A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Summary

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Abstract

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted “X”). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

Introduction

A novel invasion assay has been developed with the goal of identifying the involvement of specific hormones, cytokines and/or growth factors as chemoattractants in cancer cell invasion. The ability of tumor cells to invade through the extracellular matrix (ECM) is a hallmark of the metastatic phenotype^1-3. Invasion chambers have been extensively used as in vitro tools to study cancer cell invasion and migration and may provide knowledge as to the mechanisms of in vivo tumor invasion and metastasis. The chambers consist of cylindrical cell culture inserts nested within the wells of cell culture plates. The bottoms of the inserts are semi-permeable polycarbonate or polystyrene membranes of defined pore sizes.

In the standard invasion assay, cells are seeded onto the insert membrane with serum free media and placed into cell culture wells that are filled with serum or serum-like chemoattractants to set up a chemoattractive force. This force drives cells to move through the semi-permeable membrane (migration) or alternatively, through a coating of extracellular matrix (invasion), which can then be stained using conventional dyes to detect and quantify the number of cells. The cell number is then normalized according to the extent of migration and invasion in a noninvasive cell line. This method allows an investigator to evaluate the invasive potential of different cell types under a variety of conditions, including genetic manipulations.

Hormones, cytokines and growth factors are critical components of serum and are increasingly being shown to be associated with driving the invasive phenotype⁴. However, the nature, role, and specificity of these chemoattractive serum molecules in mediating invasion still remain elusive. The challenge remains to determine what specific factors in serum or serum-like chemoattractants are responsible for driving cancer cell invasion as well as what molecules may inhibit the invasion process. In this report, we describe a methodology to evaluate the potential involvement of hormones, cytokines and/or growth factors present in serum, as molecules driving the chemoattraction and invasion of cancer cells.

In this proposed modified protocol, charcoal stripping is used to remove hormones, cytokines and growth factors from 2% fetal bovine serum (FBS) to generate 2% charcoal stripped FBS (CS-FBS). Both 2% FBS and 2% CS-FBS are used as agents to set up the chemoattractive force to drive cancer cell invasion. Using 2% CS-FBS as a chemoattractive agent in comparison to normal 2% FBS affords several benefits including first, and most prominently, the ability to study the cancer invasion phenotype in the reduced/relative absence of hormones, cytokines and growth factors. It also enables the investigator to assess whether the collective removal of hormones, growth factors, and cytokines causes an increase or decrease in the invasive index. The assay is then designed to determine whether the addition of an individual component, designated as “X” can inhibit, decrease, increase, or restore the invasive index to its original value (see Figures 2 & 3). Although this methodology is specific for achieving the physiological levels of the component that is removed from the serum during charcoal stripping, it should also be noted that charcoal stripping can also affect signal transduction pathways. For example, it has been reported that charcoal stripping can decrease the alkaline phosphatase activity of osteoprogenitor cells and induce adipogenesis through reduction of MAPK activators⁵. Charcoal stripped media are commercially available and are based on the protocol outlined that combines charcoal with dextran and incubated with fetal bovine serum O/N⁶.

This assay was developed in response to a result reported by Zucker et al.⁷ The authors demonstrated that a mutant phenotype affecting gap junction communication demonstrated an increase in the invasion index when migrating toward media with normal 2% FBS. This increase was eliminated with the substitution of charcoal-stripped serum. From this result, the authors concluded that this mutant affected migration toward a lipophilic molecule (more specifically, a hormone, growth factor, or cytokine)⁷. It is well known that hormones, growth factors, and cytokines mediate signaling pathways involved in tumor promotion and invasion⁴. Thus, it is important for scientific investigators to determine what factor(s) are driving the tumor invasion of their particular cancer cells or mutants under study. The assay is designed to address the role of individual components at their physiological concentration as determined according to the difference between the concentration of the component “X” in normal FBS and the CS-FBS. Through the development of this assay, investigators can gain more insight into the specific invasive pathway governing their system.

Hormones, growth factors, and cytokines have often been classified as tumor promoters⁸. Some of these factors such as EGF are used as direct sources for chemoattractants in invasion chambers⁹. Thus, it seems likely that these represent the major components of the serum that direct tumor invasion. This protocol proposes a simple, yet significant, modification to the conventional in vitro invasion assay which allows an investigator to assess the involvement of hormones, growth factors, and cytokines in mediating the invasive potential of cancer cells. However, the assay is designed to provide the investigator with an answer as to whether the procedure will be effective for their study at an early step in the analysis, so as not to use precious time and resources if deemed unnecessary. This method uses CS-FBS and relies on the investigators discretion as to which molecules to pursue as lead candidates that potentially mediate cancer cell invasion. The results from these analyses should prove to be useful in identifying which serum components serve as chemoattractants or inhibitors for the particular cell line or mutant being studied. In addition, this approach may help the investigator identify key signaling pathways either promoting or inhibiting cancer cell invasion; thus directing future drug design.

Protocol

1. Prepare the Different Media and Additional Components

1. Prior to experiment, prepare media consisting of DMEM or other specified media with the addition of either normal FBS, charcoal stripped FBS, or charcoal stripped FBS plus the component to be tested. Note that multiple components can be tested in each experiment.
2. Weigh out and dilute the hormones, growth factors, or cytokines appropriately to be dissolved in the charcoal stripped serum at the physiological concentration.

2. Prepare the Collagen Matrix on Ice

1. Prepare 2 ml collagen I matrix at 2.2 mg/ml by adding the following sterile filtered components on ice: 200 μl 10x PBS (pH 7.4), 5.4 μl 1 N NaOH, 600 μl of double distilled H₂O, and 1.2 ml collagen I (at 3.63 mg/ml).
2. Keep collagen I solution on ice until ready to plate.

3. Prepare Migration/Invasion Plates for Assay

1. For each cell line to be tested, use one 24-well chamber plate in which 12-wells contain inserts. Use the additional 12 wells that do not contain inserts for adding the chemoattractant media and transferring the inserts for the experimental set up.
   NOTE: A collagen matrix on plates with a polyethylene teraphthalate (PET) membrane and 8 μm pore size is optimal for the cell lines use here. However, a matrigel matrix in precoated plates can also be substituted with the pore size decreased according to the cell line being investigated.
2. Clearly label the plate, using 3 wells per condition being analyzed (FBS migration, CS-FBS migration, FBS invasion, and CS-FBS invasion as well as FBS-migration for a control, noninvasive cell line). Assay migration by movement through pores in a PET membrane, and assay invasion by movement through a collagen or matrigel matrix and then through pores in the membrane. Use different color markers for each cell condition to aid in the plating process.

4. Dispense the Invasion Matrix

1. Carefully pipette 75 μl of the collagen matrix solution into the inserts to be used for invasion assays. Use caution to avoid bubbles. Disperse bubbles by applying an inverted pipette tip to the surface.
2. Transfer the plate with the collagen-coated inserts to a 37 °C and 5% CO₂ incubator for 30 min to enable the gel to solidify.

5. Plate the Cells onto the Membrane or Invasion Matrix

1. Meanwhile, trypsinize cells and add media with 10% FBS. Spin cells at 200 x g for 5 min on a table top centrifuge and rinse 3x in serum free media.
2. Resuspend in serum free media. Count cells with a hemocytometer or automated slide counter. Add serum free media to a final concentration of 5 x 10⁴ cells/ml.
3. When the collagen matrix has solidified (after 30 min), add 700 μl of media with either 2% defined FBS or charcoal-stripped FBS to each well. Of the 12 inserts per plate:
   • 3 inserts have collagen and wells with media + 2% FBS
   • 3 inserts have collagen and wells with media + 2% CS-FBS
   • 3 inserts have no collagen and wells with media + 2% FBS
   • 3 inserts have no collagen and wells with media + 2% CS-FBS
   1. Use additional plates depending on the number of factors being tested and with a no collagen control corresponding to each condition.
4. Add cell suspension to the inserts at 5 x 10⁴ cells/ml, plating 500 μl cells per insert in all migration and invasion inserts.
5. Incubate the cells for 22 hr at 37 °C.

6. Quantify the Number of Migrating and Invading Cells

1. Set up staining of wells using methanol fixative, eosin, and hemotoxylin, in separate wells.
2. Use cotton swabs to remove cells and matrix from each well. Rrepeat with second swab application for each well.
3. With forceps, dip each insert 5 times for 1 sec into each of the 3 solutions in succession.
4. Allow inserts to dry O/N.
5. Either i) remove filters with a scalpel, cutting carefully around the edges and mount on a slide with coverslip and immersion oil, or ii) allow the inserts to dry O/N inverted and use the inserts directly for microscopy.
6. The next day, view slides or inserts under a microscope with a 20X objective and take 5 images from different regions of the filter. To improve consistency, take 4 outer fields and one center.
7. Count cells for all conditions using the ImageJ software and apply to the formulas below.
8. Determine the percent invasion as follows:
   Mean # of cells invading through collagen I insert = a
   Mean # of cells migrating through control insert = b
   % Invasion = (a / b) * 100
9. Determine the Invasion Index in 2% FBS as follows:
   % invasion of cells being assayed (in 2% FBS) = c
   % invasion of control noninvasive cells in (2% FBS) = d
   Invasion Index (FBS) = (c / d)
10. Determine the Invasion Index in 2% CS-FBS as follows:
    % invasion of cells being assayed (in 2% CS-FBS) = e
    % invasion of control noninvasive cells in (2% CS-FBS) = f
    Invasion Index (CS-FBS) = (e / f)

7. Repeat Experimental Protocol Comparing Charcoal-stripped FBS to Charcoal-stripped FBS + X_n with Multiple Factors Combined

1. Repeat the procedure multiple times as needed using different components for “X” or a combination of components.
2. Apply the calculations to determine the contribution of each factor “X” to the migration and invasion effects.

Results

The invasion index is calculated for each condition according to normalization to a noninvasive cell line. For our experiments, we use the 1205Lu melanoma cell line and established variant stable cell lines as our invasive lines as well as the premalignant noninvasive variant, WM793 from which the 1205Lu cells were derived¹⁰ which serves as a logical control. We also utilize collagen I as the invasion matrix because that is the primary component of the dermis. This is in accordance with a previous study whereb...

Discussion

Tumor metastasis is a multistep process. The cells must break through the basement membrane, intravasate into either the lymphatic system or blood microvessels, in which they are transported to distant sites. The tumor cells then extravasate and colonize into a macrometastasis¹². Progression through the epithelial to mesenchymal transition (EMT), tumor invasion, and metastasis has been enhanced by steroid hormones^13,14, growth factors^15-18, and cytokines^19-21. These molecules a...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BioCoat Control Inserts with 8.0 µm PET membrane	Corning	354578	12 in each of two 24-well plates. PET = polyethylene teraphthalate
Collagen I, rat tail	Corning	354236	Source = rat tail tendon, purity = 90%
Fixative; Diff-Quick	Thermo Fisher Scientific	NC9844047	Methanol-based fixative, hematoxylin/eosin
Fetal bovine serum	Life Technologies	16000	Designated as FBS
Charcoal stripped fetal bovine serum	Life Technologies	12676	Designated as CS-FBS
Fetal bovine serum	Thermo Scientific Hyclone	SH30070	Designated as FBS
Charcoal stripped fetal bovine serum	Thermo Scientific Hyclone	SH30068	Designated as CS-FBS