Acellular and Cellular Lung Model to Study Tumor Metastasis

Summary

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Abstract

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Introduction

Cancer metastasis is the culprit behind most cancer-related deaths and poses the ultimate challenge in the effort to fight cancer. The overall goal of this method is to design a protocol for a four-dimensional (4-D) cell culture which has a dimension of flow, in addition to the three-dimensional (3-D) cell growth. It represents the three distinct phases of the metastasis process [i.e., the primary tumor, circulating tumor cells (CTCs), and metastatic lesions].

Over the past three decades, scientists around the world yielded an unparalleled wealth of information to understand the mechanisms underlying the metastatic progression in d....

Protocol

The protocols for animal experiments were approved by the Institutional Animal Care and Use Committee at the Houston Methodist Research Institute and carried out in accordance with all regulations, applicable laws, guidelines, and policies.

1. Rat Lung Harvest

1. Anesthetize a 4- to 6-week-old male Sprague-Dawley rat by an intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) in its flank. Ensure anesthesia by checking for an absence of movement when the hind limb toe is pinched with forceps. 
2. After 10 min, shave the chest and abdomen and wipe the skin with a chlorhexidine swab.

Results

The lung harvested from rat maintains the intact vasculature and alveoli¹¹ (Figure 3A and 3B). Upon decellularization, the extracellular matrix components of an acellular lung, such as collagen, fibronectin, and elastin, are preserved¹¹ (Figure 3C, 3D, 3E, and 3F). The decellularization leads to a complete removal o.......

Discussion

The ex vivo 4-D lung provides an opportunity to study tumor growth and metastasis in a laboratory set-up. A native lung matrix is a complex system that provides support to normal tissue and maintains cell-cell interactions, cell-matrix interactions, cellular differentiation, and tissue organization. It provides an opportunity to add any tumor microenvironment components to study their effects on tumor growth and the interaction with other cells.

The lung harvest is the critical step .......

Materials

Name	Company	Catalog Number	Comments
Sprague Dowley rat	Harlan	206M	Male
Chlorhexidine swab	Prevantics, NY, USA	NDC 10819-1080-1
Heparin	Sagent Pharmaceuticals, Schaumburg, IL, USA	NDC 25021-400-10
18-gauge needle	McMaster Carr, USA	75165A249
2-0 silk tie	Ethicon, San Angelo, TX, USA	A305H
Masterflex L/S pump	Cole-Parmer, Vernon Hills, IL, USA	EW-07554-80
Masterflex L/S pump head	Cole-Parmer, Vernon Hills, IL, USA	EW-07519-05
Masterflex L/S pump cartridge	Cole-Parmer, Vernon Hills, IL, USA	EW-07519-70
Tygon Tube	Cole-Parmer, Vernon Hills, IL, USA	14171211
MasterFlex Pump tube	Cole-Parmer, Vernon Hills, IL, USA	06598-16
Female luer lock connectors	Cole-Parmer, Vernon Hills, IL, USA	45508-34	75165A249
Male luer lock connectors	Cole-Parmer, Vernon Hills, IL, USA	45513-04
black nylon ring	Cole-Parmer, Vernon Hills, IL, USA	EW-45509-04
Intravenous set	CareFusion	41134E
Sodium Dodecyl Sulfate (SDS)	Fisher Scientific	CAS151-21-3
Triton X-100	Sigma-Aldrich	X100-1L
Antibiotics	Gibco	15240-062
Silicone oxygenator	Cole-Parmer, Vernon Hills, IL, USA	ABW00011	Saint-GoBain-
Wire mesh	1164610105	Lowes	New York Wire
Female luer Lug Style TEE	Cole-Parmer, Vernon Hills, IL, USA	45508-56
Male luer integral lock ring to 200series Barb	Cole-Parmer, Vernon Hills, IL, USA	45518-08
Female luer thread style coupler	Cole-Parmer, Vernon Hills, IL, USA	45508-22
Clave connector	ICU Medical	11956
Hi-Flo ™4-way Stopcock w/swivel male luer lock	smith Medical	MX9341L
MasterFlex Pump tube	Cole-Parmer, Vernon Hills, IL, USA	06598-13	for cannula