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Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

ABSTRACT: The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo. Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo. Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro. Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

SUBMITTER: Wu FH

PROVIDER: S-EPMC5667975 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPatients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA(+)CD44(+) phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC.Methods and findingsXenogeneic tumors initiated with CoCSC were allowed to reach approximately 400 mm(3), at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent.ConclusionsCoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy.

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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Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

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