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Spontaneously slow-cycling subpopulations of human cells originate from activation of stress response pathways

ABSTRACT: Slow-cycling subpopulations exist in bacteria, yeast, and mammalian systems. In the case of cancer, slow-cycling subpopulations have been proposed to give rise to drug resistance. However, the origin of slow-cycling human cells is poorly studied, in large part due to lack of markers to identify these rare cells. Slow-cycling cells pass through a non-cycling period marked by low CDK2 activity and high p21 levels. Here, we use this knowledge to isolate these naturally slow-cycling cells from a heterogeneous population and perform RNA-sequencing to delineate the transcriptome underlying the slow-cycling state. We show that cellular stress responses – the p53 transcriptional response and the integrated stress response – are the most salient causes of spontaneous entry into the slow-cycling state.

ORGANISM(S): Homo sapiens

PROVIDER: GSE122927 | GEO | 2019/02/28

REPOSITORIES: GEO

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Project description:SUMMARY: Dormant or slow cycling tumour cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. Since their isolation from human tumors has been a long-stalled technical challenge preventing a direct study of their functional properties, we have adapted the pulse-chase expression and labeling approach using H2BeGFP (Tumbar et al., 2004, DOI:10.1126/science.1092436) to a doxycycline-inducible all-in-one lentiviral vector. In this manner we were able to label and isolate by FACS live slow cycling cancer cells (SCCC) from human colorectal, glioma and melanoma patient-derived tumors as well as from minitumors grown in matrigel (SW1222-cells) or in sphere culture (e225, e216 glioma models). We determined the differentially expressed transcriptional profiles in paired comparisons of slow-cycling cancer cell (SCCC) versus rapid cycling cancer cell (RCCC) sub-populations. H2BeGFP-retention revealed slow cycling as a transient cell state rather than a cellular entity. SCCC showed cancer-initiation potential and enhanced chemoresistance. Cells in this slow cycling status presented a distinctive non-genetic and cell-autonomous gene expression profile shared across different tumour types. We identified TET2 epigenetic enzyme as key factor controlling SCCC numbers and survival. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labelled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We demonstrate the enhanced chemoresistance of SCCC, revealed 5hmC as a biomarker for their clinical identification, and identified TET2 as a potential drug-target for SCCC elimination that could extend patients' survival.Experimental Design: Cells from human patient-derived xenografts (PDX) and human cancer cell lines were infected with a doxycycline (DOX)-inducible all-in-one lentivirus coding for the histone2B-eGFP fusion protein. The models were as following: T70 (colorectal cancer); MMPG3 and MMLN9 (melanoma); e225 and e216 (glioma) and human SW1222 colorectal cancer cell line as an infected pool as well as a clone (Clone2) and the following SW1222 derivate cell lines: SW1222-shCTRL (stable expression of nonsilencing shRNA), SW1222-shTET2 (stable expression of TET2-specific shRNA).H2BeGFP-infected cells were isolated and grown/propagated and injected subcutaneously in the flanks of immunocompromised NOD-Scid mice as described (Puig et al., Clin. Cancer Res. 2013; doi: 10.1158/1078-0432.CCR-12-1740.). H2BeGFP-infected SW1222 cells and derivate cell lines were grown in matrigel drops as minitumors. H2BeGFP-infected patient-derived melanoma MMPG3 and MMLN9 minitumor and glioma models e216 and e225 were grown as sphere cultures in their respective conditions as indicated.H2BeGFP expression was induced by DOX treatment in all experimental systems in a pulse-chase fashion and H2BeGFP fusion protein integrated into the host genome labeling all cells. Consecutive cell divisions of proliferating cells diluted and finally eliminated the fluorescent mark in an exponential decay fashion (RCCC or sRCCC), whereas slow-cycling cells (SCCC) retained the fluorescent stain, allowing FACS separation and isolation. Total RNA was separately extracted from both sub-populations of cells of each system and submitted to microarray analyses for characterization.

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Spontaneously slow-cycling subpopulations of human cells originate from activation of stress response pathways

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