Experimental Research On Construction Of MR Molecular Probe With Prostate Cancer-specific Aptamer As Well As Its Application Of Diagnosis And Therapy In Vitro And In Vivo

Objective1.To screen EpCAM-specific aptamer of prostate cancer,and to identify its binding affinity for EpCAM-positive prostate cancer cells.2.To construct the molecular probe of Aptamer^EpCAM-GoldMag-tBid with EpCAM-specific aptamer and immunoproapoptotic molecules of tBid mediated with GoldMag nanoparticles;to identify its binding affinity for EpCAM-positive prostate cancer cells and the ability of inducing apoptotic death in prostate cancer cells.3.To establish the prostatic tumor-bearing nude mice models.To evaluate the targeted binding affinity and pro-apoptotic activity of Aptamer^EpCAM-GoldMag-tBid to prostate cancer tissues and cells,and further to identify its ability of MR imaging in vitro and in vivo.Methods1.Flow cytometry and Western blot were used to detect the expression of EpCAM in prostate cancer cells of LNCap,PC-3 and DU145.2.Random ssDNA libraries flanked by fixed primer annealing sites necessary for enzymatic amplification and primer pairs were designed for SELEX selection.HEK293T cells transfected by slow virus expression vector pCDH-EpCAM were used as the binding target.The procedure of cell-SELEX involved 12 rounds of repeated selection cycles to enhance the binding affinity of ssDNA products.Two aptamers including Ep1 and Ep2were eventually selected after identified by cloning and DNA sequencing.3.Flow cytometry and immunofluorescence assay were used to evaluate the binding affinity of Ep1 and Ep2 for EpCAM-positive prostate cancer cells,respectively.4.Based on the characteristics of Au-coated iron oxide nanoparticles,GoldMag nanoparticles could conjugate with EpCAM-specific aptamers and immunoproapoptotic molecules of tBid to construct the molecular probes of Ep1-GoldMag-tBid and Ep2-GoldMag-tBid.5.Flow cytometry and immunofluorescence assay were used to evaluate the binding affinity of Ep1-GoldMag-tBid and Ep2-GoldMag-tBid for EpCAM-positive prostate cancer cells,respectively.Flow cytometry was used to evaluate the apoptosis-inducing activity of the two molecular probes towards EpCAM-positive prostate cancer cells in vitro.6.EpCAM-positive prostate cancer cells of LNCap,PC-3 and DU145 were respectively incubated with Ep1-GoldMag-tBid overnight and fixed by agarose gel.MR examinations were performed in the molecular probe-treated cells.The signal intensity of all cells samples on T2WI was measured and compared.7.PC-3 cell suspension was injected into axillary fat pad of forelimb in male nude mice to establish the prostatic tumor-bearing nude mice models.8.Immunohistochemical staining was used to identify the expression of EpCAM in tumor tissues and major organs including heart,liver,spleen,lung and kidney of tumor-bearing nude mice.9.The prostatic tumor-bearing nude mice were administrated injection of Ep1-GoldMag-tBid through orbital venae angularis.The tumor tissues and major organs including heart,liver,spleen,lung and kidney were resected to make frozen tissue sections.Immunofluorescent staining assay was used to evaluate the binding affinity of Ep1-GoldMag-tBid for tumor tissues.10.Tumor-bearing nude mice were divided into three groups randomly,and treated with different doses of Ep1-GoldMag-tBid and PBS.The growth of the tumor treated with high dose of Ep1-GoldMag-tBid（high-dose group）,low dose of Ep1-GoldMag-tBid（low-dose group）and PBS（control group）were recorded daily,and tumor volumes were calculated.11.The tumor tissues and major organs including heart,liver,spleen,lung and kidney of tumor-bearing nude mice in the three groups were respectively resected to make paraffin-embedded tissue sections.HE staining was used to observe the effect of Ep1-GoldMag-tBid on major organs of tumor-bearing nude mice.12.The prostatic tumor-bearing nude mice underwent MR examinations,and then were administrated injection of Ep1-GoldMag-tBid through orbital venae angularis.After 6hours and 12 hours subsequent to injection,MR examinations were respectively performed in the molecular probe-treated nude mice.The signal intensity of tumors on T2WI before injection as well as 6 and 12 hours following injection was measured and compared.Results1.Flow cytometry and Western blot were used to detect the expression of EpCAM in prostate cancer cells of LNCap,PC-3 and DU145,which showed that all the three types of prostate cancer cells and HEK293T cells transfected by pCDH-EpCAM were EpCAM positive,while HEK293T cells transfected by empty vector were EpCAM negative.2.Cell-SELEX was performed to screen EpCAM-specific aptamers of prostate cancer from random ssDNA libraries.The selection procedure involved 12 rounds of repeated selection cycles.Flow cytometry showed that fluorescence intensity of ssDNA products binding to HEK293T cells transfected by pCDH-EpCAM gradually increased with the increase of selection cycles.Two aptamers including Ep1 and Ep2 were eventually selected after identified by cloning and DNA sequencing.3.Flow cytometry and immunofluorescence assay were respectively used to evaluate the binding affinity of Ep1 and Ep2 for EpCAM-positive prostate cancer cells,which showed that both aptamers could specifically recognize and bind to EpCAM-positive prostate cancer cells.4.The molecular probes of Ep1-GoldMag-tBid and Ep2-GoldMag-tBid were successfully constructed by GoldMag nanoparticles conjugated with EpCAM-specific aptamers and immunoproapoptotic molecules of tBid.Flow cytometry and immunofluorescence assay were used to evaluate the binding affinity of Ep1-GoldMag-tBid and Ep2-GoldMag-tBid for EpCAM-positive prostate cancer cells,which showed that both molecular probes could specifically recognize and bind to EpCAM-positive prostate cancer cells.Flow cytometry was used to evaluate the apoptosis-inducing activity of the two molecular probes towards EpCAM-positive prostate cancer cells in vitro,which showed that both molecular probes could kill EpCAM-positive prostate cancer cells by inducing apoptotic death with stronger pro-apoptotic activity of Ep1-GoldMag-tBid.5.MR imaging of prostate cancer cells treated with Ep1-GoldMag-tBid showed decreased signal intensity on T2WI with statistically significant differences between the experimental group and the control group.6.Prostatic tumor-bearing nude mice models were successfully established.Immunohistochemical staining was used to identify the expression of EpCAM in tumor tissues and major organs including heart,liver,spleen,lung and kidney of tumor-bearing nude mice,which showed that the tumor tissues were EpCAM positive,while the major organs were EpCAM negative.7.Immunofluorescent staining assay was used to evaluate the binding affinity of Ep1-GoldMag-tBid for tumor tissues,which showed that Ep1-GoldMag-tBid could specifically bind to tumor tissues instead of normal tissues and organs.8.Three groups of tumor-bearing nude mice were respectively treated with high dose of Ep1-GoldMag-tBid,low dose of Ep1-GoldMag-tBid and PBS.During the therapy,tumors of the three groups all continued to grow,but tumor growth rate of the two experimental groups were both slower than that of the control group.In addition,the tumor growth was the slowest in the high-dose group.9.The tumor tissues and major organs of tumor-bearing nude mice in the three groups were respectively resected to make paraffin-embedded tissue sections.HE staining showed that there were no pathological morphological changes in major organs of tumor-bearing nude mice after the therapy of high dose of Ep1-GoldMag-tBid.10.MR examinations were performed in the tumor-bearing nude mice before and 6 hours and 12 hours after injection of Ep1-GoldMag-tBid.MRI scan showed that the signal intensity of tumors on T2WI decreased after 6 hours following injection,and slightly increased after 12 hours following injection.The signal intensity of the latter was still significantly lower than that before the injection with statistically significant differences between the experimental group and the control group.ConclusionOur study constructed the molecular probes of Aptamer^EpCAM-GoldMag-tBid by GoldMag nanoparticles conjugated with EpCAM-specific aptamers and immunoproapoptotic molecules of tBid.It could specifically recognize and bind to EpCAM-positive prostate cancer cells in vitro and in vivo,and induce apoptosis in tumor cells selectively with dose-dependent inhibitory effect,without harming the surrounding normal tissues and major organs of tumor-bearing nude mice.In addition,Aptamer^EpCAM-GoldMag-tBid could be used as a mlecular imaging agent for detection of EpCAM-positive prostate cancer tissues and cells in MRI.Our study is of great importance for specific diagnosis and targeting therapy of EpCAM-related prostate cancer,which would serve as a theoretical foundation for accurate diagnosis and targeting therapy of other malignant tumors in the future.