The Anti-tumor Efficacy Of Nanosecond Pulsed Electric Fields For Subcutaneous Transplanted Malignant Melanoma Tumors In Nude Mice

Objective:To evaluate the effect of nsPEF on the A375-GFP melanoma xenograftin nude miceMethods:The GFP gene was transfected into human melanoma A375cells bylentiviral vector. And the multiplicity of infection in the group with highesttransfection efficiency which was nearly close to100%was screened byfluorescence microscope and then the cell stably expressing GFP protein atthat optimum transfection conditions was constructed, which was namedA375-GFP. Cell proliferative activities of A375-GFP and A375cells weredetected by MTT assay and cell doubling time method, and the cell cycledistributions were tested by flow cytometry analysis. A375-GFP melanomaxenografts in nude mice were constructed, which were randomly dividedinto treatment group and control group. The melanoma xenografts intreatment group were exposed to the electric pulse parameters (200ns in duration,20kV/cm in amplitude,2000pulses and at a pulse frequency of5pulse/s, four directions, treatment at every3d and a total of2times), whilethose in control group were exposed to the sham treatment. In vivofluorescence image analysis system was used to observe the fluorescencechanges of melanoma xenograft at48h and10d after treatment.90d afterpulse delivery, local therapeutic efficacy was pathologically evaluated byHE stain. The changes of scar were recorded by digital camera.Results:1. A375-GFP cell line stably expressing GFP protein was successfullyconstructed.2. After72h of cell growth, no significant difference was found in ODvalue between A375-GFP(1.023±0.810)and A375cells(1.066±0.514)detected by MTT assay (P＞0.05). And no significant difference wasidentified in doubling time between A375-GFP(28.9±1.51h)and A375(29.04±1.04h)cells (P＞0.05). Flow cytometry analysis showed nosignificant difference in cell cycle distributions(P＞0.05).3. A375-GFP melanoma xenografts in nude mice were successfullyconstructed.4. The fluorescence of tumor detected by In vivo fluorescence imageanalysis system in treatment group decreased mostly at48h after treatmentand was not observed at10d after treatment, while that in control groupincreased gradually. Treatment group showed significant difference in fluorescence value as compared with control group(F=1055.11，P<0.0001).At90d after pulse delivery，Surgical excision of the therapy regionsconfirmed a complete pathologic response. Within3-4days after nsPEFtreatment, a hard scab formed at the treatment region. The scab falled offby the end of the second week. As time went on, the scar gradually becamefaded and all xenograft tumors were disappeared without recurrence.Conclusion:1. The cell line with stable, efficient and long-term GFP expressioncan be obtained by lentiviral transfection technology.2. A375-GFP cell can reflect the proliferation ability of A375for thatGFP transfected by lentiviral vector does not impact the proliferationactivities of A375cell.3. A375-GFP melanoma xenograft in nude mice can be successfullyconstructed. From the experiment, we learned that nsPEF can bring a goodtherapeutic effect with advantages of little scar and simple operation. Itmay provide a new approach for the clinical minimum invasivenesstreatment of superficial tumors.