Name: Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System
Uploaded: 2023-09-15T14:50:00
Description: Watch this Scientific Journal Video about Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System at JoVE.com

introducing human cells into a microfluidic device to investigate tumor progression in a vascularized micro-tumor system

Utarbeidelse av en mikrofluidikkbasert vaskularisert human mikrotumormodell

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.

Forfatter Spotlight: Å lage humane vaskulariserte mikrosvulster som modeller for translasjonell kreftforskning 

Etablering av en fysiologisk human vaskularisert mikrotumormodell for kreftforskning

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.

Watch this Scientific Journal Video about Introducing Human Cells into a Microfluidic Device to Investigate Tumor Progression in a Vascularized Micro-Tumor System at JoVE.com

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

Introduksjon av humane celler i en mikrofluidisk enhet for å undersøke tumorprogresjon i et vaskularisert mikrotumorsystem

For å begynne, plasser UV-steriliserte plater med høy gjennomstrømning og trombinalikoter i vevskulturhetten. Legg den tilberedte cellefibrinblandingen på is for å bremse koaguleringen. Bruk en P20-pipette til å trekke seks mikroliter av cellefibrinblandingen.Legg deretter pipettespissen forsiktig i trombinalikvinen og bland to ganger uten å introdusere luftbobler. Løft platen med høy gjennomstrømning i vinkel og sett pipettespissen raskt inn i en av lasteportene. Skyv pipettestempelet til første stopp i en jevn og flytende bevegelse og injiser cellefibrinblandingen inn i vevskammeret.Sørg for at gelen krysser helt gjennom kammeret. Legg platen flatt uten å fjerne pipettespissen eller forskyve pipetten. Fjern pipettespissen med en hånd fra P20 og la den ligge i lasteporthullet.Etter to minutter, fjern pipettespissene med forsiktige vridningsbevegelser fra lasteportene og legg lokket på platen. Inkuber platen i 15 til 20 minutter ved 37 grader Celsius for gelpolymerisering. Plasser den mikrofluidiske enheten på mikroskoptrinnet for å observere jevn fordeling av celler gjennom kammeret uten luftbobler.Sett P20-pipettespissen inn i M1 eller M3 og fjern fire mikroliter laminin sakte for å belegge hele topppanelet. Fjern pipettespissen som vist tidligere, og inkuber platen ved 37 grader Celsius i 5 % karbondioksid i 10 minutter. Tilsett 275 mikroliter EGM-2 komplette medier til de frakoblede reservoarene av brønner som ligger i rad A og B.Sett inn P200-pipettespissen i medieinnløpshullet i brønnene som inneholder 275 mikroliter EGM-2 og sakte utvise 75 mikroliter medium, se media reise gjennom kanalen og boble opp på den andre siden.Etter at pipettespissen er fjernet, skyves det gjenværende mediet fra spissen inn i mediebeholderen. Tilsett 50 mikroliter medier for å dekke de lave sidebrønnene helt i rad G og H.Inkuber platene i en til to timer som demonstrert. Under et mikroskop, identifiser eventuelle bobler i mediekanalene og fjern dem ved å gjeninnføre 75 mikroliter media i kanalene.Kontroller middels inntak eller utløp for luftbobler med det blotte øye. Sett deretter P200-pipettespissen inn i hullet og trekk boblen ut ved å løfte stempelet. I vaskulariserte mikroorganer eller VMOer fordeles endotelceller i utgangspunktet jevnt i vevkammeret, men på dag to begynte de å strekke seg ut og luminisere.Ved dag fire anastomoserte endotelcellene med de ytre mikrofluidiske kanalene, og dannet et kontinuerlig vaskulært nettverk. Den stramme vaskulære barrierefunksjonen ble bekreftet ved å perfusere FITC-dextran gjennom det mikrovaskulære nettverket med minimal lekkasje. Perfusing MDA-MB-231 kreftceller i VMOene resulterte i deres adherens til endotelforingen og påfølgende ekstravasasjon i det ekstracellulære rommet, og dannet flere mikrometastaser innen 24 timer etter perfusjon.Gjennom time-lapse mikroskopisk avbildning ble ekstravasasjonen av T-celler i det ekstracellulære rommet til VMOene observert over 45 minutter. I vaskulariserte mikrotumorer med fullt dannede kar festet T-celler seg raskt til vaskulærveggen ved perfusjon.

Research

JoVE Journal

Cancer Research

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

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 ...