Molecular Mechanisms Of Short-time Low-dose Rate Irradiation Of Iodine-125 Seeds On Perineural Invasion In Pancreatic Cancer

Pancreatic cancer (PanCa) is the fifth or fourth leading cause of cancer deaths in developed countries with an estimated 5-year overall survival of less than 5%.Poor prognosis results from the aggressive nature of the disease, with as many as 80% of patients at the time of diagnosis harboring unresectable cancer.The aggressive nature has been linked to local recurrence, lymph node metastasis, liver metastasis, peritoneal dissemination, and perineural invasion (PNI). PanCa is characterized by extremely high frequency of PNI ,which can be as high as 80%-90%, even 100%. Initial intrapancreatic PNI may lead to invasion of extrapancreatic nerve plexus.Recognition of the pathway of extrapancreatic spread and its significance in relation to PanCa progression has implications in surgical treatment of PanCa. Cancer cells spreading in the perineural space even at an early clinical stage also cause local recurrence in the retroperitoneum because of residual tumor cells in the perineural space after surgical resection.Moreover, many previous clinicopathologic reports showed that PNI in PanCa was one of the most significant poor prognostic factors and associated with poorer survival.Better understanding of the perineural invasion molecular mechanisms could bring about effective control of PNI-related morbidities such as local recurrence and extrapancreatic spread.More recently, studies have demonstrated that PNI may involve reciprocal signaling interactions between tumor cells and nerves and that these invading tumor cells may have acquired the ability to respond to proinvasive signals within the peripheral nerve milieu.The neurotrophins are the best characterized family of neurotrophic factors and comprise nerve growth factor(NGF), brain-derived neurotrophic factor(BDNF), neurotrophin 3 (NT-3) and neurotrophin 4/5(NT-4/5).It is their potent effects on neuronal growth that have made the neurotrophins prime candidates for study in the PNI invasion pathway.There is a growing body of literature implicating these molecules as well as other neurotrophic factors in cancer.There is an up-regulation in neurotrophin expression by tumor cells as well as intratumoral nerves in pancreas cancer.Tumor cells cannot migrate through extracellular matrix or nerve sheath without expressing proteinases.Matrix metalloproteinases (MMPs),in particular,the gelatinases (MMP-2 and MMP-9) seem to play a pivotal role in PNI.Expression of either epidermal growth factor (EGF) or its receptor epidermal growth factor receptor(EGFR) correlates with the presence of lymph node or distant metastases and decreased survival in PanCa. EGFR expression within tumor cells was a much better predictor of poor prognosis than EGF expression.The application of traditional external radiotherapy has been limited in PanCa for its low sensibility and its side effects due to the low toleration of surrounding normal structures including stomach, duodenum, small bowel, large intestine, liver, kidney and spinal cord.Iodine-125 interstitial brachytherapy is recognized as a method to deliver additional localized radiation dose while limiting radiation to surrounding normal tissue, characterized by small trauma, high radiation dose rate in target area, lasting effect, mild injury to peripheral normal tissue, and easy radioprotection,and has been an established and popular option for the management of some kinds of solid tumors concluding advanced PanCa.However, there are few reports about the effects of Iodine-125 seeds on molecular mechanisms of perineural invasion in PanCa.Understanding intervention of Iodine-125 seeds in perineural invasion at the molecular level is an important step towards the therapeutic targets of Iodine-125 seeds treatment for PanCa PNI.According to in vitro and in vivo models of PNI in PanCa and Iodine-125 seeds low-dose rate irradiation model system in literatures,we created a novel in vitro and a novel in vivo Iodine-125 seeds low-dose irradiation model system for examining the intervention of Iodine-125 seeds low-dose irradiation in interactions between nerve and PanCa cells. The present study was based on the above-mentioned progresses and consisted of five main parts. They are as follows:1,The construction of in vitro PanCa PNI model.Objective:In order to observe the interactions between nerve and PanCa cells and the feasibility of the coculture model was tested.Methods:This model was established by coculturing the dorsal root ganglion (DRG) with PanCa cells.DRGs of four-week-old SD rat were dissected under sterile conditions.The DRG was cut and placed in RPMI media with antibiotics.human PanCa cell line Capan-2 and Panc1 were selected for this study.EHS Matrigel was used as an extracellular matrix.In the experiments,the coculture was performed by placing a single DRG in 100μL of Matrigel in culture dishes,which were placed on ice to maintain the liquidity of Matrigel.Then,10⁵ cells per dish were seeded in the Matrigel.The cells and the DRG were immobilized in the Matrigel by warming the culture to 37°C.The Capan-2/DRG were routinely cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum in a humidified atmosphere of 95% O₂ and 5% CO₂.Capan-2 only and DRG only were cultured as controls.Results:A symbiotic phenomenon was noted when nerves were cocultured with PanCa cells.PanCa cells formed more colonies in the presence of nerves,whereas nerves in return produced more neurites when they cocultured with cancer cells.After 3days and 5days,Capan-2 cells with DRG displayed more colony area than these cells alone;neurite outgrowth from the DRG in the presence of Capan-2 were more compared with DRG nerve cultures alone.Retrograde migration of PanCa cells along the contacting neurites was observed subsequently to neurite-cancer contact, and PanCa cells were capable of migrating to reach the DRG/nerve show that retrograde extension of PanCa cells migrates into the ganglion/nerve through neurites and finally surrounds the ganglion/nerve.Conclusion:These findings indicate that PNI can be developed between PanCa cells and nerves, which may have mutual growth advantages. The coculture model is feasible.2,The construction of a novel in vitro Iodine-125 seeds short-time low-dose rate irradiation PanCa PNI model.Objective:In order to observe the intervention of Iodine-125 seeds short-time low-dose rate irradiation in interactions between nerve and PanCa cells and the feasibility of the model was tested.Methods:This model included two parts,coculture model and irradiation device. The coculture model is as above-mentioned.The irradiation device was designed by a five-diameter cell culture dish with solidified paraffin wax.The arrangement consist s of nine seeds,confined within a 30 mm diameter cycle.Eight seeds are equally spaced within recesses around the circumference of 30 mm diameter×1 mm thick polysterine cell dish lid.A ninth seed is cionfined to the center within a 6 mm diameter hole.The disc and seeds are"sandwched"between two other pieces of polystyrene sunch that the seeds are secure and constrained from moving.The matrigel layer containing the PanCa cell and DRG above the seeds is 5 mm thick and is designed to support the cell culture dish.When in use,the irradiator is located within an incubator.The incubator is sited within a radiation controlled area wth restricted access.There is also a steel cabinet in this area for storing irradiator when not in use.Manpulations of the cell dishes are undertaken using tongs and wearing lead-lined gloves and lead-lined clothes. The experiment is consist of 2 groups,the irridiation groups and non-irridiation groups.PanCa/DRG, PanCa only,DRG only were cultured in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum in a humidified atmosphere of 95% O2 and 5% CO2. were cultured as controls.Results: In non-irridiation groups,a symbiotic phenomenon was noted as above-mentioned,but in irradiation groups, the symbiotic phenomenon was inhibited, PanCa cells formed less colonies compared with those in non-irradiation groups.It was showed constrained under irradiation condtions that retrograde extension of PanCa cells migrates into the ganglion/nerve through neurites.Conclusion:These findings indicate that PNI of PanCa can be developed between PanCa cells and nerves, which may have mutual growth advantages.The intervention of Iodine-125 seeds short-time low-dose rate irradiation inhibits interactions between nerve and PanCa cells,and the model is feasible.3,The analysis of PNI-related factors of in vitro Iodine-125 seeds short-time low-dose rate irradiation PanCa PNI model.Objective:To observe the effects of Iodine-125 seeds short-time low-dose rate irradiation on molecular mechanisms of PNI in PanCa.Methods:1,The DRG,Capan-2/Panc1colonies were photographed at a magnification of×40 using inverted microscopy.Neurite outgrowth and cell colony growth were quantitated using the image analysis system.2,The concentration of NGF, EGF and TGFαin media supernatant and matrigel solution was tested by ELISA respectively.3.The expression of NT-3,NGF and EGFR mRNA was tested by RT-PCR.Results:1)As above-mentioned,a symbiotic phenomenon was noted when nerves were cocultured with PanCa cells in non-irradiation groups,and in irradiation groups,the symbiotic phenomenon was constrained.2)In irradiation groups,NGF concentration of Capan-2&DRG was higher than that in control groups, NGF concentration of Panc-1 only was lower than that in control groups.The concentration of TGFαof PanCa&DRG in irradiation groups is lower than that in control groups.3)The irradiation of Iodine-125 can decrease NGF mRNA expression in both Capan-2 and Panc-1cells; The irradiation increases NT-3 mRNA expression in Capan-2 cells and Panc1&DRG,but decrease NT-3 mRNA expression in Panc1cells only;The irradiation decreases EGFR mRNA expression in Capan-2 cells,but there was no effects on Panc1 cells.Conclusion:These findings indicate that Iodine-125 seeds short-time low-dose rate irradiation inhibits morphologically interactions between nerve and PanCa cells and biological behavior of PanCa cell.However,the results of ELISA assays indicated that Iodine-125 seeds short-time low-dose rate irradiation could increase the invasiveness.RT-PCR indicates irradiation could decrease the invasiveness,but the conclusion was not confirmed.4,The construction of in vivo Iodine-125 seeds short-time low-dose rate irradiation PanCa PNI model.Objective:To establish a in vivo PanCa PNI model and a novel in vivo Iodine-125 seeds short-time low-dose rate irradiation PanCa PNI model and the feasibility of the models was tested.Methods:The NOD/SCID mice were anesthetized and 106 viable human Capan-2 cells in 10μl of cell suspension were injected into the pancreas tail using an inoculator with a needle. The pancreas was relocated into the abdominal cavity, and the peritoneum and skin were closed with surgical suture.4 weeks after injection, when the tumor grew up to about 0.8-1.0 cm in long diameter, the 20 mice with PanCa underwent Iodine-125 seeds implantation surgically as above-mentioned and were separated randomly into two groups: 0Gy,2Gy.2 weeks after Iodine-125 irradiation,the mice were sacrificed, and the pancreas, stomach, duodenum, liver,lymph nodes, lungs, and other organs of suspected tumor involvement or metastasis were harvested.Results: The in vivo Iodine-125 seeds short-time low-dose rate irradiation PanCa PNI model was established successfully.PNI was observed 4/10 in irradiation group and 6/10 in control group.The tumor size was smaller in irriadiation group(723.56±73.26 mm3) than that in control group(1195.43±127.45 mm3).Conclusion:These findings indicate that PNI of PanCa can be developed between PanCa cells and nerves,and the model is feasible and successful.There was a tumor-reduced effect for Iodine-125 irradiation.5,The analysis of PNI-related factors of in vivo Iodine-125 seeds short-time low-dose rate irradiation PanCa PNI model.Objective:In observe the interactions between nerve and PanCa cells and the effects of Iodine-125 seeds short-time low-dose rate irradiation on molecular mechanisms of PNI in PanCa.Methods:First,MMP-2 protein expression was tested by immunohistochemistry and Western Blot assay.Second,EGFR expression was detected by RT-PCR and immunohistochemistry assay.Finally,NT-3 and NGF expression was examined by RT-PCR and immunohistochemistry assay.Results: Iodine-125 seeds short-time low-dose rate irradiation 1)down-regulates expression of MMP-2,NGF and NT-3;2)up-regulates expression of and EGFR.Conclusion:These factors are PNI-related to PanCa,our findings indicated that the effects of Iodine-125 seeds short-time low-dose rate irradiation on PNI in PanCa is complicated.Iodine-125 seeds short-time low-dose rate irradiation could inhibit expression of some factors,but increases simultaneously expression of other factors.However,NTs and MMP-2 are well-known PNI-related factors,it was suggested that Iodine-125 seeds short-time low-dose rate irradiation could inhibit PNI in PanCa.Through this study, the subjects come to the following conclusions:1.The construction of in vitro PanCa PNI model is successful.PNI of PanCa can be developed between PanCa cells and nerves, which may have mutual growth advantages. The coculture model is feasible.2.A novel in vitro Iodine-125 seeds short-time low-dose rate irradiation PanCa PNI model is created.The intervention of Iodine-125 seeds short-time low-dose rate irradiation inhibits morphologically interactions between nerve and PanCa cells and biological behavior of PanCa cell.3. Successfully constructed the a novel in vivo Iodine-125 seeds short-time low-dose rate irradiation PanCa PNI model,and demonstrated a tumor-reduced effect of Iodine-125 seeds irradiation.5.There appeared effects of Iodine-125 seeds short-time low-dose rate irradiation on PNI in PanCa,but it was complex.Iodine-125 seeds short-time low-dose rate irradiation could inhibit expression of some PNI-related factors,but increases simultaneously expression of other PNI-related factors.NTs and MMP-2 are well-known PNI-related factors,the present study suggested that Iodine-125 seeds short-time low-dose rate irradiation could inhibit PNI in PanCa.