P4 Distribution of Cetuximab in Models of Human Lung Cancer

Transcription

1 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tübingen, Robert Bosch Foundation Stuttgart, Germany FORSYS Partner Project Predictive Cancer Therapy Project Meeting P4 Distribution of Cetuximab in Models of Human Lung Cancer Thomas E. Mürdter

2 Model substance Cetuximab Commercially available (Erbitux) antibody against EGFR (c-erbb-1) Dosing schedule in vivo: once weekly initial dose of 400 mg/m 2 Subsequently 250 mg/m 2 Steady state plasma concentrations: maximum µg/ml trough µg/ml

3 ELISA for Quantification of Cetixumab Calibration range: ng/ml Dilution of perfusion buffer samples: 1:10,000 to 1:100,000 Calibration: Validation: 1,60 450nm 140 1,40 1,20 1,00 0,80 0,60 0,40 found added Intra-assay (n=5) Inter-assay (n=5) 1 µg/ml / / µg/ml / / ,20 0, Concentration [ng/ml]

4 ELISA for Quantification of Cetixumab Sample preparation for tissues Homogenization with detergent tissue + 10-fold volume of PBS/Tween 20 FastPrep 3 * 30 sec centrifuge tif Dilution of supernatant 1:10,000 to 1:100,000

5 Localization of labelled Cetuximab Fluorescence labels Autofluorescence of lung tissue following different treatment 5,5 5,0 Ex 488 nm Fluoresce ence [AU] 4,5 4,0 3,5 30 3,0 2,5 2,0 1,5 10 1,0 0,5 OregonGreen-488 lung tissue autofluorescence 0, wavelength λ[nm]

6 Localization of labelled Cetuximab Fluorescence labels Samples from lung perfusion experiment with OregonGreen labelled Cetuximab normal lung tissue tumor tissue t = 0 min t = 105 min t = 105 min OregonGreen + Autofluorescence Autofluorescence Fibres DRAQ5 nuclei

7 Binding of Cetuximab to different cell lines Lung tumor cell lines Cancer associated fibroblasts Negative controls A CAF LAMA A CAF Megol H CAF SUPB15 H CAF K /08 Primary tumor cells FITC-IgG control OregonGreen-Cetuximab

8 Localization of labelled Cetuximab Digoxigenin labels Labeling with digoxigenin active ester Immunohistochemical staining of tissue sections using monoclonal anti digoxigenin-alkaline phosphatase antibody FACS-Analysis A using fluorescence labelled ll secondary antibody

9 OregonGreen vs Digoxigenin Labelling OregoGreen-Cetuximab 1. Digoxigenin-Cetuximab g 2. FITC-Anti-digoxigenin A549 BAF3 negative control K562 FITC-IgG I control OregonGreen-Cetuximab Mouse Anti-Digoxin-IgG-FITC control 1. Cetuximab-Digoxingenin 2. Mouse Anti-Digoxin-IgG-FITC

10 Growth inhibitory effect of Cetuximab Comparison of different cell lines - Incubation of BAF3 cells in presence of different concentrations of Cetuximab (3 d) -MTT-Assay Positive results No effect on proliferation 100 MCF10 BAF3 EGF-junkies Lung cancer cell lines A549 NCI-H460 NCI-H23 NCI-A427 f control] Viability [% of BAF3 Primary tumor cells 5705/08 P21 Cancer associated fibroblasts 3925/09 CAF 2089/09 CAF Cetuximab [µg/ml] Leukemia cell line K562 EGF deprivation

11 Growth inhibitory effect of Cetuximab Comparison of different labelled Cetuximab - Incubation of BAF3 cells in presence of different Cetuximab preparations (3 d) -MTT-Assay 100 trol] ility [% of cont Viabi BAF3 0 unlabelled Digoxigenin OregonGreen EGF deprivation control

12 Lung perfusion model respirator CO 2 waterbath lung tumor reservoir T m p po 2 pco 2 pump ph data acquisition ph-depending automated dosage of CO 2 Reduction of death volume Online monitoring of lung mass

13 Lung perfusion model

14 Lung perfusion model On line data aquisition 8.50 Settings of CO 2 -dosage 7.38 delta delta Preparation: Connections: Init. lung weight: lower lobe 2 arteries ]Connections: bronchus 255 g ph ph pco Time [min] Init. Perf. Volume: 850 ml Init. Cet conc.: 100 µg/ml Flow: 110 ml/min Flow: 150 ml/min tery pressure [mm m Hg] Pulmonary art Time [min] pco2 [mm Hg] weight [g g]

15 Lung perfusion model Kinetics and distribution rfusion buffer [µ µg/ml] Cetuximab in pe Cet time [min] Cetuxima ab in peripohera l lung tissue [µg /g] Decrease of Cetuximab in perfusion buffer t ½ perfusion lung weight [min] volume [ml] [g] n.c , ,

16 Lung perfusion model Kinetics and distribution t = end of perfusion ( min) Samples from peripheral lung and perfusion buffer Cetuxim mab [µg/g] n= t = end of perfusion ( min) + ~2 h Samples from central lung, peripheral and central tumor 10 0 perf buffer Lu peripher Lu central Tu peripher Tu central lung tumor

17 Lung perfusion model Distribution immunohistochemical investigations Peripheral lung tissue after 70 min perfusion with Cetuximab-Digoxigenin Formalin fixated - Paraffin embedded Staining with mouse anit-digoxigenin-peroxidase 500 µm

18 Lung perfusion model Distribution immunohistochemical investigations Staining with mouse anti-digoxigenin-peroxidase Perfusion with Cetuximab-Digoxigenin t = 0 min t = 70 min 100 µm

19 Lung perfusion model Distribution immunohistochemical investigations Cetuximab Anti-digoxigenin Blood vessels CD34 10 mm

20 Lung perfusion model Distribution immunohistochemical investigations Cetuximab Anti-digoxigenin Blood vessels CD µm

21 Lung perfusion model Distribution immunohistochemical investigations Cetuximab Anti-digoxigenin Blood vessels CD µm

22 Lung perfusion model Distribution immunohistochemical investigations Cetuximab Anti-digoxigenin Blood vessels CD µm

23 Short term culture of tumour tissue slices Procedure tumour tissue consent of the patient, surgery routine diagnosis histology (HE) tissue punch Krumdieck tissue slicer slices: -thickness: 200µm -diameter: 5mm 24 well plate 1 slice/well Short term incubation with Oregon Green-488 labelled Cetuximab Staining of nuclei Confocal laser microscopy

24 OregonGreen-488 labelled Cetuximab Incubation of lung cancer tissue slices Incubate viable tumor slices with 100 µg/ml Oregon Green-488 labelled cetuximab in medium Wash with PBS with 1%BSA Confocal laser microscopy In 6 / 7 tumor tissues less than 5% of cells showed positive staining i No changes in proliferations rates (Ki67) No changes in cell death (TUNEL)

25 Outlook Lung perfusion Human lung perfusion experiments Cetuximab Staining of tumor tissue for EGFR-overexpression Rat lung perfusion experiments with Cetuximab Tumour tissue slice culture Binding of TRAIL fusion proteins (following labelling with digoxigenin) Influence of TRAIL fusion proteins on cell death and proliferation

26 Isolated rat lung perfusion Approx. 10 perfusions w/o substance Approx. 10 perfusions with small molecules l No sampling of tissue during perfusion Problem to display whole vascular system not yet solved

27 Outlook Lung perfusion Human lung perfusion experiments Cetuximab Staining of tumor tissue for EGFR-overexpression Rat lung perfusion experiments with Cetuximab Tumour tissue slice culture Binding of TRAIL fusion proteins (following labelling with digoxigenin) Influence of TRAIL fusion proteins on cell death and proliferation