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

Summary

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.

Abstract

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.

Introduction

Breast cancer is the most frequently diagnosed cancer in women and the second leading cause of cancer-related deaths¹. Breast cancer'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's disease typically spreads or metastasizes to 5 main sites, including the bone, lungs, lymph node, liver, and brain^4-6. Many theories have been developed to explain this process, but only a few have withstood the test of time. Ewing'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's 1889 "seed and soil" hypothesis suggested that additional molecular interactions were responsible for survival and growth of metastases, whereby cancer cells ("seeds") can only establish themselves and proliferatein organ microenvironments that produce appropriate molecular factors ("soil")⁸. Almost a century later, Leonard Weiss undertook a meta-analysis of previously published autopsy data and confirmed Ewing'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'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 metastasis^10-12, 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.

Protocol

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)

1. 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.
2. Euthanize 6-12 week old mouse by CO₂ inhalation. Mice should be left in the CO₂ chamber for approximately 1 - 2 min or until the mouse stops moving and breathing. Successful euthanasia can be further confirmed by a lack of heartbeat when checked manually with a finger. Avoid cervical dislocation as this method may rupture blood vessels of the neck leading to difficulty removing axillary lymph nodes.
   Note: Previous work has specifically used healthy female nude mice, Hsd:Athymic Nude-Foxn1^nu13.
3. In a sterile tissue culture hood, place the mouse on its back on a polystyrene foam pad, spread the limbs and use pins to keep them in place.
4. Using sterile forceps and scissors, cut the abdominal skin at the midline at the genitalia and cut upwards toward the mouth. Gently pull back the abdominal skin from the abdominal muscles and pin in place on the polystyrene foam pad.
5. Locate the axillary, brachial, and inguinal lymph nodes.
   Note: Lymph nodes are usually surrounded by fatty tissue. The inguinal lymph nodes are the easiest to locate as they are found superficially at the junction of two blood vessels on the pulled back abdominal skin. Axillary and brachial lymph nodes are located deeper within the tissue and require gentle maneuvering of tissue.
   1. After you have located the lymph nodes, use the scissors to gently and carefully cut the lymph nodes away from the skin, fat and vessels and remove them from the mouse. To confirm proper dissection, roll the forceps over the removed tissue. If a hard lump exists when rolling the forceps over the tissue, then a lymph node has likely been removed successfully.
   2. Place removed lymph nodes in ice cold PBS.
6. Using the forceps and scissors, open the abdominal cavity by cutting through the exposed abdominal wall in an upward motion towards the chest. Carefully cut through the sternum, exposing the thoracic cavity.
7. Locate the diaphragm below the lungs and cut the diaphragm. It should pull towards the ribs due to tension.
8. Lift the lungs from underneath and cut the underlying tissue towards the trachea. This allows the lungs to be removed freely from the thoracic cavity. Remove the heart and lungs en bloc and place in ice cold PBS. The heart can be removed from the lungs here or just before weighing in Step 2.
9. Remove the pins, keeping the mouse in place on the polystyrene foam pad. Turn the mouse over and cut the skin of the lower back all the way across from flank to flank.
10. Using a sterile piece of gauze to hold the torso of the mouse, peel the back skin of the mouse over the legs and feet of the mouse.
11. Using the same piece of sterile gauze, hold the lower leg in place, carefully break the ankle joint of the mouse foot and peel the skin over the joint proximally towards the knee joint.
12. Using scissors, remove the tibia free from the knee joint and place in ice cold PBS.
13. Repeat steps 1.11) to 1.12) with opposite limb.
14. Using forceps, hold the femur in place, cut away surrounding muscle tissue using the scissors and remove the femur, placing it in ice cold PBS.
15. Repeat step 1.14) with opposite limb.
16. Using a new piece of sterile gauze, hold the head of the mouse in place. Using forceps and scissors, gently remove the skin to expose the skull. Using scissors, carefully cut the occipital skull from the top center in a straight and downward line to expose the posterior brain.
17. Using forceps, scoop underneath the brain towards the anterior and remove the whole brain. Place the brain in ice cold PBS.
18. Repeat steps 1.1) to 1.17) for at least four mice.

2. Organ Weighing

1. Following organ isolation, weigh each PBS tube containing lung and brain tissue using an electronic balance.
2. Calculate the weight difference by subtracting the pre-isolation weight (PBS only) from the weight of the PBS tube + organs (lung and brain).
3. Determine the amount of media needed to resuspend tissue fragments from the calculated weight difference.
   Note: Lung and brain tissue are weight normalized by resuspension in a 4:1 media:tissue ratio (vol/wt).

3. Generation of Lung- and Brain- Conditioned Media

1. In a sterile tissue culture hood, invert PBS tubes containing lung or brain three times to remove residual blood from organs and aspirate PBS containing blood. Repeat with fresh cold PBS until solution appears clear with no evidence of blood.
2. Place lungs and brains in separate 60 mm² glass petri dishes. Using two sterile scalpel blades, mince lungs or brains by repeatedly slicing back and forth until tissue fragments are approximately ~ 1 mm³ in size.
3. Resuspend tissue fragments in appropriate volume (determined previously in step 2.3) of Dulbecco's Modified Eagle's Medium (DMEM):F12 media supplemented with 1x concentrated mitogen supplement and penicillin (50 µg/ml)/streptomycin (50 µg/ml).
4. Add resuspended lung or brain tissue fragments to one well of a 6-well plate.
5. Incubate tissue fragments in media for 24 hr at 37 °C and 5% CO₂. Following incubation, collect conditioned media for each tissue and further dilute by adding three equivalent volumes of fresh media in a 50 ml conical tube.
6. Centrifuge at 1,000 x g in diluted conditioned media for each organ at 4 °C for 15 min to remove large tissue debris. Collect media supernatant and filter through a 0.22 µm syringe filter.
7. Pool conditioned media from each organ (i.e., lung with lung and brain with brain) from multiple mice to account for mouse-to-mouse variability. Aliquot and store conditioned media at -80 °C until use.

4. Generation of Bone Marrow-conditioned Media

1. In a sterile tissue culture hood, trim excess tissue from the bone and remove epiphyses (end pieces) from the bones with scissors.
2. Using a 27 ½ G needle, flush medullary cavity of bones by pushing 1 ml of PBS through the center of each bone. This will allow you to collect bone marrow stromal cells (BMSC)into a fresh tube containing PBS.
3. Centrifuge at 1,000 x g for 5 min at 4 °C and wash BMSC twice with PBS. Resuspend bone marrow stromal cells in 20 ml of bone stromal cell growth media (DMEM:F12 media supplemented with 1x concentrated mitogen supplement, penicillin (50 µg/ml)/streptomycin (50 µg/ml) and 10% fetal boven serum (FBS)).
4. Plate 10 ml of resuspended bone marrow stromal cells in one T75 flask.
   Note: Combine and plate cells from every 2 mice in each T75 flask; for four mice, two T75 flasks are needed (approximately 1 x 10⁷ cells/flask).
5. Incubate bone marrow stromal cells in bone stromal cell growth media for 24 hr at 37 °C and 5% CO₂. Following incubation, remove media from both T75 flasks and put into new T75 flask, label this flask as "Floater Flask". Add fresh bone stromal cell growth media to previous 2 flasks and incubate all 3 flasks at 37 °C and 5% CO₂.
6. After cells reach approximately 70% confluency (approximately 5 - 7 days) passage cells. To do this, remove medium and wash cells twice with PBS (3 ml each wash). Remove PBS and add 3 ml of trypsin/EDTA solution, ensuring that trypsin covers the entire surface of the flask. After cells lift off the flask (~ 2 - 3 min), stop trypsinization reaction by adding 3 ml bone stromal cell growth media). Centrifuge 900 x g for 5 min at 4 °C, discard media, and resuspend cells in 10 ml of fresh bone stromal cell growth media.
7. Pool cells from all flasks and passage 1:5 into three new T75 flasks and incubate at 37 °C and 5% CO₂. After cells once again reach 70%, repeat step 4.6 and pool and passage all adherent cells a second time. Re-plate all cells to four T75 flasks and incubate 37 °C and 5% CO₂. Bone stromal cell growth media should be used for all steps.
8. Allow cells to reach confluence, wash cells three times with PBS and add bone stromal cell collection media (DMEM:F12 media + 1x concentrated mitogen supplement + penicillin (50 µg/ml)/streptomycin (50 µg/ml); 10 ml/T75), making sure that this media is free of FBS. Collect bone marrow-conditioned media 72 hr later, filter through a 0.22 µm filter, pool, aliquot, and store at -80 °C until use.
9. If desired, confirm the phenotype of the isolated bone marrow stromal cells (BMSC) using antibodies against mouse Sca-1, CD105, CD29, and CD73, CD44, using standard flow cytometry techniques as described previously¹³.

5. Generation of Lymph Node-conditioned Media

1. In a sterile tissue culture hood, invert PBS tube containing lymph nodes three times to remove residual blood from lymph nodes and aspirate bloody PBS. Repeat with fresh cold PBS until solution appears clear with no evidence of blood.
2. Place lymph nodes in 60 mm² glass petri dishes. Using two sterile scalpel blades, mince lymph nodes by repeatedly slicing back and forth until tissue fragments are approximately 1 mm³ in size.
3. In a conical tube, resuspend tissue fragments in 10 ml of Roswell Park Memorial Institute 1640 (RPMI1640) media supplemented with penicillin (50 µg/ml)/streptomycin (50 µg/ml), 5 x 10^-5 M sterile β-mercaptoethanol (1.75 µl/500 ml media) and 10% FBS.
4. Centrifuge at 900 x g for 5 min at 4 °C and resuspend all cells in 30 ml media. Add 5 ml media/well in a 6-well plate and incubate for 7 days at 37 °C and 5% CO₂.
5. Following the 7 day incubation, discard media, wash adherent cells with 5 ml warm PBS and add 5 ml fresh media to each well. Allow cells to grow to confluency (approximately 5 - 7 days), trypsinize, pool all cells, and passage as described in step 4.6. Re-plate cells to 6-well plate. Repeat this step three times.
6. After three passages, allow cells to grow to confluency, wash wells three times with PBS and add 2 ml/well of DMEM:F12 + 1x concentrated mitogen supplement and penicillin (50 µg /ml)/streptomycin (50 µg/ml), ensuring that this media is free of FBS. Collect lymph node-conditioned media after 72 hr, pool, aliquot, and store at -80 °C until use.
7. If desired, confirm the phenotype of the isolated lymph node stromal cells (LNSC) using antibodies against mouse CD45 and gp38, using standard flow cytometry techniques as described previously¹³.

6. Use of Organ-conditioned Media for Downstream Assays Related to Metastatic Behavior of Cancer Cells

1. Once the organ-conditioned media has been generated as described in steps 1 - 5, use it to carry out different downstream cell and molecular biology assays in order to determine the influence of soluble organ-derived factors on the metastatic behavior of cancer cells. Examples of standard assays (described elsewhere in the literature) include protein arrays¹³, cellular growth assays¹³, cellular migration assays¹³, tumorsphere formation¹⁴, and RT-PCR¹⁵. The original base media used to generate the organ-conditioned media (i.e., unconditioned DMEM:F12 media supplemented with 1x concentrated mitogen supplement and penicillin (50 µg/ml)/streptomycin (50 µg/ml) should be used as a negative control.

Results

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...

Discussion

Metastasis is a complex process by which a series of cellular events are ultimately responsible for tissue invasion and distant tumor establishment^4,30,31. 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 ("getting there") and growth in that organ ("growing there"). Many studies have previously focused on identifying key molecular characteristics associated with the canc...

Materials

Name	Company	Catalog Number	Comments
50 ml conical tubes	Thermo Scientific (Nunc)	339652	Keep sterile
1x Phosphate-buffered saline	ThermoFisher Scientific	10010-023	Keep sterile
Nude mice	Harlan Laboratories	Hsd:Athymic Nude-Foxn1nu	Use at 6 - 12 weeks of age
Polystyrene foam pad	N/A	N/A	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.
Forceps	Fine Science Tools	11050-10	Keep sterile
Scissors	Fine Science Tools	14058-11	Keep sterile
Gauze pads	Fisher Scientific	22-246069	Keep sterile
60 mm2 glass petri dishes	Sigma-Aldrich	CLS7016560	Keep sterile
Scalpel blades	Fisher Scientific	S95937A	Keep sterile
DMEM:F12	Life Technologies	21331-020	Warm in 37 °C water bath before use, keep sterile
1x Mito + Serum Extender	BD Biosciences	355006	Referred to as "concentrated mitogen supplement" in the manuscript. Keep sterile
Penicillin-Streptomycin (10,000 U/ml)	Life Technologies	15140-122	Keep sterile
Rosewell Park Memorial Institute 1640 (RPMI 1640)	Life Technologies	11875-093	Warm in 37 °C water bath before use, keep sterile
Fetal Bovine Serum	Sigma-Aldrich	F1051-500ML	Keep sterile
Trypsin/EDTA solution	ThermoFisher Scientific	R-001-100	Warm in 37 °C water bath before use, keep sterile
6-well tissue culture plates	Thermo Scientific (Nunc)	140675	Keep sterile
0.22 μm syringe filters	Sigma-Aldrich	Z359904	Keep sterile
T75 tissue culture flasks	Thermo Scientific (Nunc)	178905	Keep sterile
Transwells	Sigma-Aldrich	CLS3464	Keep sterile, use for migration assays
Anti-mouse Sca-1	R&D Systems	FAB1226P	use at 10 µl/106 cells
Anti-mouse CD105	R&D Systems	FAB1320P	use at 10 µl/106 cells
Anti-mouse CD29	R&D Systems	FAB2405P-025	use at 10 µl/106 cells
Anti-mouse CD73	R&D Systems	FAB4488P	use at 10 µl/106 cells
Anti-mouse CD44	R&D Systems	MAB6127-SP	use at 0.25 µg/106 cells
Anti-mouse CD45	eBioscience	11-0451-81	use at 5 µl/106 cells
Anti-mouse gp38	eBioscience	12-5381-80	use at 10 µl/106 cells
β-mercaptoethanol	Sigma-Aldrich	M6250	Keep sterile
Protein arrays	RayBiotech Inc.	AAM-BLM-1-2	Use 1 array per media condition (including negative control), in triplicate