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 ar....

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 co....

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