Experimental Studies Of¹⁸F-FDG And¹⁸F-FLT Cell Binding In Early Valuating The Inhibitory Effect Of Radiotherapy In Non-small-cell Lung Cancer Cell

Objective: To explore a useful and stable method of¹⁸F-FDG or¹⁸F-FLT cell bindingof non-small-cell lung cancer cell A549. Early assessment the inhibitory effect ofradiotherapy in non-small-cell lung cancer cell A549by¹⁸F-FDG and¹⁸F-FLT cell bindingexperiment and MTT and flow cytometry.Methods: Several different binding conditions were tested.（1）The cells number were5×10⁴～5×10⁶/hole; The reaction time was20～120min;The radioactivity of¹⁸F-FDGand¹⁸F-FLT was1.85～29.6KBq; The glucose concentration was0～11.1mmol/L.Thecount of CPM（B） in cells and CPM（F） in supernatant were measured by the γ equipment.The binding efficiency of non-small-cell lung cancer cell A549with¹⁸F-FDG or¹⁸F-FLTwas calculated.（2）The human non-small-cell lung cancer A549cells were exposed to asingle fraction of X-ray at different dose of radiation. The binding inhibition efficiency of¹⁸F-FDG and¹⁸F-FLT were calculated at24hours and48hours after irradiation.（3） MTTwas utilized to measure the inhibitory efficiency of A549cell with different dose ofradiation at24hours and48hours after irradiation.（4） Flow cytometry was applied to detectthe A549cell cycle changes at24hours and48hours after irradiation.Results:（1） The binding rates of¹⁸F-FDG were（7.65±1.05）%,（13.83±1.02）%,（21.76±1.35）%,（39.83±0.81）%,（41.43±1.86）%, while¹⁸F-FLT were （4.95±0.42）%,（6.64±1.36）%,（12.86±0.88）%,（18.76±0.70）%,（21.49±1.52）%at the conditionof cells density ranges from5×10⁴to5×10⁶per hole，incubating time100minutes,¹⁸F-FDGand¹⁸F-FLT radioactivity3.7KBq;The difference of¹⁸F-FDG binding rate was nosignificant between1×10⁶and5×10⁶group （P>0.05）. The difference of¹⁸F-FDG binding rate was significant between the others （P<0.05）. The difference of¹⁸F-FLT binding ratewas not significant between1×10⁵and5×10⁴group （P>0.05）. The difference of¹⁸F-FLTbinding rate was significant between the others （P<0.05）. The binding rates of¹⁸F-FDGwere （15.16±1.27）%,（19.27±1.43）%,（26.71±1.35）%,（29.35±1.66）%,（39.83±0.81）%,（40.41±2.28）%, while¹⁸F-FLT were （5.54±1.28）%,（9.36±1.25）%,（11.52±1.62）%,（14.12±2.75）%,（18.76±0.70）%,（20.52±1.89）%respectively,at the condition of1×10⁶cells,incubating time from20to120minutes,¹⁸F-FDG and¹⁸F-FLT radioactivity3.7KBq;The difference of¹⁸F-FDG binding rate was not significant between20min and40min,60min and80min,100min and120min group （P>0.05）. The difference of¹⁸F-FDGbinding rate was significant between the others （P<0.05）. The difference of¹⁸F-FLTbinding rate was not significant between40min and60min,100min and120min group（P>0.05）. The difference of¹⁸F-FLT binding rate was significant between the others（P<0.05）. At the condition of1×10⁶cells,incubating time100minutes,radioactivity rangesfrom1.85to29.6KBq,The binding rate of¹⁸F-FDG and¹⁸F-FLT were （35.61±3.14）%,（39.83±0.81）%,（37.35±3.44）%,（38.63±2.93）%,（38.12±3.25）%, and （16.72±1.96）%,（18.76±0.70）%,（17.62±1.34）%,（18.39±1.02）%,（17.56±2.13）%accordingly; Thedifference of¹⁸F-FDG and¹⁸F-FLT binding rate was not significant in all groups （P>0.05）.At the condition of1×10⁶cells, incubating time100minutes,radioactivity3.7KBq,glucoseconcentration from0to11.1mmol/L,the binding rates of¹⁸F-FDG and¹⁸F-FLT were（37.47±1.54）%,（7.39±2.05）%,（5.13±1.36）%,（3.38±1.69）%, and （17.51±1.35）%,（16.38±1.82）%,（16.42±2.03）%,（17.87±0.93）%accordingly;The binding rate of¹⁸F-FDG decreased when the glucose concentration raised. The binding rate of¹⁸F-FDGwas significant in all groups （P<0.05）. The binding rate of¹⁸F-FLT without significantdifference among the groups （P>0.05）.（2） The¹⁸F-FDG binding inhibition rates of A549cell were（1.28±1.04）%,（3.01±1.66）%,（3.27±1.83）%,（6.32±3.32）%and （3.93±2.17）%,（5.97±2.65）%,（9.46±2.03）%,（13.38±1.88）%after24hours and48hours irradiation of different dose. At24hours after irradiation, the binding inhibition rate was not significant between2Gy and 4Gy group,4Gy and6Gy group （P>0.05）,while the difference was significant between theothers （P<0.05）;At48hours after irradiation, the binding inhibition rate was not significantbetween2Gy and4Gy group （P>0.05）, while the difference was significant between theothers （P<0.05）. The¹⁸F-FLT binding inhibition rates of A549cell were （4.14±4.36）%,（12.53±3.35）%,（19.44±3.14）%,（28.29±6.03）%and （3.16±2.27）%,（8.42±4.59）%,（12.70±3.55）%,（15.54±2.11）%after24hours and48hours irradiation of different dose.The binding inhibition rate was significant among the groups of24hours after irradiation（P<0.05）;At48hours after irradiation, the binding inhibition rate was not significantbetween6Gy and8Gy group （P>0.05）, while the difference was significant between theothers （P<0.05）.（3） With the method of MTT, the difference of the inhibition rates of A549cell were（12.95±0.92）%,（13.26±2.67）%,（17.02±1.32）%,（20.80±0.88）%and （17.72±1.64）%,（28.27±1.32）%,（32.52±0.95）%,（34.57±1.48）%after24hours and48hours irradiationof different dose. The inhibition rate of A549cell grows complying with increase of theirradiation doses and times after radiotherapy. At24hours after irradiation, the inhibitionrate was not significant between2Gy and4Gy group （P>0.05）, while the difference wassignificant between the others （P<0.05）.At48hours after irradiation, the difference of theinhibitory rate was significant in the groups （P<0.05）.（4） Flow cytometry was applied to detect the A549cell cycle changes.The fraction ofA549cells in S phrase were32.76%,31.28%,18.55%,11.48%,3.18%and12.29%,11.26%,11.03%,10.97%,10.19%respectively,after24hours and48hours irradiation ofdifferent dose. The fraction of A549cells in G2phrase were37.29%,42.19%,48.11%,65.59%,79.99%and37.85%,40.27%,44.01%,44.50%,55.04%respectively.Conclusions:1.On certain conditions,¹⁸F-FDG and¹⁸F-FLT binding rates of A549cells increased as the cell number and incubating time grew，irrelative with radioactivity,¹⁸F-FDG uptake rates decreased when glucose concentration raised while¹⁸F-FLT uptakerates were irrelative with glucose concentration.2.The¹⁸F-FDG and¹⁸F-FLT bindinginhibition rates of A549cell grows complying with increase of the irradiation doses and times after radiotherapy. The binding inhibition rate was positively correlated with theMTT inhibition rate.3.An increased fraction of A549cells in G2/M phrase was observedafter different dose of irradiation. The¹⁸F-FLT uptake rate is positively correlated with thefraction of S phrase.