Drug-Selected Human Lung Cancer Stem Cells: Cytokine Network, Tumorigenic and Metastatic Properties

Background: Cancer stem cells (CSCs) are thought to be responsible for tumor regeneration after chemotherapy, although direct confirmation of this remains forthcoming. We therefore investigated whether drug treatment could enrich and maintain CSCs and whether the high tumorogenic and metastatic abilities of CSCs were based on their marked ability to produce growth and angiogenic factors and express their cognate receptors to stimulate tumor cell proliferation and stroma formation. Methodology/Findings: Treatment of lung tumor cells with doxorubicin, cisplatin, or etoposide resulted in the selection of drug surviving cells (DSCs). These cells expressed CD133, CD117, SSEA-3, TRA1-81, Oct-4, and nuclear β-catenin and lost expression of the differentiation markers cytokeratins 8/18 (CK 8/18). DSCs were able to grow as tumor spheres, maintain self-renewal capacity, and differentiate. Differentiated progenitors lost expression of CD133, gained CK 8/18 and acquired drug sensitivity. In the presence of drugs, differentiation of DSCs was abrogated allowing propagation of cells with CSC-like characteristics. Lung DSCs demonstrated high tumorogenic and metastatic potential following inoculation into SCID mice, which supported their classification as CSCs. Luminex analysis of human and murine cytokines in sonicated lysates of parental- and CSC-derived tumors revealed that CSC-derived tumors contained two- to three-fold higher levels of human angiogenic and growth factors (VEGF, bFGF, IL-6, IL-8, HGF, PDGF-BB, G-CSF, and SCGF-β). CSCs also showed elevated levels of expression of human VEGFR2, FGFR2, CXCR1, 2 and 4 receptors. Moreover, human CSCs growing in SCID mice stimulated murine stroma to produce elevated levels of angiogenic and growth factors. Conlusions/Significance: These findings suggest that chemotherapy can lead to propagation of CSCs and prevention of their differentiation. The high tumorigenic and metastatic potentials of CSCs are associated with efficient cytokine network production that may represent a target for increased efficacy of cancer therapy. © 2008 Levina et al

Immunological and Nonimmunological Effects of Indoleamine 2,3-Dioxygenase on Breast Tumor Growth and Spontaneous Metastasis Formation

The role of the tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO1), in tumor escape and metastasis formation was analyzed using two pairs of Ido1+ and Ido1− murine breast cancer cell lines. Ido1 expression in 4T1 cells was knocked down by shRNA, and Ido1 expression in NT-5 cells was upregulated by stable transfection. Growth of Ido1− tumors and spontaneous metastasis formation were inhibited in immunocompetent mice. A higher level of cytotoxic T lymphocytes was generated by spleen cells from mice bearing Ido1− tumors than Ido1+ tumors. Tumor and metastatic growth was enhanced in immunodeficient mice, confirming an intensified immune response in the absence of Ido1 expression. However, Ido1+ tumors grow faster than Ido1− tumors in immunodeficient SCID/beige mice (lacking T, B, and NK cells) suggesting that some Ido1-controlled nonimmunological mechanisms may be involved in tumor cell growth regulation. In vitro experiments demonstrated that downregulation of Ido1 in tumor cells was associated with decreased cell proliferation, increased apoptosis, and changed expression of cell cycle regulatory genes, whereas upregulation of Ido1 in the cells had the opposite effects. Taken together, our findings indicate that Ido1 expression could exert immunological and nonimmunological effects in murine breast tumor cells

Multiplex analysis of adhesive molecules, MMPs and cancer antigens in the lysates of xenografted parental H460 and CSC-derived tumors.

<p>Sonicated extracts were prepared from H460- and CSC-derived tumors growing in SCID mice (5 tumors per group) and concentrations of various tumor-producing factors were analyzed using multiplex kits. Only factors with significant differences in their concentrations (at least p<0.05) are included.</p

Analysis of β-catenin intracellular distribution in H460 cells and DSCs.

<p>Cells were fixed and incubated with Alexa Fluor® 488 phalloidin or with primary Abs against β-catenin and with secondary Alexa Fluor 488 conjugated Abs. Next cells were stained with Hoechst33342. Cell images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Compartment Analysis BioApplication Software Module and the Target Activation BioApplication Software Module. <i>A, Images of H460 cells and DSCs immunofluorescently stained for β-catenin (A).</i> B, <i>An average fluorescence intensity of nuclear β-catenin in H460 (black line) and DSCs (grey line)</i>.C, <i>An average fluorescence intensity of cellular phosphor- β-catenin in H460 (black line) and DSCs (grey line). D, Cytoskeleton images of H460 cells and DSCs immunofluorescently stained for phalloidin and Hoechst33342.</i></p