Aptamer Selection Against MCF-7 Cells And Applications For Molecular Subtyping Of Breast Cancer And Biomarker Discovery

Precise molecular subtyping of breast cancer plays a pivotal role in the personalized treatment of breast cancer.Currently,the molecular subtyping of breast cancer based on the differential expressions of biomarkers in the tumor tissues is not very accurate and can no longer meet the requirement of breast cancer management.Such challenges are mainly focused on two aspects: on one hand,the widely employed gold standard technique for molecular subtyping of breast cancer is immunohistochemistry(IHC),which is complex,costly,time-consuming,labor-consuming and heavily dependent on the experience of operators,and thus greatly affects the accuracy of diagnosis and is prone to misdiagnosis or missed diagnosis;on the other hand,there is a lack of specific tumor biomarkers for precise molecular subtyping of breast cancer.Therefore,it is in need to discover new biomarkers with higher specificity for deeper and more precise molecular subtyping of breast cancer.As an emerging nucleic acid based molecular probe,aptamer is a short,single-stranded nucleic acid sequence screened by SELEX technology in vitro,whose functions are very similar to antibodies.Compared with antibodies,aptamer has a couple of preferable advantages such as wider-range of targets,higher specificity and binding affinity,lower cost,easier modification,higher stability and non-immunogenicity.Aptamer selection using cells as the targets also known as Cell-SELEX can obtain aptamers without prior knowledge of the specific molecular target on the cell surface,which can be used as molecular probes or recognition ligands for differential diagnosis and targeted therapy of corresponding diseases,and also can be used for the identification of biomarkers on the cell surface,thus discovering new biomarkers.Therefore,in this paper,an improved Cell-SELEX technology with multiple counter selection using several kinds of control cells rather than using a single control cell in traditional Cell-SELEX was conducted,which chose typical Luminal A breast cancer cells MCF-7 as the target cells,HER2-positive breast cancer cells SK-BR-3,triple negative breast cancer cells MDA-MB-231 and human normal mammary epithelial cells MCF-10 A as the control cells to screen specific DNA aptamers against MCF-7 cells,with expectation to provide a new molecular probe for the precise molecular subtyping of breast cancer and then identify the molecular target of the aptamer to provide a new specific biomarker and therapeutic target for the high-efficient and precise molecular subtyping and targeted therapy of breast cancer.The main contents of this paper are as follows:1.Aptamer selection against MCF-7 cells and their identificationA Cell-SELEX technology with multiple counter selection was conducted using typical Luminal A breast cancer cells MCF-7 as the target cells,HER2-positive breast cancer cells SK-BR-3,triple negative breast cancer cells MDA-MB-231 and human normal mammary epithelial cells MCF-10 A as the control cells.After successive 17 rounds of screen,a well enriched secondary library specifically binding to MCF-7cells was obtained,which was then cloned and sequenced and a total of 10 aptamer candidates were obtained,among which four aptamer sequences were identified with well bind ability to the target MCF-7 cells.2.Characterization of aptamers and applications for molecular subtyping of breast cancerThe specificity and binding affinity of the above four aptamers were systematically investigated,and an aptamer named MF3 with the best specificity and favorable binding affinity was chosen for further applications,whose Kd value was82.25 ± 25.14 n M.Then,temperature and enzyme digestion on its binding ability to the target MCF-7 cells as well as its stability were studied.Aptamer MF3 exhibited similar binding ability to the target MCF-7 cells at 4 °C,25 °C and 37 °C;its molecular target on the cell membrane was demonstrated to be a membrane protein;showed well stability in the 1640 cell culture medium containing 10% FBS at 37 °C,which could be preserved in the medium for 4 h and is longer than that of the random library(2 h).Furthermore,the aptamer MF3 was applied for the differential diagnosis of breast cancer molecular subtypes.The results indicated that aptamer MF3 could differentiate:(1)MCF-7 cells from SK-BR-3 cells,MDA-MB-231 cells and MCF-10 A cells.Since those four cells belong to Luminal A,HER2-positive,triple-negative breast cancer cells and human normal mammary epithelial cells respectively,aptamer MF3 is endowed with the ability to specifically differentiate those three breast cancer subtypes and normal breast tissues;(2)xenografted tumors of MCF-7 cells from those of MDA-MB-231,SK-BR-3 cells and their tissue sections;(3)Luminal A clinical breast cancer tissues from Luminal B(HER2+),HER2-enriched and triple negative clinical breast cancer tissues,as well as para-carcinoma tissues and normal breast tissues.3.Aptamer truncation and applications for molecular subtyping of breast cancerAptamer MF3 was systematically truncated and optimized based on its secondary structure,and a truncated aptamer MF3 Ec with a length of 58 nt was obtained.Aptamer MF3 Ec was also able to distinguish Luminal A breast cancer from HER2-positive and triple-negative breast cancer,including cells,tissues,and xenografted tumors in animal models.In the clinical experiments using breast cancer tissue sections,MF3 Ec also showed similar differential ability among breast cancer molecular subtypes,para-carcinoma tissues and normal breast tissues to the original sequence MF3,which could distinguish Luminal A breast cancer tissue sections from Luminal B(HER2+),HER2-enriched,triple negative,para-carcinoma and normal breast tissue sections.Compared with MF3,aptamer MF3 Ec exhibited higher binding affinity to MCF-7 cells,whose binding affinity was about 4 times of that of MF3 with a low Kd value of 18.95 ± 2.90 n M.Secondly,MF3 Ec was more stable than MF3.MF3 Ec could be stably preserved in the 1640 cell culture medium containing 10%FBS at 37 °C for about 12 h,which was much longer than that of MF3 for about only4 h.Meanwhile,MF3 Ec also showed better tumor targeting ability and longer retention time in the tumor sites than MF3.MF3 Ec was able to stay at the tumor sites of MCF-7 cell-bearing mice for about 5 h,which is longer than that of MF3 for about3 h.In the clinical experiment using clinical breast cancer tissue sections,aptamer MF3 Ec showed better performance against Luminal A breast cancer tissue sections than MF3,the positive rate of Luminal A breast cancer tissue sections stained with MF3 Ec was as high as 81.82%,while the positive rate of those stained with MF3 was only 70%.Therefore,the truncated aptamer MF3 Ec has better performance than MF3 and is more potential for clinical applications.4.Aptamer-based biomarker discovery of breast cancerAptamer-based protein pull down technology was conducted to capture and purify the molecular target of aptamer MF3 Ec on the cell membrane of MCF-7 cells.Subsequently,LC-MS/MS,Western blot and RNAi technology were conducted and PHB2 protein expressed on the cell membrane was identified as the specific molecular target of aptamer MF3 Ec.PHB2 protein on the cell membrane may be a potential biomarker specifically expressed in Luminal A breast cancer tissues,which provides a new molecular biomarker and therapeutic target for the precise molecular subtyping and targeted therapy of clinical breast cancer.