Non-invasive Imaging to Assess Transplanted Islets Alvin C. Powers - PowerPoint PPT Presentation

US Postal Stamp, issued 2001 Non-invasive Imaging to Assess Transplanted Islets Alvin C. Powers Vanderbilt University

Today � Rationale and challenges for imaging pancreatic islets � Overview of imaging modalities being used � Bioluminescence to assess transplanted pancreatic islets

Pancreatic Islets Are an Imaging Challenge Pancreas Human Islet Purified Human Islets α cells β cells δ cells Photo courtesy of David Harlan Photo courtesy of David Harlan Islets size ( < 250 µ m) is less than resolution of imaging modalities (CT, MRI)

Non-Immune Barriers to Improving Islet Transplantation � Transplanted, intra-hepatic islets are relatively inaccessible � Techniques to non-invasively assess or image islet mass are not available. � Difficult to study islet survival following transplantation (must rely on islet function). � Cannot assess interventions to sustain or improve islet survival after transplantation

Features of Ideal Islet Imaging Modality � Non-invasive and allow for serial, in vivo measurements in the same animal or person � Non-toxic to islet cells � Useful for study of islets in native pancreas and after transplantation � Applicable to murine models � Adaptable for human imaging

So what do you want to know from islet imaging? � Should it measure islet cell mass or number? Should it reflect beta cell number of islet cell number? � Should it reflect function or health of beta cells? The metabolic state and insulin secretory capacity of the beta cell will fluctuate in different physiologic and pathophysiologic conditions. � Is spatial resolution of islets important? Islet mass ≠ Islet function

Approaches to Image Pancreatic Islets Pancreas � Utilize an islet-specific protein or process � Introduce a reporter into islet cells

Features/Approaches That Could be Useful for Imaging � Unique glucose metabolism (GLUT2, glucokinase) K + Ca ++ � Islet or beta cell-specific (or enriched) cell surface markers � High concentration of zinc SUR ATP/ADP Insulin Ca ++ Metabolism Glucokinase Glucose Glucose GLUT2

Features/Approaches That Could be Useful for Imaging � Unique glucose metabolism (GLUT2, glucokinase) � Islet or beta cell-specific (or enriched) cell surface markers � High concentration of zinc � In pancreas, islets are highly vascularized with a fenestrated endothelium � MRI � PET Islet in Mouse Pancreas Islet in Mouse Pancreas � Bioluminescence Imaging

Non-invasive Assessment of Islets Using Bioluminescence US Postal Stamp, issued 2001

Bioluminescence Reaction Luciferase + Luciferin + ATP + O 2 Luciferase-Luciferin + AMP + PP i Luciferase-Luciferin + AMP + O 2 Oxyluciferin* + CO 2 + AMP Oxyluciferin + h ν Oxyluciferin* Photons Luciferase Nucleus Oxygen ATP D-Luciferin

Islet Transplantation Model Islet Transplantation Model NOD- SCID Adapted from JDRF figure Adapted from JDRF figure

NOD-SCID Mouse Model � Lack B- and T-Lymphocytes � NOD background further reduces immunity because of NK cell deficiency � Accept xenografts � Do not develop insulitis or diabetes � Allow long-term expression of adenoviral DNA � Species-specific insulin assay to distinguish human insulin from endogenous mouse insulin

Bioluminescence of Bioluminescence of Transplanted Islets Transplanted Islets Culture Culture Image with Image with CCD Camera CCD Camera Adenovirus Adenovirus encoding encoding luciferase luciferase Quantify Quantify Photon Photon Transplant Transplant Emission Emission

In vitro Bioluminescence in Human islets A. Islets (IEQ)/well B. 0 1000 50 (no virus) 16 r 2 = 0.9808 n = 3 wells 12 8 4 0 0 250 500 750 1000 1250 100 500 1000 # of Islets/well

Imaging Transplanted Islets Kidney Capsule Liver Luminescent Kidney Capsule Liver Luminescent (human islets) (mouse islets) Rod (human islets) (mouse islets) Rod 1500 500 IEQ 1000 IEQ 2000 IEQ Photon counts 0

Imaging Transplanted Islets Kidney Capsule Liver Kidney Capsule Kidney Capsule Liver Kidney Capsule (human islets) (mouse islets) (luminescent rod) (human islets) (mouse islets) (luminescent rod) 1500 2000 500 IEQ 1000 IEQ 2000 IEQ Photon counts Photon counts 0 0

Standardization of Imaging Using Luminescent Bead A C 1.2 1.2 Intensity Normalized to 0 Degrees Intensity Normalized to 0 Degrees 1 1 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0 0 -60 -60 -40 -40 -20 -20 0 0 20 20 40 40 60 60 Angle of Rotation [Degrees] Angle of Rotation [Degrees] B D Camera Camera 1.2 1.2 Intensity Normalized to 0 Degrees Intensity Normalized to 0 Degrees 1 1 0.8 0.8 θ θ 0.6 0.6 0.4 0.4 0.2 0.2 0 0 -60 -60 -40 -40 -20 -20 0 0 20 20 40 40 60 60 θ θ Angle of Rotation [Degrees] Angle of Rotation [Degrees]

Absorption of Photons by Surrounding Tissues 400 400 500 500 600 600 700 700 800 800 Wavelength [nm] Wavelength [nm] 5 cm D E 2000 500 Photon Photon Counts Counts Camera Aperture Air 50 cm 0 0 F 0.3 0.3 Ratio of In Vitro Intensity to In Ratio of In Vitro Intensity to In Skin 0.25 0.25 0.025 cm 0.2 0.2 Vivo Intensity Vivo Intensity Renal Renal Renal 0.15 0.15 Hepatic Hepatic Liver Bead 0.1 0.1 0.1 cm 0.05 0.05 0 0 Hepatic 0 0 1 1 2 2 3 3 4 4 5 5 6 6 7 7 Bead Weeks Post Implant Weeks Post Implant

Bioluminescence of Islets in Kidney or Liver Kidney Liver • 200 mouse islets • Imaged two weeks post-transplant

Bioluminescence is Influenced by Site of Transplantation Renal Intensity Hepatic Intensity (in vitro/in vivo) (in vitro/in vivo) Bead 0.2384 + 0.0261 0.0645 + 0.0140 100 islets 0.0476 0.0116 200 islets 0.0284 0.0112

Bioluminescence of Transplanted Murine Islets 120000 Liver In vivo bioluminescence 100000 Kidney (photon counts) 80000 60000 40000 20000 0 50 100 200 # of Murine Islets Transplanted

Transplanted Human Islet Number and Luminescence 20000 2000 r 2 = 0.9946 16000 1600 n = 3 - 4 mice 12000 1200 8000 800 r 2 = 0.9755 n = 3 - 4 mice 4000 400 0 0 0 500 1000 1500 2000 2500 # of Islets Transplanted

Bioluminescence of Transplanted Islets � Dependent of level of luciferase expression within mouse or human islets; (requires ATP and oxygen and viable islets) � Optical scattering properties of tissue in which luciferase-expressing islet reside and tissues through which emitted light must exit the animal influence bioluminescence. � If these are considered in calculations, bioluminescence reflects transplanted islet # and function.

Bioluminescence of Transplanted Murine Islets Beneath Renal Capsule In vivo bioluminescence (x10 6 ) 5 4 (photon counts) 3 2 1 Tx (50 islets) 0 0 2 4 6 8 10 12 14 16 18 20 Weeks post-transplantation

Bioluminescence of Transplanted Murine Islets Intrahepatic In vivo bioluminescence (x10 6 ) 5 4 (photon counts) 3 2 1 Tx (125 islets) 0 -1 0 1 2 3 4 5 6 7 8 9 Weeks post-transplantation

Decline in Bioluminescence of Transplanted Islets � Large decline ( > 60%) suggesting islet loss in first week after transplantation � Beginning 2 weeks post-transplant, greater loss from intra-hepatic islets of all types � Liver is unfriendly site? � Destroyed by immune attack against luciferase or adenoviral/primate proteins? � Islets no longer express luciferase � Cell division and progeny cells no longer express luciferase

Alternative Approaches � Lentivirus (D. Kaufman, UCLA) � AAV virus � Transgenic expression of luciferase

Bioluminescence to Assess Transplanted Islets � Non-invasive � Sensitive (detect 25-50 transplanted mouse islets) � Photon generation likely reflects islet cell number (and maybe islet function) � Poor spatial resolution � Allows for serial measurements of intra- hepatic islets � Allows testing of interventions to increase or sustain transplanted islet mass

Bioluminescence as Islet Imaging Modality Non-invasive and allow for serial, in vivo Yes measurements in the same animal Non-toxic to islet cells Yes Useful for study of islets in native pancreas Probably and after transplantation (in kidney and liver) Applicable to murine models Yes Adaptable for human imaging No

Co-Registration of Multiple Imaging Modalities and Biologic Information � All imaging modalities will have limitations. � No single modality will answer all questions about islets in pancreas or transplanted islets. � Understanding islet survival and function will require integration of complementary imaging modalities and physiology in animal models and in humans. Martin Lepage, John Gore, Vanderbilt Imaging Institute

Acknowledgements � E. Duco Jansen � Marcela Brissova � Michael Fowler � John Virostko � Daniel Kaufman � Wendell Nicholson � Mark Atkinson � Masa Shiota � A. Radhika � Mark Magnuson � Alena Shostak JDRF � Maureen Gannon � Greg Poffenberger NIH � David Piston � Zhongyi Chen VA � John Gore � Craig Hauck � David Harlan � Jeanelle Kantz � Boaz Hirschberg � Chunhua Dai � Graeme Bell � P. Brahmachary � Soo Young Park � Qing Cai