Name: analysis of cancer cell invasion and anti-metastatic drug screening using hydrogel micro-chamber array (hmca)-based plates
Uploaded: 2018-10-25T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates at JoVE.com

This work is supported by the bequest of Moshe Shimon and Judith Weisbrodt.

1. HMCA Plate Embossing
NOTE: The complete process for the design and fabrication of polydimethylsiloxane (PDMS) stamp and HMCA imaging plate used in this protocol is described in detail in our previous articles15,16. The PDMS stamp (negative shape) is used to emboss the HMCA (positive shape) which consists of approximately 450 MCs per well (Figure 1A). As demonstrated in Figure 1B</stro...

<ol>
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It is well documented that living organisms, characterized by their complex 3D multicellular organization are quite distinct from the commonly used 2D monolayer cultured cells, emphasizing the crucial need to use cellular models which better mimic the functions and processes of the living organism for drug screening. Recently, multicellular spheroids, organotypic cultures, organoids and organs-on-a-chip have been introduced8 for the use in standardized drug discovery. However, the 3D multicellular...

Cancer death is attributed mainly to the dissemination of metastatic cells to distant locations. Many efforts in cancer treatment focus on targeting or preventing the formation of metastatic colonies and progression of systemic metastatic disease1. Cancer cell migration is a crucial step in the tumor metastasis process, thus, the research of the cancer invasion cascade is very important and a prerequisite to finding anti-metastatic therapeutics.
The use of animal models as tools for studying metastatic disease has been found to be very expensive and not always representative of the tumor in humans. Moreover, the extracellular microenvironment topology, mechanics and composition strongly affect cancer cell behavior2. Since in vivo models inherently lack the ability to separate and control such specific parameters which contribute to cancer invasion and metastasis, there is a need for controllable biomimetic in vitro models.
In order to metastasize to distant organs, cancer cells must exhibit migratory and invasive phenotypic traits which can be targeted for therapy. However, since most in vitro cancer models do not mimic the actual features of solid tumors3, it is very challenging to detect physiologically relevant phenotypes. In addition, the phenotypic heterogeneity that exists within the tumor, dictates the need for analyzing tumor migration at single-element resolution in order to discover phenotype-directed therapies, for instance, by targeting the metastasis-initiating cell population within heterogeneous tumors4.
The study of cell motility and collective migration is primarily conducted in monolayer cultures of epithelial cells on homogeneous planar surfaces. These conventional cellular models for cancer cell migration are based on the population analysis of individual cells invading through the membranes and ECM components5, but in such systems, the intrinsic differences between individual cells cannot be studied. Generating 3D spheroids either via scaffolds or in scaffold-free micro-structures is considered as a superior means to study the tumor cell growth and cancer invasion6,7,8. However, most 3D systems are not suitable to high throughput formats, and inter-spheroid interaction cannot usually be achieved since isolated individual spheroids are generated in each micro-well9. Recent studies involving the cancer migration are based on microfluidic devices3,10,11,12, however, these sophisticated microfluidic tools are difficult to produce and cannot be used for high throughput screening (HTS) of anti-invasive drugs.
Two main phenotypes, collective and individual cell migration, which play a role in tumor cells overcoming the ECM barrier and invading neighboring tissue, have been demonstrated13,14, each displaying distinct morphological characteristics, biochemical, molecular and genetic mechanisms. In addition, two forms of migrating tumor cells, fibroblast-like and amoeboid, are observed in each phenotype. Since both, invasion phenotypes and migration modes, are mainly defined by morphological properties, there is a need for cellular models that enable imaging-based detection and examination of all forms of tumor invasion and migrating cells.
The overall goal of the current method is to provide a simple and reproducible biomimetic 3D in vitro cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. Here, we introduce the HMCA-based 6-well imaging plate for the research of tumor invasion and anti-metastatic therapy. The technology enables the formation of large numbers of uniform 3D tumor spheroids (450 per well) in a hydrogel micro-chambers (MC) structure. Various ECM components are added to the spheroid array to enable the invasion of the cells into the surrounding environment. Invasion process is continuously monitored by short- and long-term observation of the same individual spheroids/invading cells and facilitates morphological characterization, fluorescent staining and retrieval of specific spheroids at any point. Since numerous spheroids share space and medium, interaction via soluble molecules between individual spheroids and their impact on one another is possible. Semi-automatic image analysis is performed by using in-house MATLAB code which enables faster and more efficient collection of large amount of data. The platform facilitates accurate, simultaneous measurement of numerous spheroids/invading cells in a time-efficient manner, allowing medium throughput screening of anti-invasion drugs.

<table>
	<tbody>
		<tr>
			<td>6 Micro-well Glass Bottom Plates with 14 mm micro-well #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>P06-14-1.5-N</td>
			<td>Commercial glass bottom plates which are used for HMCA embossing</td>
		</tr>
		<tr>
			<td>UltraPure Low Melting Point Agarose</td>
			<td>Invitrogen</td>
			<td>16520100</td>
			<td>A solution of 6% agarose is warmed up to 80&#176;C before use, a solution of 1% agarose is warmed to 37&#176;C</td>
		</tr>
		<tr>
			<td>Trypsin EDTA solution B</td>
			<td>Biological Industries</td>
			<td>03-052-1A</td>
			<td>Warmed to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>DMEM medium, high glucose</td>
			<td>Biological Industries</td>
			<td>01-055-1A</td>
			<td>Warmed to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>Special Newborn Calf Serum (NBCS)</td>
			<td>Biological Industries</td>
			<td>04-122-1A</td>
			<td>Heat Inactivated</td>
		</tr>
		<tr>
			<td>DPBS (10X), no calcium, no magnesium</td>
			<td>Biological Industries</td>
			<td>02-023-5A</td>
			<td>Kept on ice before use</td>
		</tr>
		<tr>
			<td>NaOH, anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-500G</td>
			<td>Used for the preparation of 1M NaOH solution</td>
		</tr>
		<tr>
			<td>Cultrex Type I collagen from rat tail, 5mg/ml</td>
			<td>Trevigen</td>
			<td>3440-100-01</td>
			<td>Kept on ice before use</td>
		</tr>
		<tr>
			<td>Hyaluronic acid sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>H5542-10MG</td>
			<td>Kept on ice before use</td>
		</tr>
		<tr>
			<td>Fisetin</td>
			<td>Sigma-Aldrich</td>
			<td>F505-100MG</td>
			<td>Added to the culture medium, invasion inhibitor</td>
		</tr>
		<tr>
			<td>DETA/NO</td>
			<td>Enzo Life Sciences</td>
			<td>alx-430-014-m005</td>
			<td>Added to the culture medium, nitric oxide donor</td>
		</tr>
		<tr>
			<td>PI</td>
			<td>Sigma-Aldrich</td>
			<td>P4170</td>
			<td>Used at very low concenrtation without the need for washing</td>
		</tr>
		<tr>
			<td>Dymax 5000-EC UV flood lamp complete system with light shield &#38; Dymax 400 Watt EC power supply</td>
			<td>Dymax Corporation</td>
			<td>PN 39823</td>
			<td>Used for HMCA plate sterilization by UV</td>
		</tr>
		<tr>
			<td>Inverted IX81 microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>Used for automatic image acquisition</td>
		</tr>
		<tr>
			<td>Incubator for microscope</td>
			<td>Life Imaging Services</td>
			<td></td>
			<td>Essential for time lapse experiments with image acquisition, pre adjusted to 37&#176;C, 5% CO2 and keeping a humidified atmosphere</td>
		</tr>
		<tr>
			<td>Sub-micron motorized stage type SCAN-IM</td>
			<td>Marzhauser Wetzlar GmbH</td>
			<td></td>
			<td>Used to predetermine image acquisition areas, for automatic image acquisition</td>
		</tr>
		<tr>
			<td>14-bit, ORCA II C4742-98 cooled camera</td>
			<td>Hamamatsu Photonics</td>
			<td></td>
			<td>Highly sensitive, used for imaging</td>
		</tr>
		<tr>
			<td>Fluorescent filter cube for PI detection</td>
			<td>Chroma Technology Corporation</td>
			<td></td>
			<td>Filter cube specifications: excitation filter 530-550 nm, dichroic mirror 565 nm long pass and emission filter 600-660 nm</td>
		</tr>
		<tr>
			<td>The Olympus Cell^P operating software</td>
			<td>Olympus</td>
			<td></td>
			<td>Software used to control microscope, motorized stage, camera and image acquisition</td>
		</tr>
		<tr>
			<td>Matlab R2014B analysis software</td>
			<td>Mathworks</td>
			<td></td>
			<td>Used to develop in house graphic user interface for image analysis</td>
		</tr>
		<tr>
			<td>Excel software</td>
			<td>Microsoft</td>
			<td></td>
			<td>Used for data management, calculation, plot creation and statistics</td>
		</tr>
	</tbody>
</table>

analysis of cancer cell invasion and anti-metastatic drug screening using hydrogel micro-chamber array (hmca)-based plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

The unique HMCA imaging plate is used for the invasion assay of 3D tumor spheroids. The entire assay, beginning with the spheroid formation and ending with the invasion process and additional manipulations, is performed within the same plate. For the spheroid formation, HeLa cells are loaded into the array basin and settle in the hydrogel MCs by gravity. The hydrogel MCs, which have non-adherent/low attachment characteristics, facilitate the cell-cell interaction and the formation of 3D t...

Watch this Scientific Journal Video about Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates at JoVE.com

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

Research

JoVE Journal

Cancer Research

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

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Perineural invasion (PNI) is found in approximately 40% of head and neck squamous cell carcinomas (HNSCC). Despite multimodal treatment with surgery, ...

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

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A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...