MALIGNANT PLEURAL FLUIDS AID MESOTHELIOMA CELL GROWTH AND PROPAGATION

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1 MALIGNANT PLEURAL FLUIDS AID MESOTHELIOMA CELL GROWTH AND PROPAGATION HUI MIN CHEAH PhD Candidate School of Medicine and Pharmacology University of Western Australia Lung Institute of Western Australia

2 MALIGNANT PLEURAL EFFUSION (MPE) IN MALIGNANT MESOTHELIOMA >95% of malignant pleural mesothelioma (MPM) patients suffer from pleural effusion No studies on why MPM produces pleural fluid throughout the course of disease Often viewed as a mechanical effect on breathing No studies have investigated its biological role

3 MALIGNANT PLEURAL FLUID IN TUMOUR ENVIRONMENT Normal Mesothelioma

4 EVIDENCE OF BIOLOGICAL ACTIVITY Numerous growth factors, cytokines etc. - VEGF, TNF-α, IL-8, MCP-1 May be biologically active involved in cancer progression Important to examine the interaction of Cancer and pleural effusion

5 Pleural Space PROPOSED BIOLOGICAL ROLE OF MPE IN CANCER PROGRESSION Free-floating malignant cells METASTASIS PLEURAL Chemotherapeutic FLUID agents FORMATION CHEMORESISTANCE Tumour cells PROLIFERATION Capillaries VASCULAR PERMEABILITY Lymphatics

6 AIMS Define the biological effects of pleural fluids on mesothelioma cells Proliferation and tumour growth in vitro and in vivo Migration Chemoresistance

7 PLEURAL FLUID SAMPLES Obtained from pleural clinic and patients with indwelling pleural catheters Supernatant of pleural effusions were collected and stored at -80 C Samples were randomly selected Mesothelioma (n=60): Subtypes including epitheloid, biphasic, sarcomatoid

8 Pleural Space PROLIFERATION AND TUMOUR GROWTH PLEURAL FLUID FORMATION Tumour cells PROLIFERATION Capillaries Lymphatics

9 COLONY FORMATION ASSAY Stimulate MM cells with MPE (Mesothelioma) (v:v 30, 10, 3%) Pleural fluids were diluted in standard tissue culture media (DMEM) Crystal violet staining

10 PLEURAL FLUID INCREASES COLONY FORMATION WITH MPE TREATMENT 0% MPE 3% MPE 10% MPE 30% MPE

11 PROLIFERATION ASSAY Stimulate MM cells with pleural fluids 30% (v:v) for 48hrs Pleural fluids were diluted in standard tissue culture media (DMEM) -ve control (Serum Free) and +ve control (10% Fetal Calf Serum) Trypan blue counting by 1 blinded investigator

12 PLEURAL FLUID INDUCES MM CELL PROLIFERATION

13 PLEURAL FLUID INDUCES PROLIFERATION IN ALL CELL LINES TESTED

14 Subcutaneous Model IN VIVO STUDIES TUMOUR GROWTH Injected 1 X 10 6 MM cells S/C Injections of MPE (mesothelioma) or saline - for first 6 days after tumour formation (10mm 2 )

15 PLEURAL FLUID ENHANCES TUMOUR GROWTH IN SUBCUTANEOUS MODEL Injections

16 Pleural Space MIGRATION Free-floating malignant cells METASTASIS PLEURAL FLUID FORMATION Tumour cells Capillaries Lymphatics

17 MIGRATION ASSAY SCRATCH-WOUND ASSAY MM cells are grown to confluency and a wound is introduced by scratching with a pipette tip Cells are then treated with pleural fluids (v:v 30%) for 24hrs % wound closure - determined by Cellavista Analyzer (Roche)

18 24 hrs 0 hrs PLEURAL FLUID PROMOTES MIGRATION OF MM CELLS +ve control (FCS) -ve control (Serum Free) 30% MPE

19 PLEURAL FLUID PROMOTES MIGRATION OF MM CELLS

20 PLEURAL FLUID PROMOTES MIGRATION OF MM CELLS

21 Pleural Space CHEMORESISTANCE Chemotherapeutic agents CHEMORESISTANCE Tumour cells Capillaries Lymphatics

22 CHEMORESISTANCE APOPTOSIS ASSAY Drug combination Cisplatin (50μM) + Pemetrexed (25μM) Treatment Cells were treated for 48hrs in complete media with: -Drug combination + Pleural fluid 30% (v:v) -Drug combination only Cells were stained with Annexin V-FITC and 7AAD

23 PLEURAL FLUID PROVIDES PARTIAL PROTECTION AGAINST DRUG-INDUCED CELL DEATH

24 SUMMARY Malignant pleural effusions have potent biological activities: Increase proliferation in vitro and in vivo Stimulator of migration in all MM cell lines tested Potential involvement in chemoresistance Proof-of-principle study Further validation in vivo

25 ACKNOWLEDGEMENTS SUPERVISORS W/Prof. Y C Gary Lee Prof. Jenette Creaney Dr. Sally Lansley PLEURA GROUP MEMBERS Ai Ling Tan Dr. Julius Varano FUNDING BODIES RAC Sir Charles Gairdner Group University of Western Australia Lung Institute of Western Australia

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