Genetic And Biological Characteristics Of Subclones Isolated From A2780and SKOV3Cell Lines Of Human Ovarian Cancer By Distinct Invasion Capacity

BackgroundsUnequivocally, ovarian cancer is the most lethal gynecologic malignancy, and metastasis and recurrence were the leading cause of death for patients with such disease.The concept of intratumoral heterogeneity has been blamed for treatment failure in ovarian carcinoma and other malignancies. The divergence of cell subpopulations in tumor confers a heterogeneous genomic tapestry with implications for clinical prognosis, including the acquisition of metastatic potential and resistance to chemotherapy. Next-generation sequencing technology has enabled the study and quantification of the molecular diversity in cancer.Objectves1To isolate and single out the single-cell subclones from A2780and SKOV3cell lines of human ovarian cancers by distinct invasion capacity;2To test and identify the disparities of invasiveness and migratory capacity between the targeted subclones by various vitro and vivo experiments;3To investigate the disparities of genetic mutation between the targeted subclones by exome Seq and RNA Seq.Materials and Methods1By limiting dilution method, the cell suspension of A2780and SKOV3was diluted into one cell per well and cultured in96-well plate respectively. The cells were cultured under the routine conditions. Only the single-cell subclones were reserved and amplified. Transwell insert assay+MTT assay was repeatedly performed to detect disparities of invasive and migratory capacity among the subclones which were cultured and amplified successfully. According to the result, the subclones with highest and lowest metastatic potential and with relative stable biological properties from A2780and SKOV3were named as A-H、A-L、S-H and S-L respectively.2The four subclones, A-H/A-L and S-H/S-L, were used as the subjects for all the following experiments. MTT assay and flow cytometry (FCM) were adopted to detect the difference of cell proliferation and cell cycle distribution between A-H/S-H and S-H/S-L. FCM was performed to detect the apoptosis rate in the targe subclones. Real-time PCR and western blot were used to assay the expression of Bcl-2、caspase3/7, at both mRNA and protein levels. Then the anti-apoptosis ability was evaluated by this way. The expression of TrkB, related to anoikis and Beclinl, related to autophagyat both mRNA and protein levels was also tested to compare the disparities of ability of anti-anoikis and activity of autophagy. MTT assay was used to detect the sensitivity of the targeted subclones to cisplatin and taxol. The targeted subclones were respectively injected subcutaneouslyin nude mice, in order to compare their ability in tumor formationin vivo.3The karyotype of the targeted subclones was analyzed. Chromosome preparations were examined afterboth conventional Giemsa staining. Exome Seq and RNA Seq were conducted in the targeted subclones.Results1By limiting dilution method and under the routine culture conditions, a total of45subclones amplified from single cell were successfully obtained fromA2780. For SKOV3, there were40subclones acquired in the end. The results of Transwell insert assay+MTT assay proved the existence of heterogeneous subpopulations within one tumor cell line. A-H/S-H had significantly higher invasiveness and migratory capacity than A-L/S-L.2Compared to their corresponding counterparts, A-H and S-H showed significantly stronger ability of proliferation, anti-apoptosis and anti-anoikis. Their autophagy activity was suppressed. Lower sensitivity to chemotherapy and higher capacity in tumor formation in vivo were presented simultaneously.3At chromosomal level, the targeted subclones also displayed a significant heterogeneity between the corresponding counterparts, yet homogeneity was presented in their interior. After the progressive selection and filtering of the data obtained from exome sequencing, a total of66gene mutation sites was acquired including57Indels and9SNPs. Ten of them was selected randomly for further validation by Sanger sequencing. In the end, CNTNAP1was found a mutation in5’-UTR region in A-H and S-H, yet negative mutation in A-L and S-L. Several reports in the literature had showed that the variants and abnormal expression of CNTNAP1were associated to tumorigenesis and tumor progression. The result of RNA Seq showed that a total of25different genes were found including20upregulating in the transcriptomes of A-H and S-H, and5upregulating in the transcriptomes of A-L and S-L (P<0.05, Fold change>2). The activation of PI3K/AKT/mTOR pathway and the abnormal expression of the related genes were validated in the targeted subclones by real time PCR and western blot.Conclusions1There are heterogeneous subpopulations with distinct invasive capacity co-existing within the tumor cell lines of human ovarian cancer.2A-H/S-H andA-L/S-L are relative ideal modes to investigate the development of ovarian carcinoma.3The subclones with higher invasive capacity show significantly stronger independen viability, which possibly play more important role in the tumorigenesis, tumor metastasis and resistence to chemotherapy.4The mutation of CNTNAPland the activation of PI3K/AKT/mTOR pathway are possibly associated with the higher invasive capacity of subpopulations in the cell lines of human ovarian cancer.