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T1855 Molecular Imaging in Mouse Colon Tumors with Protease-Activatable Probe for Early Tumor Detection
tumors in distal colon with an average number of tumors of 7.9 ± 2.1 (number of tumors/ mouse). Histopathologically, cancer-to-adenoma ratio was significantly higher at week 16 than at week 6 (cancer:adenoma=7:1 vs. 0:7, p<0.05). NIRF imaging demonstrated that all tumors were visible with the MMP-activatable probe. Target-to-background ratios (TBRs) of cancer and adenoma to adjacent normal mucosa were 5.61 and 3.06 respectively. Sensitivity and specificity for tumor detection with light imaging by two experienced endoscopists were 79% and 44%, whereas, those with NIRF imaging were 82% and 67%. Results of immunohistochemistry and western blotting confirmed MMP overexpression in the tumor compared with adjacent mucosa. Conclusions Mouse colon tumor model with AOM and DSS is useful for molecular imaging study covering evolutional stages of carcinogenesis. Intensity of NIRF imaging by a protease-activatable probe is related to each tumor stage. MMP expression in colon tumors correlates with fluorescence imaging. This strategy may be used for early diagnosis of colon tumors.
Morphological Feature Quantification of Colonic Crypt Patterns Using Microscope-Integrated OCT Xin Qi, Yinsheng Pan, Zhilin Hu, Michael V. Sivak, Joseph Willis, Kayode Olowe, Andrew M. Rollins Background: Optical coherence tomography examination of the upper and lower GI tract has shown promising technological advances over the last decade. However one limitation preventing implementation in clinical settings is the difficulty in interpreting images with large amounts of information in a timeframe suitable to facilitate an endoscopist. Reliable analytic methodologies to rapidly process and interpret images obtained at endoscopy do not currently exist. A computerized model to accurately quantify OCT obtained architectural features of colonic crypts could serve as a proof of principle that this concept is viable. Aim: To demonstrate the use of computerized algorithms to quantify the morphological features of colonic crypts. Methods: Samples of fresh colon tissues (10 normal and 10 aberrant crypt focus (ACF)) were obtained from colectomies. The samples were stained with methylene blue and microscopic images were recorded to approximately simulate In Vivo imaging with magnification chromoendoscopy. In addition, 3D OCT volumes were recorded from the same sites using an integrated OCT scanner. The crypts within the micrographs were automatically segmented using marker-based watershed morphological processing. The morphological features of the segmented crypts were extracted and quantified. For the correlated 3D OCT volumes, the crypts were first segmented in each en-face plane of the stack. Then the segmented crypts were visualized in 3D by volume rending. Finally 3D central axes of the crypts, called skeletons, were extracted and the orientations of the skeletons were quantified by centroid-searching. Results: For typical grossly normal colonic tissues, the mean area of segmented crypts was 4213µm2; the mean major axis length was 89µm; the mean minor axis length was 60µm; the mean eccentricity was 0.67; the mean standard deviation angle between the en-face plane and the skeletons was 2.5 degree. For typical aberrant crypt foci (ACF) colonic tissues, the mean area of segmented crypts was 10267µm2; the mean major axis length was 145µm; the mean minor axis length was 88µm; the mean eccentricity was 0.74; the mean standard deviation angle between the en-face plane and the skeletons was 12.7 degree. There were significant morphological feature differences between normal and ACF colonic crypts. Conclusion: These methods can quantify morphological features of colonic crypts, and the results correspond well to known differences between the crypt features of normal and aberrant crypts. Further development of this approach to OCT derived data analysis may facilitate the evolution of this technology into clinical practice.
T1856 Phospho-HSP20 Inhibits Translocation of PKCα Into the Caveolae: Real Time Monitoring of Movement of PKCα and Caveolin-1 in Human Colonic Circular Smooth Muscle Sita Somara, Robert R. Gilmont, Paul M. Jenkins, Jeffrey R. Martens, Khalil N. Bitar Background: Membrane translocation of PKCα is crucial for contraction of colonic smooth muscle cells (CSMC). Caveolae, caveolin-rich lipid rafts membrane microdomains, are essential for the docking of PKCα. Objective: To determine in real time the effect of HSP20 phosphorylation status on the translocation of PKCα into the caveolae. Methods: 1. CSMC were cultured from human sigmoid colon. 2. Human CSMC were transfected with DStagged wild type caveolin-1 cDNA (pDsRed-WT-cav1) and with GFP-tagged PKCα cDNA. To study the effect of HSP20, cells were also transfected with either phosphomimic-HSP20 cDNA (16D) or non-phosphomimic-HSP20 cDNA (16A). 3. Transfected CSMC were grown on a cover slip to monitor in real time the movement of PKCα and cav-1 using confocal microscopy. Results: A. Human CSMC: Resting State: PKCα and Cav-1 were distributed evenly throughout the cell while Cav-1 was denser at the membrane. Effect of Ach: In response to Ach stimulation, PKCα rapidly moved to the membrane where it colocalized with Cav-1. Effect of PdBU: In response to PdBU stimulation, PKCα translocated faster (<3sec) to membrane where it colocalized with Cav-1. B. Human CSMC transfected with 16AHSP20: Resting State: A percentage of PKCα was membrane associated while Cav-1 was distributed throughout the cell but was denser at the membrane. Effect of PdBU: In response to PdBU stimulation, all of the PKCα translocated to the membrane and colocalized with Cav-1. C. Human CSMC transfected with 16DHSP20: Resting State: PKCα and Cav-1 were distributed evenly throughout the cell while cav-1 was denser at the membrane. Effect of PdBU: In response to PdBU, no translocation and localization of PKCα to membrane was observed. Instead PKCα moved into non-plasma membrane vesicle like structures. Summary: Real-time monitoring of live human CSMC suggests that in response to contractile stimulation, PKCα rapidly translocates to the membrane and colocalizes with Cav-1. Ectopic expression of non-phosphomimic HSP20 resulted in elevated membrane levels of PKCα in resting cells. PKCα translocation to the membrane upon stimulation occurred normally. Ectopic expression of phosphomimic-HSP20 inhibited PKCα translocation. Conclusions: Translocation of PKCα to caveolae in response to contractile stimulation is rapid and dynamic. Expression of phosphomimic HSP20 inhibited PKCα translocation suggesting that HSP20 phosphorylation plays a crucial role in PKCα translocation. Supported by Grant NIH RO1 DK 042876 and R01 DK057020
T1854 Intracellular Compartment-Specific Detection of H2O2 in Liver Cells Using a Fluorescent Fusion Protein Gunda Millonig, Hongnian Guo, Helmut_Karl Seitz, Sebastian Mueller Introduction: H2O2 is the central reactive oxygen species in the liver that acts as important signaling molecule within and between liver cells. Increased levels of H2O2 in distinct cellular compartments are an important feature of various liver pathologies including all forms of hepatitis (e.g. ASH, NASH) or ischemia-reperfusion injury. Thus far, it has been impossible to specifically detect H2O2 within hepatocytes and subcellular compartments. Widely used dyes such as DCF (dichloro-dihydro-fluoresceine) or its esters are non-specific and may lead to various artifacts. Methods and Results: We here use a fusion protein of the bacterial H2O2 sensor OxyR and YFP that changes its fluorescence spectrum at 405 and 485nm upon binding of H2O2 (HyPer, Evrogen, Moscow, Russia). In addition to mitochondrial and cytosolic expression of HyPer, we generated a peroxisome-targeted plasmid. Hyper was transiently expressed in Huh7 cells cultured in 96-well plates for fluorescence measurements. We established a high throughput method to analyze H2O2 levels in cultured living hepatocytes in real-time over 2 hours. Extracellular H2O2 > 10 mM as bolus or as continuous release by glucose oxidase showed an increase in cytosolic but not mitochondrial H2O2 levels. Inhibition of catalase by 3-amino-1,2,4-triazole increased fluorescence in peroxisomal, mitochondrial and cytoplasmatic H2O2 , whereas increased production of superoxide radicals after treatment with antimycin A did not change fluorescence. Expression of catalase cDNA decreased intracellular fluorescence levels markedly. Conclusion: The OxyR-YFP fusion protein HyPer is the first specific dye to measure H2O2 in distinct intracellular compartments (cytosol, mitochondria, peroxisomes). Our optimized high throughput procedure on attached cultured cells will allow to study intracellular H2O2 release in hepatocytes under various pathological conditions. This work was supported by the Dietmar Hopp Foundation
T1857 Morphological Versus Functional Imaging to Detect Recurrences After Resection of Liver Metastases of Digestive Endocrine Tumors (Lmdet) S. Scigliano, Frédérique Maire, Jean Louis Stievenart, R. Lebtahi, Reza Kianmanesh, Alain Sauvanet, Marie-Pierre Vullierme, Anne Couvelard, Pascal Hammel, Jacques Belghiti, Philippe B. Ruszniewski, Dominique Le Guludec Aims : to study the outcome of patients (pts) after resection of LMDET, and to assess diagnostic accuracy of somatostatin receptor scintigraphy (SRS) and morphological imaging (MI) for the detection of recurrences. Patients and Methods : between 1992 and 2006, 41 pts (16 males, median age 51 years, range 21-75) had resection of well-differenciated LMDET. The primary, located mainly in the pancreas (17 pts) or the small bowel (12 pts), had been resected. 16 pts (39%) had a functioning tumor. No extrahepatic tumor was detected on preoperative imaging nor during surgery (except resection of lymph nodes). Morphological investigation (MI), including abdominal CT scan, and/or hepatic MRI and thoracic CT if necessary, was performed post-operatively and every 6 months. SRS was performed yearly. Confirmation of recurrences was confirmed by pathology in case of reintervention, or by imagery during follow-up. Results : median post-operative follow-up was 76 months (range 11-174). Recurrence was documented in 32 pts (78 %), mainly in the liver (24 pts), lymph nodes (5pts), bone (5 pts) and/or lung (3 pts), 19 (2-79) months after liver surgery. Seven pts (17 %) died from tumor progression, 47 (16-118) months after surgery. 3 year-overall and disease-free survival was 95 and 25 %, respectively. During follow-up, 136 imaging procedures were performed in the 41 pts. SRS and MI (performed within a maximum of 15 days) gave concordant results in 64 % of the cases. Sensitivity, specificity, PPV, NPV and diagnostic accuracy of SRS to detect recurrences were 89, 94, 98, 75 and 91 %, respectively. Corresponding figures for MI were 68, 91, 95, 50 and 74 %, respectively. SRS detected extra-abdominal tumors in 11 pts (bone 5, mediastinal lymph nodes 3, thyroid 3, lung 1, pelvic 1). In 11 of the 32 pts with recurrences (34 %), lesions (liver 5, bone 3, abdominal or pelvic lymph nodes 5, mediastinal lymph nodes 3, pancreas 1) were evidenced 15.5 (5-31) months earlier by SRS than by MI. Conclusion : recurrences are frequent (78 %) in pts operated on for LMDET with curative intent. SRS has a high sensitivity and NPV for the detection of recurrences during post-operative follow-up, mainly for extra-abdominal
T1855 Molecular Imaging in Mouse Colon Tumors with Protease-Activatable Probe for Early Tumor Detection Soon Man Yoon, Seung-Jae Myung, Hyun Mi Kim, Yeon Mi Ryu, Kyeongsoon Park, Kwangmeyung Kim, Ick Chan Kwon, Young Soo Park, Dae Hyuk Moon, Do Hoon Kim, Mi Young Do, Byong Duk Ye, Jeong-Sik Byeon, Suk-Kyun Yang, Jin-Ho Kim Background/Aims Early detection of small primary tumors remains the mainstay of successful cancer therapy and improves survival rates. Matrix metalloproteinases (MMPs) revealed to participate in the early stages of tumorigenesis and primary tumor growth and are overexpressed in a number of cancers including colon cancer. The aims of the current study were to evaluate an efficient animal model for early and small colon tumors and to assess the feasibility of protease-activatable probe for tumor detection. Methods We used a mouse model for colon tumor with a combined treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS). Male BALB/c mice (n=10, age 5 weeks) were given a single intraperitoneal administration (10 mg/kg body weight) of a colon carcinogen, AOM, followed by 2% DSS in drinking water for 7 days. At week 6, 8, 12 and 16 respectively, mice were injected via tail vein with MMP-activatable probe (MMPSenseTM680, VisEn Medical, Inc., MA, USA) followed by surgical exposure of the colon. Excised colons were evaluated with near infrared fluorescence (NIRF) imaging, histologic examination, and immunohistochemistry and western blotting. Results All mice treated with AOM and DSS developed colon
A-577
AGA Abstracts
AGA Abstracts
T1853
Morphological Feature Quantification of Colonic Crypt Patterns Using Microscope-Integrated OCT Xin Qi, Yinsheng Pan, Zhilin Hu, Michael V. Sivak, Joseph Willis, Kayode Olowe, Andrew M. Rollins Background: Optical coherence tomography examination of the upper and lower GI tract has shown promising technological advances over the last decade. However one limitation preventing implementation in clinical settings is the difficulty in interpreting images with large amounts of information in a timeframe suitable to facilitate an endoscopist. Reliable analytic methodologies to rapidly process and interpret images obtained at endoscopy do not currently exist. A computerized model to accurately quantify OCT obtained architectural features of colonic crypts could serve as a proof of principle that this concept is viable. Aim: To demonstrate the use of computerized algorithms to quantify the morphological features of colonic crypts. Methods: Samples of fresh colon tissues (10 normal and 10 aberrant crypt focus (ACF)) were obtained from colectomies. The samples were stained with methylene blue and microscopic images were recorded to approximately simulate In Vivo imaging with magnification chromoendoscopy. In addition, 3D OCT volumes were recorded from the same sites using an integrated OCT scanner. The crypts within the micrographs were automatically segmented using marker-based watershed morphological processing. The morphological features of the segmented crypts were extracted and quantified. For the correlated 3D OCT volumes, the crypts were first segmented in each en-face plane of the stack. Then the segmented crypts were visualized in 3D by volume rending. Finally 3D central axes of the crypts, called skeletons, were extracted and the orientations of the skeletons were quantified by centroid-searching. Results: For typical grossly normal colonic tissues, the mean area of segmented crypts was 4213µm2; the mean major axis length was 89µm; the mean minor axis length was 60µm; the mean eccentricity was 0.67; the mean standard deviation angle between the en-face plane and the skeletons was 2.5 degree. For typical aberrant crypt foci (ACF) colonic tissues, the mean area of segmented crypts was 10267µm2; the mean major axis length was 145µm; the mean minor axis length was 88µm; the mean eccentricity was 0.74; the mean standard deviation angle between the en-face plane and the skeletons was 12.7 degree. There were significant morphological feature differences between normal and ACF colonic crypts. Conclusion: These methods can quantify morphological features of colonic crypts, and the results correspond well to known differences between the crypt features of normal and aberrant crypts. Further development of this approach to OCT derived data analysis may facilitate the evolution of this technology into clinical practice.
T1856 Phospho-HSP20 Inhibits Translocation of PKCα Into the Caveolae: Real Time Monitoring of Movement of PKCα and Caveolin-1 in Human Colonic Circular Smooth Muscle Sita Somara, Robert R. Gilmont, Paul M. Jenkins, Jeffrey R. Martens, Khalil N. Bitar Background: Membrane translocation of PKCα is crucial for contraction of colonic smooth muscle cells (CSMC). Caveolae, caveolin-rich lipid rafts membrane microdomains, are essential for the docking of PKCα. Objective: To determine in real time the effect of HSP20 phosphorylation status on the translocation of PKCα into the caveolae. Methods: 1. CSMC were cultured from human sigmoid colon. 2. Human CSMC were transfected with DStagged wild type caveolin-1 cDNA (pDsRed-WT-cav1) and with GFP-tagged PKCα cDNA. To study the effect of HSP20, cells were also transfected with either phosphomimic-HSP20 cDNA (16D) or non-phosphomimic-HSP20 cDNA (16A). 3. Transfected CSMC were grown on a cover slip to monitor in real time the movement of PKCα and cav-1 using confocal microscopy. Results: A. Human CSMC: Resting State: PKCα and Cav-1 were distributed evenly throughout the cell while Cav-1 was denser at the membrane. Effect of Ach: In response to Ach stimulation, PKCα rapidly moved to the membrane where it colocalized with Cav-1. Effect of PdBU: In response to PdBU stimulation, PKCα translocated faster (<3sec) to membrane where it colocalized with Cav-1. B. Human CSMC transfected with 16AHSP20: Resting State: A percentage of PKCα was membrane associated while Cav-1 was distributed throughout the cell but was denser at the membrane. Effect of PdBU: In response to PdBU stimulation, all of the PKCα translocated to the membrane and colocalized with Cav-1. C. Human CSMC transfected with 16DHSP20: Resting State: PKCα and Cav-1 were distributed evenly throughout the cell while cav-1 was denser at the membrane. Effect of PdBU: In response to PdBU, no translocation and localization of PKCα to membrane was observed. Instead PKCα moved into non-plasma membrane vesicle like structures. Summary: Real-time monitoring of live human CSMC suggests that in response to contractile stimulation, PKCα rapidly translocates to the membrane and colocalizes with Cav-1. Ectopic expression of non-phosphomimic HSP20 resulted in elevated membrane levels of PKCα in resting cells. PKCα translocation to the membrane upon stimulation occurred normally. Ectopic expression of phosphomimic-HSP20 inhibited PKCα translocation. Conclusions: Translocation of PKCα to caveolae in response to contractile stimulation is rapid and dynamic. Expression of phosphomimic HSP20 inhibited PKCα translocation suggesting that HSP20 phosphorylation plays a crucial role in PKCα translocation. Supported by Grant NIH RO1 DK 042876 and R01 DK057020
T1854 Intracellular Compartment-Specific Detection of H2O2 in Liver Cells Using a Fluorescent Fusion Protein Gunda Millonig, Hongnian Guo, Helmut_Karl Seitz, Sebastian Mueller Introduction: H2O2 is the central reactive oxygen species in the liver that acts as important signaling molecule within and between liver cells. Increased levels of H2O2 in distinct cellular compartments are an important feature of various liver pathologies including all forms of hepatitis (e.g. ASH, NASH) or ischemia-reperfusion injury. Thus far, it has been impossible to specifically detect H2O2 within hepatocytes and subcellular compartments. Widely used dyes such as DCF (dichloro-dihydro-fluoresceine) or its esters are non-specific and may lead to various artifacts. Methods and Results: We here use a fusion protein of the bacterial H2O2 sensor OxyR and YFP that changes its fluorescence spectrum at 405 and 485nm upon binding of H2O2 (HyPer, Evrogen, Moscow, Russia). In addition to mitochondrial and cytosolic expression of HyPer, we generated a peroxisome-targeted plasmid. Hyper was transiently expressed in Huh7 cells cultured in 96-well plates for fluorescence measurements. We established a high throughput method to analyze H2O2 levels in cultured living hepatocytes in real-time over 2 hours. Extracellular H2O2 > 10 mM as bolus or as continuous release by glucose oxidase showed an increase in cytosolic but not mitochondrial H2O2 levels. Inhibition of catalase by 3-amino-1,2,4-triazole increased fluorescence in peroxisomal, mitochondrial and cytoplasmatic H2O2 , whereas increased production of superoxide radicals after treatment with antimycin A did not change fluorescence. Expression of catalase cDNA decreased intracellular fluorescence levels markedly. Conclusion: The OxyR-YFP fusion protein HyPer is the first specific dye to measure H2O2 in distinct intracellular compartments (cytosol, mitochondria, peroxisomes). Our optimized high throughput procedure on attached cultured cells will allow to study intracellular H2O2 release in hepatocytes under various pathological conditions. This work was supported by the Dietmar Hopp Foundation
T1857 Morphological Versus Functional Imaging to Detect Recurrences After Resection of Liver Metastases of Digestive Endocrine Tumors (Lmdet) S. Scigliano, Frédérique Maire, Jean Louis Stievenart, R. Lebtahi, Reza Kianmanesh, Alain Sauvanet, Marie-Pierre Vullierme, Anne Couvelard, Pascal Hammel, Jacques Belghiti, Philippe B. Ruszniewski, Dominique Le Guludec Aims : to study the outcome of patients (pts) after resection of LMDET, and to assess diagnostic accuracy of somatostatin receptor scintigraphy (SRS) and morphological imaging (MI) for the detection of recurrences. Patients and Methods : between 1992 and 2006, 41 pts (16 males, median age 51 years, range 21-75) had resection of well-differenciated LMDET. The primary, located mainly in the pancreas (17 pts) or the small bowel (12 pts), had been resected. 16 pts (39%) had a functioning tumor. No extrahepatic tumor was detected on preoperative imaging nor during surgery (except resection of lymph nodes). Morphological investigation (MI), including abdominal CT scan, and/or hepatic MRI and thoracic CT if necessary, was performed post-operatively and every 6 months. SRS was performed yearly. Confirmation of recurrences was confirmed by pathology in case of reintervention, or by imagery during follow-up. Results : median post-operative follow-up was 76 months (range 11-174). Recurrence was documented in 32 pts (78 %), mainly in the liver (24 pts), lymph nodes (5pts), bone (5 pts) and/or lung (3 pts), 19 (2-79) months after liver surgery. Seven pts (17 %) died from tumor progression, 47 (16-118) months after surgery. 3 year-overall and disease-free survival was 95 and 25 %, respectively. During follow-up, 136 imaging procedures were performed in the 41 pts. SRS and MI (performed within a maximum of 15 days) gave concordant results in 64 % of the cases. Sensitivity, specificity, PPV, NPV and diagnostic accuracy of SRS to detect recurrences were 89, 94, 98, 75 and 91 %, respectively. Corresponding figures for MI were 68, 91, 95, 50 and 74 %, respectively. SRS detected extra-abdominal tumors in 11 pts (bone 5, mediastinal lymph nodes 3, thyroid 3, lung 1, pelvic 1). In 11 of the 32 pts with recurrences (34 %), lesions (liver 5, bone 3, abdominal or pelvic lymph nodes 5, mediastinal lymph nodes 3, pancreas 1) were evidenced 15.5 (5-31) months earlier by SRS than by MI. Conclusion : recurrences are frequent (78 %) in pts operated on for LMDET with curative intent. SRS has a high sensitivity and NPV for the detection of recurrences during post-operative follow-up, mainly for extra-abdominal
T1855 Molecular Imaging in Mouse Colon Tumors with Protease-Activatable Probe for Early Tumor Detection Soon Man Yoon, Seung-Jae Myung, Hyun Mi Kim, Yeon Mi Ryu, Kyeongsoon Park, Kwangmeyung Kim, Ick Chan Kwon, Young Soo Park, Dae Hyuk Moon, Do Hoon Kim, Mi Young Do, Byong Duk Ye, Jeong-Sik Byeon, Suk-Kyun Yang, Jin-Ho Kim Background/Aims Early detection of small primary tumors remains the mainstay of successful cancer therapy and improves survival rates. Matrix metalloproteinases (MMPs) revealed to participate in the early stages of tumorigenesis and primary tumor growth and are overexpressed in a number of cancers including colon cancer. The aims of the current study were to evaluate an efficient animal model for early and small colon tumors and to assess the feasibility of protease-activatable probe for tumor detection. Methods We used a mouse model for colon tumor with a combined treatment of azoxymethane (AOM) and dextran sodium sulfate (DSS). Male BALB/c mice (n=10, age 5 weeks) were given a single intraperitoneal administration (10 mg/kg body weight) of a colon carcinogen, AOM, followed by 2% DSS in drinking water for 7 days. At week 6, 8, 12 and 16 respectively, mice were injected via tail vein with MMP-activatable probe (MMPSenseTM680, VisEn Medical, Inc., MA, USA) followed by surgical exposure of the colon. Excised colons were evaluated with near infrared fluorescence (NIRF) imaging, histologic examination, and immunohistochemistry and western blotting. Results All mice treated with AOM and DSS developed colon
A-577
AGA Abstracts
AGA Abstracts
T1853