Tu1873 Systemic Delivery of Fiber Redesigned Infectivity-Selective Oncolytic Adenovirus for...
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three Rel-subunits are critical mediators of NF- κB dependent anti-apoptotic signalling in PDAC by subunit specific regulation of specific target genes. Tu1873 Systemic Delivery of Fiber Redesigned Infectivity-Selective Oncolytic Adenovirus for Pancreatic Cancer Yoshiaki Miura, Joohee Han, Julia Davydova, Masato Yamamoto Systemic therapies for cancer, including pancreatic cancer, have provided only limited efficacy for patients to date. Adenovirus (Ad) has frequently been used as a backbone of oncolytic viral agents. However, unlike loco-regional therapy, systemic application of cancer gene therapy mandates different level of selectivity of gene delivery. So far, insufficient cancer selectivity at the stage of infection has greatly hampered efficacy of Ad-based therapy upon systemic administration. The controlled distribution and selective transduction of the vector would overcome the obstacles for systemic delivery and enable efficient systemic treatment of cancer. We have recently developed transductionally-targeted Ads by high-throughput screening of high-diversity (10^9-level) Ad-library. Mesothelin (MSLN) is highly over- expressed in pancreatic cancers. Ad with redesigned AB-loop targeted to MSLN (AdML- VTIN) was successfully isolated from the high throughput screening of Ad library by infectivity for MSLN-expressing 293 cells (293-MSLN). The in vitro binding of AdML-VTIN corre- sponded to MSLN-expression of each cell line, and the suppression of MSLN-expression with siRNA or antibody blocked the binding to MSLN-expressing cells, which indicates the vector is targeted to mesothelin. In MSLN-positive cells (Panc-1), this virus exhibited higher binding ability than that of Ad with 5/3 modified fiber which is known to exhibit highest infectivity in pancreatic cancer cells. The in vivo antitumor effect was compared in Panc-1 (MSLN-positive) and MiaPaCa-2 (MSLN-negative) subcutaneous xenografts. The injection of the MSLN-retargeted oncolytic Ad showed significant antitumor effect against Panc-1 tumors, and disappearance of tumors was observed in 4 out of 8 mice. Contrarily, the same virus showed no antitumor effect in MiaPaCa-2. Viral DNA amount in the tumors correlated to the anti-tumor effect. Upon systemic administration in the nude mice with Panc-1 subcutaneous xenografts, AdML-VTIN showed significantly lower liver sequestration com- pared to the wild-type Ad5, and the copy number of this retargeted virus was significantly (1000-fold) higher in the tumor. These data indicates that this vector enables efficient systemic delivery and treatment of MSLN-expressing pancreatic cancer xenografts. In this study, our MSLN-targeted Ad vector exhibited selective infectivity to MSLN-positive pan- creatic cancer cells in vitro and in vivo. Moreover, this virus showed dramatically augmented delivery to the tumors and significantly reduced liver sequestration upon systemic administra- tion. This new genetically modified Ad, which is transductionally retargeted to pancreatic cancer, may embody a next generation systemic therapy for this devastating disease. Tu1874 Earlier Age of Presentation of Pancreatic Adenocarcinoma in Individuals With Chronic Pancreatitis Arpan Mohanty, Nilesh H. Shah, Michelle A. Anderson, Aaron R. Sasson, Kristen J. Vogel, Simon Sherman, David C. Whitcomb, Randall Brand BACKGROUND: Pancreatic cancer (PC) screening is reserved for the subset of the general population at high risk for PC. Patients with chronic pancreatitis (CP) are at increased risk for PC; a recent meta-analysis reported a relative risk of 13.3 [Best Pract Res Clin Gastroent- erol. 2010 Jun;24(3)]. The exact mechanism that links the two diseases is unknown but may be caused by a shared exposure to risk factors such as cigarette smoking and alcohol consumption and/or facilitation of tumor initiation and promotion by inflammation. We hypothesized that if the latter mechanism was true, the inflammatory state induced from CP would lead to an earlier age of onset of PC development. The goal of this study was to compare the age of onset and stage of PC in patients with a history of CP to those without CP. METHODS: Prospective data on 660 individuals with PC from the multi-center Pancreatic Cancer Collaborative Registry was reviewed for a history of CP. Identified CP cases were matched with controls based on gender, alcohol use and tobacco use. Propensity score matching was used to match, with replacement, each case with its 3 closest controls. Tobacco use was classified as non-smoker; ongoing use of tobacco: cessation of tobacco use , 10 years prior to diagnosis of PC; and tobacco use . 10 years prior to diagnosis of PC. Alcohol users were classified as past or current users of alcohol or non-users. When more than 3 controls were an exact match (ties) for a particular case, the ties were all used and weighted equally. The average treatment effect on the treated (ATT) for age of PC diagnosis and PC stage were calculated. RESULTS: A total of 16 CP patients from the 660 PC cases were identified. 56% of CP patients were males. After matching, CP patients were diagnosed 4.9 years (95% CI: 0.6-9.2 years) earlier than controls (p=0.025). There was no statistical difference in PC stage (I/II vs. III/IV) between control and CP cases. CONCLUSIONS: A history of CP appears to have an independent effect on age of onset of PC with a ~5 year earlier diagnosis, supporting the role of inflammation in PC carcinogenesis. A lack of difference in PC stage, suggests that this result is not related to more frequent abdominal imaging in the CP group. Findings from this study should be factored into any future PC surveillance recommendations for CP patients. Further studies are warranted to determine if shared genetic susceptibility factors involving inflammatory pathways are present in CP and PC patients. Tu1875 Fibroblast Activation Protein (FAP) Expression of Cancer-Associated Fibroblast Predicts Poor Prognosis in Patients With Pancreatic Adenocarcinoma Tomoya Kawase, Koji Yoshida, Keisuke Hino [Background and Aim] In many solid tumors, the stroma is increasingly recognized to be important in promoting tumor proliferation, invasion, metastasis, and chemoresistance. Especially, several studies using mouse pancreatic ductal adenocarcinoma(PDAC)model revealed that fibroblast activation protein(FAP) played an important role in tumorgenesis S-869 AGA Abstracts by reducing the surrounding immunity. FAP, or seprase, is a 170kDa dimer surface glycopro- tein and expressed on cancer associated fibroblasts(CAF). The aim of this study was to investigate the relationship between FAP expression of CAF and the prognosis in patients with PDAC. [Materials and Methods] During 2008-2012 paraffin-embedded specimens of PDAC were collected from 50 patients under the approval by the ethics committee of the institution. Standard immunohistochemical detection of FAP was performed using anti-FAP primary antibody. The expression levels of FAP were classified into 4 degrees (negative, 1+, 2+, 3+) and compared with patients' clinical course. [Results] FAP of CAF was detected in 92%(46/50) of specimens and its expression level was 1+ in 16, 2+ in 19 and 3+ in 11 specimens. The expression level of FAP was negatively correlated with overall survival of patients (p,0.001). Furthermore multivariate analysis showed FAP expression level, clinical staging of pancreatic cancer extracted as significant factors associated with overall survival. [Conclusion] The present results indicated that FAP expression of CAF is one significant factors associated with overall survival of patients with pancreatic ductal adenocarcinoma. Tu1876 Feasibility of Pathway Analysis in EUS-FNA Obtained Tissue of Pancreatic Cancer Patients: A Step Towards Personalized Based Medicine Wesley K. Utomo, Vilvapathy Narayanan, Katharina Biermann, Casper H. van Eijck, Marco J. Bruno, Maikel P. Peppelenbosch, Henri Braat Introduction: Pancreatic cancer (PC) remains one of the most lethal common malignancies worldwide. The five year survival of all stages combined is only 5%. Deregulation of several core signaling pathways have been identified in PC and distinct pathways are affected between different patients. Hence, personalized based medicine is an attractive approach in the treatment of PC. Here, we aim to study the feasibility of pathway analysis using tissue obtained by Endoscopic Ultrasound-Fine Needle Aspiration (EUS-FNA); the activation of mammalian target of Rapamycin (mTOR) in specific by the measurement of phosphorylated S6 (pS6), a downstream protein of the mTOR pathway. Experimental design: Immunohisto- chemical (IHC) staining of pS6 on Formalin Fixed Paraffin Embedded (FFPE) material from PC resection specimens was performed. Forty nine slides(33 tumor regions and 36 healthy regions) were stained and scored by an expert pathologist. To study mTOR activation and inhibition by rapamycin(Rap), PC cell lines (BxPC3, SU8686) were analyzed by means of western blot (cleaved caspase 3, to measure apoptosis, and pS6), cell viability assays and flow cytometry (pS6). Single cell suspensions obtained through EUS-FNA of pancreatic tumors were stained for cytokeratin 8/18 (CK8/18) and pS6 expression and subsequently analyzed using flow cytometry. Results: The variability of IHC stained FFPE material was compared by means of the coefficient of variation of 63.73% in tumor regions (n=33) compared to 35.69% in healthy regions (n=36). Levels of pS6 were 6-30 fold lower after 2 hours of incubation with 0.1 μM Rap. Cell viability were 43% and 38% after 3 days of 12.5 μM Rap. On flow cytometry analysis of cell lines, Rap reduced the Mean Fluorescence Intensity (MFI) in CK8/18 positive cells of pS6 by 75%(BxPC3) and 80%(SU8686). Validation in fresh EUS-FNA tissue by flow cytometry analysis showed higher expression of pS6 in 2 out of 6 (33%) confirmed PC. MFI reductions of 80% and 83% of pS6 in CK8/18 positive populations were observed after incubation with 0.1 μM Rap. Absolute numbers of CK8/18 positive, pS6 positive cells also decreased by 98% and 86.4%. Conclusions: As hypothesized, a great variation in the expression of pS6 exists between PC patients in FFPE material. This finding might indicate a possible subpopulation with higher activation of the mTOR pathway in PC patients. In PC cell lines, Rap reduced cell viability in a dose dependent manner and inhibits pS6 expression without leading to apoptosis. Validation in EUS-FNA tissue showed a marked reduction of pS6 expression after incubation with Rap. The results of this study show the feasibility of pathway analysis in EUS-FNA tissue which is a first step towards personalized based medicine in PC. Further studies in EUS-FNA tissue are being undertaken to determine other activated signaling pathways in PC. Tu1877 Changes in Vascularization and Innervation Patterns in PanIN Lesions As Revealed by Novel Three-Dimensional Imaging Techniques Ya-Yuan Fu, Jennifer M. Bailey, Janivette Alsina, Subhash Kulkarni, Shiue-Cheng Tang, Steven D. Leach, Pankaj J. Pasricha Background & Aims: Pancreatic Intraepithelial Neoplasia (PanIN) is a pancreatic cancer precursor lesion. Due to the opacity of pancreas, microtome-based planar microscopy is a routinely used method to observe PanIN lesions. However, this technique limits our understanding of the morphology and development of PanIN and pancreatic cancer by restricting tissue visualization in a 2-dimensional (2-D) space, rather than rendering a true 3-dimensional (3-D) image of an intact developing PanIN. In order to identify and generate the intact 3-D image of PanIN for facilitating better and early diagnosis of pancreatic cancer, we aimed to (1) apply Ulex europaeus agglutinin I (UEA-I) as a new cell surface marker to specifically label the PanIN lesion, (2) characterize the altered vascular and neural networks by vessel painting the pancreatic tissue with wheat germ agglutinin (WGA) and immunostain- ing, and (3) combine the fluorescent staining with 3-D microscopy to observe the native PanIN lesions at a subcellular level. Methods: We used two independent models of murine PanIN. The first involves the expression of Kras from the progenitor cells of the pancreas: Pdx-1:Cre; LsL-KrasG12D mice (9 months old). The second involves expression of Kras from adult acinar cells: Mist1:CreERT2;LsL-KrasG12D;mTmG mice (analyzed 6 weeks after administration of tamoxifen), and control mice (Mist1:CreERT2;mTmG). The mice were perfused with fluorescent conjugated WGA to reveal the blood vessels of pancreas. The innervation was labeled by immunostaining (using pan-neuronal marker PGP9.5). We used two independent methods to label PanIN. First, the mTmG background enables the visualiza- tion of GFP+ cells expressing KrasG12D. Second, UEA-I was used as a novel cell surface marker to label the PanINs. The labeled tissues were treated with the optical-clearing solution FocusClear (Gastroenterology, 139:p1100-1105, 2010) to prepare transparent specimens for confocal microscopy. Results: Comparisons of staining for various lectins revealed UEA- I to be a specific label for PanIN lesions. This allowed us to develop robust 3-D visualization of PanIN lesions and their relationship to blood vessels and nerves. We discovered an increase in vessel density in PanIN-bearing mice relative to normal pancreas. At the same AGA Abstracts
Transcript of Tu1873 Systemic Delivery of Fiber Redesigned Infectivity-Selective Oncolytic Adenovirus for...
three Rel-subunits are critical mediators of NF- κB dependent anti-apoptotic signalling inPDAC by subunit specific regulation of specific target genes.
Tu1873
Systemic Delivery of Fiber Redesigned Infectivity-Selective OncolyticAdenovirus for Pancreatic CancerYoshiaki Miura, Joohee Han, Julia Davydova, Masato Yamamoto
Systemic therapies for cancer, including pancreatic cancer, have provided only limited efficacyfor patients to date. Adenovirus (Ad) has frequently been used as a backbone of oncolyticviral agents. However, unlike loco-regional therapy, systemic application of cancer genetherapy mandates different level of selectivity of gene delivery. So far, insufficient cancerselectivity at the stage of infection has greatly hampered efficacy of Ad-based therapy uponsystemic administration. The controlled distribution and selective transduction of the vectorwould overcome the obstacles for systemic delivery and enable efficient systemic treatmentof cancer. We have recently developed transductionally-targeted Ads by high-throughputscreening of high-diversity (10^9-level) Ad-library. Mesothelin (MSLN) is highly over-expressed in pancreatic cancers. Ad with redesigned AB-loop targeted to MSLN (AdML-VTIN)was successfully isolated from the high throughput screening of Ad library by infectivityfor MSLN-expressing 293 cells (293-MSLN). The in vitro binding of AdML-VTIN corre-sponded to MSLN-expression of each cell line, and the suppression of MSLN-expressionwith siRNA or antibody blocked the binding to MSLN-expressing cells, which indicates thevector is targeted to mesothelin. In MSLN-positive cells (Panc-1), this virus exhibited higherbinding ability than that of Ad with 5/3 modified fiber which is known to exhibit highestinfectivity in pancreatic cancer cells. The in vivo antitumor effect was compared in Panc-1(MSLN-positive) and MiaPaCa-2 (MSLN-negative) subcutaneous xenografts. The injectionof the MSLN-retargeted oncolytic Ad showed significant antitumor effect against Panc-1tumors, and disappearance of tumors was observed in 4 out of 8 mice. Contrarily, the samevirus showed no antitumor effect in MiaPaCa-2. Viral DNA amount in the tumors correlatedto the anti-tumor effect. Upon systemic administration in the nude mice with Panc-1subcutaneous xenografts, AdML-VTIN showed significantly lower liver sequestration com-pared to the wild-type Ad5, and the copy number of this retargeted virus was significantly(1000-fold) higher in the tumor. These data indicates that this vector enables efficientsystemic delivery and treatment of MSLN-expressing pancreatic cancer xenografts. In thisstudy, our MSLN-targeted Ad vector exhibited selective infectivity to MSLN-positive pan-creatic cancer cells in vitro and in vivo. Moreover, this virus showed dramatically augmenteddelivery to the tumors and significantly reduced liver sequestration upon systemic administra-tion. This new genetically modified Ad, which is transductionally retargeted to pancreaticcancer, may embody a next generation systemic therapy for this devastating disease.
Tu1874
Earlier Age of Presentation of Pancreatic Adenocarcinoma in Individuals WithChronic PancreatitisArpan Mohanty, Nilesh H. Shah, Michelle A. Anderson, Aaron R. Sasson, Kristen J. Vogel,Simon Sherman, David C. Whitcomb, Randall Brand
BACKGROUND: Pancreatic cancer (PC) screening is reserved for the subset of the generalpopulation at high risk for PC. Patients with chronic pancreatitis (CP) are at increased riskfor PC; a recent meta-analysis reported a relative risk of 13.3 [Best Pract Res Clin Gastroent-erol. 2010 Jun;24(3)]. The exact mechanism that links the two diseases is unknown butmay be caused by a shared exposure to risk factors such as cigarette smoking and alcoholconsumption and/or facilitation of tumor initiation and promotion by inflammation. Wehypothesized that if the latter mechanism was true, the inflammatory state induced fromCP would lead to an earlier age of onset of PC development. The goal of this study was tocompare the age of onset and stage of PC in patients with a history of CP to those withoutCP. METHODS: Prospective data on 660 individuals with PC from themulti-center PancreaticCancer Collaborative Registry was reviewed for a history of CP. Identified CP cases werematched with controls based on gender, alcohol use and tobacco use. Propensity scorematching was used to match, with replacement, each case with its 3 closest controls. Tobaccouse was classified as non-smoker; ongoing use of tobacco: cessation of tobacco use , 10years prior to diagnosis of PC; and tobacco use . 10 years prior to diagnosis of PC. Alcoholusers were classified as past or current users of alcohol or non-users. When more than 3controls were an exact match (ties) for a particular case, the ties were all used and weightedequally. The average treatment effect on the treated (ATT) for age of PC diagnosis and PCstage were calculated. RESULTS: A total of 16 CP patients from the 660 PC cases wereidentified. 56% of CP patients were males. After matching, CP patients were diagnosed 4.9years (95% CI: 0.6-9.2 years) earlier than controls (p=0.025). There was no statisticaldifference in PC stage (I/II vs. III/IV) between control and CP cases. CONCLUSIONS: Ahistory of CP appears to have an independent effect on age of onset of PC with a ~5 year earlierdiagnosis, supporting the role of inflammation in PC carcinogenesis. A lack of difference inPC stage, suggests that this result is not related to more frequent abdominal imaging in theCP group. Findings from this study should be factored into any future PC surveillancerecommendations for CP patients. Further studies are warranted to determine if shared geneticsusceptibility factors involving inflammatory pathways are present in CP and PC patients.
Tu1875
Fibroblast Activation Protein (FAP) Expression of Cancer-AssociatedFibroblast Predicts Poor Prognosis in Patients With PancreaticAdenocarcinomaTomoya Kawase, Koji Yoshida, Keisuke Hino
[Background and Aim] In many solid tumors, the stroma is increasingly recognized to beimportant in promoting tumor proliferation, invasion, metastasis, and chemoresistance.Especially, several studies using mouse pancreatic ductal adenocarcinoma(PDAC)modelrevealed that fibroblast activation protein(FAP) played an important role in tumorgenesis
S-869 AGA Abstracts
by reducing the surrounding immunity. FAP, or seprase, is a 170kDa dimer surface glycopro-tein and expressed on cancer associated fibroblasts(CAF). The aim of this study was toinvestigate the relationship between FAP expression of CAF and the prognosis in patientswith PDAC. [Materials and Methods] During 2008-2012 paraffin-embedded specimens ofPDAC were collected from 50 patients under the approval by the ethics committee of theinstitution. Standard immunohistochemical detection of FAP was performed using anti-FAPprimary antibody. The expression levels of FAP were classified into 4 degrees (negative, 1+,2+, 3+) and compared with patients' clinical course. [Results] FAP of CAF was detected in92%(46/50) of specimens and its expression level was 1+ in 16, 2+ in 19 and 3+ in 11specimens. The expression level of FAP was negatively correlated with overall survival ofpatients (p,0.001). Furthermore multivariate analysis showed FAP expression level, clinicalstaging of pancreatic cancer extracted as significant factors associated with overall survival.[Conclusion] The present results indicated that FAP expression of CAF is one significantfactors associated with overall survival of patients with pancreatic ductal adenocarcinoma.
Tu1876
Feasibility of Pathway Analysis in EUS-FNA Obtained Tissue of PancreaticCancer Patients: A Step Towards Personalized Based MedicineWesley K. Utomo, Vilvapathy Narayanan, Katharina Biermann, Casper H. van Eijck,Marco J. Bruno, Maikel P. Peppelenbosch, Henri Braat
Introduction: Pancreatic cancer (PC) remains one of the most lethal common malignanciesworldwide. The five year survival of all stages combined is only 5%. Deregulation of severalcore signaling pathways have been identified in PC and distinct pathways are affectedbetween different patients. Hence, personalized based medicine is an attractive approach inthe treatment of PC. Here, we aim to study the feasibility of pathway analysis using tissueobtained by Endoscopic Ultrasound-Fine Needle Aspiration (EUS-FNA); the activation ofmammalian target of Rapamycin (mTOR) in specific by the measurement of phosphorylatedS6 (pS6), a downstream protein of the mTOR pathway. Experimental design: Immunohisto-chemical (IHC) staining of pS6 on Formalin Fixed Paraffin Embedded (FFPE) material fromPC resection specimens was performed. Forty nine slides(33 tumor regions and 36 healthyregions) were stained and scored by an expert pathologist. To study mTOR activation andinhibition by rapamycin(Rap), PC cell lines (BxPC3, SU8686) were analyzed by means ofwestern blot (cleaved caspase 3, to measure apoptosis, and pS6), cell viability assays andflow cytometry (pS6). Single cell suspensions obtained through EUS-FNA of pancreatictumors were stained for cytokeratin 8/18 (CK8/18) and pS6 expression and subsequentlyanalyzed using flow cytometry. Results: The variability of IHC stained FFPE material wascompared by means of the coefficient of variation of 63.73% in tumor regions (n=33)compared to 35.69% in healthy regions (n=36). Levels of pS6 were 6-30 fold lower after 2hours of incubation with 0.1 μM Rap. Cell viability were 43% and 38% after 3 days of 12.5μM Rap. On flow cytometry analysis of cell lines, Rap reduced the Mean FluorescenceIntensity (MFI) in CK8/18 positive cells of pS6 by 75%(BxPC3) and 80%(SU8686). Validationin fresh EUS-FNA tissue by flow cytometry analysis showed higher expression of pS6 in 2out of 6 (33%) confirmed PC. MFI reductions of 80% and 83% of pS6 in CK8/18 positivepopulations were observed after incubation with 0.1 μM Rap. Absolute numbers of CK8/18positive, pS6 positive cells also decreased by 98% and 86.4%. Conclusions: As hypothesized, agreat variation in the expression of pS6 exists between PC patients in FFPE material. Thisfinding might indicate a possible subpopulation with higher activation of the mTOR pathwayin PC patients. In PC cell lines, Rap reduced cell viability in a dose dependent manner andinhibits pS6 expression without leading to apoptosis. Validation in EUS-FNA tissue showeda marked reduction of pS6 expression after incubation with Rap. The results of this studyshow the feasibility of pathway analysis in EUS-FNA tissue which is a first step towardspersonalized based medicine in PC. Further studies in EUS-FNA tissue are being undertakento determine other activated signaling pathways in PC.
Tu1877
Changes in Vascularization and Innervation Patterns in PanIN Lesions AsRevealed by Novel Three-Dimensional Imaging TechniquesYa-Yuan Fu, Jennifer M. Bailey, Janivette Alsina, Subhash Kulkarni, Shiue-Cheng Tang,Steven D. Leach, Pankaj J. Pasricha
Background & Aims: Pancreatic Intraepithelial Neoplasia (PanIN) is a pancreatic cancerprecursor lesion. Due to the opacity of pancreas, microtome-based planar microscopy isa routinely used method to observe PanIN lesions. However, this technique limits ourunderstanding of the morphology and development of PanIN and pancreatic cancer byrestricting tissue visualization in a 2-dimensional (2-D) space, rather than rendering a true3-dimensional (3-D) image of an intact developing PanIN. In order to identify and generatethe intact 3-D image of PanIN for facilitating better and early diagnosis of pancreatic cancer,we aimed to (1) apply Ulex europaeus agglutinin I (UEA-I) as a new cell surface marker tospecifically label the PanIN lesion, (2) characterize the altered vascular and neural networksby vessel painting the pancreatic tissue with wheat germ agglutinin (WGA) and immunostain-ing, and (3) combine the fluorescent staining with 3-D microscopy to observe the nativePanIN lesions at a subcellular level. Methods: We used two independent models of murinePanIN. The first involves the expression of Kras from the progenitor cells of the pancreas:Pdx-1:Cre; LsL-KrasG12D mice (9 months old). The second involves expression of Krasfrom adult acinar cells: Mist1:CreERT2;LsL-KrasG12D;mTmG mice (analyzed 6 weeks afteradministration of tamoxifen), and control mice (Mist1:CreERT2;mTmG). The mice wereperfused with fluorescent conjugated WGA to reveal the blood vessels of pancreas. Theinnervation was labeled by immunostaining (using pan-neuronal marker PGP9.5). We usedtwo independent methods to label PanIN. First, the mTmG background enables the visualiza-tion of GFP+ cells expressing KrasG12D. Second, UEA-I was used as a novel cell surfacemarker to label the PanINs. The labeled tissues were treated with the optical-clearing solutionFocusClear (Gastroenterology, 139:p1100-1105, 2010) to prepare transparent specimensfor confocal microscopy. Results: Comparisons of staining for various lectins revealed UEA-I to be a specific label for PanIN lesions. This allowed us to develop robust 3-D visualizationof PanIN lesions and their relationship to blood vessels and nerves. We discovered anincrease in vessel density in PanIN-bearing mice relative to normal pancreas. At the same
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