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....

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....

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

List of materials used in this article

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