial Progenitor CellsStemBioSysN/A
Collagen I, rat tailEnzo Life Sciences
Collagenase from Clostridium histolyticum (type 4)Sigma-AldrichC5138
Corning Hank’s Balanced Salt Solution, 1X without calcium and magnesiumCorning21-021-CV
Corning DMEM with L-Glutamine, 4.5g/L Glucose and Sodium PyruvateCorning10013CV
DAPISigma-AldrichD9542
DPBS, no calcium, no magnesiumGibco14190144
EGM-2 Endothelial Cell Growth Medium-2 BulletKitLonzaCC-3162
Fibrinogen from bovine plasmaNeta ScientificSIAL-341573
Fibronectin human plasmaSigma-AldrichF0895
Fluorescein isothiocyanate–dextran (70kDa)Sigma-AldrichFD70S-1G
Gelatin from porcine skinSigma-AldrichG1890
Hyaluronidase from sheep testes (type 4)Sigma-AldrichH6254
Laminin Mouse ProteinGibco23017015
Leica TCS SP8LeicaN/A
MDA-MB-231ATCCHTB-26
NHLF – Normal Human Lung FibroblastsLonzaCC-2512
Nikon Eclipse TiNikonN/A
Paraformaldehyde 4% in 0.1M Phosphate BufferSaline, pH 7.4Electron Microscopy Sciences 15735-90-1L
PBMCs - Peripheral blood mononuclear cellsLonzaCC-2702
PBS, pH 7.4Gibco10010049
Premium Grade Fetal Bovine Serum (FBS), Heat InactivatedAvantor Seradigm97068-091
ProLong Gold Antifade MountantInvitrogenP10144
Quick-RNA Microprep KitZymo ResearchR1051
Thrombin from bovine plasmaSigma-AldrichT4648
Triton X-100 (Electrophoresis),Fisher BioReagentsBP151-100
TrypLE Express Enzyme (1X), phenol redGibco12605028
Trypsin-EDTA (0.05%), phenol redGibco25300062
VasculifeLifeline Cell TechnologyLL-0003

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