Preliminary Study Of TOLD 3.0T Magnetic Resonance Imaging In Assessment Of Hypoxia In Rabbit VX₂ Liver Tumor Model

BackgroundTumor vasculature often fails to maintain normal oxygenation levels due to the high rate of tumor cell proliferation and dysfunctional vascularization.Adaptation to hypoxia appears to be an important step in tumor progression.Hypoxia is a common feature of most solid tumors,reflecting an imbalance of oxygen delivery and consumption.Hypoxia is a common feature of most solid tumors,reflecting an imbalance of oxygen delivery and consumption.Hypoxia presents a ’Janus face’ in tumor biology.On the one hand,it is associated with restrained proliferation and with differentiation,but on the other,it promotes adaptive processes leading to tumor aggressiveness,progression,and acquired resistance to treatment.During cancer progression,neoplastic cell proliferation induces changes in microcirculation,which in turn induce hypoxia.Tumor tissues are more likely to cause gene duplication,transcriptional instability and gene mutation in hypoxia environment.Hypoxic tumor cells are less sensitive to radiotherapy,and are also more resistant to chemotherapy and biotherapy,leading to further progress in cancer and even failure in treatment.Hypoxia,or low oxygenation,has emerged as an important factor in tumor biology and response to cancer treatment.It has been correlated with angiogenesis,tumor aggressiveness,local recurrence,and metastasis.hypoxia is an independent prognostic marker of progression,metastasis,and disease-free survival after radiotherapy.In recent years,molecular targeted drug therapy for the treatment of liver cancer has attracted more and more attention.The only approved systematic treatment for advanced HCC is sorafenib,a multi kinase inhibitor with antiangiogenesis and antiproliferative effect.The efficacy of the treatment of advanced liver cancer has been confirmed by two breakthrough Ⅲ clinical trials.However,due to the high heterogeneity of sorafenib treatment of individual reactions,the development of sorafenianic resistance in recent years has aroused concern.Anti angiogenesis leads to hypoxic progress,aggravating cell hypoxia,resulting in antagonistic effect and resistance.If we want to improve the therapeutic effect and adjust the treatment plan in time,we find that the potential mechanism of Sorafenib resistance is critical.TOLD-MRI technology is a new magnetic resonance technology,which provide indirect estimates of tissue O2 for monitoring tumour oxygenation.The MRI longitudinal relaxation rate(R1,R1=1/T1,s-1)is sensitive to changes in the level of molecular oxygen(O2)dissolved in blood plasma or interstitial.Molecular O2 is weakly paramagnetic,which effectively acts as a T1-shortening contrast agent.The change in R1 relaxation rate is dependent on the amount of O2 dissolved in blood plasma,intracellular fluids,and extracellular fluids.Breathing hyperoxic gases increases the level of dissolved O2,since arterial oxygen saturation of haemoglobin(SaO2)is typically 98-100%at physiologic baseline.R1 has not been investigated in a large animal model tumour using a clinical field strength MR system.The purpose of this study is to evaluate the value of TOLD imaging in the diagnosis of VX2 HCC,and to explore its application value in the diagnosis of hypoxia.PART Ⅰ Assessment of Tumor Hypoxia Using Tissue Oxygen Level Dependent in a Rabbit VX2 Liver Tumor ModelObjectiveTissue oxygen level dependent(TOLD)MRI has been performed to explore the change of R,before and after oxygen inhalation in a rabbit VX2 liver tumor xenografts model.In this study,correlation was analyzed between the alteration(△R1)in tumor oxygen inhalation and the expression of HIF-1α,and the diagnostic value of TOLD was explored in staging hypoxia levels.Materials and Methods1.Rabbit VX2 liver tumor and grouping methodsEighteen New Zealand white rabbits,implanted with VX2 liver tumor,were incubated for 14 days,21 days and 28 days(six rabbits respectively).TOLD was performed on a 3.0T MRI scanner(Ingenia,Philips,Netherlands)before and after 30 min oxygen inhalation(twice respectively),using an animal coil.After that the rabbit VX2 liver tumor tissue sacrificed for pathological HIF-1α examination.2.1mage post-processingT1 maps were generated with MRmap software V1.4.R1 difference((△R1=△R1(O2)-△R1(air)))of VX2 liver tumor were measured for whole lesion(△R1whole)and non-necrotic area(△R1nonnecrotic),and the corresponding relative change△R1%whole and △R1%nonnecrotic were calculated.Immunohistochemical analysis of hypoxia-inducible factor-1α(HIF-1α)expression of rabbit VX2 liver tumor was performed on a high-powered microscope(H0-H4).3.Statistical methodsUse SPSS 20.0 statistical software package for data processing.Measurement data were expressed as mean±standard deviation(x ±s).Kruskal-Wallis H Test was used to test the diffrerence of △R1 values among each groups.Mann-Whitney U test was applied to analysis the differences between two groups.Spearman correlation test was used to evaluate the correlation between measured values and the expression of HIF-1α.Receiver operating characteristic(ROC)curve analysis was used to determine diagnostic accuracy.ResultsTwelve rabbits have completed MR scan and pathology examination(four rabbits,eight VX2 liver tumor,respectively).Significant differences(P<0.05)were found in △R1whole,△R1nonnecrotic,△R1%whole and△R1%nonnecrotic of differenttime points.No significant differences(P>0.05)were found in △R1nonnecrotic and △R1%nonnecrotic between 21 days and 28 days.Mann-Whitney U test show statistical differences in △R1whole and △R1%whole among the different time points(P<0.05).Hypoxia level was correlated with △AR1whole,△R1nonnecrotic,△R1%whole,△R1%nonnecrotic(r=-0.608,r=-0.545,r=-0.601,r=-0.585,p<0.01,respectively).Area under ROC curve(AUC)of △R1whole,△R1nonnecrotic,△R1%whole and△R1%nonnecrotic in differentiating hypoxia levels were as follows:HO VS H1-3(0.865,0.825,0.849,0.833);H0-1 VS H2-3(0.890,0.860,0.882,0.875),H0-2 VS H3(0.702,0.702,0.726,0.726).ConclusionsThe AR,decreases with the growth of rabbit VX2 liver tumor,and has a high negative correlation with the hypoxia grading of HIF-1α.Our study shows that AR,have a high diagnostic efficacy for noninvasive measurement of tumor hypoxia of VX2 liver tumor,suggesting the potential of TOLD as a non-invasive technique for the assessment of tumor hypoxia level.PART Ⅱ Assessment of Tumor Hypoxia Response to Sorafenib in Rabbit VX2 Liver Tumor Xenografts by Tissue Oxygen Level Dependent MR Imaging.ObjectiveTOLD was applied to investigate the change of R1 before and after oxygen inhalation between control group and treated group.The changes of HIF-1α level after a week of sorafenib in the treatment group were analyzed and compared with the control group.Materials and Methods1.Rabbit VX2 liver tumor and grouping methodsEighteen New Zealand white rabbits,implanted with VX2 liver tumor,were incubated for control group(PART Ⅰ 21 days,28 days)and treated group,six respectively.The treated group were given a week of sorafenib(20mg/kg/d)on 21 days after implantation.TOLD was performed on a 3.0T MRI scanner(Ingenia,Philips,Netherlands)before and after 30 min oxygen inhalation,using an animal coil.Get the VX2 liver tumor tissue for pathological examination after MRI scan.2.Image post-processingT1 maps were generated with MRmap software V1.4.R1 difference((ΔR1=ΔR1(O2)-ΔR1(air)))of VX2 liver tumor were measured for whole lesion(ΔR1whole)and non-necrotic area(ΔRInonnecrotic),and the corresponding relative changeΔR1%whole and △R1%nonnecrotic were calculated.Immunohistochemical analysis of hypoxia-inducible factor-la(HIF-1α)expression of rabbit VX2 liver tumor was performed on a high-powered microscope(H0-H4).3.Statistical methodsUse SPSS 20.0 statistical software package for data processing.Measurement data were expressed as meann±standard deviation(x ± s).Non-parametric test was used to test the diffrerence of AR,values among each groups.Mann-Whitney U test were applied to analyze the difference of HIF-1α expression between control and treatment group.ResultsThirteen rabbits have completed MR scan and pathology examination(4 rabbits and eight VX2 liver tumor for 21 days and 28 days,5 rabbits and ten VX2 liver tumor for treated group).No significant differences(P>0.05)were found between control group and treated group at week 3 in △R1whole,△AR1nonnecrotic,△R1%whole and△R1%nonnecrotic.Significant differences(P<0.01)were found in △R1 whole,△R1nonnecrotic,△R1%whole and △R1%nonnecrotic between treated group and control group at week 4,.Significant differences were also detected in treated group before and after the application of sorafenib.There was no significant difference in the pathological grade of HIF-1α between the treatment group and the control group after a week of sorafenib(P>0.5),but the HIF-1α positive cell expression rate of treated group was significantly higher than control group(28 days).The difference was statistically different(P<0.05).ConclusionsSustained treatment with sorafenib accelerated tumor hypoxia and increased expression of HIF-1α.The ΔR1 of treatment group decreased obviously after a week of sorafenib,compared to control group.This trend was consistent with the increased expression of HIF-1α.The change of ΔR1 can be detected by TOLD.As a repeatable non-invasive examination technique,TOLD can monitor and evaluate the therapeutic effects of anti-tumor drugs.It has important potential application value for individualized treatment for adjusting treatment plans.