A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

Summary

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Abstract

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

Introduction

It is established that cancer cell motility is predictive of metastatic potential, given that such behavior facilitates invasion through the basement membrane and entry into the circulatory system¹. Most research on motility has focused on how cells behave in a two-dimensional (2D) setting, though it is becoming widely recognized that the movement of cells in a three-dimensional (3D) matrix is more representative of how these same cells will actually behave in vivo². 3D culture systems are increasingly used to study cellular behaviors that range from cell morphology, to growth kinetics and drug sensitivity³. The desire to monitor cancer cell invasion within the context of a 3D milieu has led to the synthesis of previously established techniques involving the generation of 3D cell aggregates (spheroids) via the hanging drop culture method⁴, followed by embedding these spheroids into a 3D extracellular matrix (ECM) composed of collagen and basement membrane materials⁵. This method seeks to improve upon these previously established techniques by providing a streamlined approach that can be easily utilized to compare invasion under a variety of experimental conditions.

More traditional ways to assess cell motility in vitro are the scratch-wound assay and the transwell assay⁶. The former assay depicts cell motility in a 2D setting, and is therefore independent of a variety of features critical for in vivo invasion, e.g. protease activity⁷. The transwell assay can better model cell invasion when the well inserts are coated with ECM substrates but, only a single parameter, i.e. the appearance of cells on the opposite membrane surface is measured, and many nuances of cell invasion are thus not readily observable. In contrast to these techniques, the cancer cell spheroid invasion assay (Figure 1) allows for the real-time monitoring of cell invasion in a setting that is not only physiologically relevant, but also permits important cell line-specific features to be visualized, such as individual vs. collective cell migration⁸. This method also affords advantages over standard 3D culture growth assays. The generation of cellular aggregates via the hanging drop method initially constrains cell movement, so that cells will be incentivized to invade after this constraint is lifted. Furthermore, once that constraint is lifted, cell egress will proceed in a uniform direction that can then be conveniently quantitated.

The most popular ECM materials used for cancer cell spheroids assays are Matrigel and type I collagen, where each of these components has important and distinct roles in influencing metastatic behavior. Matrigel is a secreted mixture of proteins produced by Engelbreth-Holm Swarm mouse sarcoma cells, and is enriched in basement membrane proteins such as laminin, entactin, and type IV collagen⁹. For this reason Matrigel is henceforth referred to as “basement membrane materials.” These basement membrane materials provide essential ligands needed for integrin adhesion during cancer cell invasion¹⁰ in addition to many other proteins that exert a range of effects on cell behavior¹¹. In comparison, type I collagen, commonly prepared from acid digests of tendons and other dense collagenous structures¹², is a much simpler matrix material that serves as a major structural element of the connective tissue and stroma supporting tissues and organs of the body. It has been demonstrated that the physical characteristics of collagen can regulate a number of features of cell motility; for example, the alignment of collagen fibrils at the tumor-stromal interface permits cancer cells to subsequently migrate along those fibrils when invading into the stroma¹³. In the assay presented here, both type I collagen and basement membrane materials are utilized as tools to study 3D cancer cell-stroma interactions.

The effect of inhibition or stimulation of pathways that control invasion can be monitored after the cells have been embedded in the 3D matrix. Cells can be pretreated during growth in the hanging drops or upon transfer to the 3D culture, depending on whether a lengthy treatment will be required to modulate invasion. For shorter treatments, it is recommended that the drug be mixed with the spheroid suspension after collection, as well as the media that will surround the 3D cultures, to facilitate adequate drug exposure to the cells. Next, normal or tumor-associated stromal cells can be admixed with the matrix material to evaluate their role in modulating tumor cell invasion, or to determine how paracrine and autocrine signaling influences cell behavior. This idea was shown in a study where the coculture of colon cancer and endothelial cells in hanging drops led to a vascular network within the spheroids¹⁴. Finally, the ECM constituents can also be altered, as cancer cell invasion is impacted by different substrates¹⁵. The method presented below will thus provide a framework for assessing cancer cell invasion under a variety of conditions. In general it was found that not all cell lines will create spheroids in the hanging drops and epithelial-looking cell lines typically form regular spheres.

Protocol

1. Generation of Spheroids

1. Prepare single-cell suspension for hanging drop cultures by detaching adherent cancer cell cultures of ~70% confluence using a PBS wash followed by exposure to 0.05% trypsin-EDTA solution.
   1. Neutralize the trypsin solution with cell culture media and count the cells using an aliquot of the cell suspension. Note: The specific cell culture media will depend on the cell line being tested. Follow ATCC media recommendations, where the cell culture media is typically DMEM + 10% FBS. More information can be found in the Materials Table.
   2. Perform a dilution to allow for the seeding of 500-1,000 cells per 20 µl drop of cell culture media.
2. Acquire 10 cm dish and add 5 ml of sterile PBS to the bottom. Note: This step protects the hanging drops from evaporation. Lower-cost “bacteriological grade” dishes can be used in place of cell culture grade dishes, as cells will not contact the culture surface.
3. Use a multi-channel pipet to transfer 20 µl droplets of the diluted cell suspension to the inner surface of the lid.
   1. Pipet 40 drops (5 rows of 8 drops) onto the lid of the 10 cm dish.
4. Invert the lid and place over the culture dish. Incubate the hanging drop cultures at 37 °C for 72 hr to generate spheroids.
   1. Flip the lid in a confident, yet controlled manner. Note: Inverting the lid too fast or too slow may cause the droplets to shift. Some cell lines may form spheroids in 48 hr or less, while others may require more than 72 hr to produce compact aggregates.

2. Embedding of Spheroids into 3D Matrix

1. Thaw an aliquot of growth factor-reduced basement membrane materials at 4 °C overnight before embedding the spheroids.
   1. Optional: Layer wells with ECM in advance to prevent potential spheroid interaction with the tissue culture surface.
      1. Pipet 200 µl of ECM per well on a 24-well plate.
      2. Tilt the plate to ensure that the ECM covers the entire well surface.
      3. Carefully remove excess ECM with a pipet.
      4. Incubate the plate until the wells are dry: 3 hr at room temperature or overnight at 4 °C.
2. Collect the spheroids by tilting the lid of the tissue culture dish and pooling the media. Transfer the media with the spheroids into a 1.5 ml microcentrifuge tube.
3. Allow 10 min for the spheroids to settle at the bottom of the microcentrifuge tube. Spheroids should be visible by eye.
4. Mix 100 µl of the basement membrane materials with 100 µl of cold (4 °C) type I collagen in a separate pre-chilled tube. Keep the mixture at 4 °C to prevent either gel from solidifying prematurely.
   1. Use pre-chilled pipet tips if transferring small volumes.
   2. Give care when mixing the basement membrane materials and collagen type I to prevent air bubble formation. Note: Air bubbles may hinder imaging later in the protocol if they become embedded in the gel.
5. Aspirate the spheroids from the 40 µl bottom portion of the microcentrifuge tube, and combine with the basement membrane materials/type I collagen mixture. Be careful to prevent air bubble formation when mixing. Note: The solution containing of 100 µl basement membrane materials, 100 µl collagen type I, and 40 µl of the spheroids will create enough material for 4 independent 3D cultures. This can be scaled up or down.
6. Pipet 40 µl drops of the viscous mixture into the centers of wells on a 24-well plate. Keep the plate level to prevent the mixture from running into the side of the well. Note: A single column on the 24-well plate – 4 wells – is recommended for each condition to be tested.
7. Place the plate into a 37 °C incubator and leave undisturbed for 30 min. The 3D culture will polymerize during this period.
8. Slowly submerge the 3D cultures in 1 ml of cell culture media.
   1. Ensure that the media is warm when adding it into the wells to further promote gel polymerization.
   2. CRITICAL STEP: Gently add the media to the 3D cultures. Pipetting the media too quickly into the wells can lead to detachment of the 3D cultures from the tissue culture surface.

3. Monitoring and Analyzing Spheroid Invasion

1. Image invasion from the spheroids into the surrounding 3D matrix at time-points decided by the investigator. Acquire photographs using an inverted microscope with the 20x objective lens. Note: Ideal time-points will differ depending on the cell line being tested. More invasive cell lines will begin their egress from the spheroids shortly after plating and, therefore, take initial photographs within a couple of hours after plating. Generally, photographs are taken at 0 hr (after plating), 24 hr, and 48 hr. Invasion typically concludes 24 to 48 hr after plating.
2. Quantitate invasive ability using image analysis software like Image J.
   1. Analyze invasion as the cell distance from the edge of the spheroid.
      1. Use the Straight Draw Tool to mark the radius and/or maximal invasive distance.
      2. Click “Analyze” in the top menu, and then click “Measure” to display the length measurement.
   2. Analyze invasion as the total invasive area outside the spheroid.
      1. Use the Freehand Draw Tool to trace the border of the spheroid and/or total invasive area.
      2. Click “Analyze” in the top menu, and then click “Measure” to display the area measurement.
   3. Use a plugin like ROI Manager Tools to create measurements for a stack of images.

Results

Using the spheroid invasion assay (Figure 1), a panel of cancer cell lines was tested for their ability to form spheroids, as well as for the amount of cell egress exhibited after implantation in a 3D matrix consisting of basement membrane materials and type I collagen (Table 1). These results demonstrate that not every cancer cell line will create well-formed spheroids, where cell lines possessing an epithelial morphology in vitro tended to produce regular and smooth aggregates...

Discussion

This study evaluated the performance of a panel of cancer cell lines in a spheroid invasion assay (Table 1). Generally, we find that spheroid formation is enhanced in the more epithelial-looking cell lines, where the presence of cell-cell junctions promotes the formation of a spheroid-like architecture. Known cell lines that have undergone an epithelial-mesenchymal transition, like MDA-MB-231 cells do not form spheroids in the hanging drop culture most likely due to their reduced E-, N-, and P-cadherin e...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Matrigel Growth Factor Reduced	Corning	CB-40234
Collagen Type I, Rat Tail, 100 mg	Millipore	08-115
DMEM	Life Technologies	11995-065
RPMI 1640 Medium	Life Technologies	11875-093
PBS	Life Technologies	10010-023
0.05% Trypsin-EDTA	Life Technologies	25300-054
Fetal Bovine Serum, Heat Inactivated	Omega Scientific	FB-12
100 mm TC-treated Dishes	Corning Incorporated	430167
24-well TC-treated Plates	NEST Biotechnlology	702001
Olympus IX-71 Inverted Microscope
Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.