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

A Framework for Evaluating Biomarkers for Early Detection: Validation of Biomarker Panels for Ovarian Cancer

A panel of biomarkers may improve predictive performance over individual markers. Although many biomarker panels have been described for ovarian cancer, few studies used pre-diagnostic samples to assess the potential of the panels for early detection. We conducted a multi-site systematic evaluation of biomarker panels using pre-diagnostic serum samples from the Prostate, Lung, Colorectal, and Ovarian Cancer (PLCO) screening trial

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

Chemotherapy selectively enriches for self-renewing lung cancer cells.

<p>Parental H460 cells and DSCs (1000 cell/ml) were plated onto ultra low adherent plates in MC-based serum free media supplemented with growth factors and cultivated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003077#s2" target="_blank">Material and Methods</a>. Tumor spheres generated from single-cell suspension cultures of parental H460 cells and drug survived CSCs were counted after 3 weeks of culture (1 st generation), and then spheres were dissociated and replated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003077#s2" target="_blank">Material and Methods</a>. <i>A, Lung tumor spheres generated from single-cell cultures of parental H460 cells and DSCs, imaged on indicated day of culture. B, Maintenance of enhanced ability to form tumor spheres during several generations</i> of DSCs transfer (for comparison only 1-st and 5-th generation's data are presented). C, <i>Immunofluorescent images of lung tumor spheres stained for CD133, CD117 and TRA-1-81 (10X objective).</i></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

Expression of adhesion molecules, VLA-4(CD49d), VLA-5(CD49e), VLA-6(CD49f), by H460 cells and DSCs.

<p>Cells were incubated with Abs against VLA-4-FITC and VLA-6-PC5 or VLA-5-FITC and VLA-6-PC5. Cell images were acquired using the Cellomics ArrayScan HCS Reader (20X, 40X objectives) and analyzed using the Target Activation BioApplication Software Module. <i>A, Immunofluorescent images of VLA4/VLA6 (left) and VLA-5/VLA-6 (right) expression in H460 and DSCs cells (40X objective).</i> B-D, A<i>n average fluorescence intensity of VLA-4(B), VLA-5(C) and VLA-6(D) in H460 cells (black line) and DSCs (grey line).</i></p

Expression of growth factor and chemokine receptors in lung CSCs.

<p><i>A, B</i>, H460 cells and lung CSCs dissociated from spheres were plated into 96-well plates precoated with Collagen IV and cultured 8 h. Then adherent cells were immunofluorescently stained for CXCR4 (SDF-1 receptor); images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. A. <i>Immunofluorescent images of CXCR4 in H460 and CSCs cells. B. Fluorescence intensity (pix) of CXCR4 is plotted against object area. C. Expression of growth factor and chemokine receptors in lung CSCs growing in tumor spheres.</i> Lung tumor spheres were immunofluorescently stained for VEGFR1; FGFR2, CXCR1 and CXCR4 receptors; images were acquired using the Cellomics ArrayScan HCS Reader (10X objective). Immunofluorescent images of lung tumor spheres stained for VEGFR1, FGFR2, CXCR1 and CXCR4 are presented.</p

Multiplex analysis of cytokines.

<p><i>A, In vitro cytokine production by CSCs and parental human tumor H460 cells.</i> Cells were cultivated in 96-well plates for 24 h in complete RPMI 1640 medium; samples of conditioned media were collected. Cells were fixed, stained with Hoechst 33342, and cell numbers were determined using image cytometry. Concentrations of human cytokines, chemokines, growth factors, MMPs, adhesion molecules and cancer antigens were analyzed using Luminex technology. Concentrations of cytokines pg/10⁶ cells/ml were calculated. Only factors with significant differences in their concentrations are presented. B, <i>Analysis of murine cytokines in extracts of xenografted parental H460 and CSCs-derived tumors.</i> SCID mice were inoculated s.c with 5×10⁵ of parental H460 or CSCs (5 mice per group). Samples of tumors, derived from parental H460 cells and CSCs, were sonicated, and concentrations of 19 murine cytokines in cellular extracts were measured using multiplexed cytokine assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003077#s2" target="_blank">Materials and Methods</a>. Only factors with significant differences in their concentrations (at least p<0.05) are included. Results are presented as pg or ng of cytokine per mg of total tumor protein.</p

Tumorigenic and metastatic properties of H460 cells and lung CSCs.

*<p>H460 cells and CSCs were injected s.c. into SCID mice at concentrations of 5×10³–5×10⁵ cells (in 200 µl PBS) per mouse. Mice were sacrificed when tumors reach 2 cm in diameter.</p>**<p>H460 cells and CSCs were inoculated i.v. into the tail vein of SCID mice (5×10⁴ tumor cells/mouse). After 60 days mice were sacrificed, lungs were removed and fixed in the Bouin's solution, and metastatic nodules were counted under a dissecting microscope.</p

Multiplex analysis of cytokines and growth factors 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 cytokines, chemokines, angiogenic and growth factors were analyzed using multiplex kits. Only factors with significant differences in their concentrations (at least p<0.05) are included.</p