Quantitative Evaluation Of Uveal Melanoma Microcirculation Pattern Of Vasculogenic Mimicry With VEGFR1 Targeted Contrast-enhanced Ultrasound

Uveal melanoma(UM)is a malignant tumor originated from uveal melanocytes.At present,it has been found that the density and shape of microvessels in tumor tissue,especially the presence of vascular mimicry(VM),is also an important indicator for clinical judgment of disease and prognosis,but there is currently a lack of noninvasive detection and evaluation methods with good correlation and high specificity with microvascular state.The previous study defined the characteristics of the time-intensity curve of contrast enhanced ultrasound(CEUS)in the UM model,determined the specific qualitative and quantitative indexes for clinical diagnosis,and confirmed the correlation between the CEUS and the microcirculation state of the tumor.It was considered that the CEUS could quantitatively evaluate the blood supply of uveal melanoma.With the development of targeted CEUS technology,in order to enhance the CEUS imaging at the molecular level,targeted CEUS is considered to be introduced into the detection of VM in UM.It is confirmed that vascular endothelial growth factor receptor 1(VEGFR1)is related to the formation of VM of melanoma.Therefore,VEGFR1 target is selected,and the correlation between VM of UM and VEGFR1 is studied.After confirmation this correlation,VEGFR1 targeted ultrasound contrast agent is prepared,the imaging difference between non-targeted CEUS and VEGFR1 targeted CEUS is observed in the quantitative assessment of microcirculation in the model of MUM-2b melanoma,the imaging difference of VEGFR1 targeted CEUS is compared in the model of VEGFR1-ctrl and VEGFR1 knockdown((VEGFR1-KD)melanoma,and the relation of VM structure in histology and the targeted CEUS imaging is analyzed,in order to confirm the role and significance of VEGFR1 targeted CEUS in the detection of VM microcirculation and VEGFR1 molecular imaging of UM.PART 1 The establishment of the model of uveal melanoma and the study of microcirculation in the tumorObjective:To establish the UM model of human mum-2b cells line implanted in the eyes of mice,and to understand the correlation between the tumor and VM.Methods:The human UM mum-2b cell line was cultured and incubated,and implanted under the retina of nude mice.The model of mum-2b UM was established 2-3 weeks later.The microcirculation and vascular mimicry were observed by PAS / HE staining of frozen section under the microscope(×200).In vivo and in vitro immunofluorescence staining was used to observe the expression of VE cadherin and Eph A2,as well as the expression of MMP2 and MMP9,which were related with VM formation.Results:The model of UM with mum-2b cells implanted in the eyes of mice was established successfully.The tumor tissue is rich in VM,showing PAS positive loop structure with an average of 49.5 / visual field.In vivo and in vitro experiments showed that VE cadherin,Eph A2,MMP2 and MMP9,which were related to VM,were highly expressed.Conclusions:The UM model with mum2 b cells implanted in the eyes of mice is rich in VM structure microcirculation.PART 2 VEGFR1 in regulation of vasculogenic mimicry formation in uveal melanomaObjective:To investigate the involvement of VEGFR1 in vasculogenic mimicry formation in ocular uveal melanoma.Methods : The expression of VM related protein VEGFR1 was observed by immunofluorescence staining in vivo and in vitro of MUM-2b cell and UM model.The VEGFR1-sh RNA-carrying lentivirus was used to knockdown the VEGFR1 expression in MUM-2b cells(VEGFR1-KD).The expression of VE cadherin,Eph A2,MMP2,MMP9,and VEGFR1 were observed in both VEGFR1-ctrl and VEGFR1-KD in vitroby Western-Blot,the corresponding m RNA level was detected by q PCR.The ability of cells to form vessels like structure was measured by tube formation experiment in vitro.VM patterns were compared by frozen section between VEGFR1-ctrl and VEGFR1-KD uveal melanoma.Results:VEGFR1 was highly expressed in mum-2b cells and tumor models.In vitro experiments showed that VEGFR1 knockdown significantly reduced the expression of VE cadherin,Eph A2,MMP2,MMP9 and the corresponding m RNA level.VEGFR1 knockdown in the mum-2b cell line inhibited the formation of VM lumen in vitro,24 hours later,there were 6 / visual field in sh1 group,7.5 / visual field in sh3 group and 99 / visual field in control group(P < 0.001).VEGFR1 knockdown inhibited the formation and counting of VM network in the tumor model,and reduced the volume of tumor,the average diameter of VEGFR1-KD tumor was 0.61 ± 0.05 cm,compared with the control tumor(1.00 ± 0.07cm),P < 0.01.Conclusions:VEGFR1 was highly expressed in UM,and VEGFR1 knockdown could inhibit VM microcirculation formation and tumor volume.We supposed VEGFR1 could be a key point of regulating VM formation of UM.PART 3 Quantitative evaluation of uveal melanoma microcirculation pattern of vasculogenic mimicry with VEGFR1 targeted contrast-enhanced ultrasoundObjective:To investigate the application of VEGFR1 targeted contrast-enhanced ultrasound(CEUS)to quantify VM perfusion and function in ocular UM model.Methods:VEGFR1 targeted ultrasound contrast agent was constructed by binding VEGFR1 specific antibody to microbubbles(MBs)surface,with fluorescence observation of the binding between MBs and cells.The imaging difference of VEGFR1 targeted CEUS was compared in the model of VEGFR1-ctrl and VEGFR1-KD melanoma.The imaging difference between non-targeted CEUS and VEGFR1 targeted CEUS was observed in the quantitative assessment of microcirculation in the model of MUM-2b melanoma.The Sono Liver software was used to quantitativelyanalyze tumor microcirculation perfusion.The peak time,peak intensity,maximum intensity(IMAX),area under the time intensity curve,rise time(RT),mean transit time(MTT),slope and duration of ascending and descending branches of contrast agent filling in uveal melanoma circulation were calculated.And the relation of VM structure in histology and the targeted CEUS imaging was analyzed.Results:VEGFR1 targeted MBs were constructed and were able to static and specifically bind to MUM-2b melanoma cells.CEUS with VEGFR1 targeted MBs(with IMAX 230.50 ± 11.13,MTT 64.43 ± 6.33s)showed significantly enhancement of VM imaging through the whole perfusion phases compared to CEUS with Ig G MBs(with IMAX 144.60 ± 8.67,MTT 38.66 ± 3.34s),P<0.05.And VEGFR1 targeted imaging were able to detect the decrease of IMAX and MTT in VEGFR1-KD melanoma(with IMAX 158.20±26.21,MTT 36.41±4.26s)compared to control melanoma(with IMAX 265.50±23.87,MTT 59.67±5.99s),P<0.05.In the pathological section of the tumor,the number of VM lumens stained by PAS was 13.5 ± 3.5/visual field for VEGFR1-KD,and 49.5 ± 0.5/visual field for the control(P < 0.01),which was consistent with the result of VEGFR1 targeting CEUS.Conclusions:VEGFR1 targeted CEUS could be a reliable method of high sensitivity and specificity in detecting VM at the molecular level in UM in vivo.These findings might help advance the further use of targeted CEUS to examine blood supply patterns in UM,and advance the development of accurate diagnosis and precise treatment efficacy in the future.