Intravoxel Incoherent Motion Diffusion Weighted Imaging And Dynamic Contrast-enhanced MRI For In Vivo Monitoring The Therapeutic Efficacy Of Nanosecond Pulsed Electric Field In Cervical Cancer Xenograft Model

Objective:To establish two kinds of novel fuctional magnetic resonance imaging (MRI) scan protocol-multi b-value diffusion weighted imaging (DWI) and dynamic contrast-enhanced MRI (DCE-MRI) for small animals and compare the diffusion and perfusion characteristics quantitatively between cervical adenocarcinoma and squamous carcinoma in nude mouse xenograft models.To explore the mechanism and appropriate intensity of nanosecond pulsed electric fields (nsPEF) inhibiting growth of cervical cancer cell lines-HeLa and SiHa in vitro and monitor early therapeutic response and long-term efficacy of nsPEF treating HeLa cervical cancer-bearing nude mice in vivo with multi b-value DWI and DCE-MRI.Methods:Human cervical cancer cell lines, Hela and Siha, were injected subcutaneously in back of Balb/c female nude mice to establish cervical adencarcinoma and squamous carcinoma xenografts. The MRI scan was conducted in3.0T MRI unit (MR750, GE, Waukesha) coupled with a35mm diameter small animal coil containing the routine T2WI, multi b-value DWI and DCE-MRI. Multi b-value DWI was acquired with11grading b values-0,20,50,100,200,400,600,800,1000,1200,1500s/mm2utilizing spin-echo sequence. For DCE-MRI, a200-phase dynamic serie of T1WI2D SPGR images was obtained every3s, with an intravenous bolus of Gd-DTPA given after the10th baseline data point through catheterized tail vein tube. D (pure molecular diffusion), D*(perfusion related diffusion), and f (perfusion fraction) were obtained from a bi-exponential IVIM model of multi b-value DWI. Pharmacokinetic parameters of CER(contrast enhancement ratio), Ktrans(transfer rate constant), KeP(reverse rate constant), Ve(extravascular extracellular volume fraction),fPV(fraction of plasma volume) and AUC9o(area under curve90s)were obtained from a two-compartment model of DCE-MRI. These quantitative parameters of the two different pathological cervical cancers were compared using student t-test in SPSS20.0and a less than0.05two-side p value is considered statistical significant. Cervical cancer cell lines-HeLa and SiHa were treated in three electric intensities by nsPEF with100ns pulse duration and1Hz frequency in vitro (low:20kv/cm25pulses; medium:20kv/cm40pulses; high:25kv/cm40pulses). Cell viability were tested with CCK-8kit in3time points (24h,48h and72h) and apoptosis/necrosis were examined by flow cytometry with Annexin-FITC-PI kit. Nude mice bearing HeLa cervical cancer were randomly divided in the nsPEF-treated and control groups, the former one received200pulses nsPEF treatment with100ns pulsed duration and50kv/cm intensity. Multi b-value DWI and DCE-MRI were conducted separately.10nude mice (5from nsPEF-treated and5from control group) got a continuous monitoring with DWI scan before,24hours and14days after nsPEF treatement or none; while the other7nude mice (4from nsPEF-treated and3from control group) got the DCE-MRI scan before and14days after nsPEF treatement or none. Immunohistochemical staining of CD31,CD34and TUNEL test were performed in the excised tumors to evaluate the neovasculature and apoptosis/necrosis pathologically. Quantitative parameters as described above were acquired from the functional MRI and analyzed in student t test or repeated measures of multi-variance analysis in SPSS20.0, as well a less than0.05two-side p value is considered statistical significant.Results:I.10HeLa adenocarcinoma and10squamous carcinoma nude mice with similar tumor volume got multi b-value DWI scan, HeLa mice has a trend of lower D than SiHa, but without statistical significant (0.259±0.031vs.0.363±0.064×10-3mm2/s, p=0.163). While perfusion fraction of Hela-mice is significantly higher than SiHa (0.211±0.023vs.0.145±0.012, p=0.022). The other5HeLa mice and4Siha mice were successfully taken DCE-MRI scan. Hela mice got an obviously higher prefusion related parameters than SiHa mice all with a marginal trend toward significant (CER:1.892±0536vs.0.477±0.142, p=0.057; Ktrans:0.156±0.036vs.0.058±0.013min-1; fPV:0.126±0.038vs.0.022±0.007, p=0.050; AUC90:22.32±6.19vs.6.30±1.56, p=0.060). Hela xenograft tumor shows more restricted water diffusion motion and higher perfusion of new formed vessles than SiHa tumor.II. In vitro study, CCK-8test showed proliferation inhibition of both HeLa and SiHa cell is positively correlated with intensity of nsPEF and reach the optimum rate at48h, which can almost reach90%compared to control in the highest intensity group. Flow cytometry showed nsPEF can induce early apoptosis and necrosis in HeLa and SiHa cell lines after2hours incubation. Along with electric intensity increase, necrosis played a major role in cell death, which in the highest intensity necrosis/apoptosis ratio were81%/5.9%for HeLa and85.1%/14.5%for SiHa separately.Ⅲ. Growth of tumor exposed to nsPEF has been inhibited by prominently than control group, whose tumor volume was decreased by37%while the latter one was increased by150%(p=0.025). In the multi b-value DWI team, D value showed significant rise in nsPEF group, particularly at24h time point, which however decreased gradually in the control group along with tumor progression (p=0.022), indicating D can predict early response to nsPEF therapy. Pathological results verified necrosis and apoptosis induced by nsPEF in HE stain and TUNEL assay. In DCE-MRI team, quantitative neovasculature related parameters significantly decreased14days after nsPEF treatment (△Ktrans=Ktrans-14day-Ktranspre,△Ktranscontrol=0.049±0.018min-1vs.△KtransnsPEF=-0.028±0.006min-1, p=0.005) and it correlated with lower vessel density of CD31and CD34in immunohistochemistrical stain.Conclusion:Ⅰ. Multi-b value DWI and DCE-MRI were excellent quantitative imaging biomarkers to measure nude mice tumor xenografts. Cervical adenocarcinoma has a lower pure water diffusion coefficient than squamous carcinoma in tumor-bearing model driven from a bi-exponential IVIM model of multi-b value DWI. Both multi-b value DWI and DCE-MRI indicate adenocarcinoma has much higher neovasculature perfusion than squamous carcinoma.Ⅱ. nsPEF can inhibit proliferation cervical cell line-HeLa and Siha through inducing necrosis and early apoptosis. It is apparently dose-dependent and slightly time-dependent.Ⅲ. D value from the bi-exponential IVIM model of multi-b value DWI is a sensitive quantitative imaging biomarker for evaluating early response of nsPEF treatment in cervical cancer xenograft. DCE-MRI is a robust non-invasive tool to measure amounts and functions of tumor’s angiogenesis and reaction to anti-angiogenic therapy in vivo. nsPEF can inhibit tumor growth by induce cellular apoptosis in early phase and suppress blood vessels in the long term.