Monitoring Tumor Response to Antivascular …...Monitoring Tumor Response to Antivascular Therapy...

Therapeutics, Targets, and Chemical Biology

Monitoring Tumor Response to AntivascularTherapy Using Non-Contrast IntravoxelIncoherent Motion Diffusion-Weighted MRIChangzheng Shi1, Dexiang Liu1,2, Zeyu Xiao1, Dong Zhang1, Guanfu Liu1,2,Guanshu Liu3,4, Hanwei Chen1,2, and Liangping Luo1

Abstract

Antivascular therapy is a promising approach to the treat-ment of non–small cell lung cancer (NSCLC), where an imag-ing modality capable of longitudinally monitoring treatmentresponse could provide early prediction of the outcome. In thisstudy, we sought to investigate the feasibility of using intra-voxel incoherent motion (IVIM) diffusion MRI to quantitative-ly assess the efficacy of the treatments of a vascular-disruptingagent CA4P or its combination with bevacizumab on experi-mental NSCLC tumors. CA4P caused a strong but reversibleeffect on tumor vasculature; all perfusion-related parameters—D�, f, fD�, and Ktrans

—initially showed a decrease of 30% to60% at 2 hours and then fully recovered to baseline on day 2 for

CA4P treatment or on days 4 to 8 for CA4P þ bevacizumabtreatment; the diffusion coefficient in tumors decreased initiallyat 2 hours and then increased from day 2 to day 8. We observeda good correlation between IVIM parameters and dynamiccontrast-enhanced MRI (DCE-MRI; Ktrans). We also found thatthe relative change in f and fD� at 2 hours correlated well withchanges in tumor volume on day 8. In conclusion, our resultssuggest that IVIM is a promising alternative to DCE-MRI for theassessment of the change in tumor perfusion as a result ofantivascular agents and can be used to predict the efficacy ofantivascular therapies without the need for contrast mediainjection. Cancer Res; 77(13); 3491–501. �2017 AACR.

IntroductionDespite recent advances in treatment, advanced non–small cell

lung cancer (NSCLC) remains one of the most lethal cancers. Theefficacyof currently available chemotherapy for advancedNSCLC,including platinum-based doublet, crizotinib, gefitinib, erlotinib,afatinib, bevacizumab, and pemetrexed, is still limited (1), sug-gesting an urgent need to develop effective treatments that cansignificantly improve the overall survival rates. Vascular-disrupt-ing agents (VDA) are a class of anticancer agents that can selec-tively attack the tumor vasculature and cause catastrophic vascularshutdown, tumor ischemia, and necrosis (2, 3). Several VDAs,including combretastatin A4 phosphate (CA4P; ref. 4), are nowbeing investigated in phase II/III clinical trials for advanced

ovarian, NSCLC, and anaplastic thyroid cancer (5, 6). Differingfrom antiangiogenic agents, another major class of vascular-targeting therapeutics that act by inhibiting the formation of aneovasculature (angiogenesis), VDAs act on the existing tumorvasculature within the first several hours (up to 24 hours)after administration. However, this acute effect on tumor per-fusion is reversible, the survival tumor cells can quickly regainvascular perfusion, and tumors regrow soon after (4). Conse-quently, VDAs are often used in combination with othertherapies, including radiotherapy (7), hyperthermia (8), che-motherapy (9), and antiangiogenic therapy (10). Antiangio-genic agents directly or indirectly inhibit proangiogenic signal-ing pathways to completely or partially block the formation ofnew blood vessels (2, 11). For example, bevacizumab (Avas-tint), a recombinant humanized monoclonal antibody, inhibitsVEGF-A and is in clinical use for metastatic colorectal cancerand NSCLC (3, 11).

Noninvasive MRI plays an important role in the evaluation ofantivascular therapies (12). As antivascular therapymainly acts onthe tumor vasculature, a reduction in tumor size would occur atsome time after therapy administration. A number of MRI meth-ods have been used to evaluate tumor responses during the courseof antivascular therapies. For example, dynamic contrast-enhanced MRI (DCE-MRI) is widely used to assess tumor perfu-sion (10, 13); diffusion-weighted imaging (DWI) is used tomonitor the changes in tumor cellularity (14, 15); and 19F MRI(16) is used to map oxygenation by imaging lipid relaxationenhancement (MOBILE; ref. 17). Among these methods, DCE-MRI has been shown to have a superior ability to render thechanges in perfusion and permeability and is themost commonlyused MRI method in antivascular therapies (18, 19). However,there is a rising concern about Gd deposition (20, 21), which,

1Medical Imaging Center, The First Affiliated Hospital of Jinan University,Guangzhou, China. 2Department of Radiology, Panyu Central Hospital, Guangz-hou, China. 3F.M. Kirby Research Center for Functional Brain Imaging, KennedyKrieger Institute, Baltimore, Maryland. 4The Russell H. Morgan Department ofRadiology and Radiological Science, Johns Hopkins University School of Med-icine, Baltimore, Maryland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

C. Shi, D. Liu, and Z. Xiao contributed equally to this article.

Corresponding Authors: Liangping Luo, The First Affiliated Hospital of JinanUniversity, No. 613 West Huangpu Road, Guangzhou 510630, China. Phone:8602-0386-88425; Fax: 8602-0386-88000; E-mail: [email protected]; andHanwei Chen, Panyu Central Hospital, No. 8 Eest Fuyu Road, Guangzhou511486, China. Phone: 8602-0348-58891; Fax: 8602-0348-58888; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-16-2499

�2017 American Association for Cancer Research.

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although rare, prevents DCE-MRI from being used frequently andrepetitively for clinical applications.

We hypothesized that intravoxel incoherent motion (IVIM)DWI can be used as a non–contrast-based alternative to tradi-tional DCE-MRI for assessing the tumor response to an antivas-cular therapy, which can be used repetitively in a short timeinterval. The IVIM method was originally developed by Le Bihanin 1986 using a biexponential model to separate the diffusion-weighted signal into a pure diffusion fraction and a perfusiondominated "pseudo-diffusion" fraction (22), permitting thesimultaneous assessment of tissue diffusion and microperfusion(22–24). Recently, in both preclinical and clinical studies, therehas been renewed interest in utilizing the perfusion-related para-meters of IVIM DWI, including the blood pseudo-diffusion coef-ficient (D�) and the perfusion fraction (f), to quantifymicrovesselperfusion in solid tumors (25–31). The IVIM method is consid-ered an attractive approach because it can assess microperfusionin the tumor without the need for exogenous contrast agents,making it possible to be used within a short time interval for theevaluation of therapeutic response, even in patients with renalinsufficiency, or in patients with contraindications to contrastagents (24, 32).

In the present study, we aimed to investigatewhether IVIMDWIcan be used as to characterize early changes in tumor perfusionand diffusion as a result of antivascular therapies, including aCA4P-based vascular-disrupting therapy and retreatment using acombination of CA4P and bevacizumab. Furthermore, we exam-ined whether IVIM parameters could be used to predict thetherapeutic responses of NSCLC tumors. As DCE-MRI is the mostwidely used MRI technology, we also studied the correlationbetween the perfusion-related IVIM-DWI parameters and DCE-MRI parameters. To the best of our knowledge, this is the firststudy to report on the use of IVIMDWI to evaluate the therapeuticeffects of antivascular therapies in an experimentalNSCLCmodel.

Materials and MethodsCell culture

The human lung carcinoma cell line A549 was obtained fromthe American Type Culture Collection (ATCC, lot# CCL-185,purchased 2015) and passaged less than 15 times prior toimplantation. The cells were cultured at 37�C in a 5% CO2

atmosphere in DMEM (Gibco), supplemented with 10% FBS,and 1% penicillin/streptomycin. Immediately prior to in vivo

implantation, the A549 cells were assessed by PCR to provideSTR profiles, fromwhich cell line authenticity was confirmed, andthe absence of mycoplasma was confirmed using an e-Myco PCRdetection kit (Boca Scientific).

Animal modelAll animal experiments were performed in accordance with

protocols approved by our institutional Animal Care and UseCommittee. Male BALB/c nude mice (4–5 weeks of age, bodyweight�20 g) were purchased from the laboratory animal centerof Sun Yat-senUniversity (Guangzhou, China) andmaintained ina specific pathogen-free environment. The mice were subcutane-ously injectedwith 2� 106 A549 cells in 0.2mL serum-freemediainto the left flank to develop NSCLC xenografts. Tumors wereallowed to grow for 14 days to reach a size of approximately 10mm in the longest dimension. This tumor size was sufficient forthe development of relatively high vascularity, allowing MRIassessment of the effect of antivascular therapies. Tumor volumeswere assessed using an MRI-based volumetric measurementmethod. In brief, for each animal, observer-defined regions ofinterest (ROI) were placed on the tumor throughout a series ofaxial fast Spin Echo (FSE) T2w images, and the number of pixels ineach ROI was counted. The tumor volume was then calculated bythe multiplication of the sum of pixel number and voxel volume(defined by the product of pixel area and slice thickness).

Antivascular treatmentA total of 57 mice with NSCLC xenograft tumors were ran-

domly allocated to three groups: groupA received saline as control(n¼ 21); group B received CA4P treatment (n¼ 18); and group Creceived CA4P plus bevacizumab treatment (n ¼ 18). Saline,CA4P (100 mg/kg; OXiGENE, Inc.), or bevacizumab (5 mg/kg,the Avastin, Roche) were administrated via tail vein injectionimmediately after baseline MRI scans. In the group C (CA4P þbevacizumab), the CA4P was administered 1 hour after thebevacizumab on the first day (33). Then bevacizumab wasadministrated daily. The schedule for treatment, MRI, and histo-logic assessments is shown in Fig. 1. In brief, MRI was conductedbefore treatment, and 2 hours, 2 days, 4 days, and 8 days after thetreatment of CA4P or vehicle. Six mice from each group wererandomly selected for longitudinal MRI assessment from day 0 today 8. At each time point, 3 mice from the non-MRI subgroup ineach group were randomly selected and sacrificed for histologic

Figure 1.

Schematic diagram of the design of theanimal experiment. At each time point,threemicewere sacrificed for histologic analysis.Throughout the study, six mice were monitoredlongitudinally for the inhibition of tumorgrowth and changes in MRI parameters.

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analysis. Threemice were randomly selected from a control groupas the baseline for histologic analyses.

MRI acquisitionAll MRI scans were conducted using a 1.5-T Signa HDxt super-

conductor clinical MR system (GE Medical System) equippedwith a human eight-channel wrist coil. Animals were anesthetizedby intraperitoneal injection of 0.3% pentobarbital and imaged inthe supine position. Transverse T2-weighted imageswere acquiredusing an FSE sequence [TR¼ 2,280 ms, TE¼ 77.6 ms, matrix size¼ 256� 192, field of view (FOV)¼ 7.0� 5.6 cm2, slice thickness¼ 2.0 mm, slice gap ¼ 0.2 mm, and NEX ¼ 2]. DCE-MRI wasacquired using a 3D fast spoiled gradient-recalled echo (SPGR)sequence (TR ¼ 40 ms, TE ¼ 2.4 ms, flip angle ¼ 35�, slicethickness¼ 2.0 mm, slice gap¼ 0.2 mm, matrix size¼ 128� 96,andFOV¼7.0�5.6 cm2).A total of 35T1w imageswere obtainedat a temporal resolution of 3 seconds before and after the injectionof Gd-DOTA (Magnevist, Bayer Schering Pharma, 0.1 mmol/kgbody weight followed by 0.3 mL of a 0.9% saline flush). Thebaseline T1 map was assessed using a variable flip angle T1mapping method, with the flip angle ¼ 3�, 6�, 9�, 12�, and15�. IVIM-DWI MRI was acquired using a free-breathing, sin-gle-shot, echo-planar imaging pulse sequence (TR ¼ 4,000 ms,TE ¼ 91.8 ms, slice thickness ¼ 2.0 mm, slice gap ¼ 0.2 mm,matrix size ¼ 128 � 96, and FOV ¼ 10 � 7 cm2) with diffusiongradients applied in three orthogonal directions (13 b values ¼0, 25, 50, 75,100, 150, 200, 400, 600, 800, 1,000, 1,200,1,500 s/mm2). The chemical shift-selective saturation techniquewas used for fat suppression.

Image analysisAfter eachMRI acquisition session, MR images were transferred

to a dedicated post-processing workstation (AW4.5, GE Health-care) for quantitative analysis. The IVIM-DWI data were analyzedby the Functool MADC program, using a biexponential modeldefined by

SI=SI0 ¼ ð1� f Þ � exp ð�bDÞ þ f � exp ð�bD�Þ

where SI0 is themean signal intensity of the ROI for a b value of 0,and SI is the signal intensity for higher b values. D represents thetrue diffusion coefficient, D� is the pseudo-diffusion coefficient,and f is the perfusion fraction. ROIs were manually drawn byoutlining the tumor border on the T2w image that showed thelargest cross-sectional tumor area.

DCE-MRI data were analyzed using the Cinetools program andthe Tofts model, and the volume transfer coefficient (Ktrans) wascalculated. A total of 90 IVIM-DWI and90DCE-MRI datasets from18 mice, each at 5 different time points (i.e., baseline, 2 hours,

2 days, 4 days, and 8 days), were analyzed by a radiologist with6-year experience in MRI.

Histologic analysisExcised tumors werefixed in 4%paraformaldehyde, embedded

in paraffin, sliced into sections of 5 mm thickness, and stainedusing a hematoxylin and eosin stain. TUNEL staining was per-formed to assess apoptotic cells using an in situ Cell DeathDetectionkit (RocheDiagnostics) according to themanufacturer'sinstruction. Ki67 staining was performed to assess cell prolifer-ation using an anti-Ki67 antibody (1:200; Santa Cruz Biotech-nology). HIF1a staining was performed to assess tumor hypoxiausing a monoclonal anti–HIF-1a antibody (1:25; Novus Biolo-gicals). Finally, CD31 staining was performed to measure micro-vascular density (MVD) using an anti-CD31 antibody (1:200; BDBiosciences). For TUNEL, Ki67, HIF1a, and CD31 analyses, tenfields per section were randomly selected and analyzed. TheMVDof the tumorwas evaluated using the "hot spot"methoddescribedby Weidner and colleagues (34). In brief, the three most vascu-larized areas on each section were selected as the hot spots underlow magnification (�40), and vessels were manually counted onthe images at high magnification (�200). The MVD was calcu-lated as the mean of three hot spot areas. The TUNEL, Ki67,HIF1a, and CD31 were expressed as the ratio (%) of positive cellsto all tumor cells using Image-Pro Plus 6.0 software (MediaCybernetics).

Statistical analysisAll numeric data were presented as themean� SD. The normal

distribution of the acquired data was evaluated using the Kolmo-gorov–Smirnov test. The computed IVIM-DWI and DCE-MRIparameters in all three groups were compared using a one-wayANOVA with least significant difference (LSD) as a post hoc test.Pearson correlation analysis was used to assess the correlationsbetween IVIM-DWI and DCE-MRI parameters at each time point,and the correlations between the relative changes in IVIM-DWIparameters at early time points (i.e., 2 hours), as well as treatmentresponses quantified by the change in tumor volume on day 8. P <0.05was considered statistically significant. All statistical analyseswere performed using the SPSS software package (Version 19.0,SPSS Inc.).

ResultsThe effects of antivascular therapies on tumor growth

Our studies showed that CA4P treatment resulted in a strikinginhibitory effect on tumor growth as early as 4 days after thetreatment. As shown in Fig. 2A, treatment groups (both CA4P andCA4P þ bevacizumab) exhibited a much slower increase rate in

Figure 2.

The therapeutic effects of CA4P alone and CA4P þbevacizumab (BV) on tumor growth. A, Tumor growthcurve quantified by tumor volumes at different time pointsafter the treatment. B, Comparison of the relative change(%) in the tumor volume in each group on the final day (day8) of the study. Comparisons among the three groups wereperformed using ANOVA with post hoc LSD tests(n ¼ 6; �� , P < 0.001). Error bars, SEM.

IVIM MRI of Tumor Response to Antivascular Therapies

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tumor volumes compared with the control group. As shownin Fig. 2B, the relative increase in tumor volume on day 8 fortheCA4P andCA4Pþbevacizumab treatment groupswere 95.3%� 13.5% and 73.2% � 22.4%, respectively. In comparison, thetumor volumes in the control group increased for more than 2-fold, i.e., 201.4%� 31.1%. The doubling time for tumor volumeswas estimated using a single exponential growth equation to be5.5 days (control), 8.6 days (CA4P), and 11.3 day (CA4P þbevacizumab), which is consistent with previous findings (33).An ANOVA with post hoc LSD tests revealed significant differencesbetween the tumor sizes of the treatment groups and that of thecontrol groups (all P values were less than 0.001), but notbetween the two treatment groups (P > 0.05, Fig. 2B). However,no group showed a decrease in tumor size, indicating that asingle injection of CA4P or CA4P þ bevacizumab, while inhibit-ing tumor growth effectively, cannot eradicate the tumorcompletely.

Histologic analysesTo evaluate the effect of antivascular treatments on NSCLC

A549 tumors, we performed CD31, Ki67, TUNEL, and HIF1a atdifferent time points after treatment with CA4P or CA4P þbevacizumab to assess MVD, cell proliferation, cell apoptosis,and tumor hypoxia, respectively. Representative immunohisto-chemical sections of CD31, Ki67, TUNEL, and HIF1a staining intheCA4Pþbevacizumabgroupat different timepoints are shownin Fig. 3A, and the quantitative analyses for each staining areshown in Fig. 3B–E. CD31 staining showed a sharp drop at 2hours after the treatment in the two treatment groups, which wasattributed to the vessel-disrupting effects of CA4P. This wasfollowed by a gradual recovery after day 2. Moreover, CD31staining also revealed a markedly lower MVD in the CA4P þbevacizumab treatment group than in the CA4P treatment group.Ki67 staining showed distinctive patterns of changes in cell

proliferation for the different groups: the control group showeda slow but continuous increase in Ki67 expression, indicatinggrowth and proliferation of tumor cells; the CA4P treatmentgroup demonstrated a negligible change in Ki67 expressionthroughout the experiment, indicating tumor cells were in anonproliferating stage due to the inhibition of CA4P on tumorgrowth. In contrast, the CA4P þ bevacizumab treatment groupshowed a substantial decrease in Ki67 expression as early as 2hours after the treatment, indicating a much higher therapeuticeffect on the proliferation of tumor cells. TUNEL staining showedthat the maximal degree of cell apoptosis occurred on day 2 aftertreatment. HIF1a staining revealed a noticeable drop in tumoroxygenation, which wasmost profound at 2 hours after treatmentin both treatment groups, and this was maintained at a higherlevel in the CA4P þ bevacizumab group but oscillated greatly inthe CA4P group.

The effects of antivascular therapies on tumor perfusion asrevealed by both DCE-MRI and IVIM methods

To evaluate the feasibility of using IVIM-DWI as a noninvasivemeans by which to assess tumor responses to antivascular ther-apies,weperformed longitudinal IVIM-DWIMRIonNSCLCA549tumors in the control group, the CA4P group, and the CA4P þbevacizumab group for up to 8 days (Fig. 4). We also used DCE-MRI as a standardmethod with which to validate the quantitativemeasurement of tumor perfusion using the IVIM-DWI method.The longitudinal measurements from IVIM-DWI and DCE-MRIparameters in each group at each time point are shown in Fig. 5,and the relative changes in each parameter in the three groups aresummarized in Table 1. In the control group (n ¼ 6), both IVIM-DWI and DCE-MRI measurements showed good intragroupreproducibility, i.e., the CVs of D� and f for the three groupswere 15.9%, 16.8%, and 14.8%, respectively, and the CV of Ktrans

was 9.8%.

Figure 3.

Immunohistochemistry of tumors at different tine points after treatments. A, Representative H&E staining (�20) and IHC images of CD31, HIF1a, Ki67, and TUNELstaining (�200) of CA4P þ bevacizumab (BV)–treated tumors before and at different time points after the treatment. The longitudinal assessmentof CD31 (B), Ki67 (C), TUNEL (D), and HIF1a (E) of tumors in the control group, CA4P treatment group, and CA4P þ bevacizumab treatment group atdifferent time points.

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Acatastrophic effect ofCA4P(withorwithoutbevacizumab)onthe tumor perfusion was revealed by the perfusion-related IVIMparameters, including D�, f, and fD� (Fig. 5A–C and Table 1). Forthe CA4P treatment group, the average D�, f, and fD� were firstdecreased dramatically to 40.8%, 35.5%, and 61.6% of theirbaseline values, respectively, at 2 hours after the treatment. Thiseffect was then reversed slowly and, as early as day 2, no significantdifference couldbeobserved in theseperfusion-relatedparametersbetween the CA4P-treated groups and the control group (P >0.05). On day 8, only a net decrease of 10.7%, 18.1%, and 27.3%was observed for D�, f, and fD�, respectively, compared with thebaseline values. In contrast, the decrease in perfusion was much

stronger and longer in the CA4Pþ bevacizumab treatment group.While all the perfusion-related parameters were also reversedslowly on day 2, they were still significantly lower than those ofthe control group and the CA4P treatment group (P < 0.05). Thedifference in the perfusion-related parameters between the CA4Pgroups and the other groups became insignificant after day 4.

As shown in Fig. 5D, a similar trend in the tumor perfusioncould be seen using DCE-MRI. For example, the Ktrans in thetreated tumors decreased significantly by 43.5% and 55.3% in theCA4P and CA4P þ bevacizumab groups, respectively, at 2 hoursafter the treatment. The decrease in Ktrans was then reversedgradually. Compared with the control group, both the

Figure 4.

T1 weighting image and parametric maps (D, D� , f, fD, and Ktrans) of a representative CA4P þ bevacizumab–treated tumor before and at different timepoints after the treatment.






CA4P-treated and (CA4Pþ bevacizumab)-treated groups showeda significantly different Ktrans till day 2 (P ¼ 0.003 and P < 0.001,respectively). However, no significant difference in Ktrans wasobserved after day 4, except on day 8, where a significantly highKtrans in the CA4P groups was observed. On day 8, the averageKtrans in the CA4P þ bevacizumab group was still 5% lower than

their baseline value, indicating a great synergy between thevascular-disrupting effect of CA4P and the inhibitive effect onthe recovery of collapsed tumor vessels and formation of neo-vasculature by bevacizumab.

We also assessed the changes in the diffusion parameter, D, inthe IVIMmodel, in the treatment groups. As shown in Fig. 4E, the

Figure 5.

Longitudinal changes of IVIM parameters D� (A), f (B), fD�(C), and D (E), and the DCE-MRI parameter Ktrans (D) in the control, CA4P, and CA4P þ bevacizumab(BV) groups, respectively, before and after treatment. Data points are shown in mean � SD.

Table 1. Relative changes (%) in IVIM-DWI and DCE-MRI parameters of NSCLC A549 tumors in the control group, CA4P group, and CA4P þ bevacizumab groupbefore and after treatment

Group A Group B Group C Pa

Parameters Time Control CA4P CA4P þ BV B vs. A C vs. A B vs. C

2 h �7.2 (�22.4–2.9) �39.7 (�60.2 to �26.3) �40.8 (�66.2 to �31.1) <0.001 <0.001 NSDDa (%) 2 d 2.9 (�10.8–23.7) �4.8 (18.1–24.1) �18.7 (�24.9–24.1) NS 0.009 NS

4 d �4.3 (�42.1–22.5) �5.2 (�26.7–29.9) �20.1 (�28.2–0.3) NS NS NS8 d 6.0 (�37.2–32.0) �4.4 (�23.5–21.3) �10.7 (�26.6–14.8) NS NS NS

Df (%) 2 h �9.7 (17.3–�1.3) �28.4 (�32.3 to �24.7) �35.5 (�44.1 to �20.7) <0.001 <0.001 NS2 d �9.9 (�17.2–3.43) �9.6 (�17.3 to �0.5) �24.2 (�29.4 to �19.0) NS 0.001 0.0014 d �10.1 (�28.5 to �1.4) �20.4 (�30.6 to �5.6) �26.7 (�38.9 to �19.4) NS 0.007 NS8 d �13.2 (�36.9–2.2) �13.2 (�24.8 to �5.2) �18.1 (�30.4 to �7.7) NS NS NS

D(fDa) (%) 2 h �16.3 (�30.5–1.3) �56.7 (�71.0 to �46.1) �61.6 (�81.0 to �46.8) <0.001 <0.001 NS2 d �7.1 (�19.1 to �18.8) �14.7 (�23.3–2.5) �38.3 (�45.6 to �25.3) NS <0.001 0.0024 d �13.0 (�59.1–19.1) �24.7 (�43.8–2.5) �41.2 (�54.8 to �29.1) NS 0.024 NS8 d �7.5 (�45.1–21.3) �16.8 (�33.9–11.1) �27.3 (�36.4 to �5.9) NS NS NS

D Ktrans(%) 2 h �7.5 (�14.6 to �1.0) �43.5 (�61.0 to �30.2) �55.3 (�78.1 to �39.3) <0.001 <0.001 NS2 d �8.3 (18.2–5.2) �27.5 (�39.9 to �16.8) �32.8 (�46.7 to �21.3) 0.003 <0.001 NS4 d �8.9 (�21.5–3.9) �7.7 (�21.8–8.2) �16.6 (�35.6 to �6.7) NS NS NS8 d 2.8 (�13.6–10.3) 30.1 (3.4–51.9) �5.2 (�21.1–0.7) 0.001 NS <0.001

NOTE: Data were means with ranges in parentheses.Abbreviations: BV, bevacizumab. NS, nonsignificant.aANOVA with LSD tests post hocwas used to determine the differences of the parameters among the three groups. A significance threshold of P < 0.05 was used.

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Dvalues in both groups B andCwere slightly decreased at 2 hours(P ¼ 0.009 for group B and P ¼ 0.007 for group C), and thenmarkedly increased after day 2 (P ¼ 0.004 for group B and P <0.001 for group C) and remained at a high level on day 8 (P ¼0.131 for group B and P¼ 0.005 for group C) compared with thecontrol group. Interestingly, although the differences were notstatistically significant, the average increases in D in the CA4P þbevacizumab treatment group were always higher than those inthe CA4P group at each time point.

Heterogeneous distribution can be seen among all metrics inthe obtained MRI maps. Therefore, we also investigated thechanges on these parameters in the rim and core of each tumorusing a simple segmentation method as shown in Supplemen-tary Fig. S1. As expected, the pronounced changes in diffusionparameters occurred in the tumor core (SupplementaryFig. S2), whereas all the perfusion-related parameters showedmuch stronger changes in the tumor rim (SupplementaryFigs. S3–S6).

Correlations between IVIM parameters and DCE-MRIparameter or tumor responses

To validate the usefulness of IVIM-DWI parameters in moni-toring the changes in tumor perfusion, we performed analyses tostudy of the correlation between the perfusion measured usingIVIM and that by DCE-MRI. As shown in Fig. 6, moderate butsignificant correlations were observed between the three perfu-sion-related IVIM parameters and Ktrans: D�: r ¼ 0.6934 (P <0.0001), f: r ¼ 0.6453 (P < 0.0001), and fD�: r ¼ 0.7127 (P <0.0001), respectively.Moreover, the analyses based on segmentedtumor regions (Supplementary Figs. S4–S6) clearly showed animproved correlation between the changes in the perfusion-relat-ed IVIMmetrics withKtransmeasured usingDCE-MRI in the tumorrim than those in the whole tumor.

To investigate the usefulness of IVIM-DWI parameters in earlypredicting the tumor response to an antivascular treatment, wealso carried out analyses to study the correlation between theIVIM parameters at early time points and the tumor responses,

Figure 6.

Correlations of IVIM DWIwith DCE-MRI and tumor responses.A, The correlation between the Ktrans measured using DCE-MRI and perfusion-related IVIM parametersD� (A), f (B), and fD� (C), respectively. D, The correlation between the relative changes in tumor volume on day 8 and the relative perfusion-related IVIMparameter fD� at 2 hours. E, The correlation between fD� at 2 hours and the relative changes of immnunohistochemical markers in the course of the study. F, Thecorrelation between the relative changes in tumor volume on day 8 and the relative changes of the diffusion-related IVIM parameter D on day 2. G, Thecorrelation between D on day 2 and the relative changes of immnunohistochemical markers in the course of the study.






quantified by the changes in tumor volume, on day 8. As shownin Fig. 6D, the changes in the perfusion-related parameter fD� at 2hours after treatment showed statistically significant correlationswith the relative changes in tumor volume on day 8, with aPearson correlation coefficient of 0.8663 (P ¼ 0.0003). Whencompared with the changes of all four immunohistochemicalmarkers in the time course of the study (Fig. 6E), fD� at 2 hoursafter the treatment candefinitively separate both treatment groupsfrom the control group, reflecting the changes in tissue perfusion.It should be noted that the measured perfusion changes in thetumor rimhadan improved correlationwith the changes in tumorvolume (i.e., r ¼ 0.93, P < 0.0001, Supplementary Fig. S7G),indicating that further improvements can be achieved usingappropriate image segmentation methods to take the tumorheterogeneity into account.

As shown in Fig. 6F, the changes in the diffusion parameter Donday 2 also had a strong negative correlationwith the changes intumor volume on day 8, with r ¼ �0.8853 (P ¼ 0.0001). Figure6G shows that D on day 2 can effectively separate the CA4P þbevacizumab group from the control group in a similar way ofimmunohistochemical markers. However, D alone cannot beused to discriminate CA4P group and control group effectively.It should be noted that the measured diffusion changes in thetumor core had an improved correlation with the changes intumor volume (i.e., r¼�0.8592, P¼ 0.0003, Supplementary Fig.S7H), indicating that, again, further improvements can beachieved using appropriate image segmentation methods to takethe tumor heterogeneity into account.

DiscussionThe purpose of the present study was to investigate the

feasibility of using IVIM to assess the changes in tumor perfu-sion and diffusion in response to antivascular therapies. Itshould be noted that the current study used an A549 NSCLCmouse model. It is because that, even orthotopic models suchas Lewis lung adenocarcinoma (3LL) can be used, xenografts ofhuman tumor cells such as A549 and H460 indeed have beenused more commonly to investigate vascular disruption inNSCLC xenografts in preclinical studies (35, 36), which haveled to a number of clinical trials. Moreover, as exemplified byseveral recent clinical studies (37, 38), the IVIM technique usedin the current study indeed is ready for imaging lung cancers inclinical settings. We used the most widely studied vascular-disrupting agent CA4P that would result in a significant inhi-bition of tumor growth (4, 7–9). Our result was in a goodagreement with previous studies that, when combined with theantiangiogenic agent bevacizumab, the antitumor effect can bemuch stronger than the CA4P alone, by the synergy of thevascular-disrupting effect and inhibition of new vessels (10,33). The change in tumor perfusion was confirmed by DCE-MRI, a tracer-based MRI method that is being widely used inantivascular therapies as an imaging biomarker (13, 18). Theinhibition of tumor growth correlated well with the strongdecrease in tumor perfusion at 2 hours after treatment. Thechanges in the tumor vasculature and those on a cellular levelwere confirmed by immunohistochemistry. Using this well-established and validated animal model, we showed that IVIMis capable of assessing the changes in both tumor perfusion anddiffusion in response to antivascular treatments, without theneed for contrast media injection.

The biexponential IVIM model can be used to estimate perfu-sion-related parameters, including the blood pseudo-diffusioncoefficient (D�), indicative of blood flow, and the perfusionfraction (f), indicative of the fractional volumeof active capillariesin the tumor, by fitting to fit DWI data acquired at multiple bvalues (22), which provides a simple and practical way to quan-titatively assess the perfusion in small vessels and capillaries,namely microperfusion (23–31). In contrast, DCE-MRI measuresclassic perfusion, which is the pharmacokinetics of the injectedtracer, and the volume transfer constant, Ktrans, represents tissueperfusion and permeability (39). Therefore, the perfusion studiedby IVIMand that byDCE-MRI are intrinsically different,which canexplain the discrepancy reported in some studies (40, 41). How-ever, many other studies have shown that there is, indeed, a goodcorrelation between IVIM parameters and the Ktrans measured byDCE-MRI (26). Our results also suggested that IVIM parameterswere well correlated with DCE-MRI with Pearson correlationcoefficients on the order of 0.6–0.7. Among the parametersstudied, fD� showed the strongest correlation (r ¼ 0.7127).Because tumor perfusion assessed byDCE-MRI is themost widelyused biomarker in antivascular therapies (13, 39), our currentstudy implies that IVIM DWI, as a non–contrast-enhanced altera-tive to DCE-MRI, is a promising approach for monitoring thetumor response to antivascular therapies, and can be used repet-itively at short time intervals, without concerns for Gd depositionsafety, which is particularly helpful to patients with impairedrenal function (6).

It should be noted that there was a non-negligible hetero-geneous spatial distribution in all MRI metrics (Figs. 4 and 5;and Table 1). The nature of this heterogeneity is the tumorheterogeneity within an individual tumor and the response ofdifferent parts to an antivascular treatment. Therefore, our MRImetrics should be considered as a gross assessment for thechanges in tissue perfusion and diffusion globally. Not only themean values but also standard deviations should be consideredin order to interpret the data accurately. Ideally, a carefulmechanistic validation using a high-resolution small animalMRI scanner can guarantee the spatial correlation between thepathologic changes and MRI findings, which is however beyondthe scale of current study.

As early as 2 hours after the administration of CA4P, allperfusion-related parameters, including D�, f, fD�, and Ktrans,showed a sharp decrease of about 30% to 60%. The destructionof the tumor vasculature was confirmed by immunohistochem-icalfindings usingCD31 staining. A strong hypoxia due to the lossof blood supply was also confirmed byHIF1a staining. As a resultof the shutdown of blood perfusion, tumor cells began to developapoptosis and the portion of proliferating cells decreased. Boththe measures of IVIMDWI and DCE-MRI could reflect the changein perfusion, indicative of the acute effect of CA4P. There was nosignificant difference in the perfusion parameters between theCA4P group and CA4P þ bevacizumab group, indicating theeffect of CA4P is dominant, whereas the effect bevacizumab isnegligible in the early stage. In this stage, D obtained from IVIMmodel strongly decreased, likely attributable to the decrease ofextracellular space (reduced overall water diffusivity) as a result ofischemic swelling cells (31), which is consistent with our histo-logic findings and in good agreement with previous studies onrodentmodels of rhabdomyosarcoma (42) and liver tumors (31).

On day 2, both immunohistochemistry (CD31 and HIF1astaining) and MRI results showed the reversion of tumor

Shi et al.





perfusion. There was no significant difference in IVIM parameters(D�, f, and fD�) between the CA4P treatment group and thecontrol group, whereas there was a significant difference in Ktrans

between them. This observation can be explained by either therecovery of collapsed capillaries or the development of newcapillaries, which led to an increase in microperfusion, but notclassic perfusion, as large vessels were still not fully opened. Incontrast to the CA4P treatment alone, the differences in the IVIMparameters (D�, f, and fD�) between the CA4P þ bevacizumabtreatment group and the control group were still significant,confirming that the function of bevacizumab was to inhibit theformation of new blood vessels (11). Interestingly, only IVIMparameters, but not Ktrans, showed a significant differencebetween the CA4P and CA4Pþ bevacizumab groups. At this timepoint, histologic analysis showed a recovered CD31 and HIF1aexpression, the greatest degree of cell apoptosis (TUNEL staining),and strongly inhibited cell proliferation (Ki67 staining). All theimmunohistochemical analyses showed a noticeable differencebetween the two treatment groups, suggesting a significant dif-ference on the cellular level. The IVIMmeasurement also revealeda markedly increased D caused by cell death (both necrosis andapoptosis), edema, and further tumor repopulation (2, 5, 31,42, 43). For example, Loveless and colleagues reported thatdiffusion parameters but not DCE-MRI parameter Ktrans servedas a better biomarker for antiangiogenic therapies (44). Our resultalso proved the strong correlation between the diffusion param-eter Dwith the changes in tumor volume on day 8, suggesting thatthe D diffusion parameter is a complementary biomarker to theperfusion parameters.

After day 4, both immunohistochemistry and MRI resultsshowed the regrowth of blood vessels and the recovery of bloodflow in the treatment groups. Among all the perfusion-relatedparameters, only the relative changes in f (and fD�) in the CA4Pþbevacizumab group showed a significant difference comparedwith the control group. Higher D values were still observed in thetwo treatment groups, which is consistent with the TUNEL stain-ing and Ki67 staining.

The present study suggests that fD� (both relative change andabsolute value) at 2 hours can be used as early predictors of theefficacy of antivascular treatments. This finding is consistent withprevious studies on the antiangiogenic agent, sorafenib (45), andthe vascular disrupting agent, CKD-516 (31), on liver cancers.Ourstudy also indicated that the relative changes in f and fD� on day 2could predict tumor resistance to vascular-disrupting agents.When combined with antiangiogenic agents, such as bevacizu-mab, the antivascular effects of CA4P were greatly augmented,which is in good agreement with previous reports (33, 46). In ourstudy, only the relative changes in f and fD� on day 2 couldeffectively predict the different efficacies of CA4P and CA4P þbevacizumab treatment.

Moreover, our studies demonstrated that the early changes inmicroperfusion (fD� at 2 hours) and diffusion (D on day 2)measured by IVIM DWI correlated well with immunohisto-

chemical changes. Specifically, Fig. 6E and G shows that fD�(2hours) and D (2 days) correlated negatively with CD31 andKi67 but positively with TUNNEL and HIF1a, which are con-sistent with the previously reports (31, 42, 44, 47). It is wellknown that D and fD� reflect different aspects of the tumorresponse to antivascular treatments, which are complementaryto each other: D (2 days) represents the early tumor responsesat cellular level on the first 2 days (25, 47) and fD� (2 hours)represents the acute tumor vascular responses in first 2 hours.Compared with D (2 days), fD� (2 hours) of control group andthose of treatment groups were more separable, indicatingtreatments caused much stronger effects on tumor microperfu-sion (i.e., MVD and microcirculation) than on diffusion char-acteristics (tissue cellularity, extracellular space tortuosity, andcell membrane integrity) in the early stage (47). Nevertheless, D(2 days) and fD� (2 hours) might be used combinedly for theearly and accurate prediction of the tumor response to anti-vascular treatments.

In conclusion, our results suggest that IVIMDWI is a promisingalternative to DCE-MRI for assessing the change in tumor perfu-sion by antivascular agents. In addition, the diffusion parametermeasured using IVIM DWI provides insights into the changes intumor cellularity, which is complementary to perfusion measure-ments, thus providing a better understanding of tumor responseto antivascular treatment. The relative changes in f and fD� at earlytime points can be used to predict the efficacy of antivasculartherapies without the need for contrast media injection.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: C. Shi, Z. Xiao, H. Chen, L. LuoDevelopment of methodology: H. Chen, L. LuoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Liu, Z. Xiao, D. Zhang, H. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Shi, D. Liu, D. Zhang, G. Liu, G. LiuWriting, review, and/or revision of the manuscript: Z. Xiao, D. Zhang, G. Liu,G. Liu, H. Chen, L. LuoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D. Zhang, L. LuoStudy supervision: H. Chen, L. Luo

Grant SupportC. Shi, Z. Xiao, and L. Luo received Science and Technology Planning

Project of Guangdong Province, China (2014A020212673) and CharacteristicInnovative Project of Guangdong Province Department of Education(2014KTSCX021).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 17, 2016; revised February 15, 2017; accepted May 3,2017; published OnlineFirst May 9, 2017.

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2017;77:3491-3501. Published OnlineFirst May 9, 2017.Cancer Res Changzheng Shi, Dexiang Liu, Zeyu Xiao, et al. Non-Contrast Intravoxel Incoherent Motion Diffusion-Weighted MRIMonitoring Tumor Response to Antivascular Therapy Using

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