Name: generation of organ-conditioned media and applications for studying organ-specific influences on breast cancer metastatic behavior
Uploaded: 2016-06-13T15:00:00
Description: Watch this Scientific Journal Video about Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior at JoVE.com

This work was supported by grants from the Canadian Breast Cancer Foundation-Ontario Region, the Canada Foundation for Innovation (No. 13199), and donor support from John and Donna Bristol through the London Health Sciences Foundation (to A.L.A.). Studentship and fellowship support were provided by the Ontario Graduate Scholarship program (Province of Ontario, to G.M.P. and J.E.C.), the Canada Graduate Scholarship-Master's program (to M.M.P), the Canadian Institutes of Health Research (CIHR)-Strategic Training Program (to M.M.P., G.M.P and J.E.C.) and the Pamela Greenaway-Kohlmeier Translational Breast Cancer Research Unit at the London Regional Cancer Program (to M.M.P., G.M.P., J.E.C. and Y.X.). A.L.A. is supported by a CIHR New Investigator Award and an Early Researcher Award from the Ontario Ministry of Research and Innovation.

All animal studies were conducted in accordance with the recommendations of the Canadian Council on Animal Care, under protocols approved by the Western University Animal Use Subcommittee.
1. Organ Isolation (Lung, Brain, Bone, Lymph Node)
<ol>
	<li>Prepare four sterile 50 ml conical tubes (one for each organ to be isolated) containing approximately 30 ml of sterile phosphate-buffered saline (PBS). Pre-weigh each tube of PBS using an electronic balance.</li>
	<li>Euthanize 6-12 week old mous...

<ol>
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Metastasis is a complex process by which a series of cellular events are ultimately responsible for tissue invasion and distant tumor establishment4,30,31. The ex vivo model system presented here can be utilized to study two important aspects of metastatic progression: cancer cell homing or migration to a specific organ (&#34;getting there&#34;) and growth in that organ (&#34;growing there&#34;). Many studies have previously focused on identifying key molecular characteristics associated with the canc...

Breast cancer is the most frequently diagnosed cancer in women and the second leading cause of cancer-related deaths1. Breast cancer&#39;s high mortality rate is mainly due to the failure of conventional therapy to mitigate and eliminate metastatic disease; approximately 90% of cancer-related deaths are due to metastasis2. Understanding the underlying molecular mechanisms of the metastatic cascade is paramount to the development of therapeutics effective in both early and late-stage breast cancer.
Past research has helped elucidate the multistep nature of breast cancer metastasis and it is hypothesized that the outcome of both cancer progression and metastasis is largely dependent upon interactions between cancer cells and the host environment3. Clinical observations indicate that many cancers display organ tropism, i.e., the tendency to preferentially metastasize to specific organs.In the case of breast cancer, a patient&#39;s disease typically spreads or metastasizes to 5 main sites, including the bone, lungs, lymph node, liver, and brain4-6. Many theories have been developed to explain this process, but only a few have withstood the test of time. Ewing&#39;s theory of metastasis, proposed in the 1920s, hypothesized thatthe distribution of metastasis was strictly due to mechanical factors; whereby tumor cells are carried throughout the body by normal defined physiological blood flow patterns and simply arrest in the first capillary bed they encounter7. In contrast, Stephen Paget&#39;s 1889 &#34;seed and soil&#34; hypothesis suggested that additional molecular interactions were responsible for survival and growth of metastases, whereby cancer cells (&#34;seeds&#34;) can only establish themselves and proliferatein organ microenvironments that produce appropriate molecular factors (&#34;soil&#34;)8. Almost a century later, Leonard Weiss undertook a meta-analysis of previously published autopsy data and confirmed Ewing&#39;s prediction that many metastatic tumors detected at the time of autopsy were found in the anticipated proportions that would be expected if metastatic organ tropism was determined by blood flow patterns alone. However, in manyinstances there were fewer or more metastases formed at certain sites then would be expected by Ewing&#39;s proposed mechanical factors9. These accounts and theories suggest that specific organ microenvironments play a critical role in the dissemination patterns and subsequent growth and survival of many cancers, including breast cancer.
Past research efforts have mainly focused on tumor-cell derived factors and their contribution to the organ tropism observed in breast cancer metastasis10-12, however little research has explored factors derived from the organ microenvironment that may provide a favorable niche for the establishment of breast cancer metastases. This is largely attributable to the technical challenges of studying components of the organ microenvironment in vitro. 
The current article describes a comprehensive ex vivo model system for studying the influence of soluble components of the lymph node, bone, lung, and brain on the metastatic behavior of human breast cancer cells. Previous studies have validated this model system by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential13. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and to evaluate the role of organ-derived soluble factors in driving the process of cancer metastasis.

<table border="0" cellpadding="0" cellspacing="0" width="1195">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">50 ml conical tubes</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td style="width:223px;">339652</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">1X Phosphate-buffered saline</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:223px;">10010-023</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Nude mice</td>
			<td style="width:159px;">Harlan Laboratories</td>
			<td style="width:223px;">Hsd:Athymic Nude-Foxn1nu</td>
			<td>Use at 6-12 weeks of age</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:339px;">Polystyrene foam pad</td>
			<td style="width:159px;">N/A</td>
			<td style="width:223px;">N/A</td>
			<td style="width:475px;">The discarded lid (~4 x 8 inches or larger) of a polystyrene foam shipping container can be used for this purpose. Sterilize by wiping with ethanol.</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:339px;">Forceps</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:223px;">11050-10</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:339px;">Scissors</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:223px;">14058-11</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:339px;">Gauze pads</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:223px;">22-246069</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:339px;">60 mm2 glass petri dishes</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">CLS7016560</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:339px;">Scalpel blades</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:223px;">S95937A</td>
			<td style="width:475px;">Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">DMEM:F12</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">21331-020</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">1 x Mito+ Serum Extender</td>
			<td style="width:159px;">BD Biosciences</td>
			<td style="width:223px;">355006</td>
			<td style="width:475px;">Referred to as &#34;concentrated mitogen supplement&#34; in the manuscript. Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">15140-122</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Rosewell Park Memorial Institute 1640 (RPMI 1640)</td>
			<td style="width:159px;">Life Technologies</td>
			<td style="width:223px;">11875-093</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Fetal Bovine Serum</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">F1051-500ML</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">Trypsin/EDTA solution</td>
			<td style="width:159px;">ThermoFisher Scientific</td>
			<td style="width:223px;">R-001-100</td>
			<td>Warm in 37 &#176;C water bath before use, keep sterile&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">6-well tissue culture plates</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td>140675</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">0.22 &#956;m syringe filters</td>
			<td>Sigma-Aldrich</td>
			<td>Z359904</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:339px;">T75 tissue culture flasks</td>
			<td style="width:159px;">Thermo Scientific (Nunc)</td>
			<td style="width:223px;">178905</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Transwells</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">CLS3464</td>
			<td>Keep sterile, use for migration assays</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse Sca-1</td>
			<td>R&#38;D Systems</td>
			<td>FAB1226P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD105</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB1320P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD29</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB2405P-025</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD73</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">FAB4488P</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD44</td>
			<td>R&#38;D Systems</td>
			<td style="width:223px;">MAB6127-SP</td>
			<td>use at 0.25 &#181;g/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse CD45</td>
			<td>eBioscience</td>
			<td style="width:223px;">11-0451-81</td>
			<td>use at 5 &#181;l/106 cells</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:339px;">Anti-mouse gp38</td>
			<td>eBioscience</td>
			<td style="width:223px;">12-5381-80</td>
			<td>use at 10 &#181;l/106 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">&#946;-mercaptoethanol&#160;</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:223px;">M6250&#160;</td>
			<td>Keep sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:339px;">Protein arrays</td>
			<td style="width:159px;">RayBiotech Inc.</td>
			<td style="width:223px;">AAM-BLM-1-2</td>
			<td>Use 1 array per media condition (including negative control), in triplicate</td>
		</tr>
	</tbody>
</table>

generation of organ-conditioned media and applications for studying organ-specific influences on breast cancer metastatic behavior

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused on identifying factors inherent to breast cancer cells that are responsible for this observed metastatic pattern (termed organ tropism), however much less is known about factors present within specific organs that contribute to this process. This is in part because of a lack of in vitro model systems that accurately recapitulate the organ microenvironment. To address this, an ex vivo model system has been established that allows for the study of soluble factors present within different organ microenvironments. This model consists of generating conditioned media from organs (lymph node, bone, lung, and brain) isolated from normal athymic nude mice. The model system has been validated by demonstrating that different breast cancer cell lines display cell-line specific and organ-specific malignant behavior in response to organ-conditioned media that corresponds to their in vivo metastatic potential. This model system can be used to identify and evaluate specific organ-derived soluble factors that may play a role in the metastatic behavior of breast and other types of cancer cells, including influences on growth, migration, stem-like behavior, and gene expression, as well as the identification of potential new therapeutic targets for cancer. This is the first ex vivo model system that can be used to study organ-specific metastatic behavior in detail and evaluate the role of specific organ-derived soluble factors in driving the process of cancer metastasis.

Generation of Organ-conditioned Media
An overview diagram/schematic of the process of organ isolation and generation of conditioned media is presented in Figure 1, with representative photographic images of the procedure shown in Figure 2. It should be noted that when this protocol was first under development, liver was included in our analysis because it is a common site of bre...

Watch this Scientific Journal Video about Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior at JoVE.com

Generation of Organ-conditioned Media and Applications for Studying Organ-specific Influences on Breast Cancer Metastatic Behavior

This manuscript describes an ex vivo model system comprised of organ-conditioned media derived from the lymph node, bone, lung, and brain of mice. This model system can be used to identify and study organ-derived soluble factors and their effects on the organ tropism and metastatic behavior of cancer cells.

Breast cancer preferentially metastasizes to the lymph node, bone, lung, brain and liver in breast cancer patients. Previous research efforts have focused ...

The overall goal of this procedure is to generate conditioned media using mouse lymph nodes, bones, lungs, and brains to identify and study organ derived soluble factors and their effects on the metastatic behavior of cancer cells. This method can help answer key questions in the metastasis field, such as, how do organ specific factors influence cancer cell growth, migration, and gene expression? This technique provides a straightforward ex vivo platform representative of organ specific in vivo soluble factors that influence the phenotype and behavior of cancer cells.To isolate the lymph nodes, begin by placing a six to twelve week old mouse on a polystyrene foam pad in a sterile tissue culture hood, and secure the limbs in an outstretched position. Next, use forceps and scissors to make a midline incision in the abdominal skin from the genitalia to the mouth. Then, gently pull the skin from the abdominal muscles and pin it to the dissecting pad.Using scissors, carefully remove the lymph nodes from the skin, fat, and vessels, placing the nodes in 30 milliliters of ice cold PBS as they are harvested. When all of the lymph nodes have been collected, use the forceps and scissors to carefully cut through the exposed abdominal wall in an upward motion through the sternum, exposing the thoracic cavity. Cut the diaphragm.Lift the lungs from underneath, and cut the underlying tissue toward the trachea. Then, transfer the lungs into a new container of ice cold PBS. The heart can be removed from the lungs during this step or prior to organ weighing.To harvest the leg bones, place the mouse in the prone position, and cut the skin across the lower back from flank to flank. Next, use a sterile piece of gauze to grasp the torso and the skin over the legs and feet. Using the same piece of gauze, hold the lower leg while carefully breaking the ankle joint of one foot, and peel the skin over the joint proximally towards the knee.Then, using scissors, remove the tibia from the knee joint and place the leg bone in ice cold PBS. After harvesting the second tibia, hold the femur in place with forceps and use scissors to cut away the surrounding muscle tissue. Then, place the leg bone in ice cold PBS.To harvest the brain, use a new piece of sterile gauze to grasp the head, and gently remove the skin with scissors and forceps to expose the skull. Next, use scissors to carefully the occipital bone from the top center of the skull in a straight and downward line to expose the posterior brain. Then, scooping underneath the tissue toward the anterior of the skull, transfer the whole brain into ice cold PBS.To generate the lung conditioned medium, invert the tubes of lung tissue three times to remove any residual blood from the organs. Then, replace the wash with fresh cold PBS and repeat until the saline is blood free. Next, transfer the lungs into one 60 square millimeter glass Petri dish per mouse, and use two sterile scalpel blades to mince the tissues with repeated back and forth slicing.When the tissue fragments are approximately one millimeter cubed in size, re-suspend the pieces in the appropriate volume of DMEM:F12 medium supplemented with antibiotics, and incubate the tissues in one well of a six well plate per mouse at 37 degrees Celsius and five percent CO2. After 24 hours, transfer the entire contents of each well into individual 50 milliliter conical tubes, and dilute the conditioned media with three equivalent volumes of fresh DMEM:F12 medium per tube. Centrifuge the tubes to remove any large pieces of tissue debris.Then, pool the conditioned media through a 0.22 micron syringe strainer into a single 50 milliliter conical tube. To generate bone marrow conditioned medium, transfer the bones under a sterile tissue culture hood, and trim the excess tissue from the bone. Use scissors to remove the epiphyses.Then, use a 27 and a half gauged needle to flush one milliliter of PBS through the medullary cavity of each bone into a tube of fresh PBS. When all of the bone marrow stromal cells have been collected, wash the cells three times in PBS. After the last wash, re-suspend the pellet in 20 millilters of bone stromal cell growth medium supplemented with 10%fetal bovine serum, and seed 10 milliliters of cells per T75 flask.When the cells have reached approximately 70%confluency, wash the cultures two times with three milliliters of PBS. Then, detach the cells with three milliliters of 0.025%trypsin/EDTA solution, taking care that the trypsin covers the entire flask surface. After two to three minutes, arrest the enzymatic reaction with three milliliters of bone stromal cell growth medium, and pool the cells in a 50 milliliter tube.Spin down the cells. Then, re-suspend the pellet in 10 milliliters of fresh medium. Now, pass into the cultures at a one to five ratio into three new T75 flasks until they reach 70%confluency.Then, wash the cells three times with PBS as just demonstrated, and feed the cultures with serum free bone stromal cell collection medium supplemented with mitogen and antibiotics for 72 hours. After the third day of culture, pool the media through a 0.22 micron strainer. The stromal cells should be adherent in culture with an elongated and fibroblastic morphology observed in the lymph node stromal cell cultures, and a smaller and more rounded morphology exhibited by the bone marrow stromal cell cultures.Flow cytometric analysis confirms that the lymph node stromal cells are largely CD45 negative, and gp38 positive. Bone marrow stromal cells should be positive for CD-44 and CD-29, weakly positive for CD-105 and Sca-1, and negative for CD-73. Human breast cancer cells with varying genetic backgrounds exhibit differential migration and growth patterns towards specific organ conditions.For example, in this representative experiment, the bone-seeking 231-BoM variant preferentially migrates to the bone marrow conditioned medium over the brain and lung conditioned media. Similarly, the lung-seeking 231-LM2 variant preferentially migrates to lung conditioned medium over the bone marrow and brain conditioned media. Further, RT qPCR analysis demonstrates that the exposure of parental MDA-MB-231 breast cancer cells to bone or lung conditioned medium induces an up regulation of the genes associated with bone or lung specific metastases of the breast cancer cells in vivo.Once mastered, the tissue harvesting and conditioned medium set ups can be completed in two to three hours if they are performed properly. The sub culturing steps on subsequent days take 15 to 30 minutes per day. While attempting these procedures, it is important to remember to keep everything sterile.Following the conditioned medium generation, other methods like protein arrays, cell growth and migration assays, tumorsphere formation, and RT-PCR can be performed to answer additional questions about the effect of organ specific factors on cancer cell behavior. After its development, this technique paved the way for researchers in the field of breast cancer research to explore organ specific metastatic behavior in an ex vivo model system. After watching this video, you should have a good understanding of how to generate conditioned media using the bones, lungs, and brains of mice.Don't forget that working with sharp dissection tools can be hazardous, and that precautions, such as using precise and careful dissection technique, should always be taken while performing this procedure.

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