Establishment of a malignant pleural effusion mouse model: pathogenesis pathways

A malignant pleural effusion (MPE), a significant clinical problem, may indirectly be caused by the cancer or its treatment, predominantly lung and breast adenocarcinoma or may appear as pleural effusion unrelated to the cancer (1). More specifically, the development of malignant mesothelioma or, most commonly, pleural metastasis of different cancers, are the main causes for resulting MPE. Systemic spread of disease and reduction of life expectancy and quality are the main implications of MPE (1).

Management of MPE centers on palliation of symptoms because no available treatments prolong survival (1). However, the limited treatment options which are nonspecific and often ineffective, include, treatment of the primary tumor, draining of pleural cavity and pleurodesis, the best method so far to control re-accumulation of the pleural fluid (2).

It is a fact that the pathogenesis of MPE is not fully understood. However, recently several considerable efforts have been made towards this target. These efforts include the development of animal models of MPE which have contributed significant advances in the understanding of the pathogenesis of MPE. In most studies, mice have been used and occasionally rats and rabbits. The different models vary in terms of the immune status of hosts, selection of tumor cells, and location/pathway of the inoculation of tumor cells (3).

The most common practice in animal models of MPE is the introduction of tumor cells or tissue into the pleural cavity of a recipient animal (host). Although it is different from the spontaneous metastasis of tumor cells from a primary site to the pleural cavity, it provides a useful way for research on the subsequent behaviors (including tumor angiogenesis, tumor growth, invasion, metastasis, and the formation of MPE) of tumor cells after they reach the pleural cavity (4).

A novel approach is by spontaneous genetically induced carcinogenesis in the absence of any exposure to asbestos (5) in contrast to traditionally requirement of some form of asbestos exposure, either via inhalation or intrapleural delivery (6,7). To facilitate tumor take of allogeneic (most of the times human) cancer cells or tissues in an alien host, animal models of MPE have mostly been developed as in immune-deficient hosts, including severe combined immunodeficient (SCID) and athymic (nude; natural cytotoxicity receptor, NCR-deficient) mice as well as immune-deficient rats (3). In recent studies immunocompetent hosts implanted with syngeneic tumors, have been used, such as when the VX2 tumor is propagated in the pleural space of New Zealand White (NZW) rabbits (8-10) or the MethA fibrosarcoma is injected into the pleural cavities of syngeneic Balb/c mice (11), and investigators reported that syngeneic models are superior to those employing immunecompromised animals (12,13).

Furthermore, tumor cell suspensions or tissue can be done either by tying cancerous tissues in the pleural space of animals, which is tedious and time-consuming, or by injection via fine needles which is easier and more reproducible (3). In addition, as far as histologic type and organ of origin is concerned, the greatest applicability is found when animal models employ cancer types greatly associated with MPE, such as lung and breast adenocarcinoma and lymphoma (2). The majority of studies have reported intraperitoneal rather than intrapleural introduction of tumors, which is easier, however, intrapleural (orthotopic) models are advantageous in the study of MPE (3).

Finally, delivery of tumor cells to the pleural space can be achieved in three ways: (I) by intravenous injection with subsequent blood-borne translocation to lung vasculature and lung/pleural outgrowth (14) by intratracheal; (II) intrabronchial, intrapulmonary; (III) subpleural injection with subsequent outgrowth into the pleural space (15) and by direct introduction into the pleural cavity (8-10,16,17).

A very important step in the study of animal models of MPE is the determinations of tumor progression which is shown either by cachexia (16,18), or mostly by determination of pleural fluid accumulation (8-10,17-20). Pleural tumor dissemination can be assessed by counting the number/size of pleural tumor implantations (15-18). In addition, the number or fraction of animals surviving is also an important end-point (5,16-18).

Imaging modalities are easy and rapid assessment of pleural effusion and tumor and include photography (14,17,18,20), radiography, computed tomography magnetic resonance and positron emission imaging (21,22), as well as bioluminescent/biofluorescent imaging of relevant tumor cell reporters (21,22).

Since according to recent studies tumor-induced inflammation, new vessel formation (angiogenesis), and vascular hyperpermeability are important in MPE pathogenesis (18,20,22,23), determinations relevant to these biologic processes may serve as additional end-points in animal models of MPE.

Inflammation can be measured by the closed confines of the pleural space, the tumor tissue, and the blood of MPE animals (3,18). Angiogenesis is quantified by immunolabeling of endothelial cells with factor VIII-related antigen or CD34 with subsequent assessment of the amount/density of new vessels in pleural tumor tissue (17-20,22). In addition, angiogenic mediators such as VEGF are an important end-point in the pathogenesis of MPE (23).

Vascular permeability is also a very important phenomenon for understanding the pathogenesis of MPE. It can be determined by several ways, such as the measurement of protein or albumin in pleural fluid or blood (3). Recent studies indeed, have shed light in the pathogenesis of MPE. More specifically, researchers concluded that the VEGF and its receptor 1 (VEGF1) play a significant role in MPE formation (20,23). Others reported that autocrine IL-6/Stat3/VEGF signaling pathway may also be activated in patients with MPE (19).

In another study, IL-5 was identified as a promoter to MPE formation by adenocarcinoma through effects on the MPE-associated inflammatory response (12). The same group experimented with aminobiphosphonates, such as zoledronic acid (ZA), which has potent indirect antitumor effects and concluded that ZA limited the expression of pro-inflammatory and angiogenic mediators, as well as the activity of small GTP proteins Ras and RhoA, in tumor cells in vivo and in vitro. They suggested that this intervention should be considered for testing in clinical trials (18).

Other investigators concluded that tumor-derived osteopontin plays an important role in MPE formation (17), and that vinorelbine may improve the final outcome (24). More groups found that pleural fibrinolytic activity and MYO18B gene repression is an important pathway in the pathogenesis of MPE (8-10,25). Recently, researchers tried to establish a MPE nude mouse model by trans-pleural inoculation of Lewis lung carcinoma cell lines expressing enhanced green fluorescent protein (LLC-EGFP) by observing the tumor growth with in vivo fluorescence imaging system (4). They suggest that this fluorescent model is a powerful and reliable tool in the investigation of pleural metastasis of lung cancer.

In conclusion, the study of MPE models has elucidated MPE pathobiology and has provided proof-of-concept evidence for the paracrine effects of genetic alterations in cancer cells on the host. Preclinical models of MPE could result in finding several potential therapeutic targets against this condition and hopefully will halt or even prevent MPE in cancer patients.

Acknowledgements

Dr. Paul Zarogoulidis would like to thank the organizing committee of Chinese Association of Bronchology and Interventional Pulmonology (CABIP) for the invitation to present: “Management of malignant pleural effusion by suicide gene therapy in advanced stage lung cancer” in the 1^st annual Conference of CABIP in Shanghai 29^th June - 1^st July 2012.

Disclosure: The authors declare no conflict of interest.

References

1. Muduly D, Deo S, Subi Ts, et al. An update in the management of malignant pleural effusion. Indian J Palliat Care 2011;17:98-103.
2. West SD, Davies RJ, Lee YC. Pleurodesis for malignant pleural effusions: current controversies and variations in practices. Curr Opin Pulm Med 2004;10:305-10.
3. Stathopoulos GT, Kalomenidis I. Animal models of malignant pleural effusion. Curr Opin Pulm Med 2009;15:343-52.
4. Ma X, Sun Y, Wang S, et al. Establishment of a malignant pleural effusion mouse model with Lewis lung carcinoma cell lines expressing enhanced green fluorescent protein. Zhongguo Fei Ai Za Zhi 2012;15:317-23.
5. Jongsma J, van Montfort E, Vooijs M, et al. A conditional mouse model for malignant mesothelioma. Cancer Cell 2008;13:261-71.
6. Kroczynska B, Cutrone R, Bocchetta M, et al. Crocidolite asbestos and SV40 are cocarcinogens in human mesothelial cells and in causing mesothelioma in hamsters. Proc Natl Acad Sci U S A 2006;103:14128-33.
7. Robinson C, van Bruggen I, Segal A, et al. A novel SV40 TAg transgenic model of asbestos-induced mesothelioma: malignant transformation is dose dependent. Cancer Res 2006;66:10786-94.
8. Hatton MW, Southward SM, Ross BL, et al. Relationships among tumor burden, tumor size, and the changing concentrations of fibrin degradation products and fibrinolytic factors in the pleural effusions of rabbits with VX2 lung tumors. J Lab Clin Med 2006;147:27-35.
9. Hatton MW, Southward SM, Ross BL, et al. Angiostatin II is the predominant glycoform in pleural effusates of rabbit VX-2 lung tumors. J Lab Clin Med 2002;139:316-23.
10. Hatton MW, Southward SM, Legault KJ, et al. Fibrinogen catabolism within the procoagulant VX-2 tumor of rabbit lung in vivo: Effluxing fibrin (ogen) fragments contain antiangiogenic activity. J Lab Clin Med 2004;143:241-54.
11. Kimura K, Nishimura H, Matsuzaki T, et al. Synergistic effect of interleukin-15 and interleukin-12 on antitumor activity in a murine malignant pleurisy model. Cancer Immunol Immunother 2000;49:71-7.
12. Stathopoulos GT, Sherrill TP, Karabela SP, et al. Host-derived interleukin-5 promotes adenocarcinoma-induced malignant pleural effusion. Am J Respir Crit Care Med 2010;182:1273-81.
13. Psallidas I, Karabela SP, Moschos C, et al. Specific effects of bortezomib against experimental malignant pleural effusion: a preclinical study. Mol Cancer 2010;9:56.
14. Boehle AS, Dohrmann P, Leuschner I, et al. An improved orthotopic xenotransplant procedure for human lung cancer in SCID bg mice. Ann Thorac Surg 2000;69:1010-5.
15. Ohta Y, Kimura K, Tamura M, et al. Biological characteristics of carcinomatosa pleuritis in orthotopic model systems using immune-deficient rats. Int J Oncol 2001;18:499-505.
16. Astoul P, Colt HG, Wang X, et al. Metastatic human pleural ovarian cancer model constructed by orthotopic implantation of fresh histologically-intact patient carcinoma in nude mice. Anticancer Res 1993;13:1999-2002.
17. Cui R, Takahashi F, Ohashi R, et al. Osteopontin is involved in the formation of malignant pleural effusion in lung cancer. Lung Cancer 2009;63:368-74.
18. Stathopoulos GT, Moschos C, Loutrari H, et al. Zoledronic acid is effective against experimental malignant pleural effusion. Am J Respir Crit Care Med 2008;178:50-9.
19. Yeh HH, Lai WW, Chen HH, et al. Autocrine IL-6-induced Stat3 activation contributes to the pathogenesis of lung adenocarcinoma and malignant pleural effusion. Oncogene 2006;25:4300-9.
20. Yano S, Herbst RS, Shinohara H, et al. Treatment for malignant pleural effusion of human lung adenocarcinoma by inhibition of vascular endothelial growth factor receptor tyrosine kinase phosphorylation. Clin Cancer Res 2000;6:957-65.
21. Stathopoulos GT, Zhu Z, Everhart MB, et al. Nuclear factor-kappaB affects tumor progression in a mouse model of malignant pleural effusion. Am J Respir Cell Mol Biol 2006;34:142-50.
22. Stathopoulos GT, Kollintza A, Moschos C, et al. Tumor necrosis factor-alpha promotes malignant pleural effusion. Cancer Res 2007;67:9825-34.
23. Zebrowski BK, Yano S, Liu W, et al. Vascular endothelial growth factor levels and induction of permeability in malignant pleural effusions. Clin Cancer Res 1999;5:3364-8.
24. Cui R, Yoshioka M, Takahashi F, et al. Vinorelbine is effective for the malignant pleural effusion associated with lung cancer in mice. Anticancer Res 2008;28:1633-9.
25. Edakuni N, Ikuta K, Yano S, et al. Restored expression of the MYO18B gene suppresses orthotopic growth and the production of bloody pleural effusion by human malignant pleural mesothelioma cells in SCID mice. Oncol Res 2006;16:235-43.