| Objectives:Triple negative breast cancers(TNBCs)account for about 15% of breast cancers,which have a higher risk of earlier metastasis and are associated with dismal prognosis.The membrane protein Nectin-4 has been found to be overexpressed in various tumors including TNBCs.Our research aims to synthesize a nuclide molecular imaging probe basing on 99 mTclabelling with anti-Nectin-4 monoclonal antibody(m Ab Nectin-4),and to investigate the feasibility of this probe-guided SPECT/CT imaging for breast cancer diagnosis and in vivo Nectin-4 assessment.Methods:m AbNectin-4 was radiolabeled with 99 mTc by SHNH.The radiolabeling yield and radiochemical purity of 99mTc-HYNIC-m Ab Nectin-4 were measured by instant thin layer chromatography(ITLC)before and after PD-10 purification respectively.Two breast cancer cell lines(MDA-MB-468,TNBC,Nectin-4 overexpression;and MCF-7,non-TNBC,Nectin-4 underexpression)were used in this study.Cell uptake and cell blocking assays were performed to evaluate the specificity of binding to Nectin-4 in vitro.Nude mice bearing xenografts of MADA-MB-468 and MCF-7 were established.The radioactive probe 99mTcHYNIC-m Ab Nectin-4 was administered via the tail vein for each mouse(The MDA-MB-468 blocked group mice were treated with excess unlabeled m Ab Nectin-4 1-2 h prior to probe administration).SPECT/CT images of all tumor-bearing mice were acquired at 3 h,6 h,12 h,24 h and 36 h after 99mTc-HYNIC-m Ab Nectin-4 injection,respectively.The tumor-bearing mice of each group were sacrificed at 36 h post probe injection and selected organs were collected to evaluate the biodistribution.The corresponding tumor/muscle(T/M)and tumor/blood(T/B)ratio were also calculated.Results:The results of western blot,cytometry,confocal laser scanning microscopy,and immunohistochemistry verified that the expression level of Nectin-4 in MDA-MB-468 cells or tumor tissue was higher than that in MCF-7 counterparts.An antibody-based radioprobe 99mTc-HYNIC-m Ab Nectin-4 was successfully synthesized with high radiolabelling yield(73.03% ± 6.95%)and radiochemical purity(95.03% ± 2.40%).99mTc-HYNIC-m Ab Nectin-4 could be accumulated by MDA-MB-468 cells specifically in vitro with the highest uptake of 4.27% ± 0.09 % at 8 h,which was significantly higher than that of MCF-7 control cells(1.07% ± 0.15%,p < 0.001)and MDA-MB-468 blocking group cells(0.79 % ± 0.03%,p < 0.001).The SPECT/CT images illustrated the MDA-MB-468 tumor had obviously higher 99mTcHYNIC-m Ab Nectin-4 uptake in comparison to MCF-7 tumor.The tumors of MDA-MB-468 blocking group had relatively lower activity accumulation,which was suggesting that this radioprobe obtained excellent detection capability and specificity.The results of biodistribution study demonstrated that the MDA-MB-468 tumors had significantly higher uptake of radiolabeled probe with 15.32 ± 1.04 %ID/g(36 h)comparing to MCF-7 tumors(3.02 ± 0.20 %ID/g,p < 0.001),which could be inhibited by excess m Ab Nectin-4(4.33 ± 0.48 %ID/g,p < 0.001).In addition,the mice bearing MDA-MB-468 tumor exhibited superior T/M and T/B ratio(13.19 ± 4.44 and 1.37 ± 0.09)comparing to the MCF-7 group(4.16 ± 1.18 and 0.47 ± 0.05)and MDA-MB-468 blocking group(4.09 ± 0.66 and 0.38 ± 0.03),respectively.Conclusion: In this study,we successfully synthesized an antibody-based radioprobe 99mTcHYNIC-m Ab Nectin-4.The in vitro and in vivo specific binding to Nectin-4 were confirmed by cell uptake assays and SPECT imaging,respectively.SPECT imaging here provides a noninvasive strategy for assessing Nectin-4 expression in tumors,and is expected to provide image guidance for Nectin-4-targeted therapy for various Nectin-4-overexpressing cancers,including TNBC.Objectives: Chemotherapy remains the main available systemic regimen for triple-negative breast cancer(TNBC).However,drug resistance limits the therapeutic efficacy,and novel therapeutic strategies for TNBC are urgently needed.Recent studies have found that Nctin-4 is highly expressed in TNBC,and the previous section of this study verified that m Ab Nectin-4 has good targeting and affinity for Nectin-4 overexpressed TNBC tumor,and corresponding SPECT/CT imaging can successfully verify and assess Nectin-4 expression.Photothermal therapy(PTT)is a promising non-invasive strategy for cancer therapy.In this section,a photothermal agent m Ab Nectin-4-ICG based on Nectin-4 targeting was synthesized.Through the tumor targeting of m Ab Nectin-4,ICG can be accurately delivered to tumor tissue to realize the PTT for TNBC.Methods: The photothermal agent m Ab Nectin-4-ICG was synthesized by coupling the antibody m Ab Nectin-4 with ICG-NHS-ester,and its optical properties were measured by an UV spectrophotometer.After co-incubating m Ab Nectin-4-ICG with MDA-MB-468 TNBC cells,the cytotoxicity of the agent was detected,and fluorescence(FL)imaging was performed to verify the in vitro targeting.The photothermal conversion efficiency and photothermal stability of m Ab Nectin-4-ICG following 808 nm laser irradiation were examined by thermal camera.MDA-MB-468 cells treated with m Ab Nectin-4-ICG were underwent laser irradiation at different ICG concentrations,power densities,and irradiated times to evaluate the in vitro PTT efficacy.In vivo FL images was obtained to test the tumor targeting of the agent,and to determine the optimal treatment time window for PTT.The TNBC tumor-bearing mice were randomly divided into 4 groups,namely m Ab Nectin-4-ICG + laser group,free ICG + laser group,saline + laser group and saline only group.Post 24 hours to tail vein administration of m Ab Nectin-4-ICG/ICG/saline,the tumor was treated with laser irradiation and the tumor temperature were recorded.Mice in each group were followed up for 30-day duration to monitor the living status,body weight and tumor volume.On the 30 th day after treatment,the blood and selected organs were harvested,and biological toxicity was evaluated by hematological tests and organ H&E staining.Results:The m AbNectin-4-ICG was successfully synthesized with an UV absorption peak located at 790 nm.Stable fluorescence property was detected till 48 h away from light.Only low cytotoxicity of m Ab Nectin-4-ICG was observed when when ICG concentration below 20 μ g/m L.Cells incubated with m AbNectin-4-ICG had significantly enhanced FL uptake,suggesting excellent in vitro cell targeting.The m Ab Nectin-4-ICG solution has favorable photothermal conversion efficiency,which is enhanced with the elevation of ICG concentration and laser power density.The results of in vitro PTT experiments demonstrated that only m Ab Nectin-4-ICG plus 808 nm laser group had obvious cell killing effect,which was obviously enhanced with the increase of ICG concentration,laser power and irradiated time.In vivo FL images illustrated that the TNBC tumors have prominent m Ab Nectin-4-ICG accumulation,and peaked at 24 h;whereas no obvious tumor FL signal was detected in the tumors of free ICG group and the saline group mice.Tumor temperature of the m Ab Nectin-4-ICG + laser group could be significantly elevated with the laser irradiation,exceeding the injury temperature threshold(42°C);while in tumor-bearing mice injected with free ICG and normal saline,the elevated tumor temperature was not significant.Compared with the control group,under an m Ab Nectin-4-ICG-mediated NIR-PTT regimen,TNBC xenograft tumor growth was obviously suppressed and completely ablated.No obvious abnormality in the hematological examinations and H&E staining was explored in each group,indicating negligible biotoxicity.Conclusion: The photothermal agent m Ab Nectin-4-ICG based on Nectin-4 targeting has favorable optical properties,photothermal conversion efficiency,biocompatibility and specific targeting.By targeted delivery of ICG to tumor tissue,m Ab Nectin-4-ICG could achieve an efficient PTT regimen to delay and suppress tumor growth by inducing hyperthermia.Combined with the guidance of Nectin-4-targeting SPECT/CT imaging in the previous section of this study,this photothermal agent-mediated PTT regimen offers a simple theranostic strategy and may contribute to improve the TNBC patients’ management. |
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