Unknown,Transcriptomics,Genomics,Proteomics

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Gene Expression Profiles of tumor sorted fractions demarcating genetic instability using DNA-RNA content based cell cycle analysis in ovarian cancer xenograft tumor

ABSTRACT: Our earlier report states role of genetic instability and aneuploid cells in drug resistance and tumor recurrence. Hence segregating tumor based on DNA content is a good model to study genetic in stability. We have earlier reported isolation of cell line (A4T) from a patient with Grade IV serous adenocarcinoma (Bapat et al. Cancer Research. 2005). Xenograft generated by injecting A4T cells in NOD/SCID mice exhibited genetic instability (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). We generated xenograft by injecting 2.5x106 A4T P65 s.c in NOD/SCID mice. Mice were observed for tumor formation and tumors were harvested at day 28 (4 weeks) and processed for collagenase digestion for preparation of single cell suspension. Further cells were subjected to staining with Hoechst (33342) for 45min at 37ËC followed by pyronin Y for 45min at 37ËC and processed for FACS sorting. Staining profile was acquired on BD FACS Aria II across UV laser for Hoechst and PE channel for PyroninY with linear scale. Details of the Hoechst pyronin Y based ploidy and cell cycle analysis are published in our earlier reports (Kusumbe and Bapat Cancer Research. 2009; Naik et.al Oncogene 2015). DNA content of cell line injected is considered as euploid while the higher genetic content is called as aneuploid. Euploid and aneuploid were sorted with 99% purity and 1x105 cells from each fraction was subjected to RNA isolation, labeling and microarray analysis. RNA of each fraction was labeled with Cy3 and designated as A4Euploid and A4Aneuploid. Three samples of each were chosen for hybridization.

ORGANISM(S): Homo sapiens

SUBMITTER: Sharmila Bapat

PROVIDER: E-GEOD-74852 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Study question: Could extracellular vesicles (EVs) secreted by aneuploid embryos a) serve for development of RNA biomarkers for preimplantation genetic testing for aneuploidy (PGT-A) and b) elicit a relevant transcriptomic response in decidualised primary endometrial stromal cells (dESCs)? Summary answer: Aneuploid embryo EVs a) contain genes PPM1J, LINC00561, ANKRD34C and TMED10 in differential abundance from euploid EVs and b) induce up-regulation of MUC1 transcript in dESCs. What is known already: PGT-A is popular as it is thought to facilitate transfer of euploid embryos to increase implantation probability but the technology is controversial, as it requires an invasive embryo biopsy with an elusive long-term biosafety. The development of non-invasive methods to screen out aneuploid embryos is paramount. It is also critical to decode the embryo-endometrial dialog underlying implantation failure. We have previously reported that IVF embryos secrete EVs that can be internalised by ESCs, conceptualising that successful implantation to the endometrium is facilitated by EVs, which may additionally serve as biomarkers of ploidy status. Study design, size, duration: Embryos destined for biopsy on days 5-7 for PGT-A were grown under standard conditions. Spent media (30μl) were collected from euploid (n=175) and aneuploid (n=140)embryos at cleavage (days 1-3) stage and from euploid (n=187) and aneuploid (n=142) embryos at blastocyst (days 3-5) stage. Media samples from n=35 cleavage embryos were pooled in order to obtain five euploid and four aneuploid pools. Similarly, media samples from blastocyst were pooled to create one euploid and one aneuploid pool. ESCs were obtained from five women undergoing diagnostic laparoscopy. Participants/materials, setting, methods: EVs were isolated from pools of media derived from euploid and aneuploid day 3 embryos with differential centrifugation and EV-RNA sequencing was performed following the SMARTer Stranded Total RNA-Seq approach. ESCs were decidualised (E2:10nM, P4:1uM, cAMP:0.5 mM twice every 48 hours) and treated for 24 hours with 50 ng/ml euploid or aneuploid EVs extracted from blastocyst media. RNA sequencing was performed on ESCs following the Illumina Truseq RNAseq protocol. Main results and the role of chance: Aneuploid cleavage stage embryos secreted EVs that were less abundant in RNA fragments originating from the genes PPM1J (log2fc=-5.13, p=0.011), LINC00561 (log2fc=-7.87, p=0.010) and ANKRD34C (log2fc=-7.30, p=0.017) and more abundant in TMED10 (log2fc=1.63 p=0.025) compared to EVs from euploid embryos. Decidualisation per se induced downregulation of MUC1 (log2FC=-0.54, p=0.0028) in ESCs as prerequisite for the establishment of receptive endometrium. The expression of MUC1 transcript in decidualised ESCs was significantly increased following treatment with aneuploid compared to euploid embryo-secreted EVs (log2FC=0.85, p=0.0201). Limitations, reasons for caution: The findings of the study may require validation utilising a second cohort of EVs samples. Wider implications of the findings: The discovery that the RNA cargo of EVs secreted from aneuploid cleavage stage embryos is diverse from that of euploid embryos potentiates the development of non-invasive methodology for PGT-A. The upregulation of MUC1 in dESCs following aneuploid embryo EV treatment proposes a new mechanism underlying implantation failure. Funding: The study was supported by a MSCA fellowship awarded to SM by the European Commission (CERVINO grant agreement ID: 79620) and by a BIRTH research grant from Theramex HQ UK Ltd

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Gene Expression Profiles of tumor sorted fractions demarcating genetic instability using DNA-RNA content based cell cycle analysis in ovarian cancer xenograft tumor

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