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MIPS Stanford University Molecular Imaging Program at Stanford
School of Medicine Department of Radiology
Raman Endoscopy for Delineation of Non-Muscle-Invasive Bladder
Cancer
Ryan Davis Department of Radiology
Gambhir lab
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Bladdercancerisaworldwidepublichealthconcern:
• 165,000peoplediedofbladdercancerworldwidein2012• In2012,5yearprevalencewas1,300,000people• EarlystagebladdercanceristreatedwithtransurethralresecAon
http://www.nature.com/nrc/journal/v15/n1/fig_tab/nrc3817_F1.html http://seer.cancer.gov/statfacts/html/urinb.html
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Bladdercancerhasoneofthehighestrecurrenceratesofallcancers
• Disease free interval between recurrences is often 1-2 years.
• Recurrence is caused by disease left behind after transurethral resection or field defect.
• Several prospective clinical trials have shown that improved detection (lower miss rate) of fluorescence cystoscopy (FC) compared to white light cystoscopy (WLC) reduces the recurrence rate.
New and improved cystoscopy techniques are essential for improving bladder cancer delineation and reducing recurrence
White Light Cystoscopy
Fluorescence Cystoscopy
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WeproposeamolecularcystoscopystrategybasedonSurfaceEnhancedRamanSca8eringnanopar:cles
toimprovedelinea:onofbladdertumors
Gold (60 nm)Raman dye
Silica Coating
= fluorophore
= antibody
1 2 3
4 5
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RamanSca8eringistheinelas:csca8eringoflightbyvibra:onalmodesofmolecularbonds
Fluorescence Raman Scattering Surface Enhanced Raman Scattering
Raman scattered: ~1:10 million Raman scattered: ~1:10-1:100
Khaywah M. et al. 2015
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Gambhir/Contag labs have a endoscope and microscope capable of detecting Raman scattering
Raman microscope
Raman Endoscope. Designed by Ellis Garai in Contag lab.
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WeuseRamanspectroscopybecauseofitspoten:altomul:plexseveralmoleculartargets
Pros/cons of Surface-enhanced Raman Scattering (SERS) nanoparticles for cystoscopy
• Pros• Multiplexing (sharp spectral lines)• Low autofluorescence contamination• Can detect 1-10 pM particles on tissue
• Cons• Requires nanoparticle• Requires specialized cystoscope/endoscope• Limited by quality of biomarker/antibody
Anti-CD47Raman Dye #1
Anti-c-METRaman Dye #2
Anti-CA9Raman Dye #3
Control antibodyRaman Dye #4
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Overviewofmethods/results
Selection of targets: literature review, searching protein & antibody databases
Validation of Ab binders for target
NP functionalization with Ab, testing for specific binding
Apply to human tissue samples, unmix spectra, and visualize
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Overviewofmethods/results
Selection of targets: literature review, searching protein & antibody databases
Validation of Ab binders for target
NP functionalization with Ab, testing for specific binding
Apply to human tissue samples, unmix spectra, and visualize
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unstained
Anti-CD47-dye
Anti-CD47 + blocking Ab
a
b
c
d
An:-CD47valida:onstudiesweresuppor:veofac:vetarge:ngtoCD47
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An:-CA9valida:onstudiesweresuppor:veofac:vetarge:ngtoCA9
β-actin
CA9 + GFP (85kDa)
CA9-eGFP plasmid - + +
β-actin
CA9 (50 kDa)
CoCl2 Time (hr)
+ + + - 26 20 14 0
a
b
c
a) Western blot of HCT116 cells transfected with CA9+eGFP fusion
b) Flow cytometry of HeLa cells induced to express CA9 with CoCl2
c) Western blot of HeLa cells induced to express CA9 with CoCl2
d) Flow cytometry of HCT116 cells transfected ith CA9-eGFP plasmid showed significant binding over controls.
d
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Overviewofmethods/results
Selection of targets (literature review, searching protein & antibody databases)
Validation of Ab binders for target
NP functionalization with Ab, testing for specific binding
Apply to human tissue samples, unmix spectra, and visualize
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a) Binding of CD47-targeted NPs to cells in suspension. KO of CD47 or control antibody caused 38 and 25 times less NP binding (median fluor.) compared to positive control. b&c) Titrations of crosslinker and Ab concentrations were performed to optimize Specific binding ratio (SBR = (WT-KO)/WT).
a b c
Nanopar:clesfunc:onalizedwithan:-CD47ac:velytargetedcellsinsuspension
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Overviewofmethods/results
Selection of targets (literature review, searching protein & antibody databases)
Validation of Ab binders for target
NP functionalization with Ab, testing for specific binding
Apply to human tissue samples, process spectra & images, visualize
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Sample 2Sample 1
SummaryofBladder:ssuesamplesSample 3 Sample 4
photo
Raman(antiCD47
minus control)
Surgeon’s report Normal
urothelium and muscle
Tumor (t), possible inflammation (i),
healthy/muscle (h)
i
th
h
t
Tumor (t), healthy/muscle (h)
h
t
No nanoparticles used. Measured
background signal
Tumor (t), healthy/muscle (h).(no H&E)
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Conclusions
• Binders successfully target CD47 and CA9 in flow and Western blot assays, c-MET binder in progress
• Conjugating CD47 binder to SERS particles actively targets CD47
• Nanoparticles bind to tissue, but further work is needed to see if binding correlates to cancer on histology
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Microscopeandendoscopecandetectpar:cles,althoughendoscopehas~10xlowerSNR.
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Twomainpossibili:esforNP/Abconjuga:on
+
SH
Maleimide-DL65010,000x
SH
SHSH
SH
SHSH
HS
+
SH
SHSH
SH
HS
S440S440-DL650
SH
SHSH
SH
HS+
SM-(PEG)1215,000x
Antibody2,000xS440-DL650
S440-DL650-Ab
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Poten:alsolu:on:PEGgylatesurfaceanduselongercrossilnker
S440-DL650-Ab(using PEG12
crosslinker instead of PEG4)
+
S440-DL650-Ab(using PEG12
crosslinker instead of PEG4)
I believe this will improve the stability and specific binding ability of my particles!