CD44 is an adhesion molecule expressed in cancer stem-like cells. Here, we show that a CD44 variant (CD44v) interacts with xCT, a glutamate-cystine transporter, and controls the intracellular level of reduced glutathione (GSH). Human gastrointestinal cancer cells with a high level of CD44 expression showed an enhanced capacity for GSH synthesis and defense against reactive oxygen species (ROS). Ablation of CD44 induced loss of xCT from the cell surface and suppressed tumor growth in a transgenic mouse model of gastric cancer. It also induced activation of p38(MAPK), a downstream target of ROS, and expression of the gene for the cell cycle inhibitor p21(CIP1/WAF1). These findings establish a function for CD44v in regulation of ROS defense and tumor growth.

Here we report a microfluidics method to enrich physically deformable cells by mechanical manipulation through artificial microbarriers. Driven by hydrodynamic forces, flexible cells or cells with high metastatic propensity change shape to pass through the microbarriers and exit the separation device, whereas stiff cells remain trapped. We demonstrate the separation of (i) a mixture of two breast cancer cell types (MDA-MB-436 and MCF-7) with distinct deformabilities and metastatic potentials, and (ii) a heterogeneous breast cancer cell line (SUM149), into enriched flexible and stiff subpopulations. We show that the flexible phenotype is associated with overexpression of multiple genes involved in cancer cell motility and metastasis, and greater mammosphere formation efficiency. Our observations support the relationship between tumor-initiating capacity and cell deformability, and demonstrate that tumor-initiating cells are less differentiated in terms of cell biomechanics.cell mechanics | cytoskeleton | genomic profiling C ell deformability is commonly measured using magnetic twisting cytometry, particle tracking rheometry, optical tweezers, micropipette aspiration, atomic force microscope, and other derivative cell stretching or poking methods (1-4). Applications of these methods to stem cells have revealed the greater deformability of the cytoskeleton and nucleoskeleton in less differentiated cells, whereby deformability generally decreases during differentiation to mature cells (5-8). Research on cancer cell deformability has also consistently revealed that increased deformability is correlated with increased metastatic potential (9-15).Despite the success achieved using cell deformability measurements, isolation of cells with differential deformabilities remains a great challenge (10, 16). Microfabrication-assisted technology, using microscale arrays of round or rectangular posts, channels, or other simple patterns, has the potential to solve this problem (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). Here, we focused on the mechanical properties of cancer cells in designing a unique cell purification system for the purpose of generating subpopulations enriched in highly deformable cells. We used microfabrication technology and obtained a subpopulation of SUM149 breast cancer cells with stem-cell-like deformability and mammosphere formation capability.The separation device, a mechanical separation chip (MS-chip), employs artificial microbarriers in combination with hydrodynamic force to separate deformable from stiff cells (Fig. 1A). Both the microbarrier structures and the fluidic parameters are essential to the cell-enrichment process. The most notable feature of the device is the precise placement of the microbarriers to impede the passage of stiff cells. Published in vivo observations suggest that the minimum crossable barrier for cancer cells is ∼8 μm or less (28,29). Here, we took those barrier dimensions into account in designing the MSchip to separate cells based on perfusion through constrictions. As...

Similar to normal tissue stem cells, cancer stem cells (CSCs) are thought to be quiescent or slow-cycling and, thereby, insensitive to chemo-and radiotherapies. CD44, a cell surface component that interacts with the extracellular matrix, has been found to be highly expressed in CSCs of several solid tumors. However, the relevancy between CD44+ cells and slow-cycling cells and the underlying mechanisms for the emergence of CD44 + CSCs during tumorigenesis have not been elucidated. Here we show that a gastric gland residing at the squamo-columnar junction (SCJ) in normal mouse stomach contains CD44 + stem cell-like slow-cycling cells and that this characteristic CD44 + gland was expanded by prostaglandin E2 (PGE 2 ) and Wnt signaling in K19-Wnt1 ⁄ C2mE mouse, a genetic mouse model for gastric tumorigenesis. The analysis of three transgenic mouse lines, K19-Wnt1, K19-C2mE and K19-Wnt1 ⁄ C2mE, revealed that the expansion of CD44 + SCJ cells is triggered by PGE 2 -mediated signaling and is prominently enhanced by the addition of Wnt activation. Furthermore, each expanded CD44 + gland in gastric tumor of K19-Wnt1 ⁄ C2mE mouse contains a few BrdU label-retaining quiescent or slow-cycling cells, suggesting that the CD44 + SCJ cells in normal mouse are candidates for the cell-of-origin of gastric CSCs. These observations suggest that PGE 2 -mediated inflammatory signaling and Wnt signaling cooperatively trigger the expansion of CD44 + slow-cycling stem-like cells in SCJ, leading to development of lethal gastric tumors in mice. (Cancer Sci 2010; 101: 673-678) C D44, a major cell adhesion molecule, has been implicated in a wide variety of physiological processes and pathological processes, including lymphocyte homing, wound healing, and cell migration, as well as cancer cell growth and metastasis.(1,2) CD44 has been recently detected as a cell surface marker of cancer stem cells (CSCs) in several types of cancer.(3) Moreover, a CD44 + subpopulation of cells isolated from several gastric cancer cell lines has been reported to have stem cell-like properties, which are the capacities to self-renew and to differentiate.(4) However, the underlying mechanism by which CD44 + CSC-like subpopulations emerge in the process of tumor development is largely unknown. Cancer stem cells, also known as cancer-initiating cells, are responsible for tumor initiation and maintenance. CSCs possess the ability to drive tumor growth and are inherently resistant to chemo-and radiotherapies. Recent studies have revealed that melanoma-initiating cells (5) and leukemia-initiating cells are a slow-cycling or quiescent subpopulation.(6,7) These quiescent or slow-cycling properties are thought to be a major reason why CSCs are resistant to cancer therapies targeted at proliferating cells. It has been shown that cell adhesion molecules play an essential role in the quiescence of hematopoietic stem cells by regulating adhesion in the osteoblastic niche.(8) Therefore, we hypothesized that CD44, which is highly expressed in CSCs, is involved in the regul...

Invasiveness, cellular atypia, and proliferation are hallmarks of malignant gliomas. To effectively target each of these characteristics, it is important to understand their sequence during tumorigenesis. However, because most gliomas are diagnosed at an advanced stage, the chronology of gliomagenesis milestones is not well understood. The aim of the present study was to determine the onset of these characteristics during tumor development. Brain tumor-initiating cells (BTICs) were established by overexpressing H-Ras(V12) in normal neural stem/progenitor cells isolated from the subventricular zone of adult mice harboring a homozygous deletion of the Ink4a/Arf locus. High-grade malignant brain tumors were then created by orthotopic implantation of 10(5) BTICs into the forebrain of 6-week-old wild-type mice. Micewere killed every week for 5 weeks, and tumors were assessed for cellular atypia, proliferation, hemorrhage, necrosis, and invasion. All mice developed highly invasive, hypervascular glioblastoma-like tumors. A 100% penetrance rate and a 4-week median survival were achieved. Tumor cell migration along fiber tracts started within days after implantation and was followed by perivascular infiltration of tumor cells with marked recruitment of reactive host cells. Next, cellular atypia became prominent. Finally, mass proliferation and necrosis were observed in the last stage of the disease. Video monitoring of BTICs in live brain slices confirmed the early onset of migration, as well as the main cell migration patterns. Our results showed that perivascular and intraparenchymal tumor cell migration precede tumor mass formation in the adult brain, suggesting the need for an early and sustained anti-invasion therapy.