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

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

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

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

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

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

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

Increased expression of growth factor receptors (VEGFR1, FGFR2,) in lung CSCs.

<p>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 incubated with FITC-conjugated Abs against FGFR2, VEGFR1 and VEGFR2 fixed and stained with Hoechst 33342. Images were acquired using the Cellomics ArrayScan HCS Reader (20X objective) and analyzed using the Target Activation BioApplication Software Module. <i>A, Immunofluorescent images of VEGFR1 and FGFR2 in H460 and CSCs cells (20X objective). B, Fluorescence intensity (pix) of VEGFR1 and FGFR2 plotted against object area.</i> Each point represents a single cell. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003077#pone-0003077-g008" target="_blank">figures 8</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003077#pone-0003077-g009" target="_blank"></a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003077#pone-0003077-g010" target="_blank">10</a> red lines show the boundaries of the fluorescence intensity of H460 cells.</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

In vitro differentiation potential of lung cancer sphere cells and drug resistance of CSCs.

<p><i>A, Loss of stem cell marker (CD133) and increase of differentiation markers (CK8/18) by lung CSCs differentiated progenitors.</i> Parental H460 cells and CSCs from tumor spheres were seeded in collagen coated well plates and cultured for 3 weeks in complete RPMI 1640 medium supplemented with 10% FBS. Upper row - cell images in phase –contrast microscopy; in the middle - cells immunofluorescently stained for CD133 and bottom row - cells immunofluorescently stained for CK 8/18. <i>B, Self-renewing ability of differentiated lung cancer cells treated with cisplatin</i>. Relative % of cells generated tumor spheres from single-cell suspension cultures of drug selected CSCs, cells differentiated during 3 weeks and Progenitors of CSCs differentiated for 3 weeks were treated with cisplatin (1 µM) for two days. Surviving cells were transferred into low adherent plates and cultured in semisolid serum free medium supplemented with growth factors. Numbers of formed tumor spheres were determined and presented as percent of control. Control is number of spheres formed by transfer of cells derived from control tumor spheres. Number of these spheres is accepted as 100 %. <i>C, Effect of cisplatin and doxorubicin on proliferation of parental H460 cells, CSCs and their differentiated cells</i>. H460, lung CSCs and differentiated cells were plated in 96-well plates precoated with Collagen at 1×10⁴ cells/well in complete RPMI 1640 medium with 10% FBS. After 24 h doxorubicin or cisplatin was added at the indicated concentrations. Cells were cultured for 72 h, fixed, stained with Hoechst 33342 (2 µg/mL), and counted using the Cellomics ArrayScan HCS Reader.</p