Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tübingen, Robert Bosch Foundation Stuttgart, Germany FORSYS Partner Project Predictive Cancer Therapy Project Meeting 14.10.09 P4 Distribution of Cetuximab in Models of Human Lung Cancer Thomas E. Mürdter
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 170 240 µg/ml trough 40 90 µg/ml
ELISA for Quantification of Cetixumab Calibration range: 0.1 4 ng/ml Dilution of perfusion buffer samples: 1:10,000 to 1:100,000 Calibration: Validation: 1,60 Absorption @ 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 1.03 +/- 0.17 0.97 +/- 0.14 50 µg/ml 61.9 +/- 5.9 52.2 +/- 7.6 0,20 0,00 0 1 2 3 4 5 Concentration [ng/ml]
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
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 0,0 500 550 600 650 700 wavelength λ[nm]
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
Binding of Cetuximab to different cell lines Lung tumor cell lines Cancer associated fibroblasts Negative controls A427 198 CAF LAMA A549 18199 CAF Megol H23 447 CAF SUPB15 H460 13336 CAF K562 5705/08 Primary tumor cells FITC-IgG control OregonGreen-Cetuximab
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
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
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 90 80 70 60 BAF3 Primary tumor cells 5705/08 P21 Cancer associated fibroblasts 3925/09 CAF 2089/09 CAF 50 10-2 10-1 10 0 10 1 10 2 10 3 Cetuximab [µg/ml] Leukemia cell line K562 EGF deprivation
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 90 80 70 60 60 BAF3 0 unlabelled Digoxigenin OregonGreen EGF deprivation control
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
Lung perfusion model
Lung perfusion model On line data aquisition 8.50 Settings of CO 2 -dosage 7.38 delta 0.1 7.32 delta 0.03 125 8.25 100 Preparation: Connections: Init. lung weight: lower lobe 2 arteries ]Connections: bronchus 255 g ph 8.00 7.75 750 7.50 7.25 ph pco 2 7.00 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time [min] 50 500 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 40 30 20 10 0 10 20 30 40 50 60 70 80 90 100 110 120 130 0 200 Time [min] 75 50 25 450 400 350 300 250 pco2 [mm Hg] weight [g g]
Lung perfusion model Kinetics and distribution rfusion buffer [µ µg/ml] Cetuximab in pe 100 90 80 70 60 50 40 30 20 Cet04 10 10 0 0 0 10 20 30 40 50 60 70 80 time [min] 100 90 80 70 60 50 40 30 20 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. 2000 75 9,4 875 255 5,9 1000 430
Lung perfusion model Kinetics and distribution t = end of perfusion (70 130 min) Samples from peripheral lung and perfusion buffer Cetuxim mab [µg/g] 70 60 n=3 50 40 30 20 t = end of perfusion (70 130 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
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
Lung perfusion model Distribution immunohistochemical investigations Staining with mouse anti-digoxigenin-peroxidase Perfusion with Cetuximab-Digoxigenin t = 0 min t = 70 min 100 µm
Lung perfusion model Distribution immunohistochemical investigations Cetuximab Anti-digoxigenin Blood vessels CD34 10 mm
Lung perfusion model Distribution immunohistochemical investigations Cetuximab Anti-digoxigenin Blood vessels CD34 100 µm
Lung perfusion model Distribution immunohistochemical investigations Cetuximab Anti-digoxigenin Blood vessels CD34 100 µm
Lung perfusion model Distribution immunohistochemical investigations Cetuximab Anti-digoxigenin Blood vessels CD34 100 µm
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
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)
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
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
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