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

Summary

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.

Abstract

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.

Introduction

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 extr....

Protocol

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 articles^15,16. 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, each of the MCs has a shape of a truncated upside-down square-shaped pyramid (height: 190 µm, small base area: 90 µm x 90 µm, ....

Results

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.......

Discussion

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.......

Materials

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