Experimental Study On Stent-Magnetic Hyperthermia For Esophageal Cancer Treatment

Background:Cancer is one of the most serious diseases, a threat to human health. Based on the GLOBOCAN2008estimates, about12.7million cancer cases and7.6million cancer deaths are estimated to have occurred in2008; of these,56%of the cases and64%of the deaths occurred in the economically developing world. Lung cancer has been the most common cancer in the world by2008, followed in terms of incidence by breast cancer, stomach cancer, prostate cancer and liver cancer. The continuing growth and aging of the world’s population mean that, even with the current (2008) rates of morbidity and mortality, the burden of cancer will continue to increase. WHO estimates that the deaths from cancer in the world will increase by72percent from2008to2030,7.6million people died of cancer in2008and will reach13million in2030.The traditional therapies such as surgery, chemotherapy, and radiation therapy are still the most effective treatment for cancer. With the advancement of technology and in-depth study, new methods for cancer treatment such as immunomodulatory agents, gene therapy and monoclonal antibody therapy were developed. However, these methods are not yet mature enough and expensive and not suitable for large-scale clinical application. The current treatments have their own limitations. Surgery has been available only for localized tumor and inability in many advanced cases. The quality of life for patients was seriously impacted by the tissue and organ defects after surgery. It is obvious that radiation and chemotherapy can cause systemic damage and may lead to poor treatment received in many patients. Comprehensive and individual treatment of cancer is a consensus currently.More than100years ago, it was recognized that tumors can be treated with heat. Hyperthermia is an important adjuvant to radiotherapy and chemotherapy. Comparing with radiotherapy and chemotherapy, it has less restriction in treating cancer and can overcome the tumor resistance to chemo and radiation. But it had some limitations difficult to overcome. First, it is difficult to control the treatment temperature precisely. Second, the tolerable temperature of normal tissue is very close to the tumor-killing temperature. Hyperthermia at higher temperature becomes unacceptable. Thirdly, the thermal conductivity of human tissue is good. It is difficult to effectively control the scope of heating and avoid the damage of the normal tissue surrounding the tumor. Fourthly, it is still difficult to achieve uniform temperature within the target volume. Conformal hyperthermia is a hot research field. BSD2000Hyperthermia System, which can non-invasively deliver localized therapeutic heating (hyperthermia) by applying radiofrequency (RF) energy to certain cancerous tumors, has entered the clinical application. But microwave and radiofrequency energy have limitations. Subcutaneous fat can absorb high-frequency electromagnetic wave and attenuate the electromagnetic energy within the patient’s body. So the early developed hyperthermia equipments such as microwave, radio frequency, infrared ray are confined to the tumors located in skin. There is no effective hyperthermia method to treat the cancer located in deep tissue yet. It is well known that low frequency electromagnetic fields can penetrate human’s body with little attenuation of energy. But it can not effectively deliver energy to the target tissue. However, if there are metal conductors within the target tissue, the situation is quite different. According to inductive heating principle, all electro-conductive materials such as metal conductor or magnetic media can be heated inductively. If the metal conductor or magnetic media was implanted in the deep tissue and an external medium or low frequency alternating magnetic field was applied to the body, a new technology of deliver targeted hyperthermia to deep tissue is achieved.Based on this idea, a number of inductive heating equipments have been developed for experimental and clinical researches. Many satisfactory results have been obtained. In2004, a novel hyperthermia-and thermoablation-system MFH300F has been developed by Jordan A and successfully clinical applied. Our laboratory began the development of the similar equipment in2003and successfully developed the first clinical machine of magnetic induction heating of china in2008. In this study, the clinical machine was applied. A series of cell and animal studies were applied to determine its characteristics of magnetic induction heating, applicable parameters and biological effects.Part I Stent-Magnetic Hyperthermia on Human Esophageal Cancer Cell Line EC-109in Vitro Chapter1Exploration of the Magnetic Field Parameters of the set TemperatureObjective:To explore the magnetic parameters and conditions what achieve and repeat the set temperatures by the clinical machine of magnetic induction heating.Methods:We kept the ambient temperature constant at37℃by a water bath circulator. The exact location of the magnetic poles was determined to ensure the cryogenic vials at the same location in the magnetic field every time. An alternating magnetic field (AMF) was applied to the cryogenic vial contained with a stent immersed by culture medium. We recoded the applied parameters and time when the set temperature reached and maintained balance. The same temperatures heated by water bath served as control. Each set temperatures was tested6times. The heating curves were drawn.Results:By adjusting the magnetic field parameters, the metallic stent can be heated rapidly in the AMF. The set temperatures reached quickly and were maintained balance for a long time. The magnetic parameters of achieving the temperature of43℃、48℃、53℃、were360mm magnetic gap, coil current45A was applied for80sec and then adjusted to15A;360mm magnetic gap, coil current45A was applied for140sec and then adjusted to16A;240mm magnetic gap, coil current45A was applied for160sec and then adjusted to20A; respectively. The water bath times of achieving the set temperature of43℃、48℃、53℃、were160sec, 170sec,180sec, respectively.Conclusions:The stent can be heated to the set temperature quickly and maintained balance for a long time in the AMF. Chapter2Effect of Magnetic Hyperthermia on the Morphology and Proliferation and Apoptosis and Necrosis of Human Esophageal Cancer cell line Eca-109Objective:To investigate the effect of magnetic hyperthermia at different temperatures on the morphology and proliferation and apoptosis and necrosis of human esophageal cancer cell line Eca-109.Methods:Eca-109cells were heated at43℃,48℃,53℃by water bath and magnetic hyperthermia. Normal cells served as control. The cell morphology changes at different time points were observed with an inverted microscope. MTT assay measures the changes in cell proliferation and viability. Flow cytometry evaluated the apoptosis and necrosis. The in situ programmed cell death was tested by TUNEL analysis. The cell morphological features of apoptosis were observed.Results:After exposed to the magnetic fields, no distinct changes in cell morphology were observed. Heated by the two heating methods, no distinct changes in cell morphology at43℃were observed in30min. The cell morphologic changes showed time and temperature dependence at48or53℃. The magnetic field alone had no effect on cell survivals and apoptotic rates (P>0.05). Heated by the two heating methods, no effect of cell proliferation or apoptotic induction at43℃was observed in30min. The cell proliferations were significantly inhibited and the percentage of apoptosis/necrosis significantly increased at48or53℃. Temperature-and time-dependence was observed (P<0.05). The time points of maximal inhibition rate were48℃for20min;53℃for10min respectively. The time points of maximal apoptotic/necrotic rates were48℃for30min;53℃for20min respectively. The TUNEL assay showed that the magnetic fields did not change the number of the stained nuclei. Heated by the two heating methods, the number of the stained nuclei significantly increased at43℃with the exposed time. A sudden decrease in the numbers of the stained nuclei was observed at48℃. The time points of the two heating methods were10min,15min respectively. As for53℃, the time points were both5min. The apoptotic/necrotic rates and the numbers of stained nuclei increased with the time after these time points.Conclusions:The magnetic fields and heating at43℃in30min have no effect on Eca-109cell. Heated by the two heating methods at48℃or53℃can temperature-and time-dependent inhibit cell proliferation and induced apoptosis/necrosis. The time points of maximal inhibition and apoptosis/necrosis rates were48℃for30min;53℃for20min respectively. These parameters can be used as experimental and clinical reference. The results of TUNEL immunohistochemistry staining of apoptosis show that the magnetic fields do not increase the numbers of stained nuclei. Heated by the two heating methods, the numbers of stained nuclei can time-dependent increase significantly at43℃and suddenly decrease at48℃,53℃. The time points of this phenomenon at48℃,53℃were10min,5min respectively. This phenomenon may suggest the suspension threshold of reversal of apoptosis combining with the results of flow cytometry and MTT assay.There is difference in heat resistant between the cells in a cell population. There are not only heat-sensitive cells but also heat-resistant cells. The mechanism and methods of prevention of heat-resistant should be further studied. Part Ⅱ To Assess Safety of Magnetic Hyperthermia in Pigs’Esophagus in the Acute Phase Objective:To assess the safety of magnetic hyperthermia in pig esophagus in the acute phase.Methods:11Guizhou miniature pigs were randomly divided into5groups as follows:normal group, esophageal stent group,43℃,48℃and53℃groups. The normal pig was killed immediately. The stent group was killed2h after stent implantation. The stent fixed with a thermocouple was implanted in a pig’s esophagus. The outside esophageal wall was found through tracheotomy and another thermocouple was fixed on it. The pigs after surgery were placed in the AMF. The temperatures of the inner and outer wall of the esophagus were recoded.Results:By adjusting the magnetic field parameters, the metallic stent can be heated rapidly in the AMF. The set temperatures reached quickly and maintained balance for a long time. The magnetic parameters of achieving the temperatures of43℃、48℃、53℃were coil current31A was applied till the temperature reached43℃and then adjusted to.23A, coil current45A was applied till the temperature reached49℃and then adjusted to35A, coil current45A was applied till the temperature reached54-55℃and then adjusted to40A, respectively. The magnetic gaps of the three groups were360mm. The equilibrium times were30min. The maximum temperature of the outer wall of the esophagus did not exceed40℃. After the cessation of the external magnetic field, the temperature can restore to37℃no more than15min. After the pig vomiting, the temperature curves of the inner and outer wall of the esophagus changed significantly. The two curves almost coincide. The maximum temperature difference of the two curves was only4℃. The esophageal biopsies revealed:no effects on the esophagus implanted with stent or heated by magnetic induction heating at43℃were found. The esophageal submucosal tissue will be congestion at48℃. Heating at53℃, some blisters formated in the esophageal mucosa and some blood cells and plasma leaked into the tissue spaces. Conclusions:After heated at48℃for30min, some reversible Changes were found in the esophagi. Esophageal magnetic hyperthermia at48℃for30min is an acceptable reference parameter. Part III Effect of Magnetic Fluid Hyperthermia in Rabbit Esophageal Transplanted Cancer ModelsObjective:To investigate the effect of magnetic fluid hyperthermia (MFH) in rabbit esophageal transplanted cancer modelsMethods:Esophageal carcinoma transplanted models were created by transplanting VX2cells to rabbit esophagus surgically. The mangetic nanopartilces modified by aminosilane (amino-MNPs) were intratumoral injected. The animals received magnetic fluid hyperthermia (MFH) in AMF at48℃for30min. The hyperthermia was repeated every three days. The untreated group, the magnetic fluid group served as controls. The eating habit, weight, systemic metastasis, tumor volume and survival were observed. Tissue sections from the three groups were stained with HE and the morphological changes were observed.Results:By adjusting the magnetic field parameters, the metallic stent can be heated rapidly in the AMF. The intratumoral temperatures could reach48℃and be maintained for30min. After the cessation of the external magnetic field, the temperature can restore to37℃no more than lOmin. The varying degrees of difficulty swallowing were observed in14days after tumor cells transplantation. The weights of all the rabbits began to fall after surgery. With the reduction in eating, the rabbits all died of malnutrition when their weights loss reached to30%. Comparing the weights between the three groups, no significant difference was found(P>0.05). Two or three rabbits had lung metastases in each group. Comparing the untreated group with the magnetic fluid group, there is no significant difference in tumor volume and survival(P>0.05). Comparing with the other two groups, no significant difference was found in tumor volume in7day after hyperthermia(P>0.05). At the time of14day after hyperthermia, the tumor volume of MFH group was smaller than the other two groups(P<0.05). Comparing with the other two groups, the MFH group had a longer survival(P<0.05).The biopsies revealed that repeated esophageal hyperthermia at48℃for30min will not cause permanent heat damage.Conclusions:MFH at48℃for30min is safe and effective. The survival of the tumor-bearing rabbits was prolonged. This parameter is an acceptable reference for clinical application...