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

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

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.

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

Kanser Araştırmaları için Fizyolojik İnsan Vaskülarize Mikro-Tümör Modelinin Oluşturulması

Watch this Scientific Journal Video about Preparing a Vascularized Micro-Tumor System Using Human Cells for Studying Tumors and Drug Responses at JoVE.com

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

Tümörleri ve ilaç yanıtlarını incelemek için insan hücrelerini kullanarak vaskülarize bir mikro tümör sistemi hazırlama 

Başlamak için, HBSS hücre ayrışma reaktifi EGM-2 ve DMEM'i 10 ila 15 dakika boyunca 37 santigrat derece su banyosuna yerleştirin. Trombin ve laminini gece boyunca 4 santigrat derecede ve fibrinojeni oda sıcaklığında çözdürün. 500 mikrolitrelik bir mikrosantrifüj tüpüne 1.5 mikrolitre trombin ekleyin.Mikroakışkanlarda sıkışan havayı çıkarmak için UV ile sterilize edilmiş yüksek verimli plakaları 30 dakika boyunca bir desikatöre yerleştirin. Hücre birleşmesini ve transdüksiyon verimliliğini doğrulamak için T75 şişesini mikroskop altına 4x büyütmede yerleştirin. Hücreleri beş mililitre HBSS ile iki kez yıkayın.Ardından, şişeye bir mililitre ayrışma reaktifi ekleyin ve bir ila iki dakika boyunca% 5 karbondioksit ile 37 santigrat derecede inkübe edin. Avuç içini kullanarak şişeye hafifçe vurun ve mikroskop altında tüm hücrelerin kaldırıldığını gözlemleyin. Hücreleri dokuz mililitre uygun ortamla yıkayın ve süspansiyonu 15 mililitrelik konik bir tüpe toplayın.Hemen küçük bir alikotu çıkarın ve hücreleri sayın. Hücre süspansiyonunu 300G ve dört santigrat derecede üç ila beş dakika santrifüjleyin. Sonra, süpernatanı çıkarın ve peleti buz üzerinde uygun ortamda yeniden süspanse edin.Verilen denklemi kullanarak, gereken hücre sayısını hesaplayın. Hücreleri mililitre başına 1 kez 10 ila 6 hücre konsantrasyonunda yeniden süspanse edin ve aşağıdaki denklemi kullanarak gereken hücre hacmini hesaplayın. Gösterildiği gibi gerekli hücre süspansiyonu hacmini santrifüjleyin.Daha sonra, hesaplanan bir fibrinojen hacminde, peleti buz üzerinde yeniden süspanse edin.

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

88 Görüntüleme. Başlamak için, HBSS hücre ayrışma reaktifi EGM-2 ve DMEM'i 10 ila 15 dakika boyunca 37 santigrat derece su banyosuna yerleştirin. Trombin ve laminini gece boyunca 4 santigrat derecede ve fibrinojeni oda sıcaklığında çözdürün. 500 mikrolitrelik bir mikrosantrifüj tüpüne 1.5 mikrolitre trombin ekleyin.Mikroakışkanlarda sıkışan havayı çıkarmak için UV ile sterilize edilmiş yüksek verimli plakaları 30 dakika boyunca bir desikatöre yerleştirin. Hücre birleşm...

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

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.

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 ...