Diagnostic des maladies du foie par spectroscopie et imagerie multimodale François Le Naour Inserm U1193, Centre HépatoBiliaire, Villejuif Médecine moléculaire et modèles animaux: pathologie hépatique Institut Pasteur 18 mai 2017
Function of the liver Glycogen synthesis & catabolism Lipid synthesis & catabolism (production of triglyceride) Lipoprotein synthesis Destruction of old red blood cells & leucocytes Production of coagulation factors Metabolism of toxins and drugs Urea synthesis Storage of vitamins Storage of elements (Fe, Cu) The liver is the organ with the most important activity in metabolism
Chronic liver diseases and cancer Normal liver Cirrhosis Hepatocellular Carcinoma Alcohol Dysmetabolic syndrome (diabete, obesity) Viral Hepatitis 6 th cause of cancer worldwide 2 nd cause of death by cancer Poor prognosis (survival < 1 year)
Chronic liver diseases and cancer Normal liver Cirrhosis Hepatocellular Carcinoma Alcohol Dysmetabolic syndrome (diabete, obesity) Viral Hepatitis 6 th cause of cancer worldwide 2 nd cause of death by cancer Poor prognosis (survival < 1 year) Need of markers for diagnostic & prognostic
Hypothèse Le développement des pathologies induit des variations de composition. La composition globale reflète l état du tissu.
A multimodal spectroscopy-based approach at synchrotron SOLEIL Liver Cryomicrotome Tissue section The synchrotron SOLEIL Brillance Accordability IR UV X-ray Microspectroscopy Spectral markers
The synchrotron SOLEIL Electrons Light Characteristics - Brightness IR UV X - Accordability
Light Electrons
X-ray fluorescence spectroscopy Jean-Philippe Braly/LookatSciences
Infrared
Infrared microspectroscopy at SMIS beamline Infrared
Multimodal spectroscopy 10 µm Elemental composition Autofluorescence Biochemical composition
Analyse qualitative de la stéatose hépatique
Chronic liver diseases Normal liver Steatosis Cirrhosis Alcohol Dysmetabolic syndrome (diabete, obesity) Viral Hepatitis
Liver steatosis Steatosis: excess of fat in hepatocytes (TG) Lipid droplets in at least 5% of cells
The synchrotron SOLEIL Electrons Light InfraRed
Infrared spectroscopy IR Polyethyleneoxide Absorbance Energy IR IR IR H C O O H H C O C N 3000-2800 cm-1 1740 cm-1 1650 / 1540 cm-1
Infrared spectroscopy on tissues IR Proteins Absorbance Lipids ADN/sugars Tissue section 3800 3400 3000 2600 2200 1800 1400 1000 Wave number (cm-1) The spectrum is resulting of the global biochemical composition
Infrared microspectroscopy on steatosis Min 1475-1710 cm -1 Proteins 3000-2800 cm-1 Lipids 1710-1780 cm-1 Esters 1710-1780 cm-1 Unsaturated Max Absorbance Absorbance 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 8 7 6 5 4 3 2 1 0 Steatotic Unsaturated C=C 3500 non steatotic 3500 Lipids CH2 & CH3 3000 3000 2500 2500 Esters C=O 2000 Wavenumber (cm-1) 2000 1500 Proteins Amides I & II Esters C=O 1500 1000 1000
Analysis of various stages of steatosis Infrared spectroscopy & Principal component analysis (PCA) Healthy Low Intermediate High Lipids: 2700-3100 cm -1
Analysis of various stages of steatosis Healthy Low Intermediate High CH3 Molecular changes in the composition CH2 C=C
Time of flight-secondary ion mass spectrometry TOF-SIMS Aggregates Bi3+ Secondary ions Desorption ionisation Molecules Tissue section
Time of flight-secondary ion mass spectrometry TOF-SIMS Bi 3+ 25 kev Without any treatment of the sample Lateral resolution : 1-2µm Mass <1500 Da Lipids
Lipidomic in situ of steatosis using ToF-SIMS Centrilobular vein Intensity per unit surface (counts x 10 3 ) b 20 10 MAG 313.3 339.3 Chol. 369.3 551.6 523.5 DAG 577.6 603.6 0.2 PC 0.4 758.6 760.6 TAG 853.8 855.8 857.8 0.0 700 800 900 Portal tract 0 300 400 500 600 700 800 900 m/z d
Lipidomic in situ of steatosis using ToF-SIMS DAG : diacyl glycerol C36:4 C36:3 C36:2 C36:1 C36:0
Lipidomic in situ of steatosis using ToF-SIMS DAG C36 unsaturated DAG C36 saturated Red = Unsaturated Green = Saturated
Multimodal spectroscopy on steatosis Infrared & ToF-SIMS Gradation of steatosis Molecular changes in the composition steatotic vesicles Enrichment in cholesterol Lipids with longer acyl chains Enrichment in unsaturated lipids Steatotic vesicles potentially highly reactive to peroxydation
Stéatose & transplantation hépatique
Chronic liver diseases and cancer Normal liver Cirrhosis Hepatocellular Carcinoma Alcohol Dysmetabolic syndrome (diabete, obesity) Viral Hepatitis 6 th cause of cancer worldwide 2 nd cause of death by cancer Poor prognosis (survival < 1 year) TRANSPLANTATION
Liver transplantation : a good treatment for liver diseases 2003-2009 Agence de la Biomédecine
Limitation of liver transplantation Shortage of grafts Year Transplantation Waiting patients 2005 1024 474 2006 1037 486 2007 1061 540 2008 1011 574 2009 1047 669 2010 1092 806 2011 1164 932 2012 1161 941 2013 nc 1104 Agence de la Biomédecine
Limitation of liver transplantation Shortage of grafts Additional grafts: living donors, graft splitting, domino program, use of marginal grafts, non-heart-beating donors Additional grafts But increased risk Quality of the graft: Steatosis
Graft steatosis Primary non-function 80% with steatosis > 60% 15% with steatosis 30-60% Delayed function 35% with steatosis 30-60% Long term poorer evolution of the graft: fibrosis, survival Ploeg Transplantation 1993
Steatosis sensibilizes liver to ischemia-reperfusion lesions through altered blood flow in liver microcirculation and enhanced peroxydation of membrane lipids
Ischemia-Reperfusion lesions in liver graft
20, 30 or 40%? Estimation of steatosis
Assessment of Hepatic Steatosis by Expert Pathologists: The End of a Gold Standard El-Badry, et al Annals of Surgery. 2009; 250(5):691-697 Cong Poor inter-observer reproducibility Par
Estimation of steatosis in the context of emergency Paraffin Frozen > 24h < 15 min A liver transplantation has to be performed in less than 8h
Histological estimation of steatosis is not correlated to the lipid content % steatosis Triglycerides (TG) (nmol/mg)
Histological estimation of steatosis - Poor inter-observer reproducibility - Poor conservation of the morphology on frozen tissue sections - Not correlated to the true lipid content
Analysis of the non-steatotic tissue Normal liver + Absorbance 7 6 5 4 3 2 1 0 3800 3400 3000 2600 2200 1800 1400 1000 3800 3400 3000 2600 2200 1800 1400 1000 Steatosis + Absorbance 7 6 5 4 3 2 1 0 Wave number (cm-1) The non-steatotic part of the tissue is NOT normal The whole tissue reflects the grade of steatosis
Implementation to the hospital Easy to use method No need of liquid nitrogen Regular glass slide Automatic calculation using the standard curve Only 1 spectrum Only 1 pixel 2 mm x 2 mm Only 1 minute! ATR infrared spectroscopy
Quantification of steatosis ATR-InfraRed spectroscopy Pixel 2 mm x 2 mm 500µm Absorbance 0.8 0.6 0.4 Lipids Proteins Ratio: lipids / proteins 0.2 3500 3000 2500 2000 1500 1000 Wavenumbers (cm -1 ) Estimation of lipids
Quantification of steatosis InfraRed microspectroscopy 25 tissue samples from liver surgical specimens - Steatosis : 5 to 90% Infrared spectroscopy allows the quantification of lipids on tissue section
Quantification of steatosis ATR-InfraRed spectroscopy
Lipid evaluation by IR microspectroscopy 700 600 Estimated Measured [TG] nmol/mg 500 400 300 200 100 0 0 1 2 3 4 5 6 7 8 9 10 11 12 Liver grafts Validation on additional 21 liver graft biopsies
Quantification of liver steatosis Reproducible Rapid method (1 min) Non expensive procedure Assessment of liver graft quality
P Dumas C Sandt I Youssef L Gadea M Réfrégiers F Jamme N Trcera S Kascakova M Danulot C Peng A Brunelle D Touboul D Debois O Laprévote C Guettier M Wavelet B Trousselle MP Bralet Denis Castaing Eric Vibert Thanks for fundings Centre Hépatobiliaire Hôpital Paul Brousse Université Paris-Sud