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

Published: June 13th, 2016



1London Regional Cancer Program, London Health Sciences Centre, 2Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, Western University, 3Department of Biochemistry, Schulich School of Medicine & Dentistry, Western University, 4Department of Oncology, Schulich School of Medicine & Dentistry, Western University, 5Lawson Health Research Institute
* These authors contributed equally

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 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.

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....

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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 mous.......

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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.......

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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 ("getting there") and growth in that organ ("growing there"). Many studies have previously focused on identifying key molecular characteristics associated with the canc.......

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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....

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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 
1 x 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

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