Lung tumor spheres have high tumorigenic potential.

<p>Tumorigenic potential of tumor sphere cells is greater than monolayer cancer cells. Monolayer and tumor sphere cells from A549 were injected s.c. into athymic nude mice at concentrations of 1×10⁴ cells/ml. Mice were sacrificed at day 60, and tumor was surgically removed and measured, and the mean values of the tumor size were shown in the table.</p

Histopathologic examination of the engrafted tumors by H&E and β-Catenin staining.

<p>Surgically removed tumor was fixed in buffered formalin and subsequently analyzed by immunohistochemistry (IHC) by standard protocol.</p

Track CSCs in tumor sphere based on their proteasome activity.

<p><b>A.</b> Frequency of ZsGreen cells in A549 and H1299 monolayer cultures. <b>B.</b> Frequency of ZsGreen cells in A549 and H12199-derived tumor spheres with accumulation of ZsGreen-cODC, and thus low proteasome activity is indicated. <b>C.</b> Number of spheres formed from the ZsGreen positive and the ZsGreen negative cells after sorting with flow cytometry into 96-well plates. ZsGree-positive and –negative cells were sorted into 96-well plate (100cells/well), after cultured in tumor sphere media for 2 weeks; number of formed spheres was counted. Student's paired, and two-tailed t-tests were performed, ***p<0.001 (three replicates per experiment).</p

Lung tumor spheres exhibit cancer stem cell features.

<p><b>A.</b> Lung tumor spheres showed increased CD133 expression. Expression of CD44 and CD133 in monolayer and tumor sphere cells, as determined by two-color flow cytometry analysis. Isotype-matched monoclonal antibodies and a preimmune second antiserum were used as negative controls. The percentage of single- and double-positive cells are indicated. <b>B.</b> Lung tumor spheres showed increased OCT3/4 expression. An average fluorescence intensity of OCT3/4 in control (grey area), monolayer cells (dotted line) and tumor spheres (solid line) is shown. Percentages of positive cells are indicated in the table.</p

Decreased proteasome activities of the 26S proteasome in A549 and H1299 tumor spheres.

<p><b>A.</b> caspase-like, <b>B.</b> chymotrypsin-like, and <b>C.</b> trypsin-like activity of monolayer culture and tumor spheres from A549 and H1299 cells was measued over the indicated times. <b>D.</b> End point analysis (120 min) of 26S proteasome activities in monolayer cultures and tumor spheres. Mean ± SD is plotted and derived from three independent experiments. Student's paired, and two-tailed t-tests were performed, *p<0.05, **p<0.01, and ***p<0.001 (three replicates per experiment).</p

Chemoresistence of monolayer and tumor sphere.

<p><b>A.</b> cisplatin, <b>B.</b> doxorubicin resistance of monolayer culture and tumor spheres were analyzed. Cells dissociated from lung tumor spheres or monolayers were plated out in 96-well plate at 2×10⁴ cells/ml, 24 h later, media containing different concentrations of cisplatin or doxorubicin was added. After 48 h culturing, 10% Alamar Blue was added and read at 544/590 nm, the percent viability was calculated relative to cells not exposed to any chemotherapeutic drugs. The results represent the means ± SD.</p

SP and NSP cells form tumor spheres with equal capacity in human adenocarcinoma cell lines.

<p><b>A.</b> Characteristic Hoechst 33342 dye staining profile demonstrating the existence of SP (polygon gated) and NSP (ellipse gated) cells in human A549 and H1299 adenocarcinoma cells. Percentage of SP cells is indicated. Cells were stained with 5 mg/ml Hoechst33342 (HO), and control cells were also co-stained with 10 mM fumitremorgin C (FTC) to specify the SP cells. Cells were sorted using a Beckman MoFlo cytometer and data were annotated using FCS Express. <b>B.</b> Phase-contrast photographs of lung tumor spheres obtained from SP and NSP cells. SP and NSP cells (1000 cell/ml) were plated onto ultra low adherent flasks in serum free media supplemented with growth factors and cultivated as described in Material and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013298#s2" target="_blank">Methods</a>. <b>C.</b> Re-stain of SP and NSP cells from A549 and H1299 with Hoechst 33342 dye. Percentage of SP cells is labeled.</p

Top 30 significant prognostic KEGG pathways related to recurrence.

<p>Top 30 significant prognostic KEGG pathways related to recurrence.</p

Validation of the 51-gene signature in four independent datasets.

<p>Kaplan-Meier survival analysis was performed in low (<i>full red line</i>) and high (<i>dashed blue line</i>) risk patient groups defined by the 51-gene classifier. AUC for survival models based on stage (<i>dashed red line</i>) or 51-gene classifier (<i>full black line</i>) was also compared. The testing dataset GSE8894 do not have available stage information and all patients in the WUSTL dataset are stage IB. So the time dependent ROC using stage information in these two datasets could not be calculated; all set at 0.5 instead. Tick marks, patients whose data were censored at last follow-up.</p

Clinical summary of patients in the analyzed datasets.

<p>Clinical summary of patients in the analyzed datasets.</p