ABSTRACT
BACKGROUND: Isolation of colorectal cancer (CRC) cell populations enriched for cancer stem cells (CSCs) may facilitate target identification. There is no consensus regarding the best methods for isolating CRC stem cells (CRC-SCs). We determined the suitability of various cellular models and various stem cell markers for the isolation of CRC-SCs. METHODS: Established human CRC cell lines, established CRC cell lines passaged through mice, patient-derived xenograft (PDX)-derived cells, early passage/newly established cell lines, and cells directly from clinical specimens were studied. Cells were FAC-sorted for the CRC-SC markers CD44, CD133, and aldehyde dehydrogenase (ALDH). Sphere formation and in vivo tumorigenicity studies were used to validate CRC-SC enrichment. RESULTS: None of the markers studied in established cell lines, grown either in vitro or in vivo, consistently enriched for CRC-SCs. In the three other cellular models, CD44 and CD133 did not reliably enrich for stemness. In contrast, freshly isolated PDX-derived cells or early passage/newly established CRC cell lines with high ALDH activity formed spheres in vitro and enhanced tumorigenicity in vivo, whereas cells with low ALDH activity did not. CONCLUSIONS: PDX-derived cells, early passages/newly established CRC cell lines and cells from clinical specimen with high ALDH activity can be used to identify CRC-SC-enriched populations. Established CRC cell lines should not be used to isolate CSCs.
Subject(s)
Colorectal Neoplasms/pathology , Neoplastic Stem Cells/pathology , Primary Cell Culture/methods , Animals , Cell Proliferation , Cell Separation/methods , Coculture Techniques , HCT116 Cells , HT29 Cells , Humans , Hyaluronan Receptors/metabolism , Mice , Mice, Nude , Neoplastic Stem Cells/physiology , Spheroids, Cellular/metabolism , Spheroids, Cellular/pathology , Tumor Cells, CulturedABSTRACT
Rictor and its binding partner Sin1 are indispensable components of mTORC2 (mammalian target of rapamycin complex 2). The mTORC2 signaling complex functions as the regulatory kinase of the distinct members of AGC kinase family known to regulate cell proliferation and survival. In the early chemotaxis studies in Dictyostelium, the rictor's ortholog has been identified as a regulator of cell migration. How rictor regulates cell migration is poorly characterized. Here we show that rictor regulates cell migration by controlling a potent inhibitor of Rho proteins known as the Rho-GDP dissociation inhibitor 2 (RhoGDI2). On the basis of on our proteomics study we identified that the rictor-dependent deficiency in cell migration is caused by upregulation of RhoGDI2 leading to a low activity of Rac and Cdc42. We found that a suppression of RhoGDI2 by rictor is not related to the Sin1 or raptor function that excludes a role of mTORC2 or mTORC1 in regulation of RhoGDI2. Our study reveals that rictor by suppressing RhoGDI2 promotes activity of the Rho proteins and cell migration.
