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Wide-field fluorescence endoscopy and dual-axis confocal microscopy for
targeted imaging of cathepsin activity in colon cancer
Steven SensarnContag Lab
SCIT Program Seminar 02/05/14
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Early Detection of Cancer:Colorectal Cancer
• Third most common form of cancer affecting men and women and second most deadly
• Regular screening prevents 60% of deaths, and 9/10 patients whose cancer is detected early are alive 5 years later (CDC.gov)
• Cancerous and precancerous growths are removed during screening
• After age 50, colonoscopy recommended once every 10 years (Cancer.org)
• Because screening is so infrequent, it is especially important not to miss even early lesions
• Wide-field fluorescence imaging with cancer-targeting molecular probes may catch early lesions
Colon
Endoscope
Fluorescence Fiberscope
Fluorescence ImageWhite-Light
Image
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Technology Overview
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Digitally displayed image
Wide-Field Fluorescence Endoscopy forColon Cancer Detection
DAC microscope
Wide-field fluorescence
Slide courtesy of Mike Mandella
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Wide-Field Fluorescence Endoscopy forColon Cancer Detection
~0.9 mm
225 Multi-Mode Illumination Fibers
6600 Imaging Fibers
~0.4 mm
231 cm
GRIN Lens
70°FOV
CCD
Filter
Boston Scientific SpyGlass Fiberscope
125 μm
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Rodent Endoscope System
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Dual-Axis Confocal (DAC) Miniature Microscope• Optical sectioning without
cutting tissue
• Image stacks: hundreds of tissue sections in one 3D acquisition
• MEMS technology and miniature optics: endoscope-compatible microscope
Micro-MirrorFiberscope
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Targeting Colon Cancer in a Mouse Model with Cathepsins
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Healthy Intestine (Surrounding Tumor)
Tumor (polyp)
DA
PI/
BM
V-1
09
(C
y5)
Pan Cathepsin Probe: BMV-109
• High cathepsin protease enzyme activity in inflammation- and tumor-associated macrophages
• BMV-109 targets active enzyme, covalently binds, and de-quenches to become fluorescent
Slide courtesy of Ehud Segal
Bogyo Lab
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• A/J mouse aged 6-8 weeks injected i.p. with 1 mg/kg azoxymethane weekly for 6 weeks
• Develops numerous polyps in the distal colon
• Recapitulates human adenoma to carcinoma sequence
• More inclined to beta catenin pathway than APC mutation pathway
Animals prepared and maintained by Gambhir Lab
Mouse Model of Colon Cancer
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Topical Staining Protocol
• Mouse anesthetized with isoflurane, and distal colon flushed with PBS enema
• BMV-109 probe administered (200 μL, 10 μM) intra-rectally using soft catheter and syringe
• Incubation for 45 min• Unbound probe washed out with PBS enemas• In-vivo imaging of descending colon/rectum and
ex-vivo imaging of entire colon
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In-Vivo Fluorescence Imaging
White Light + Color Camera
Remove Mirror + Laser On + LED Off
Laser + EMCCD (Fluorescence)
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iPhone
iPhone
iPhone
IVIS 200
IVIS 200
M1
M2
M3
Ex-Vivo Fluorescence Imaging
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Wide-Field + DAC
iPhoneFiberscope
DAC Microscope
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Biomarker-Specific Fluorescence
Polyp Polyp Polyp ColonColonColon
Vehicle (intrarectal-topical)
BMV-109 (intrarectal-topical )
BMV-109 (intravenous)
α tubulin
Cat S
Cat L
Cat X
Cat B
• Covalently bound probe can be imaged on fluorescence gel reader to confirm cathepsin labeling
• (Example above: BMV-109 administered topically and intravenously to APCMin transgenic colon cancer mice)
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Vision: A Multimodal Fluorescence Endoscope
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Multimodal Fluorescence Imaging
iPhone
IVIS 200White Light
Wide-Field Fluorescence
DAC Microscopy: Functional Cellular Labeling
DAC Microscopy: Morphological Counterstain
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Summary
• Colon cancer prevention and survival are highly dependent on effective screening
• Wide-field fluorescence endoscopy combined with targeted molecular probes offers a new mode of sensitive imaging for early detection of tumors
• Future endoscopic tools can include wide-field and microscopic fluorescence imaging to better locate and identify cancer margins
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Acknowledgements
• Fluorescence Endoscopy– Gambhir Lab
• Cristina Zavaleta
• Wansik Lee
– Bogyo Lab
• Ehud Segal
– Contag Lab
• Stephan Rogalla
• DAC Microscope Development– Contag Lab
• Jonathan Liu
• Mike Mandella
• Hyejun Ra
• Nathan Loewke
– Solgaard Lab
• Jae-Woong Jeong
• Special Thanks– Contag Lab members
– Small Animal Imaging Core Facility
• Funding and Support– SCIT Fellowship
– Network for Translational Research (NTR)
– Stanford In Vivo Cellular and Molecular Imaging Centers (ICMIC)
– Stanford Center for Children’s Brain Tumors (CCBT)
– Stanford Photomedicine Center