Preparation And Evaluation Of EphA2 Targeted Cyclic Peptide Probes For Triple-negative Breast Cancer Based On Radionuclide Molecular Imaging

ObjectiveTriple negative breast cancer(TNBC)accounts for about 15%of breast cancer,lack of highly selective biological markers,with high invasion,metastasis and recurrence rates and poor prognosis.At present,imaging examinations for the diagnosis and staging of TNBC mainly include mammography,ultrasound,and magnetic resonance imaging(MRI).However,these examinations have limitations.Nuclear medicine molecular imaging is a non-invasive imaging method with high sensitivity and specificity,18F-FDG PET/CT which is widely used in clinical is non-specific,it has been not obtained an ideal specific molecular imaging probe for TNBC imaging.Therefore,searching for a novel and highly selective biomarker is of great importance for the early diagnosis and prognosis of TNBC.Recently,EphA2 was reported tighhtly associated with malignant progression,poor prognosis and metastasis.,therefore,the EphA2 has the potential to be used as a molecular target for TNBC.This thesis was divided into two parts:in the first part,the novel radioiodine-labeled cyclic peptide probe targeting EphA2 was prepared and the specific binding to the TNBC cell line 4T1 with high expression of EphA2 was investigated in vitro and in vivo;in the second part,uhe novel 68Ga-labeled cyclic peptide probe targeting EprA2 was prepared and the specific binding to 4T1 cells and tumor was investigated in vitro and in vivo.The thesis aimed to provide a new molecular imaging target and novel radionuclide labeling probes for TNBC molecular imaging,and to supply a new strategy for monitoring the progression of EphA2 positive tumors.Part One Preparation of novel radionuclide iodine labeled cyclic peptide targeting EphA2 and evaluation based on phosphor-autoradiography in TNBC tumor-bearing miceMethods1.The expression of EphA2 in TNBC cell line 4T1 and EMT6 was verified through RTPCR and Western blot,and the effect of EphA2 on proliferation,,colony formation and migration of 4T1 cell line was investigated by CCK8 assay,and colony formation assay,wound healing assay and transwell assay under EphA2 inhibitor treatment.2.The cyclic peptide SD01 was designed,SD01 and control peptide YSA were prepared through solid phase peptide synthesis(SPPS)method,identified by High Performance Liquid Chromatography(HPLC)and Mass Spectrometry(MS);FITC conjugated FITC-SD01 and FITC-YSA were co-incubated with 4T1 cells,and their binding with 4T1 cells was observed under fluorescence microscope.3.4T1 cell lines with low TLR5 expression(TLR5-)and normal TLR5 expression(TLR5+)tagged with green fluorescent protein(GFP)were constructed through transfecting TLR5-shRNA and its negative control lentivirus,and the expression of TLR5 and EphA2 was detected by RT-PCR and Western blot.The expression of EphA2 and other related RTK molecules both in TLR5-and TLR5+4T1 cell lines was analyzed with antibody microarray.4.Radionuclide molecular probes 125I-SD01 and 125I-YSA were prepared and identified,cell uptake and radioligand saturation binding assays were performed to detect their specificity and affinity for 4T1 cell line;radionuclide molecular probe 131I-SD01 was prepared and identified.5.4T1 tumor-bearing mice models were constructed,the 125I-SD01 and 125I-YSA probes were injected into the model mice through tail vein,and the whole-body dynamic phosphorautoradiography were performed at 1,3,6,12,24h to study the targeting ability of 125I-SD01 and 120I-YSA.The models were executed at 6,12h,major tissues and organs,tumors and muscle tissues were removed and weighed,radioactivity was measured,and ID%/g was calculated for biodistribution studies,ex vivo phosphor-autoradiography of the isolated tumors were also performed.6.TLR5-and TLR5+4T1 tumor-bearing mouse models were constructed,fluorescence imaging of the tumor-bearing mice was performed.131I-SD01 was injected into the model mice through tail vein,whole-body dynamic phosphor-autoradiography was performed at 3,6,12,24h to study the targeting ability of 131I-SD01 for the EphA2 on tumors,and to observe the radioactivity accumulation of 131I-SD01 in tumor bearing mice dynamically.The models were executed at 6,12,24h,the major tissues and organs,tumors and muscle tissue were removed and weighed,radioactivity was measured,and%ID/g was calculated for biodistribution studies.Meanwhile,fluorescence imaging and ex vivo phosphor-autoradiography of the isolated tumors were performed.7.The isolated 4T1 and EMT6 tumor tissues were subjected to H&E and immunohistochemical staining to study the expression of EphA2,and the correlation between radioactivity accumulation of 125I-SD01 and 125I-YSA and expression of EphA2 was analyzed.The isolated TLR5-and TLR5+ 4T1 tumor tissues were subjected to H&E staining and immunohistochemical staining to study the expression of TLR5 and EphA2,and the correlation between radioactivity accumulation of 131I-SD01 and expression of EphA2 was analyzed.Results1.Both TNBC cell lines 4T1 and EMT6 expressed EphA2,and the expression level of EphA2 in 4T1 cells was higher than that in EMT6(p<0.01);the results of CCK8 assay,colony formation assay,wound healing assay and transwell assay showed that high expression of EphA2 significantly promoted the proliferation,colony formation and migration of 4T1 cells.2.SD01 was successfully prepared.SD01 and YSA were purified by HPLC with purity>95%,and the molecular weights were determined by MS,SD01 was 1457.66Da and YSA was 1346.55Da,which was consistent with expected results.Both FITC-SD01 and FITC-YSA could bind to 4T1 cells,and the binding ability of FITC-SD01 was stronger than that of FITCYSA(p<0.01).3.TLR5-and TLR5+4T1 cell lines with GFP tag were successfully constructed with transfection efficiency close to 100%;RT-PCR and Western blot results showed that compared with TLR5+4T1 cells,TLR5 expression was significantly decreased and EphA2 expression was significantly increased in TLR5-4T1 cells;the expression of 39 kinds of RTK related proteins in TLR5-and TLR5+ 4T1 cells were detected by antibody microarray,the results confirmed that the expression EphA2 was increased significantly when TLR5 was knock-down,and EphA2 showed the most obvious change among the 39 kinds of RTK related proteins(p<0.05).4.125I-SD01 and 125I-YSA were prepared successfully,with labeling rates>85%and radiochemical purity>95%;the stability of 125I-SD01 and 125I-YSA was still>95%in FBS and>85%in NS until 24h;the Log Do/w value of 125I-SD01 and 125I-YSA was-1.64± 0.01 and1.45± 0.01,indicated that both probes had good hydrophilicity.Radioligand saturation binding assays verified that both 125I-SD01 and 125I-YSA could bind with 4T1 cells,the Bmax(maximum binding ability)value was 4884 cpm/5×105 cells and 4084 cpm/5×105 cells respectively,which suggested that the 125I-SD01 group had more binding sites;both 125I-SD01 and 125I-YSA had high affinity for 4T1 cells,Kd(dissociation constant)value was 14.76 and 15.85 nM,with no significant difference between them.131I-SD01 was prepared successfully,with labeling rate>85%and radiochemical purity>97%;the stability of 131I-SD01 was still>95%in FBS and>94%in NS until 24h;the Log Do/w value of 131I-SD01 was-1.94±0.12,the probe had good hydrophilicity.5.Whole-body dynamic phosphor-autoradiography results showed specific uptake of 125I-SD01 and 125I-YSA by tumors in vivo,with the most obvious specific uptake at 12h,and the radioactivity Accnmulation at tumors of 125I-SD01 group was stronger than 125I-YSA group(p<0.05);ex vivo phosphor-autoradiography of isolated tumors also indicated that the tumors showed higher uptake for 125I-SD01 than 125I-YSA;the biodistribution study showed that 125ISD01 and 125I-YSA were mainly metabolized through kidney,and there was more radioactivity accumulation at tumors in 125I-SD01 group compared with 125I-YSA group,tumor uptake was 2.43±0.27%ID/g and T/NT ratio was 5.99 ± 0.37 in the 125I-SD01 group at 12h,1.79 ± 0.18%ID/g and 4.69±0.18 in the 125I-YSA group(tumor%ID/g and T/NT ratio,125I-SD01 vs 125IYSA,p<0.05).6.Fluorescence imaging of tumor-bearing mice clearly showed subcutaneous TLR5-4T1 and TLR5+4T1 tumors on both sides,which indicated that lentivirus-transfected 4T1 cells,either TLR5 knockdown or negative control,maintained biological properties in vivo and carried GFP tag.Although phosphor-autoradiography showed specifically uptake of 131I-SD01 by TLR5-and TLR5+4T1 tumors,the radioactivity accumulation at TLR5+4T1 tumors was lower obviously than that in TLR5-4T1 tumors at each imaging time point(p<0.05);ex vivo phosphor-autoradiography of isolated tumor also showed that the 131I-SD01 radioactivity uptake was higher in TLR5-4T1 tumors than in TLR5+4T1 tumors,which confirmed that TLR5 down-regulation led to higher EphA2 expression and higher uptake of 131I-SD01 probe in 4T1 tumor.Biodistribution studies showed that the probe was mainly metabolized through kidney;the T/NT ratios were 6.04 ± 0.18,8.29 ± 0.29,and 7.14 ± 0.39 in the TLR5-4T1 tumor group at 6,12,and 24h,4.97 ± 0.28,5.99 ± 0.24,and 5.59 ± 0.38 in the TLR5+4T1 tumor group respectively(T/NT ratios at 6,12,24h,TLR5-4T1 tumors vs TLR5+4T1 tumors,p<0.05).7.The H&E staining of isolated tumors showed typical tumor structures.Immunohistochemical staining showed that both 4T1 and EMT6 tumor tissues expressed EphA2,and the expression level of 4T1 was higher than that of EMT6 tumor tissues significantly(p<0.05);and radioactivity accumulation of 125I-SD01 and 125I-YSA had significant positive correlation with EphA2 expression.TLR5 expression was significantly decreased in TLR5-4T1 tumor tissues,whereas the expression of EphA2 was significantly increased compared with TLR5+4T1 tumor tissues(p<0.05);and radioactivity accumulation of 131I-SD01 had significant positive correlation with EphA2 expression.Part Two Preparation of novel Ga-68 labeled cyclic peptide targeting EphA2 and evaluation based on microPET/CT imaging in TNBC tumor-bearing miceMethods1.SD01 and YSA were prepared and identified according to the Part one.DOTA-SD01 and DOTA-YSA were prepared and identified by HPLC and MS.2.The expression of EphA2 in TNBC cell line 4T1 was verified through immunofluorescence staining.3.Ga-68 was produced using a 68Ge/68Ga generator,novel radionuclide molecular probe 68Ga-DOTA-SD01 was prepared and identified(with 68Ga-DOTA-YSA as a control),radioligand saturation binding assays were performed to explore the specificity and affinity of two probes for 4T1 cell line.4.4T1 tumor-bearing mice models were constructed,68Ga-DOTA-SD01 and 68GaDOTA-YSA were injected into the model mice through tail vein,and the microPET/CT imaging was performed at 5,10,30,60 min to study the targeting ability and imaging features of 68Ga-DOTA-SD01 and 68Ga-DOTA-YSA for 4T1 tumors;blocking group of 68Ga-DOTASD01 was established by pre-injection of excess unlabeled DOTA-SD01 to investigate the specificity of the novel cyclic peptide probe.The models were executed at 30 min,and major tissues and organs,tumors and muscle tissues were removed and weighed,radioactivity was measured,and%ID/g was calculated for biodistribution study.5.The isolated tumor tissues were subjected to EphA2-specific immunofluorescence staining to study the EphA2 expression,and the correlation between radioactivity accumulation of 68Ga-DOTA-SD01 and 68Ga-DOTA-YSA with EphA2 expression was analyzed.Results1.DOTA-SD01 and DOTA-YSA were successfully prepared,the peptides were purified by HPLC with purity>95%,and the molecular weights were determined by MS,DOTA-SDO1 was 1844.09Da,DOTA-YSA was 1732.94Da,which was consistent with expected results.2.The immunofluorescence staining proved that 4T1 cells highly expressed EphA2.3.68Ga-DOTA-SD01 and 68Ga-DOTA-YSA were successfully prepared,with labeling rates>90%and radiochemical purity>99%;the stability for 68Ga-DOTA-SD01 and 68GaDOTA-YSA was still>95%in FBS and>97%in PBS at 2h;the Log Do/w value of 68Ga-DOTASD01 and 68Ga-DOTA-YSA was-2.92±0.04 and-3.06 ± 0.01,respectively,indicated that both probes had good hydrophilicity.Radioligand saturation binding assays verified that both 68GaDOTA-SD01 and 68Ga-DOTA-YSA could bind with 4T1 cells,the Bmax(maximum binding ability)value was 6668 cpm/5×105 cells and 5026 cpm/5×105 cells respectively,68Ga-DOTASD01 had more binding sites;both 68Ga-DOTA-SD01 and 68Ga-DOTA-YSA had high binding affinity for 4T1 cells,Kd(dissociation constant)value was 12.15 and 11.79 nM respectively,with no significant difference between them.4.microPET/CT imaging showed specific uptake of 68Ga-DOTA-SD01 and 68GaDOTA-YSA by tumors,with the most obvious specific uptake was observed at 30min,the SUV values of tumors in 68Ga-DOTA-SD01 and 68Ga-DOTA-YS A group were 3.34± 0.25 and 2.65± 0.32 respectively,and the T/NT(tumor/opposite muscle)ratios were 3.12 ± 0.06 and 2.77±0.11 respectively,indicated that the radioactivity accumulation of 68Ga-DOTA-SD01 probe was stronger than 68Ga-DOTA-YSA probe(p<0.05);pre-injection with excess unlabeled DOTASD01 significantly blocked tumor radioactivity accumulation in the 68Ga-DOTA-SD01 group,which further confirming the specific radioactivity accumulation at tumor sites.5.The biodistribution study showed that the probes were mainly metabolized through kidney,and specific uptake of both 68Ga-DOTA-SD01 and 68Ga-DOTA-YSA by tumors was observed.There was much more radioactivity accumulation of 68Ga-DOTA-SD01 at the tumor,compared with 68Ga-DOTA-YSA,tumor uptake was 2.73 ± 0.34%ID/g and T/NT ratio was 3.55 ± 0.12 in the 68Ga-DOTA-SD01 group at 30min,1.77 ± 0.38%ID/g and 3.05± 0.10 in the 68Ga-DOTA-YSA group(tumor%ID/g and T/NT ratio,68Ga-DOTA-SD01 vs 68Ga-DOTAYSA,p<0.05).6.Immunofluorescence staining of isolated 4T1 tumor tissues showed that 4T1 tumor tissues expressed EphA2,and the radioactivity accumulation of 68Ga-DOTA-SD01 and 68GaDOTA-YSA had significant positive correlation with EphA2 expression.Conclusions1.TNBC cell line 4T1 highly expressed EphA2;the down-regulation of TLR5 significantly increased the expression of EphA2 in 4T1 cells and tumors;EphA2 could promote TNBC progression through promoting proliferation,colony formation and migration.2.EphA2-targeted cyclic peptide SD01 was successfully prepared.3.EphA2 targeted 125/131I-SD01,125I-YSA,68Ga-DOTA-SD01 and 68Ga-DOTA-YSA radionuclide labeled probes were successfully prepared,and confirmed.The results indicated that the probes in vitro could bind specifically to EphA2 positive TNBC cell line 4T1 specifically.The cyclic peptide had more binding sites than the control linear peptide YSA.4.Phosphor-autoradiography,microPET/CT imaging and biodistribution studies indicated that 125/131I-SD01,125I-YSA,68Ga-DOTA-SD01 and 68Ga-DOTA-YSA could specifically bind with 4T1 tumors in tumor bearing mice;compared with 125I-YSA and 68GaDOTA-YSA;125I-SD01 and 68Ga-DOTA-SD01 radioprobes were significantly superior in terms of tumor targeting and T/NT ratio.