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 different cancers that improved the prospect of a cure or progression-free survival. The clinical management of some cancers, such as breast cancer, improved significantly1; however, some cancers, such as lung cancer, still have a poor survival2. In vitro and in vivo animal models have been instrumental in generating new insights into the mechanisms that underpin the development of the disease. In the last few years, cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) have been of more interest as they preserve many relevant features of the primary human tumor3, such as growth kinetics, histological features, behavioral characteristics, and the response to therapy. However, each model has its limitations to understand the mechanism of CTC formation and metastasis to a distant organ4,5,6.
Recently, we developed a 4-D ex vivo lung cancer model by utilizing the concept of organ reengineering and perfusion-based cell culture. It mimics the human lung cancer growth by forming perfusable tumor nodules that grow over time with a similar human cancer-secreted protein production7. It represents the gene expression signature that predicts poor survival in patients with cancer and also shows a therapeutic response by tumor regression upon cisplatin treatment8,9. The lung model was further modified so that it can form metastatic lesions. The CTCs develop from a primary tumor and intravasate into the vasculature and extravasate into the contralateral lung to form metastatic lesions10. Gene expression studies suggest a distinct expression profile of the primary tumor, the CTCs, and the metastatic lesions, and the upregulation of the subset of genes required for the phenotype10. This metastatic process occurs due to the presence of biologic conditions seen in patients with cancer. The advantage of this model is the presence of a natural matrix and architecture, and a perfusion of nutrients that leads to the formation of tumor nodules. In addition, it also provides an opportunity to study the effects of different components of tumor microenvironment or drugs on tumor progression over time. This model can be used to grow a range of cancer cells (lung cancer, breast cancer, sarcoma etc.) in a laboratory set-up.
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
2. Lung Decellularization
3. Bioreactor Set-up
4. Metastasis Model and Cell Seeding
NOTE: Proceed to cell seeding with the above lung model for a primary tumor growth study. Modify the lung scaffold (acellular or cellular) as follows for the metastatic model.
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 of the native rat cells present in the lungs, which can be observed by hematoxylin and eosin (H&E) staining showing the absence of cells and by DNA analysis11 showing a reduction in DNA amount (Figure 3D and 3G). The ex vivo 4-D lung model can be used to grow any kind of adherent cells12. Lung cancer, lung fibroblast, breast cancer, and sarcoma cells were grown by seeding through the trachea11,12,13. The metastasis model allows for the collection of primary tumor tissue, CTCs, and metastatic lesions at different time intervals by lobectomy10. The H&E staining of the tissues showed intact vasculature and alveoli in both the cellular and acellular model (Figure 3H and 3I). The acellular 4-D lung model is an add-on model to study the interaction between different components of the microenvironment. The cellular 4-D lung model provides an intact lung microenvironment with no immune cells14. Immune cells can be added to this model through the vasculature to study their effect on tumor growth and the interaction with tumor cells. Reproducibility and quality of the model can be assessed by running the model in duplicate and analyzing the tissue/cells by H&E and immunohistochemistry using cell-specific markers. Figure 4 shows a schematic diagram that represents the major steps involved in creating the ex vivo 4-D lung model.
Figure 1: Required items for the decellularization unit of the lung scaffold. (A and B) 500 mL bottle caps are drilled and female Luer connectors are set up with tubing to create the decellularization bottles. (C) Another bottle cap needs only one hole to set up (D) the intravenous set for reagent flows through the pulmonary artery of the lung scaffold. The pump is set up with (F) a pump, (E) a pump head, and (G) a cartridge. (H) Pump tubing with female Luer connectors at each end, (I) connectors, and (J) tubes with male Luer connectors at both ends are required to set up (K) the decellularization unit. (L) The lung scaffolds are washed for 3 – 5 d in a closed-loop system with 1x antibiotics-antimycotic PBS. Please click here to view a larger version of this figure.
Figure 2: Bioreactor set-up for an ex vivo 4-D lung cell culture. (A and B) Bioreactor bottle (500 mL) caps are drilled and three female Luer lock connectors are inserted and fixed with a black nylon ring. (C) 10 ft of oxygenator tube is wrapped around a wire-mesh solenoid with male Luer integral lock rings on both ends. (D) A pulmonary artery cannula and (E) a tracheal cannula are prepared using an 18 G needle and pump tube. (F) A 20 mL syringe is set to the one-way trachea connector to seed the cells in the epithelial space of the lung scaffold. (G) The closed-loop bioreactor is set up inside the cell culture incubator for perfusion and tumor growth. Please click here to view a larger version of this figure.
Figure 3: Representative images of the ex vivo 4-D lung model for metastasis. (A) An intact heart-lung block is harvested from 4- to 6-week-old rat. (B) Histopathology shows the alveoli and pneumocytes. (C) Decellularization leads to the complete removal of the lung cells. (D) Histopathology shows an intact basement membrane with acellular lungs. (E and F) Movat's pentachrome and elastin stain reveals the presence of collagen, fibrin, and elastin with intact vasculature and bronchus. (G) DNA analysis shows a reduction in DNA content of more than 99%. (H) The cellular lung can be used directly in the bioreactor for cell culture and (I) a histological analysis shows the presence of tumor growth. Please click here to view a larger version of this figure.
Figure 4: Schematic diagram showing the major steps in the creation of the ex vivo 4-D lung model. In step 1, a heart-lung block is harvested from a rat. As per the study requirement, in step 2, the lungs can either be used directly as a cellular model or can be decellularized to remove any native cells. The next step involves setting up the lung model in the bioreactor and cell seeding. Finally, bioreactors are set up in the incubator for the cell culture. Please click here to view a larger version of this figure.
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 for the ex vivo 4-D lung model. A delay in administering the heparin injection after the thoracic opening may result in a blood clot in the lungs, which may affect the free flow of the reagents during the decellularization. A proper flushing with heparinized PBS until a colorless fluid comes from the vent is important, especially with the cellular model. The careful cutting of the superior vena cava is important to avoid any harm to the PA. During the decellularization process, it is very important to avoid air bubbles moving to the lung scaffold. The presence of air bubbles may slow down or stop the flow of reagents and contribute to an improper decellularization. Bubbles can be removed by sucking out the bubbles using a 10 – 20 mL syringe. It is very important to make sure the PA cannula is not twisted. During the bioreactor set-up, any twisting in the tubing may result in a pop-up at the connectors and result in a complete mess inside the incubator. To avoid twisting in the cannula, the cannula/needles can be cut and reconnected using biocompatible tubing, which provides the flexibility needed to rotate the cannula without disturbing the connections. Any leakage of culture media inside the incubator may further require a professional cleaning. While creating the metastasis model, careful suturing is required at the trachea bifurcation site. Any puncture of the lungs or trachea may lead to the leaking of cells and inconsistent results.
The acellular and cellular ex vivo 4-D lung models have several advantages over conventional 3D cell cultures to better understand cancer progression, as they provide the opportunity to study the major biological steps in metastasis with an additional dimension of flow. Both models mimic the biology of tumor growth and metastasis and provide an opportunity to collect tumor cells in different phases of cancer growth to better understand the gene signatures, the response to treatments, and the development of drug resistance. Although it is a lung matrix, it has the potential to grow a range of established cell lines and primary cells. The limitation of the ex vivo 4-D model is the requirement of > 10 million cells seeding to study the metastasis. Cells with a low proliferative potential may take a longer time to show the metastatic lesion. In addition, this model requires more aseptic conditions once the cells are seeded.
The authors have nothing to disclose.
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.
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 |