Imaging For Hepatic Fibrosis Using A Novel Collagen-binding Peptide (CBP1495) And Preliminary Research On Cyclic CBP1495 Activation By MMP-14 Of Pancreatic Cancer

Part 1 Imaging for hepatic fibrosis using a novel collagen-binding peptide?CBP1495?BackgroundHepatic fibrosis is a reparative or reactive pathological process resulted from a variety of causes that lead to increased fibrous tissues and reduced parenchymal cells in liver.Without effective control,excessive fibrosis can lead to liver cirrhosis,even liver failure and death.Hepatic fibrosis in early stage is reversible and different degrees of hepatic fibrosis require different treatments.Therefore,the early diagnosis,quantitative assessment,progress monitoring and therapeutic evaluation of fibrosis are especially important for guiding therapies of hepatic fibrosis.Imaging examination is a vital method for diagnosis and assessment of hepatic fibrosis.However,it is generally difficult to detect early fibrosis lesions and quantify fibrosis with conventional computed tomography?CT?,magnetic resonance?MR?and ultrasound.¹⁸F-FDG PET can be used to evaluate activity and prognosis of hepatic fibrosis,but it is both indirect and non-specific for fibrosis.Thus,there is no method of radionuclide imaging of hepatic fibrosis in the clinic that is both ideal and reliable.It would be an effective solution for this problem to develop a new molecular imaging probe to target the pathological feature of hepatic fibrosis.Hepatic fibrosis is characterised by the pathological accumulation of collagen?largely collagen type I?in the extracellular matrix?ECM?,and the amount of accumulated collagen type I is positively correlated to the severity of hepatic fibrosis.Thus,collagen type I is an ideal target for fibrosis imaging.Matrix metalloproteinase-2?MMP-2?can bind to collagen type I and degrade it.In our research,we found that a peptide fragment,CPKESCNLFVLKD?designated CBP1495?,in MMP-2 precursor can bind to collagen type I with a relatively high affinity.The peptides that target collagen type I are suitable for imaging probe of hepatic fibrosis in vivo because of their small size,high tissue penetration ability,short plasma half-life,and low immunogenicity.Therefore,this study intends to further identify the ability of CBP1495 targeting collagen type I,and visualise hepatic fibrosis in rat models using 99 mTc labelled CBP1495,to establish theoretical and experimental basis for diagnosis and assessment of hepatic fibrosis with this radiotracer.Methods1.The affinity of CBP1495 with dissolved collagen type I or a collagen mimetic peptide,?GPO?9,was measured by isothermal titration calorimetry assay?ITC?.The affinity of biotin-CBP1495 with embedded collagen type I was analysed by enzyme linked immunosorbent assay?ELISA?.2.biotin-CBP1495 targeting collagen type I and collagenous fibers in vitro were identified by Western blot and histological staining,respectively.3.CBP1495 was labelled with the radionuclide 99 mTc,and the labelling rate,stability in vitro,radiochemical purity?RCP?,binding rate of serum protein and oil-water partition coefficient were identified.4.The trace kinetics,distribution,dynamic and static imaging of ^99mTc-CBP1495 were performed in normal rabbits or mice.5.The rats were induced to hepatic fibrosis models by CCl4 and identified by histopathology.The ability of biotin-CBP1495 targeting hepatic fibrosis tissues were identified by histological staining.6.The imaging of hepatic fibrosis in rats was performed with ^99mTc-CBP1495 using pinhole collimator and SPECT/CT.7.The uptake ratio of ^99mTc-CBP1495 and hydroxyproline?Hyp?content in fibrotic liver of rats model were detected ex vivo,and the relationship between the uptake ratio of ^99mTc-CBP1495 and Hyp content were analysed.Results1.CBP1495 can bind to dissolved collagen type I with a Kd value of 6.54 ?M or?GPO?9 with a Kd value of 0.633 ?M,and embedded collagen type I with a Kd value of 0.861 ?M.2.biotin-CBP1495 stained the three electrophoretic bands contained in typical four bands of collagen type I by Western blot and positively visualised collagenous fibers in rat tail skin and mouse tendon by histological staining.3.The labelling efficiency of ^99mTc-CBP1495 was 95.06 ± 1.08 %?n = 6?.The RCP of ^99mTc-CBP1495 was still > 90% after 8 h and even > 85% after 24 h in human serum at 37°C or in saline at room temperature.The oil-water partition coefficient of the radiotracer was-3.473 ± 0.014?n = 3?.4.The tracer kinetics of ^99mTc-CBP1495 in normal rabbits?n = 6?conformed to a three-compartment model with the weight of 1/C.The correlation between the radioactive concentration?C,KBq/L?and the time?t,min?after intravenous injection of ^99mTc-CBP1495 was C?t?= 1474.001e-0.57 t + 358.843e-0.028 t + 92.985e-0.004 t.The dynamic and static imaging in rabbits and the distribution of ^99mTc-CBP1495 in mice indicated:?1?^99mTc-CBP1495 was excreted through urinary system not hepatobiliary system;?2?at 120 min after intravenous administration,^99mTc-CBP1495 maintained at a low level in blood,brain,thyroid gland,heart,liver,spleen,kidney,stomach,small intestine,muscle and bone,and the image of heart and liver is just little stronger than background;?3?no significant off-labelling was observed in vivo during 240 min after intravenous administration.5.Histopathological results confirmed successful construction of hepatic fibrosis rat models,and the collagenous fibers in fibrotic liver of rats can be visualized positively with biotin-CBP1495 by histological staining.6.The planar imaging by pinhole collimator showed an obviously higher uptake of ^99mTc-CBP1495 in fibrotic liver than normal liver of rats,and this result was further confirmed by SPECT/CT imaging.7.The target to non-target?fibrotic liver to heart?ratio was up to 8.70 ± 2.11?n = 6?by quantitative analysis ex vivo.The uptake ratio of ^99mTc-CBP1495 was significantly positively related with fibrosis degree?Hyp content?in fibrotic liver of rats?P < 0.0001,r2 = 0.7581?.ConclusionCBP1495 is a novel collagen-binding peptide which can bind to collagen type I or?GPO?9 with a relatively high affinity and target collagenous fibers in vitro.^99mTc-CBP1495 has a high labelling efficiency and RCP,and keeps stable in vitro and in vivo.The radiotracer is suitable for imaging in vivo due to its rapid clearance from blood,image with low background,excretion only through urinary system not hepatobiliary system.Hepatic fibrosis of rats can be visualised in vivo using ^99mTc-CBP1495 by SPECT/CT imaging,and the uptake ratio of ^99mTc-CBP1495 was significantly positively related with fibrosis degree in fibrotic liver of rats.Therefore,^99mTc-CBP1495 may be a promising molecular probe with potential clinical value for diagnosis and quantification of hepatic fibrosis.Part 2 Preliminary research on cyclic CBP1495 activation by MMP-14 of pancreatic cancerBackground Accurate localization and target volume delineation by tumor imaging are basis of precise radiotherapy for advanced pancreatic cancer.However,anatomical target volume drawed by conventional CT and MR is generally smaller than biological target volume invaded by tumor.¹⁸F-FDG PET can display the metabolically active region in cancer for precise radiotherapy,but it is generally hard to distinguish inflammation from cancer for ¹⁸F-FDG PET.Therefore,current clinical imaging methods have some limitations on directing precise radiotherapy for pancreatic cancer.It would be an effective solution to research and develop new molecular imaging probes targeting pancreatic cancer.The selection of specific targets on pancreatic cancer and design of molecular probe are the two key factors on constructing a new molecular imaging probe for pancreatic cancer.Unlike most other types of cancer,fibrosis in pancreatic cancer is a characteristic and universal phenomenon.Collagen type I is a major marker of collagenous fibers,so its expression in pancreatic cancer is also increased significantly.Moreover,collagen type I can promote invasion and metastasis of pancreatic cancer,and its high expression in pancreatic cancer is positively related with metastasis risk and poor prognosis.Thus,collagen type I is an important target for molecular imaging of pancreatic cancer.However,chronic inflammation in pancreas can be associated with fibrosis,so the imaging with probe directly targeted collagen will be not specific for pancreatic cancer.Collagen type I and MMP-14 can upregulate each other expression in pancreatic cancer.In the collagen-rich microenvironment of pancreatic cancer,MMP-14 can promote tumor proliferation,invasion and metastasis.MMP-14 is abnormally highly expressed in pancreatic cancer,but unexpressed or lowly expressed in normal pancreas and adjacent tissues of pancreatic cancer.Therefore,MMP-14 is another important target for molecular imaging of pancreatic cancer.In the first part study,we found that CBP1495 with a cleavage site of MMP-14 can bind to collagen type I with relatively high affinity.Based on this research result,we assume as follows: firstly,the collagen-binding ability of CBP1495 is inhibited by cyclizing CBP1495?c CBP1495?;secondly,after c CBP1495 is cleaved by MMP-14 expressed by pancreatic cancer,it returns to a linear peptide?c CBP1495-d?and recover the collagen-binding ability;thirdly,c CBP1495-d anchors and accumulates on the collagenous fibers in pancreatic cancer tissues;hereby,we construct a collagen-binding probe specificly activated by MMP-14.If this assumption is realised,due to the high expression of MMP-14 and collagen type I in pancreatic cancer,pancreatic cancer imaging with high specificity and target/non-target ratio would be expected to achieve.Based on the above analysis,this study intends to further identify MMP-14 expression in pancreatic cancer and its clinical value,then compare collagen-targeting ability of c CBP1495 and c CBP1495-d,and in vitro preliminarily evaluate c CBP1495 activation by MMP-14 expressed in pancreatic cancer.The study aims to provide theoretical and experimental basis for molecular imaging of pancreatic cancer in vivo using a probe constructed with c CBP1495 in further study.Methods 1.MMP-14 expression in 68 human pancreatic cancer samples and adjacent tissues were analysed by immunohistochemistry.MMP-14 expression in 8 human pancreatic cancer samples and adjacent tissues,pancreatic cancer cell line Bxpc-3 and normal pancreatic ductal epithelial cell line HPDE were detected by Western blot,and the ratio of tumor to non-tumor was semiquantitatively analysed.2.The clinical pathological data of above 68 pancreatic cancer patients were collected.The correlation between MMP-14 expression level?immunohistochemical scores?and clinical pathological features was evaluated.3.The affinity of biotin-c CBP1495 and biotin-c CBP1495-d with dissolved collagen type I or?GPO?9 was measured by isothermal titration calorimetry assay?ITC?.The affinity of biotin-c CBP1495 and biotin-c CBP1495-d with collagen type I was analysed by enzyme linked immunosorbent assay?ELISA?.4.The ability of biotin-c CBP1495 and biotin-c CBP1495-d targeting collagen type I and collagenous fibers in vitro were compared by Western blot and histological staining,respectively.5.The activity of MMP-14 cleaving biotin-CBP1495 was identified by HPLC-MS.6.The activation of biotin-c CBP1495 by MMP-14 or Bxpc-3?MMP-14 high expression?was analysed using ELISA.Results 1.MMP-14 was significantly higher expressed in pancreatic cancer tissues than in adjacent tissues by immunohistochemistry?P < 0.0001?.The tumor-to-non-tumor ratio of MMP-14 expression by Western blot in human pancreatic cancer samples and adjacent tissues was 4.54 29.31,14.13 ± 7.45?n = 8?.MMP-14 was strongly expressed in Bxpc-3 but weakly expressed in HPDE,and there was a significant difference between them?P = 0.0013?.2.MMP-14 expression in pancreatic cancer was significantly related with metastasis?lymph node or distant metastasis,P = 0.013?,TNM stage?P = 0.021?and prognosis?P = 0.034?.3.biotin-c CBP1495 could not bind to collagen type I,but biotin-CBP1495-d could bind to dissolved collagen type I with a Kd value of 31.5 ?M or?GPO?9 with a Kd value of 2.00 ?M,and embedded collagen type I with a Kd value of 5.797 ?M.4.biotin-c CBP1495-d stained the two electrophoretic bands contained in typical four bands of collagen type I by Western blot and positively visualised collagenous fibers in normal or fibrotic lung of rats but weaker than biotin-CBP1495 by histological staining.The results with biotin-c CBP1495 were negative both by Western bolt and histological staining.5.The HPLC-MS result showed that biotin-CBP1495 could be effectively cleaved by MMP-14 in vitro.6.The ELISA result indicated that biotin-c CBP1495 was activated partly in vitro after 2 h of interaction with MMP-14 or Bxpc-3.Conclusion MMP-14 is abnormally strongly expressed in pancreatic cancer with high tumor-to-non-tumor ratio.MMP-14 expression in pancreatic cancer was significantly related with metastasis,TNM stage and prognosis.Thus,MMP-14 is an ideal target for molecular imaging of pancreatic cancer.The cyclic CBP1495?c CBP1495?loses the ability of binding to collagen type I,but c CBP1495 can partly recover the collagen-binding ability after interaction with MMP-14 expressed by pancreatic cancer.Thus,this study preliminarily proves that c CBP1495 can be activated by MMP-14 or MMP-14-expressed pancreatic cancer.This study provides theoretical and experimental basis for molecular imaging of pancreatic cancer in vivo using a probe constructed with c CBP1495 in future.