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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Cancer Research

Acellular and Cellular Lung Model to Study Tumor Metastasis

Published: August 19th, 2018

DOI:

10.3791/58145

1Department of Surgery, Houston Methodist Hospital Research Institute, 2Division of Thoracic Surgery, Department of Surgery, Weill Cornell Medical College, Houston Methodist Hospital

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.

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.

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....

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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 .......

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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.......

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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 .......

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Min P. Kim received grant support from the Second John W. Kirklin Research Scholarship, American Association for Thoracic Surgery, Graham Research Foundation, Houston Methodist Specialty Physician Group Grant, and Michael M. and Joann H. Cone Research Award. We thank Ann Saikin for the language editing of the manuscript.

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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

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