Name: Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds (Video) | JoVE Journal
Uploaded: 2015-06-06T15:00:00.000+00:00
Duration: 10 min 16 s
Description: Learn a chick heart invasion assay to study cancer cell invasiveness in three dimensions using embryonic chicken heart tissue. This organ culture method enables screening of potential anti-invasive compounds for oncology research.

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5 minutes

We thank Marleen De Meulemeester for demonstrating the assay technique in the video film. B. I. R. is a Postdoctoral Research Fellow of the Research Foundation &#8211; Flanders (FWO &#8211; Vlaanderen). L.M.M. is a recipient of an Emmanuel van der Schueren grant from the Flemish League against Cancer (Vlaamse Liga tegen Kanker).

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</ol>

The authors declare that they have no competing financial interests.

During the preparation of PHFs, the fragments may not stay in suspension but adhere to the vessel wall; this can be overcome by increasing the volume of the culture medium. If the number of PHFs is too low and their size is too big, decrease the volume of the culture medium. Failure of the test cells to aggregate may be due to fluctuations in the temperature or to microbial infection. Alternatively, an inability to aggregate may be an intrinsic characteristic of the cells. During attachment of the aggregates to PHF, poor...

Invasion is the hallmark of malignant tumors. This activity not only leads to the destruction of surrounding tissues, but is also implicated in metastasis formation. Since cancer patients die from invasion and metastasis, and efficient anti-invasive treatments are still scarce, laboratory assays that mimic the invasion of tumor cells have been developed. The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and allows to study the effects of test compounds on tumor invasion.
The rationale behind the use of the assay is the actual concept that tumors are ecosystems where the neoplastic cells continuously interact with their stroma (host cells and extracellular matrix), and that through these molecular interactions invasion is fine-tuned1. So, in the assay tumor cells are confronted with living embryonic chick heart fragments2, which serve not only as substrates for invasion by the tumor cells, but also as a source of different types of stromal cells and matrix elements. The chick heart contains myocytes, fibroblasts and endothelial cells, and the extracellular matrix is composed of laminin, fibronectin and different types of collagen. In this way, the three-dimensional organ culture technique covers many cellular and molecular interactions implicated in invasion of patient tumors.
The main advantage of the chick heart assay is the implementation of stromal effects. This aspect is more complete than in other invasion assays in vitro that are based on tumor cell invasion into non-living gels composed of basement membrane3 or interstitial matrix4 molecules. The concept of confrontation between tumor cells and normal living host tissue as found in organ culture experimentation has been introduced by several authors including Wolff and Schneider in France5, Easty and Easty in the United Kingdom6, and Schleich in Germany7. Two technical advantages of the chick heart invasion assay over the cited methods is that the volume of the fragments can easily be standardized, and that they remain contractile, which allows functional integrity monitoring during organ culture. Furthermore, avian embryos are preferred, because they can easily be dissected from the sterile content of the egg. The assay has conceptual resemblance to the chick chorioallantois membrane assay8 by offering a complex stromal surrounding to the tumor cells.
The assay has successfully been applied to distinguish between invasive and non-invasive cell variants from the same human tumors such as in the MCF-7 (mammary)9 and HCT-8 (colonic)10 cell line families. The technique is useful to test potentially anti-invasive compounds as well11,12. As further explained, it can be used for the establishment of structure-activity relations of small organic molecules. The assay does, however, not take into account the contribution of immunological cells to cancer invasion. It should be stressed that the technique cannot be considered as a high-throughput analysis system, because of the high number of manipulations, the limited numbers of assay runs (maximum 30 cultures) and the long turn-around time (about 1 month).

<table><tbody><tr><td>Ringer&#039;s salt solution</td><td>Braun</td><td>CEO123</td><td></td></tr><tr><td>Bacto-agar</td><td>Becton Dickinson</td><td>214010</td><td></td></tr><tr><td>Tris-(hydroxymethyl)-aminomethaan Analar</td><td>VWR</td><td>103157P</td><td></td></tr><tr><td>BSA : albumin from bovine serum, cohn V fract.</td><td>Sigma</td><td>A4503-500G</td><td></td></tr><tr><td>MEM-Rega 3</td><td>Life Technologies&amp;nbsp;</td><td>19993013</td><td></td></tr><tr><td>Gyrotory shakers</td><td>New Brunswick Scientific</td><td>G10 and G33</td><td></td></tr><tr><td>Erlenmeyer flasks 50 ml</td><td>Novolab</td><td>92717</td><td></td></tr><tr><td>Glass Petri dishes</td><td>Novolab</td><td>68516</td><td></td></tr><tr><td>Iridectomy scissors</td><td>Rumex</td><td>11-0625</td><td></td></tr><tr><td>Macroscope with calibrated ocular grid</td><td>Wild</td><td>157702</td><td></td></tr><tr><td>Paraffin wax</td><td>International Medical products</td><td>8599956</td><td></td></tr><tr><td>Eosin Y</td><td>Sigma</td><td>230251-25g</td><td></td></tr><tr><td>Harris&#039; hematoxylin</td><td>Sigma</td><td>HHS32-1L</td><td></td></tr><tr><td>Coverslipping resin (Tissue-Tek)</td><td>Sakura</td><td>4494</td><td></td></tr><tr><td>Paraffin melting apparatus</td><td>GFL</td><td>1052</td><td></td></tr><tr><td>Microtome for paraffin sectioning</td><td>Reichert</td><td></td><td></td></tr><tr><td>Stppers for Erlemeyer flasks with gas in- and outlets</td><td>Novolab</td><td>2602421 and 260243</td><td></td></tr><tr><td>Diaminobenzidine</td><td>Sigma</td><td>D8001</td><td></td></tr><tr><td>REAX rocking table</td><td>Heidolph</td><td>54131</td><td></td></tr><tr><td>24-well tissue dishes</td><td>VWR</td><td>NUNC142475</td><td></td></tr><tr><td>Ophtalmological enucleation spoon</td><td>Rumex</td><td>16-060</td><td></td></tr><tr><td>Sharp forceps</td><td>Rumex</td><td>4-111T</td><td></td></tr><tr><td>Blunt forceps</td><td>Nickel-Electro LTD</td><td>7112</td><td></td></tr><tr><td>Erlenmeyer flasks 5 ml</td><td>Novolab</td><td>211901</td><td></td></tr><tr><td>Microscope slides washed and degreased</td><td>International Medical products</td><td>8613908</td><td></td></tr></tbody></table>

chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish between invasive and non-invasive cells, and enables study of the effects of test compounds on tumor invasion. Cancer cells - either as aggregates or single cells - are confronted with fragments of embryonic chick heart. After organ culture in suspension for a few days or weeks the confronting cultures are fixed and embedded in paraffin for histological analysis. The three-dimensional interaction between the cancer cells and the normal tissue is then reconstructed from serial sections stained with hematoxylin-eosin or after immunohistochemical staining for epitopes in the heart tissue or the confronting cancer cells. The assay is consistent with the recent concept that cancer invasion is the result of molecular interactions between the cancer cells and their neighbouring stromal host elements (myofibroblasts, endothelial cells, extracellular matrix components, etc.). Here, this stromal environment is offered to the cancer cells as a living tissue fragment. Supporting aspects to the relevance of the assay are multiple. Invasion in the assay is in accordance with the criteria of cancer invasion: progressive occupation and replacement in time and space of the host tissue, and invasiveness and non-invasiveness in vivo of the confronting cells generally correlates with the outcome of the assay. Furthermore, the invasion pattern of cells in vivo, as defined by pathologists, is reflected in the histological images in the assay. Quantitative structure-activity relation (QSAR) analysis of the results obtained with numerous potentially anti-invasive organic congener compounds allowed the study of structure-activity relations for flavonoids and chalcones, and known anti-metastatic drugs used in the clinic (e.g., microtubule inhibitors) inhibit invasion in the assay as well. However, the assay does not take into account immunological contributions to cancer invasion.

The histological sections as presented in Figure 5 show the end result of a number of successful assays. The sound histology of the cultures indicates viable cells and allows to interpret the interaction between tumor cells and normal tissue. Furthermore, no immune reaction from the normal tissue can be observed, which confirms the correct age of the chick embryos used e.g., before the immune rejection system is developed. There are no bacteria visible, which indicates absence of (gross) contamination during t...

Here, we present a protocol to study the invasion of tumor cells into living normal tissue fragments in three dimensions. This organ culture technique is mainly applied to test potentially anti-invasive drugs in vitro.

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

The overall goal of this procedure is to offer a relevant organ culture method for studying tumor invasion in three dimensions. This is accomplished by first preparing normal tissue fragments obtained from embryonic chicken hearts. In the second step, aggregates of tumor cells are prepared in suspension.The tumor cell aggregates are then individually confronted with the normal tissue fragments on a semi-solid agar bed for incubation in a suspension culture. In the final step, the confronting cultures are fixed for paraffin embedding and histological sectioning. Ultimately, the invasion of the tumor cells into the tissue fragments can be visualized by serial microscopic analysis of the tissue sections.The main advantage of this technique over existing methods like invasion assays with gels is that with this technique, tumor cells are confronted with three dimensional living tissue fragments. The implications of this technique extend to the therapy of cancer as there is an unmet need for anti invasive drugs. Generally, individuals new to this method will struggle, and the reason is that they disregard the importance of using the specific dimensions of the tissue fragment and of the tumor cell aggregates.Though this method can provide insight in cancer invasion, it can also be applied to other systems. For instance, studying invasion of peripheral tissues by inflammatory cells or the integration of stem cells into cardiac tissue. Supreme culture, the chick heart fragments begin by incubating a fertilized egg at 37 degrees Celsius for nine days.On day nine, disinfect the shell with 70%ethanol. Then in a tissue culture cabinet, use blunt forceps to open the shell. Next position an enucleation spoon around the neck of the embryo and pull the embryo out of the shell.Place the embryo in a five centimeter glass Petri dish containing five milliliters of ringer salt solution. Then holding sharp forceps in each hand. Rip open the ventral thoracic skin, remove the sternum and use iridectomy scissors to dissect out the heart and disconnect its major blood vessels.Transfer the heart to a new five centimeter glass Petri dish containing five milliliters of culture medium. Supplemented with 5%fetal bovine serum under a microscope with a calibrated ocular grid. Using microdissection scissors resect the upper cranial third of the heart to remove the atria and associated vessels.Then use sharp forceps to dissect the pericardium from the ventricles. Next, make a sagittal hemi section in the ventricles and remove the blood. Then transfer the ventricles into another five centimeter Petri dish with five milliliters of fresh culture, medium and fetal bovine serum, and cut the tissue into 0.4 millimeter diameter pieces.When all of the ventricles have been cut, rotate the dish gently to drive all of the fragments towards the center of the dish. Then use a stainless steel needle to move all of the Cora Eliana towards the periphery of the Petri dish. Now use a glass pasture pipette to transfer the heart fragments into a 50 milliliter Erla Meyer flask covered with a thin film of culture.Medium gas the flask with 5%carbon dioxide and incubate the fragments on a gyratory shaker at 37 degrees Celsius with 70 revolutions per minute for 24 hours. The next day, transfer the fragments into a Petri dish and use a needle to discard the Corporal Eliana, the dark necrotic fragments, and any tissue conglomerates. Incubate the fragments in a 50 milliliter erlenmeyer flask containing six milliliters of culture medium on the gyratory shaker for another 60 hours, two and a half days later, use the microscope and needles to select the steroidal fragments that exhibit a thin homogenous layer of fibroblastic cells and a diameter of 0.4 millimeters.Then transfer the pre cultured heart fragments to another Petri dish containing fresh culture medium. Three days before the start of the confronting culture, suspend 100, 000 test cells per milliliter in six milliliters of the appropriate culture medium in a 50 milliliter erlenmeyer flask. Then incubate the flask on a gyratory shaker at 37 degrees Celsius and 70 revolutions per minute with the appropriate percent of carbon dioxide.After 72 hours, view the aggregates under the microscope and select the steroidal cell aggregates with a diameter of 0.2 millimeters. Next, transfer eight of the 0.4 millimeter pre cultured heart fragments suspended in a minimal amount of culture medium into an embryological. Watch glass containing semi-solid agar medium.Move the fragments into a circle and use a small piece of filter paper to aspirate the excess medium. Place 10 of the 0.2 millimeters steroidal cell aggregates into the circle. After removing the excess medium, move one aggregate towards each of the pre cultured heart fragments until they make contact with each other.Now, seal the lid of the watch glass with paraffin and incubate the co culture at 37 degrees Celsius for four to 24 hours depending on the adhesive properties of the test cells to the pre cultured heart fragments. At the end of the incubation, immerse a confronting pairs in 37 degrees Celsius prewarm culture medium, and use a pasture pipette to transfer each pair into individual five milliliter Erla Meyer flasks containing 1.5 milliliters of culture medium each Then incubate the flasks on the gyratory shaker at 37 degrees Celsius and 120 revolutions per minute with the appropriate percent of carbon dioxide passed through. Two supplemental five milliliter erlenmeyer flasks filled with two milliliters of ringer salt solution for histological analysis of the confrontations.At the end of the incubation period, transfer the cultures to ringer salt solution for a few seconds to remove the serum proteins. Next, transfer the pears to individual three centimeter Petri dishes containing three milliliters of Bowen Holland solution. After two hours, rinse the cultures three times in three milliliters of demineralized water before incubating them in tap water for two hours to remove as much fixative solution as possible.Finally, transfer the pairs into three milliliters of 70%ethanol each for up to several days. In these images of successful cultures, no immune reaction by the normal tissue can be observed in either the tumor or heart tissue, confirming that the chick embryos were used before the development of the immune rejection system. There are also no bacteria visible indicating an absence of gross contamination during the culture period.Further, the rounded periphery of the sections confirms that the cultures existed in suspension without signs of any temporary adherence to the erla mire flask wall. Many compounds have been tested with this Chick heart invasion assay. For those that were structurally related, a quantitative structure activity relationship was established based on results of the assay with the data demonstrating a clear correlation between the predicted and experimental anti invasive activities.Indeed, this confusion matrix can be used to summarize the weaknesses and the strengths of the assay, giving a rough expression of the accuracy and the reproducibility of the experiment. While attempting this new procedure, it's extremely important to stick strictly to the prescribed dimensions of the tissue fragments Following this procedure. Other methods like confrontation with non-cancer cells can be performed to answer additional questions like Do inflammatory cells invade?After watching this video, you should have good understanding of how to discriminate between invasive and non-invasive tumor cells. Don't forget that working with fixatives can be extremely hazardous and precautions such as air ventilations should be taken while performing this procedure.

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Research

JoVE Journal

Medicine

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