Quantitative PCR results of <i>NANOG1</i> and <i>NONOGP8</i>.

<p>Compared to the cells under normoxia, there are elevated <i>NANOG1</i> and <i>NONOGP8</i> expressions in the cells under 7% O₂ for both cell lines, with up to 1.8-fold increase <i>NANOG1</i> expression and up to 2.5-fold <i>NONOGP8</i> increase in both cell lines; the cells under 1% O₂ express even higher levels of <i>NANOG1</i> and <i>NONOGP8</i>, with 2.6-fold and 3.1-fold increase in <i>NANOG1</i> expression in the PC-3 and DU145 cell lines, respectively, and with 6-fold and 10-fold increase in <i>NONOGP8</i> expressions in the PC-3 and DU145 cell lines, respectively.</p

Hypoxic effects on cell proliferation, colony formation and cell cycle.

<p>(A) Cell proliferation curves show no statistical difference for cell growth under different oxygen tensions (<i>P>0.05</i>). (B) Colony formation assay for both cell lines shows more colonies in the cells pre-treated at 7% O₂ for 48 hours and even more colonies in the cells pre-treated under 1% O₂ (C) Histograms for the colony formation efficiency shows statistically higher efficiency in the cells pre-treated under hypoxia (7% or 1% O₂) for both cell lines (<i>P</i><0.0001). (D) Flow cytometry shows extended G₀/G₁ stage for both cell lines which have been cultivated under 1% O₂ for 48 hours, in comparison to the cells always kept under normoxia. (E) Statistical analyses reveal significantly extended G₀/G₁ stage in the cells cultivated under hypoxia for both cell lines (<i>P</i><0.05).</p

CD44^bright cells are mainly positive for ABCG2, Oct3/4 and Nanog.

<p>(A) Double staining of CD44 and ABCG2 surface markers with flow cytometry assay shows higher levels expressions of these factors in both cell lines under hypoxia for 48 hours. (B) The CD44^bright cells under normoxia express higher levels of ABCG2, Oct3/4 and Nanog, but the CD44^dim cells under the same normoxia condition express very low levels of these factors. The CD44^bright cells pretreated under 1% O₂ for 48 hours show even higher levels of these factors compared to the CD44^bright cells cultivated under normoxia.</p

CD44^bright cells show stem-like properties.

<p>(A) For both PC-3 and DU145 cell lines, more colonies are shown in the CD44^bright cells than the CD44^dim cells under nomoxia condition, while even more colonies can be seen in the CD44^bright cells pretreated under 1% O₂ for 48 hours. (B) Histograms for the colony formation efficiency show statistically significantly higher efficiency in the CD44^bright cells than the corresponding CD44^dim cells (<i>P</i><0.0001 for both cell lines), and even higher level efficiency in the hypoxia- pretreated CD44^bright cells in comparisons with the CD44^bright cells without hypoxic pretreatment (<i>P</i><0.05 for both cell lines). (C) Sphere formation assay shows spheres in the CD44^bright cells in both normoxia and hypoxic pretreated cells while there is no qualified sphere in the corresponding CD44^dim cells for both cell lines. (D) Histograms show sphere formation efficiency in the CD44^bright and CD44^dim cells with and without hypoxia pretreatment.</p

Stem-like phenotype analyses by flow cytometry.

<p>PC-3 and DU145 cells were cultivated in 1% O₂ and 20% O₂ coditions for 48 hours to analyze stem-like phenotype through flow cytometry assay. (A) The representative images show that side population cells were induced in both cell lines after hypoxic treatment. (B) Statistic analyses show significant difference in side population. (C) Flow cytometry analyses show higher levels of ABCG2 expression intensity in both cell lines under hypoxia. (D) Histogram shows statistically significant difference in ABCG2 expression. (E) Flow cytometry analyses show higher CD44 expression intensity in both cell lines under hypoxia. (F) Histogram shows statistically significant difference in CD44 expression.</p

Hypoxia increases the expressions of Oct3/4 and Nanog.

<p>(A) In comparison to the cells cultivated at 20% O₂, the cells cultivated at 7% O₂ show higher levels of Oct3/4 and Nanog expressions, and the cells cultivated at 1% O₂ show the highest levels of Oct3/4 and Nanog expressions in PC-3 and DU145 cell lines by both RT-PCR and immunoblotting. (B) Immunocytochemistry reveals corresponding higher levels of Oct3/4 and Nanog expressions at 7% O₂ and the highest levels of Oct3/4 and Nanog expressions at 1% O₂, in comparison to the cells cultivated at 20% O₂ for both cell lines. Human seminoma tissue sections were used as positive controls for these two antibodies. All photos were originally taken at 200× and all the insets were taken at 400×.</p

Hypoxia increases the expressions of HIF-1α and HIF-2α.

<p>(A) In comparison to the cells cultivated at 20% O₂, the cells cultivated at 7% O₂ show higher levels of HIF-1α and HIF-2α, and the cells cultivated at 1% O₂ show the highest levels of HIF-1α and HIF-2α by both RT-PCR and immunoblotting. (B) Immunocytochemistry reveals higher levels of HIF-1α and HIF-2α expressions in the cells cultivated under 7% O₂ and the highest levels of HIF-1α and HIF-2α expressions in the cells cultivated under 1% O₂ for both cell lines. The breast carcinoma sections were used as positive controls for both HIF-1α and HIF-2α. All photos were originally taken at 200× and all the insets were taken at 400×.</p

Identification and Characterization of Cells with Cancer Stem Cell Properties in Human Primary Lung Cancer Cell Lines

<div><p>Lung cancer (LC) with its different subtypes is generally known as a therapy resistant cancer with the highest morbidity rate worldwide. Therapy resistance of a tumor is thought to be related to cancer stem cells (CSCs) within the tumors. There have been indications that the lung cancer is propagated and maintained by a small population of CSCs. To study this question we established a panel of 15 primary lung cancer cell lines (PLCCLs) from 20 fresh primary tumors using a robust serum-free culture system. We subsequently focused on identification of lung CSCs by studying these cell lines derived from 4 representative lung cancer subtypes such as small cell lung cancer (SCLC), large cell carcinoma (LCC), squamous cell carcinoma (SCC) and adenocarcinoma (AC). We identified a small population of cells strongly positive for CD44 (CD44^high) and a main population which was either weakly positive or negative for CD44 (CD44^low/−). Co-expression of CD90 further narrowed down the putative stem cell population in PLCCLs from SCLC and LCC as spheroid-forming cells were mainly found within the CD44^highCD90⁺ sub-population. Moreover, these CD44^highCD90⁺ cells revealed mesenchymal morphology, increased expression of mesenchymal markers <i>N-Cadherin</i> and <i>Vimentin</i>, increased mRNA levels of the embryonic stem cell related genes <i>Nanog</i> and <i>Oct4</i> and increased resistance to irradiation compared to other sub-populations studied, suggesting the CD44^highCD90⁺ population a good candidate for the lung CSCs. Both CD44^highCD90⁺ and CD44^highCD90⁻ cells in the PLCCL derived from SCC formed spheroids, whereas the CD44^low/− cells were lacking this potential. These results indicate that CD44^highCD90⁺ sub-population may represent CSCs in SCLC and LCC, whereas in SCC lung cancer subtype, CSC potentials were found within the CD44^high sub-population.</p> </div

Comparison of colony forming potential of different cell sub-populations from PLCCLs by colony formation efficiency assays.

<p><b>A.</b> Comparison of FACS sorted CD44high and CD44low/− cells from the LCC cell line LC006. Cells were seeded at 200 cells per well and grown in standard 6 well plates for ∼10 days. Upper wells: CD44high cells. Lower wells: CD44low/− cells. <b>B.</b> Comparison of FACS sorted CD44highCD90+ and CD44highCD90− cells from the SCLC cell line LC004 (upper plate) and the LCC cell line LC006 (lower plate). Cells were seeded at 200 cells per well and grown in standard 6 well plates for ∼10 days. In each 6-well plate: upper wells: CD44highCD90+ cells; lower wells: CD44highCD90− cells. X denotes empty wells.</p

Comparison of morphological and phenotypic features between the PLCCLs and corresponding tumor tissues.

<p><b>A.</b> H&E staining of the 4 representative primary lung cancer cell lines for different lung cancer subtypes: the SCLC cell line LC004; the LCC cell line LC006; the AC cell line LC007 and the SCC cell line LC021. The cells derived from the four subtypes of lung cancer showed heterogeneity in cellular and nuclear morphology. Cells of each primary cell line had epithelial morphology. Photomicrograph magnification, ×200. <b>B.</b> Analysis the expression of P53, Ber-EP4 and CD44 in the 4 primary cell lines and their corresponding archival patients' tumor tissues. All the primary cell lines investigated showed diffuse positive staining for P53 except the original SCC tissue of cell line LC021. Epithelial membrane antigen Ber-EP4 was widely positive in all the original lung cancer tissues and diffuse positive in all the primary cell lines. CD44 showed diffuse positive staining with different intensity in the primary cell lines and their corresponding archival patients' cancer tissues except weakly positive in the original tumor tissue of the AC cell line LC007. Sections from paraffin blocks containing human seminoma and breast cancer specimen were used as positive controls for antibodies P53, Ber-EP4 and CD44, respectively. Photomicrograph magnification, ×200.</p