Table 1.
Summary of the primary cultures included in this study.
Table 2.
Antibodies used in flow cytometry analysis.
Figure 1.
Typical epithelial cells were obtained at high purity under the serum-free condition.
A. The SCLC cell line LC004; B. The LCC cell line LC006; C. The AC cell line LC007; D. The SCC cell line LC021. All the representative images were from primary cultured lung cancer cell lines at the second passage. Photomicrograph magnification, ×200.
Figure 2.
Comparison of morphological and phenotypic features between the PLCCLs and corresponding tumor tissues.
A. 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. 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.
Figure 3.
Identification of CD44high cells in the PLCCLs by flow cytometry analysis.
A small sub-population of CD44high cells could be identified in the SCLC cell line LC004 (6.63%) (A); the LCC cell line LC006 (2.79%) (B); the AC cell line LC007 (6.69%) (C) and the SCC cell line LC021 (14.95%) (D), respectively. Left panel: isotype control Ab; right panel: CD44 Ab. Data shown are from representative experiments (n>3).
Figure 4.
Comparison of colony forming potential of different cell sub-populations from PLCCLs by colony formation efficiency assays.
A. 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. 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.
Figure 5.
Flow cytometry analysis of CD44 and CD90 expression in the PLCCLs.
Staining by anti-CD44 FITC and anti-CD90 APC. All the cell lines showed heterogeneous staining and could be divided into 4 sub-populations: CD44highCD90+, CD44highCD90−, CD44low/−CD90+ and CD44low/−CD90−. For the SCLC cell line LC004 (A), the frequency of CD44highCD90+ cell was 16.6%, and the CD44highCD90− cells was 8.2%. For the LCC cells line LC006 (B), the frequency of CD44highCD90+ cells was 1.1%, the CD44highCD90− cells was 2.5%. For the AC cell line LC007 (C), the frequency of CD44highCD90+ cells was 9.4%, and the CD44highCD90− cells was 23.4%. For the SCC cell line LC021 (D), the frequency of CD44highCD90+ cells was 2.3%, the CD44highCD90− cells was 1.1%.
Figure 6.
Analysis of the expression of stem cell and EMT related genes in different sub-populations from the PLCCLs.
Detectable expression levels of the genes were found in all sorted sub-populations from the cell lines LC004 (A), LC006 (B) and LC021 (C). PCR reaction without template served as a negative control. The relative expression of target genes was related to the expression of PGK1 and normalized to the unsorted control cells. X axis shows the target genes, Y axis shows the relative expression level (RQ). The error bars reflect the variation within the triplicates, P<0.05. (B). The FACS sorted cell sub-populations derived from the cell line LC006 were cultured in the serum-free culture system for 1–2 weeks and revealed different cell morphology for different sorted sub-populations: (a) CD44highCD90+ cells; (b) CD44highCD90− cells; (c) CD44low/−CD90+ cells, and (d) CD44low/−CD90− cells. Photomicrograph magnification, ×200.
Figure 7.
Relative irradiation resistance of different sorted cell populations from the cell line LC006. A.
The relative resistance to irradiation of the FACS sorted CD44high and CD44low/− cells derived from the LCC cell line LC006 was compared in an X-ray radiation resistance assay. The inhibition ratios were compared among the different cell populations after irradiating the monolayer cultures at 1, 2, 3 and 4Gy. The CD44high cells displayed the higher resistance to X-ray radiation at each dose tested. P<0.01. B. relative resistance to irradiation of the four different FACS sorted cell populations derived from the LCC cell line LC006 was compared in an X-ray radiation resistance assay. Four populations: CD44highCD90+, CD44highCD90−, CD44low/−CD90+ and CD44low/−CD90− cells were sorted into 96-well plates at 500 cells per well in 10 replicates. The inhibition ratios were compared among the four sub-populations after irradiation of the monolayer layer of the cultures at 1, 2 and 4Gy. The CD44highCD90+ cells displayed the highest resistance to irradiation at 2 and 4G. P<0.01.