Experimental And Clinical Research Of Isolation By Size Of Epithelial Tumor Cells (ISET) Methodology

OBJECTIVE:Systematic study of isolation by size of epithelial tumor cells (ISET) methodology in vitro, animal experiments and clinical trials. To establish a kind of method of ISET in the process of blood circulation to detect, screen and separate circulating tumor cells. This pilot study will serve as a basis for a much larger clinical trial.METHOD:1.The method for the technical processes of ISET technology in vitro established with H520-pEGFP cells. H520-pEGFP cells will mix into the physiological saline and the blood sample from healthy volunteers respectively; detect to ascertain the recovery, the sensitivity and the specificity by ISET.2.To establish an animal model of tumor cells metastasis in the blood circulating, and detect the CTCs in the peripheral blood of animals by ISET technology.3.To design an ISET screening device, detect the CTCs in the peripheral blood of96patients with pulmonary disease. To observe the sensitivity and specificity of ISET screening device used in clinical testing. To monitor the presence of CTCs in one patient’s peripheral circulation undergoing standard chemotherapy and radiotherapy.4.To design an ISET hemoperfusion system, detect the CTCs in the blood circulating of rabbit with VX2tumor, observe and compare sensitivity and security of ISET screening device and ISET hemoperfusion system.RESULTS:1.The pEGFP-N3plasmid was transfected into H520tumor cells successfully. The recovery rate of mixed tumor cells detected by ISET technology remained about97.57±1.33in physiological saline and79.53±2.63in blood sample from healthy volunteers. We could detect1tumor cell in1ml peripheral blood while no positive cells were detected in healthy control, showing the high sensitivity and specificity of the ISET technology.2.The NOD-SCID mice model and rabbit model of tumor cells metastasis in the blood circulating have been established respectively.3samples in3NOD-SCID mice and3samples in17rabbit were detected with the CTCs by ISET technology.3.The ISET screening device was designed and built successfully to detect the CTCs in the peripheral blood of96patients with pulmonary disease.2samples in67cancer patients and0sample in29non-neoplastic patients were detected with the CTCs.2samples in4patients of stage IV and0sample in63patients of stage Ⅰ-Ⅲ were detected with the CTCs. However, CTCs were not detected in all6healthy samples by control. The detection rate of CTCs in4patients of stage IV was higher than that in63tumor patients of stage Ⅰ-Ⅲ (P<0.05).4.The persistence of CTCs in peripheral circulation in the patient of stage IV were monitored by ISET screening device.5.The ISET hemoperfusion system was designed and built successfully to detect the CTCs in the blood circulating of rabbit with VX2tumor. The detection rate of CTCs by ISET hemoperfusion system was higher than that by ISET screening device (P<0.05),82.35and17.65respectively (p<0.05).Conclusion:1.The ISET technology to detect CTCs was convenient and stable with high sensitivity and good specificity. It does not rely on any large-scale experimental equipment.2.CTCs have been detected in animal models of tumor cells metastasis in the blood circulating at the first time in the international, indicating that ISET technology has a high specificity.3.ISET screening device suitable for clinical detecting CTCs and easy to assemble and carry.4.The ISET technology exhibit high specificity in clinical trials, but the sensitivity is poor.5.We can evaluate efficacy of chemotherapy and radiotherapy and other cancer-targeting therapies by using ISET technology that has great potential for mechanistic studies of ISET-isolated CTC biology.6.A new ISET hemoperfusion system could be designed to increase sensitivity to CTCs greatly and reduce the number of CTCs directly during the blood flow at the first time in the international.The ISET hemoperfusion system gives us new insights into the diagnosis of malignant tumor and monitoring adjuvant therapy.