¹⁸F-FMISO PET And ¹⁸F-FLT PET In Assessment Of Dynamic Changes In Hypoxia,reoxygenation And Proliferation During Fractionated Radiotherapy In Murine Tumors

Part 1 Experimental study of ¹⁸F-FMISO PET in evaluating tumor hypoxia and biological changes of radiotherapyObjective:The uptake of ¹⁸F-FMISO(¹⁸F-Fluoromisonidazol)was observed under normal and hypoxia conditions in vitro;The -fluoromisonidazole(¹⁸F-FMISO)-based micro positron emission tomography/computed tomography（PET/CT）was used to evaluate the changes of tumor hypoxia in vivo;Immunohistochemical analysis was used to study the radiobiological changes of tumors;The purpose of this study was to investigate the feasibility and superiority of ¹⁸F-FMISO PET/CT in tumor hypoxia imaging and biological evaluation of radiation efficacy.Method:1.In vitro experiment The aim of the study was to evaluate the uptake of hypoxic imaging tracer ¹⁸F-FMISO under normal and hypoxic conditions.The cell density was adjusted to 5×10⁵ cells/100μL,and were divided into normal group and hypoxic group.Cells in the normoxic group were incubated in a conventional CO₂ incubator with 21%O₂,74%N₂,37℃and 5%humidity.Hypoxic condition was established in a modular incubator chamber（Billups Rothenberg）.Perkin Elmer gamma counter was used to measure the radioactive counting in each group.The cellular radionuclide uptake rate was calculated according to the formula[X（CPM）-O（CPM）]/R（CPM）%.One-way analysis of variance was used to observe the uptake of radionuclides in hypoxic and normoxic group.2.In vivo experiment The effect of radiotherapy on MDA-MB-231 xenograft tumor was observed in vivo based on tumor volume and survival time of tumor bearing mice.Rat axillary subcutaneous tumor transplantation model was established and randomly divided into control group and radiotherapy group,with 16 tumors in each group.The control group was irradiated every other day,2 Gy each time,and the cumulative radiation dose was 6Gy.Tumor volume of each rat was calculated every other day and the growth curves were plotted.Survival analysis was performed on the death time of each tumor-bearing rats.¹⁸F-FMISO PET/CT imaging was performed before and 24 h after radiotherapy.Visual analysis combined with semi-quantitative analysis were used for PET/CT image interpretation.The maximum standardized uptake value（SUVmax）was calculated by measuring the maximal concentration of radioactivity in a region of interest.The tumor tissue SUVmax（T）,the contralateral normal muscle SUVmax（N）,and the ratio of the two values（T/N）were calculated as the PET/CT metric for statistical analysis.After the PET/CT imaging was completed,eight tumor models from the control group and IR group,respectively,were sacrificed for immunohistochemical study.The changes of tumor biological indicators like HIF-1α,CAIX,Glut-1,Ki67,PCNA were calculated.Pearson correlation analysis was used to study the relationship between T/N and survival time,tumor volume and tumor biomarkers in tumor-bearing mice.Result:1.In vitro experiment To observe the differences of radiotracers uptake in normal oxygen and hypoxic cells,we performed time course analyses of cellular radiotracer uptake in MDA-MB-231 cells.The results showed that the uptake rates of ¹⁸F-FMISO in hypoxia cells were significantly higher than those in oxygenated cell（P=0.021,0.000,and 0.000,respectively）.The uptake of ¹⁸F-FMISO in hypoxic cells increased overtime,which suggested higher uptake of ¹⁸F-FMISO in hypoxia cells compared to oxygenated cells.2.Animal experiment Before and 24 h after radiotherapy,The Gross tumor volume（GTV）in control group were（0.62±0.05,1.91±0.43）cm³,respectively.GTV in radiation group were（0.65±0.04,1.53±0.31）cm³,respectively.The difference between the two groups was statistically significant.There was significantly difference of tumor inhibitory effect between radiation group and control group.Survival time of tumor-bearing rats in control group and radiation group were 29.25±3.34（20-35）d,38.52±4.54（25-48）d,the survival differences between the two groups were statistically significant via Kaplan-Meier analysis（χ²=14.28,P<0.01）.3.¹⁸F-FMISO PET/CT imaging ¹⁸F-FMISO PET/CT imaging was performed of each tumor-baring rat before and 24 h after treatment.Before radiotherapy,T/N of tumor-bearing rats was（1.91±0.54）.The criterion of tumor hypoxia based on T/N was set at 1.2.TMR>1.2 was defined as the hypoxic tumor.The proportion of hypoxic tumor before treatment was 90.6%（29/32）.24 h after radiotherapy,the T/N in radiation group was（1.52±0.33）,and the proportion of hypoxic tumor was 31.2%（5/16）,while that in control group was（2.68±1.13）,and the proportion of hypoxic tumor was 100%（16/16）.Hypoxia volume（HV）was defined as the volume of the GTV with a T/N larger than 1.4 detected by PET/CT.Before radiotherapy,the HV in tumor-bearing mice was（0.42±0.26）cm³,accounting for 65.6%（0.42/0.64）of GTV.After radiotherapy,the HV in tumor-bearing mice was（0.31±0.17）cm³,accounting for 37.3（0.31/0.83）of GTV.The HV in tumor-bearing mice in control group was（1.24±0.42）cm³,accounting for 86.7%（1.24/1.43）of GTV.In addition,the GTV increased slightly in the IR group and significantly in the control group.4.Immunohistochemical analysis Immunohistochemical analysis was carried out to assess tumor biological indicators like HIF-1α,CAIX,Glut-1,Ki67,PCNA.The percentage of HIF-1αpositive cells in control group and radiation group were 80.25±6.16,39.76±8.47%,respectively;The percentage of CAIX positive cells in control group and radiation group were 78.25±7.16,35.76±6.17%,respectively;The percentage of Ki67 positive cells in control group and radiation group were 78.15±5.15,32.33±6.11%,respectively;The percentage of GLUT-1positive cells in control group and radiation group were 71.25±4.34,36.19±5.64%,respectively;The percentage of PCNA positive cells in control group and radiation group were 68.15±6.15,22.33±5.61%,respectively.Significant differences of these tumor biological indicators were observed between groups.5.Statistical analysis Pearson correlation analysis was used to study the correlation between T/N and the positive expression of tumor marker HIF-1α、CAIX、Glut-1、Ki67、PCNA.The results showed that T/N was significantly correlated with the expression of CAIX（r=0.828,p=0.005）,and was moderate correlated with the expression of Ki67 and PCNA（r=0.412,p=0.041;r=0.511,p=0.036）,However,no significant correlation was detected between T/N and HIF-1αor Glut-1（r=0.413,p=0.078;r=0.511,p=0.097,respectively）。Conclusion:The hypoxia PET tracer ¹⁸F-FMISO can selectively concentrate in hypoxic cells.Radiotherapy can effectively inhibit the growth rate of tumor and prolong the survival time of tumor bearing mice.Tumor markers such as HIF-1α,CAIX,Glut-1,Ki67 and PCNA were significantly down-regulated in tumor tissues after radiotherapy.As an index for ¹⁸F-FMISO uptake,T/N can effectively monitor tumor hypoxia status and reflect tumor proliferation to a certain extent.¹⁸F-FMISO PET/CT imaging can specifically monitor tumor hypoxia and evaluate the efficacy at early stage,and can be used as a non-invasive quantitative marker for tumor hypoxia and radiation-related changes.Part 2 Dual-Tracer Assessment of Dynamic Changes in Reoxygenation and Proliferation Decrease During Fractionated Radiotherapy in Murine TumorsObjective:Detection of reoxygenation and the degree of oxygen increase in tumors during fractionated radiotherapy is of great importance for dose adjustment in follow-up radiotherapy.Tumor hypoxia and reproliferation during tumor radiotherapy are important reasons for poor radiotherapy efficacy and long-term recurrence and metastasis of tumors.Oxygen effect and the reoxygenation and reproliferation of hypoxic cells are important theories in clinical radiobiology,which are closely related to clinical efficacy.In the current study,using an experimental murine tumor model,we investigated tumor reoxygenation and tumor proliferation changes during radiotherapy with ¹⁸F-FMISO PET/CT and ¹⁸F-FLT PET/CT prior to,during,and following fractionated radiotherapy（FRT）,with the aim to detect the relationship between tumor reoxygenation and tumoricidal effects during radiotherapy.Method:Head and neck squamous cell cell line（FADU）xenograft tumor model was established and randomly divided into control group（n = 5）,and radiotherapy group（n = 16）,which was exposed to 3 Gy daily to a maximum dose of 40 Gy with a VARIAN 23 EX medical linear accelerator（Varian Medical Systems,USA）.All mice were scanned with both ¹⁸F-FMISO and ¹⁸F-FLT PET/CT prior to（Pre-FRT,0 Gy）,during（Inter-FRT,21 Gy）,and after FRT（Post-FRT,40 Gy）.To evaluate tumor hypoxia,maximum standardized uptake（SUVmax）and the tumor-to-normal muscle ratio（TNR）were used in this study for quantitative assessment of ¹⁸F-FMISO PET/CT data.SUVmax was determined by assessing the maximal radioactivity in a region of interest（ROI）.The TNR was derived as the tumor’s SUVmax divided by the SUVmax of noncancerous tissue.Hypoxia volume（HV）was the volume with TNR≥1.25.Tumor reoxygenation was analyzed according to TNR and HV changes.To assess tumor proliferation,we measured the tumor’s SUVmax and calculated the proliferation target volume（PTV）in the ¹⁸F-FLT PET/CT image.The PTV was the volume with SUVmax >1.4.In ¹⁸F-FMISO and ¹⁸F-FLT PET/CT images,the reduction rates from Pre-FRT to Inter-FRT and Inter-FRT to Post-FRT were calculated.Result:1、Gross tumor volume（GTV）change The tumor average diameter was 6.8 ± 0.4 mm pre-irradiation,corresponding to an average volume of 144 mm3.GTV increased during the first week of irradiation and began to decrease only after 18 Gy（6th day of radiotherapy）.GTV increased on average by 24.3% from pre-irradiation（144 ± 29 mm3）to a cumulative dose of 18 Gy（179 ± 46 mm3）,with a mean overall decrease of 45% by treatment end（50 Gy;90 ±18 mm3）2、PET/CT data during radiotherapy In ¹⁸F-FMISO PET/CT imaging: Both ¹⁸F-FMISO-SUVmax and ¹⁸F-FMISO-TNR were markedly reduced from Pre-FRT to Post-FRT in the IR group.In addition,the reduced rates of SUVmax and TNR from Pre-FRT to Inter-FRT（30.3% and 27.9%,respectively）were significantly higher than those from Inter-FRT to Post-FRT（14.5% and 18.3%,respectively）.The mean reduction rate of HV from Pre-FRT to Inter-FRT（85%）was significantly elevated as compared to that from Inter-FRT to Post-FRT（71.4%）.Conversely,all the above PET parameters in the control group showed a significant continual increase during the study,which was due to the increased oxygen depletion and rapid tumor proliferation.In ¹⁸F-FLT PET/CT imaging: ¹⁸F-FLT-SUVmax was significantly and continually decreased from Pre-FRT to Post-FRT;however,the reduction rate from Pre-FRT to InterFRT（36.8%）was similar to that from Inter-FRT to Post-FRT（51.0%;p=0.087）.the PTV reduction rate from Pre-FRT to Inter-FRT（21.2%）was remarkably lower as compared to that from Inter-FRT to Post-FRT（82.7%）.Conversely,all the above PET parameters in the control group showed a significant continual increase during the study,which was due to the increased oxygen depletion and rapid tumor proliferation.3、Associations of uptake of radiotracers with tumor volume（HV,PTV and GTV） Both SUVmax（FMISO）and TNR（FMISO）were significantly associated with HV（r=0.686,p=0.024 and r=0.763,p=0.016,respectively）,but showed no associations with GTV（r=0.325,p=0.178 and r=0.145,p=0.216,respectively）.SUVmax（FLT）was significantly correlated with PTV（r=0.842,p=0.009）,but showed no correlation with GTV（r=0.211,p=1.435）.Conclusion:Reoxygenation of hypoxic tumors occurs early after radiotherapy initiation.In contrast,cell proliferation inhibition associated with tumoricidal effects takes place gradually with the course of radiotherapy.Finally,¹⁸F-FMISO and ¹⁸F-FLT PET/CT are sensitive and noninvasive tools for monitoring tumor reoxygenation and activity during fractionated radiotherapy.