Quantitative Evaluation Of Tumor Angiogenesis And CA4P Early Efficacy With Ultrasound Molecular Imaging

Tomato Lectin-decorated microbubbles,a contrast agent for molecular ultrasound imaging of tumor vasculature.Bacground and Objective Tumor neovasculature is characterized by immature disorganized tortuous blood vessels,where blood flow is non-uniform and slow.Traditionally,vascular endothelium can be observed histologically,in thin sections,by using Lycopersicon Esculentum(Tomato)Lectin,labeled with a fluorescent dye.We may thus expect that lectincarrying microbubbles(lectin-MB)may attach to endothelial cells in the vasculature.We hypothesize that adhesion and retention of lectin-MB ultrasound contrast in the tumor vasculature may be more efficient than in the normal vasculature,due to slower blood flow and lower shear associated with the tumor vasculature;therefore,specific accumulation of this contrast agent in the tumor vasculature,but not in the normal vessels,could be achieved and applied for tumor imaging.The aim of this study is to test this hypothesis in vivo by performing contrast ultrasound imaging in a murine tumor model.Methods Biotinylated microbubbles were prepared from a micellar dispersion of distearoyl phosphatidylcholine(DSPC),PEG stearate and DSPE-PEG-Biotin(2:2:0.1 mass ratio)in normal saline.Perfluorobutane gas was dispersed in this aqueous micellar phase with a probetype sonicator,to generate biotinylated microbubbles,stabilized with a lipid monolayer shell.Free unincorporated lipid was removed from microbubbles by repeated centrifugal flotation wash(~50g,10 min).Biotinylated Texas Red labeled Lycopersicon Esculentum(Tomato)Lectin(Vector Laboratories,Burlingame,CA)was attached to the surface of MBs via a streptavidin spacer.Control microbubbles were carrying biotinylated nonspecific rat Ig G2(Ig G-MB).Both targeted and control microbubble formulations were subjected to additional flotation to remove particles > 4 um size,to minimize nonspecific retention.The attachment and detachment of Lectin-MB and Ig G-MB to the Mouse lung microvessel endothelial cell were assessed in a parallel-plate flow chamber.Lectin-MB drawn through the flow chamber seeded with endothelial cell at different shear stress.Mice(n = 5)received subcutaneous injections of 500,000 MC38 cells(murine colon adenocarcinoma,a generous gift of Dr.J Schlom,NCI)in the hind leg,and imaging was performed once tumor size reached ~1 cm.Intravenous injections of 107 Lectin-MB and control Ig G-MB were made in random order.Ultrasound imaging was performed with 15L8 probe of Siemens/Acuson Sequoia c512 system in contrast pulse sequencing CPS mode(7 MHz,MI 0.2),and video clips recorded for 10 min following iv bolus of MB.On the extracted image frames,regions of interest(tumor and control contralateral leg muscle)were drawn and mean signal intensity quantified with Image J(NIH).At 10 min time point microbubbles within the beam elevation were destroyed by a short(2-sec)higher-intensity ultrasound pulse,so the level of residual circulating bubbles could be determined.Net signal from targeted bubbles was then determined by subtraction of the signal intensity before and after microbubble destruction.This signal was normalized by the ×microbubble contrast signal at peak time(~30 sec following iv),to compensate the results for the slight variation of injected contrast dose.Results Compared the Ig G-MB,static adhersion experiment showed that more Lectin-MB attached to endothelial cell(number of adherent microbubble per cell is 8.11 ± 0.55,versus 0.18 ± 0.01).Concentration of Lectin-MB and the shear stress are the independent influence factor to efficacy of adherent microbubble The quantitative analysis of the ultrasound signals for Lectin-MB group captured at 10 min showed significant and specific contrast enhancement in the tumor.On the images,tumor area was clearly delineated.Lectin-MB signal in the tumor(relative signal intensity 31.52 ± 10.24)was much greater than in the normal muscle in contralateral leg(6.16 ± 3.31)(p < 0.01).Nonspecific control Ig G-MB accumulated in the tumor at a rather low level(5.91 ± 2.88)when compared with Lectin-MB(p < 0.01).There is no significant difference between Ig G-MB and Lectin-MB in normal tissue(p > 0.01)Conclusion We have observed specific and statistically significant accumulation of tomato lectin-MB in the tumor vasculature,when compared with normal contralateral leg muscle,or with control non-targeted MB,likely via efficient adhesion and retention of targeted bubbles in slow low-shear flow in the tumor vasculature.This may become a simple non-invasive tool to delineate tumors by ultrasound molecular imaging.Quantitative Evaluation of CA4 P Efficacy in a Tumor Model with Dynamic Contrast-Enhanced UltrasoundBacground and Objective Combretastatin A4 phosphate(CA4P)is a vascular disrupting agent that rapidly shuts down blood supply to tumors.Early monitoring of tumor perfusion plays a crucial role in determining the optimal strategy to managing treatment and guiding future therapy.The aim of this study was to investigate the potential value of dynamic contrast-enhanced ultrasound(CEUS)in quantitative evaluation of tumor perfusion at an early stage in CA4 P therapy.Method Central and peripheral perfusion of tumors was detected by CEUS pre-treatment(0 h)and 2,12 and 48 h after CA4 P injection.Two perfusion parameters,maximum intensity(IMAX)and time to peak(TTP),were calculated from the time–intensity curve.After CEUS,the efficacy of CA4 P was immediately confirmed by immunofluorescence assay and hematoxylin and eosin,Hoechst 33342 and fluorescein isothiocyanate–lectin staining.Result CEUS showed that in the center region of tumors,IMAX gradually decreased from 0 to 12 h and regrew at 48 h(p < 0.01).TTP increased only at 2 h.In the peripheral regions,IMAX did not change obviously from 0 to 12 h(p > 0.05)and just increased at 48 h(p < 0.01).The TTP of peripheral regions had the same tendency to vary tendency as that of center regions.In addition,microvascular density(MVD),vascular perfusion and necrotic area of the tumor were quantitatively analyzed.A close correlation between IMAX and MVD was observed in the center areas of tumors(r = 0.72,p < 0.01),whereas the correlation between IMAX and MVD in peripheral areas was weak(r = 0.37,p < 0.01).However,IMAX was positively correlated with tumor perfusion in both center and peripheral areas of tumors(r = 0.82,p < 0.01,and r = 0.63,p < 0.01,respectively).Consequently,IMAX was a reliable indicator of tumor perfusion evaluation by CEUS.Conclusion The use of CEUS to quantify tumor perfusion could a promising method for the early detection of tumor responses in anti-vascular treatment.