Name: A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion (Video) | JoVE Journal
Uploaded: 2015-04-24T14:00:00.000+00:00
Duration: 6 min 36 s
Description: Learn a modified in vitro invasion assay to examine how hormones, cytokines and growth factors drive cancer cell invasion. This method compares invasion indices across cell variants using charcoal stripped media.

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5 minutes

The authors acknowledge funding sources from D&#8217;Youville College School of Pharmacy, Buffalo, NY.

1. Prepare the Different Media and Additional Components
<ol>
	<li>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.</li>
	<li>Weigh out and dilute the hormones, growth factors, or cytokines appropriately to be dissolved in the charcoal stripped serum at the physiological concentration.</li>
</ol>
2. Prepare the Collagen Matrix on Ice
<ol>
	<li>Prepare 2 ml collagen I matrix at 2.2 mg/ml by adding the following sterile filtered components on ice: 200 &#956;l 10x PBS (pH 7.4), 5.4 &#956;l 1 N NaOH, 600 &#956;l of double distilled H2O, and 1.2 ml collagen I (at 3.63 mg/ml).</li>
	<li>Keep collagen I solution on ice until ready to plate.</li>
</ol>
3. Prepare Migration/Invasion Plates for Assay
<ol>
	<li>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 &#956;m 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.</li>
	<li>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.</li>
</ol>
4. Dispense the Invasion Matrix
<ol>
	<li>Carefully pipette 75 &#956;l 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.</li>
	<li>Transfer the plate with the collagen-coated inserts to a 37 &#176;C and 5% CO2 incubator for 30 min to enable the gel to solidify.</li>
</ol>
5. Plate the Cells onto the Membrane or Invasion Matrix
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>When the collagen matrix has solidified (after 30 min), add 700 &#956;l of media with either 2% defined FBS or charcoal-stripped FBS to each well. Of the 12 inserts per plate:
	<ul>
		<li>3 inserts have collagen and wells with media + 2% FBS</li>
		<li>3 inserts have collagen and wells with media + 2% CS-FBS</li>
		<li>3 inserts have no collagen and wells with media + 2% FBS</li>
		<li>3 inserts have no collagen and wells with media + 2% CS-FBS</li>
	</ul>
	<ol>
		<li>Use additional plates depending on the number of factors being tested and with a no collagen control corresponding to each condition.</li>
	</ol>
	</li>
	<li>Add cell suspension to the inserts at 5 x 104 cells/ml, plating 500 &#956;l cells per insert in all migration and invasion inserts.</li>
	<li>Incubate the cells for 22 hr at 37 &#176;C.</li>
</ol>
6. Quantify the Number of Migrating and Invading Cells
<ol>
	<li>Set up staining of wells using methanol fixative, eosin, and hemotoxylin, in separate wells.</li>
	<li>Use cotton swabs to remove cells and matrix from each well. Rrepeat with second swab application for each well.</li>
	<li>With forceps, dip each insert 5 times for 1 sec into each of the 3 solutions in succession.</li>
	<li>Allow inserts to dry O/N.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Count cells for all conditions using the ImageJ software and apply to the formulas below.</li>
	<li>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</li>
	<li>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)</li>
	<li>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)</li>
</ol>
7. Repeat Experimental Protocol Comparing Charcoal-stripped FBS to Charcoal-stripped FBS + Xn with Multiple Factors Combined
<ol>
	<li>Repeat the procedure multiple times as needed using different components for &#8220;X&#8221; or a combination of components.</li>
	<li>Apply the calculations to determine the contribution of each factor &#8220;X&#8221; to the migration and invasion effects.</li>
</ol>

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The authors have nothing to disclose.

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 hormones13,14, growth factors15-18, and cytokines19-21. These molecules a...

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 phenotype1-3. 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 &#8220;X&#8221; can inhibit, decrease, increase, or restore the invasive index to its original value (see Figures 2 &#38; 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 &#8220;X&#8221; 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.

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

a modified in vitro invasion assay to determine the potential role of hormones, cytokines and/or growth factors in mediating cancer cell invasion

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

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 &#8220;X&#8221;). 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.

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...

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.

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

The overall goal of the following experiment is to establish an in vitro assay for examining the role of hormones, cytokines, and or growth factors in driving cancer cell invasion. This is achieved by determining whether there is a difference between the invasion index of different cell variants or mutants as a second step. The invasion assay is repeated using charcoal stripped media, which removes any hormones, growth factors, or cytokines from the serum.The invasion assay is then repeated with the addition of a specific known factor to the charcoal stripped medium to determine whether migration toward that factor enhances or inhibits invasion. The results show the changes in the invasion index for specific cell populations that occur during migration toward known experimental factors based on the normalization of the results against the invasive index of non-invasive cell populations. I first got the idea for this method when I was performing a traditional invasion assay, and I noticed that there was a significant difference between the invasion indexes for different cell mutants.The main advantage of this technique over existing methods like the traditional invasion assay, is that with this technique can specifically investigate the role of hormones, specific growth factors and cytokines in promoting or inhibiting cell invasion. This method may be helpful in answering key questions in the field of cancer research, including which factors lead to cancer metastasis. The Implications of this technique may provide a therapeutic approach to future drug design For each cell line to be tested, use a 24 well plate with 12 inserts.Then label the plates according to the conditions to be analyzed. Next carefully, dispense 75 microliters a freshly prepared collagen matrix solution into each insert that will be used for an invasion assay taking care not to make bubbles. Then incubate the plates with the collagen coated inserts at 37 degrees Celsius and 5%carbon dioxide for 30 minutes to allow the gel to solidify.After detaching the experimental cells with trypsin, add medium supplemented with 10%FBS to inactivate the enzyme and wash the cells three times in PBS. Then resuspend the pellet in serum free medium and count the cells. Add serum free media to a final concentration of 50, 000 cells per milliliter when the collagen matrix has solidified.After 30 minutes, add 700 microliters of medium with either 2%defined FBS 2%charcoal stripped FBS or 2%charcoal stripped FBS plus the additional component into each. Well add 500 microliters of cell suspension in serum free medium into the top of each migration or invasion. Insert, incubate the assays for 22 hours at 37 degrees Celsius.Then add methanol, fixative, eoin, and hematin into three of the empty wells of the chamber plate. Next, remove the cells and matrix from the top of each insert with two sequential cotton swabbing. Then using forceps.Dip each insert five times for one second each time into each of the three staining solutions in succession, and allow the inserts to dry overnight inverted. The next day. Use a 20 x objective to acquire images of the cells on the inserts by directly viewing the stained inserts.Five images are taken for each sample including four from the outer fields of the membrane and one from the center. Finally, using image J software, count the cells for each image and calculate the invasion index as normalized to a non-invasive cell line. This model demonstrates the potential results that may be encountered during an invasion assay and how to interpret the corresponding data.Each circle illustrates a representative result for a stained filter from one of the three conditions tested, and each dot represents a cell that passed through the invasion matrix and was stained on the bottom of the membrane. The invasion index is then calculated for each condition according to its normalization to a non-invasive cell line. For example, in this representative experiment, three hormones were tested to identify a possible x factor.Two of the hormones had no effect on the invasion index, but one was identified to exhibit inhibitory properties Once mastered. This technique can be done in approximately four hours on day one and one hour on day two if all steps are performed properly. While using this technique, it's important to properly label each individual cell insert carefully.We used a color coded system and different dots to carefully label each cell insert Following this. Other methods like in vivo, mouse models for metastasis could be used to validate the in vitro results. After watching this video, you should have a good understanding of how to assay the physiological relevance of factors such as hormones, growth factors, and cytokines as either enhancers or inhibitors of cell invasion in an in vitro model.Thank you for your interest in our video and good luck with your experiments.

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Learn a modified in vitro invasion assay to examine how hormones, cytokines and growth factors drive cancer cell invasion. This method compares invasion indices across cell variants using charcoal stripped media.

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

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

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