Targeting CD44 And EpCMA With A Bispecific Aptamer Inhibits Ovarian Cancer In Vitro And In Vivo

Ovarian cancer is the most deadly cancer among all gynecologic malignancies.Ovarian cancer is often detected at an advanced stage with wide peritoneal metastasis,due to the lack of early stages specific symptoms.Epithelial ovarian cancer(EOC)constitutes 85%-90%cases of ovarian cancer.The combination of debulking and platinum-based chemotherapy is the standard treatment for advanced OC.Although patients initially respond favorably,most of them will develop chemoresistance and eventually demise with peritoneal metastasis.Therefore,it is urgently needed to explore the molecular target-directed therapies which inhibit the peritoneal metastasis.Epithelial cell adhesion molecules(EpCAM)is overexpressed in human epithelial cancer.It has been confirmed that EpCAM participates in cancer progression and metastasis by regulating the process of EMT.Moreover,EpCAM positive cells are well recognized to possess tumor-initiating potential and EpCAM has thus been used as a key marker of ovarian cancer stem cells.CD44,as a transmembrane glycoprotein,is the principal receptor for hyaluronan(HA).The binding of HA with CD44 induces tumor cell proliferation,differentiation,invasion,and migration,leading to the progression and metastasis of tumors.CD44 has been identified as an important molecule in ovarian cancer progression.These findings strongly support the notion that CD44 and EpCAM is the ideal therapeutic target for ovarian cancer.Aptamers are ssDNA or ssRNA that can bind target with high affinity and specificity.The nature of aptamer as a small oligonucleotide implicates that it offers any advantages over the antibody such as cell-free chemically synthesis,non-immunogenicity,high tissue penetration,thermostable and low cost.Single EpCAM aptamer has been generated,which possesses the similar binding affinity as antibodies and is efficiently internalized through receptor-mediated endocytosis.Single CD44 aptamer has also been produced and can target cells with high CD44expression effectively.Importantly,single CD44 aptamer has been shown the capacity for drug delivery.However,the therapeutic effect of these two aptamers against tumorigenesis has not been reported.In this study,we constructed an RNA-based bispecific aptamer which can simultaneous targeting of CD44 and EpCMA,and detected the inhibitory effect on the growth of ovarian cancer cells and intraperitoneal xenograft in nude mice.Object:We constructed a bispecific aptamer that simultaneous targeting of CD44 and EpCAM,and illuminated the inhibitory effect on the growth of ovarian cancer cells and intraperitoneal xenograft in nude mice.The study offers a promising therapeutic agent against ovarian cancer.Methods:1.Immunohistochemistry was used to evaluate the status of CD44 and EpCAM expression in human epithelial ovarian cancer tissues.Furthermore,we analyzed the association of protein expression with clinicopathological features and the possible relevance between CD44 and EpCAM.2.Construction of a bispecific CD44-EpCAM-Aptamer and detection of binding capability,targeting specificity,serum stabilityCD44 and EpCAM aptamers were individually synthesized by in vitro transcription with PCR products as templates.Two RNAs were mixed at molar ratio1:1 and annealed to form one bispecific CD44-EpCAM molecule by heated at 94°C for 3 min.ELISA was used to examine the binding of bispecific CD44-EpCAM-Aptamer with human recombinant CD44 and EpCAM protein.We also test the serum stability of bispecific CD44-EpCAM-Aptamer.To confirm target specificity of bispecific CD44-EpCAM-Aptamer,the binding patterns of ovarian cancer cell lines(OVCAR8,SKOV3,ES2,OCC1)stained with bispecific CD44-EpCAM-Aptamer was assessed with flow cytometry.Flow cytometry was used to identify the binding capability between bispecific CD44-EpCAM-Aptamer and CD44/EpCAM silenced OVCAR8 cells.ELISA was used to determine the effect of bispecific CD44-EpCAM-Aptamer on innate immunogenicity.3.The inhibitory effect of bispecific CD44-EpCAM-Aptamer on ovarian cancer in vitro and in vivoIn vitro:CCK-8 was used to assess the cytotoxicity of bispecific CD44-EpCAM-Aptamer on ovarian cancer cells(OVCAR8,SKOV3,ES2,OCC1).Western blot was used to examine the level of Cleaved Caspase-3 in OVCAR8 and ES2 cells treated with bispecific CD44-EpCAM-aptamer for varying concentrations.Flow cytometry was used to further confirm the occurrence of apoptosis.The apoptotic pattern was also demonstrated from fluorescence microscope imaging.In vivo:Luciferase stable OVCAR8 cell line was developed by our laboratory.OVCAR8-luc cells(5x10~6)in the log phase were intraperitoneally injected into the athymic nu/nu female mice,5~8 days after implantation,the intraperitoneal ovarian cancer xenograft model was constructed.The ovarian cancer xenograft models were treated with bispecific CD44-EpCAM-Aptamer-Cy5.Cy5 fluorescence of mice and bioluminescence images were detected with Xenogen IVIS100 imaging system to analyze the biodistribution and tumor targeting capability.Mice were intraperitoneally administered with PBS,single EpCAM aptamer,single CD44 aptamer,combination of single EpCAM and CD44 aptamer,and bispecific CD44-EpCAM-aptamer at 2nmole for one month.Bioluminescence imaging were detected with Xenogen IVIS100 imaging system to evaluate and quantitative analyze the metastasis of ovarian cancer.To elucidate the molecular mechanism underlying bispecific CD44-EpCAM aptamer-led inhibition in intraperitoneal tumor growth,we performed H&E staining and immunohistochemistry staining of Cleaved Caspase-3,Ki67,E-cadherin,N-cadherin and TUNEL on tumor implants collected from sacrificed mice.We collected all major organs including spleen,liver,kidney,heart,intestine and muscle from sacrificed mice and carried out H&E staining on these organs to assess the toxicity of bispecific CD44-EpCAM-Aptamer.Results:1.The expression of CD44 and EpCAM in human epithelial ovarian cancer tissues and the association of protein expression with clinicopathological features and the relevance between CD44 and EpCAM⑴The level of CD44 and EpCAM expression were increased in human epithelial ovarian cancer when compared with normal ovary.⑵The immunohistochemical expression of CD44 and EpCAM in human epithelial ovarian cancer significantly correlated with FIGO stage and tumor differentiation(histological grade),lymph node metastasis(P<0.05).However,CD44and EpCAM expression exhibited no relationship to age,histological type(P>0.05).⑶EpCAM protein level positively correlated with CD44 protein level,demonstrating a significantly positive association between the two molecules.2.Construction of a bispecific CD44-EpCAM-Aptamer and detection of binding capability,targeting specificity,serum stability⑴We fused single CD44 and EpCAM aptamers together with a 23bp double stranded RNA adaptor and purposely left 2-3 bases unpaired between adaptor and aptamer in order to give each single aptamer spatial space to form a 3-D structure.The molecular weight of fused aptamer is 54.4Kd,which is large enough to avoid rapid renal depletion,and thus possess an ideal bioavailability.⑵We next examined the binding of bispecific CD44-EpCAM aptamer by ELISA.The results suggest that bispecific CD44-EpCAM aptamer can efficiently bind both CD44 and EpCAM.⑶We incorporated 2’-fluoro-pyrimidines into entire RNA during the process of in vitro transcription,increased nuclease resistance and serum stability.Bispecific2’-F-CD44-EpCAM-Aptamer was incubated with final 50%human serum at 37°C for2~24h.Bispecific 2’-F-CD44-EpCAM-Aptamer kept its integrity without degradation for 6h,and almost 45%of aptamer still remained at 24h.⑷The results confirm target specificity of bispecific CD44-EpCAM-Aptamer with different high CD44 and EpCAM expression cell lines(OVCAR8,SKOV3,ES2,OCC1),and demonstrated that the binding of bispecific CD44-EpCAM-Aptamer to cells is indeed through CD44 and EpCAM molecules.⑸Bispecific CD44-EpCAM-Aptamer is not immunogenic and triggered no innate immune response.We treated human peripheral mononuclear cells with bispecific CD44-EpCAM aptamer for 24 h.ELISA showed that,at the concentration up to 8μM,there was no detectable elevation in the level of IFNαover the untreated control.3.The inhibitory effects of bispecific CD44-EpCAM-Aptamer on ovarian cancer cells and intraperitoneal xenograft in nude miceIn vitro:⑴The bispecific CD44-EpCAM-Aptamer was effective in the ability to inhibit cell growth and specifically suppress ovarian cancer high CD44 and EpCAM expression cell growth.None of the aptamer affected growth of HEK293T cells,that low CD44 and EpCAM expression.⑵Western Blot showed that treatment of bispecific CD44-EpCAM aptamer led to the appearance of Cleaved Caspase-3(molecular weight 17Kd and 19Kd)in OVCAR8 and ES2 cells at 72h.Flow cytometry result showed that the population of late stage apoptotic cell population(Annexin V+/PI+)was 1.56%in control OVCAR8 cells,this population was increased to 18.89%in cells treated with 2μM bispecific CD44-EpCAM aptamer.Similarly,population of late apoptotic cells was increased from 2.54%in control to 19.58%in ES2 cells treated with 2μM bispecific CD44-EpCAM aptamer.Fluorescence microscope imaging demonstrated bispecific CD44-EpCAM-Aptamer treated OVCAR8 and ES2 cells have increased apoptosis signals of green(Annexin V)and red(PI).The bispecific CD44-EpCAM-Aptamer was effectively induce human ovarian cancer cells apoptosis in a dose-dependent manner.In vivo:⑴Bioluminescence imaging of whole body demonstrated the distribution of tumor cells and confirmed that tumor cells have spread on entire peritoneum after8-day implantation of OVCAR8-luc tumor cells.⑵Bispecific CD44-EpCAM-Aptamer has tumor targeting capability.Cy5-labeled bispecific CD44-EpCAM-Aptamer and Cy5-labeled non-targeting control MG aptamer were intraperitoneally injected to tumor bearing mice.After 4h aptamer injection,non-targeting aptamer has shown the decreased Cy5 fluorescence compared with bispecific CD44-Ep CAM-Aptamer,and was almost invisible post 8h injection,while bispecific CD44-EpCAM-Aptamer still kept strong Cy5 fluorescence at 8h.And bispecific CD44-EpCAM-Aptamer(Cy5)has greatly co-localized with spread tumors(bioluminescence).⑶The bispecific CD44-EpCAM aptamer suppressed intraperitoneal tumor outgrowth much more significantly than single CD44 and EpCAM aptamer either alone or in combination.The staining intensity of Cleaved Caspase-3 and TUNEL was much greater in bispecific CD44-EpCAM-Aptamer treated tumors than all other treatment groups,while the staining intensity of proliferation marker Ki67 was greatly decreased in bispecific CD44-EpCAM-Aptamer treated tumors.These results suggest that bispecific CD44-EpCAM-Aptamer led inhibition of intraperitoneal tumor progression results from triggering tumor cell apoptosis and inhibiting tumor cell proliferation.Upon treated with bispecific CD44-EpCAM-Aptamer,the expression of E-Cadherin are increased and N-Cadherin are reduced.That indicates that the inhibition of metastasis of CD44-EpCAM aptamer is also though reversal of EMT.⑷The nucleic acid-based bispecific CD44-EpCAM-Aptamer has no toxicity to the host and was well tolerated by the host.Conclusion:CD44 and EpCAM would be a promising joint molecular therapeutic target for ovarian cancer.The bispecific CD44-EpCAM-Aptamer is capable of blocking CD44and EpCAM simultaneously.Bispecific CD44-EpCAM-Aptamer was effectively inhibit human ovarian cancer cell growth and induce apoptosis,and suppressed intraperitoneal ovarian cancer tumor growth.Our study not only offers a promising therapeutic agent against advanced ovarian cancer but also provides a methodology to develop bispecific aptamers from individual single aptamers.