Journal: Molecular and Cellular Biology

Article Title: Human Marrow Stromal Cells Downsize the Stem Cell Fraction of Lung Cancers by Fibroblast Growth Factor 10

doi: 10.1128/MCB.00871-13

Figure Lengend Snippet: Additive FGF10 reduces the proportion of lung cancer stem cells. Cancer cells were cultured in the presence of recombinant FGF10 or vehicle (PBS [control]) for 10 days (A and B) or 48 h (C to E). (A and B) Sphere-forming assay of H1299 and A549 lung cancer cells. Representative spherical images of H1299 cells (A) and the number of formed spheres in a well (B) are shown. Scale bars, 200 μm. (C) OCT3/4 and SOX2 gene expression in H1299 and A549 cells. By quantitative RT-PCR, the levels of gene expression in FGF10-treated cells were analyzed relative to those in control cells. (D) Representative phase distribution of cell cycle with the percentage of H1299 lung cancer cells in the G0 phase. (E) Proportion of five cancer cells in the G0 phase. For panels D and E, after the FGF10 treatment, cancer cells were stained with 7-AAD–pyronin Y for the cell cycle analysis. Data are presented as the mean ± standard error per group (n = 5 for panel B and n = 3 for panels C and E). Asterisks indicate significant differences compared with the control vehicle.

Article Snippet: Using an ultra-low-attachment 96-well plate, 100 A549 cells were seeded and cultured in 100 μl of serum-free DMEM–F-12 containing 10 ng/ml human recombinant epidermal growth factor (EGF) (R&D Systems) and 10 ng/ml human recombinant basic fibroblast growth factor (bFGF) (R&D Systems) with 50 ng/ml recombinant human FGF10 (rFGF10) or an equal volume of vehicle (PBS) on day 1.

Techniques: Cell Culture, Recombinant, Expressing, Quantitative RT-PCR, Staining, Cell Cycle Assay

Journal: Molecular and Cellular Biology

Article Title: Human Marrow Stromal Cells Downsize the Stem Cell Fraction of Lung Cancers by Fibroblast Growth Factor 10

doi: 10.1128/MCB.00871-13

Figure Lengend Snippet: FGF signaling reduces the proportion of lung cancer stem cells. (A) Semiquantitative RT-PCR analysis of epithelial-specific FGF receptor isoforms FGFR1b and FGFR2b, in five cancer cells. (B) Semiquantitative RT-PCR analysis for FGFR1b-reacting FGFs (coded for by the FGF1, FGF2, FGF3, FGF7, FGF10, and FGF22 genes) in MSCs from three unrelated donors. For panels A and B, expression of β-actin mRNA (ACTB) was used as a control. (C and D) Functional analysis of recombinant FGF proteins. Cancer cells were cultured for 48 h with recombinant FGF10, FGF1, or vehicle (PBS [control]) and were stained with Hoechst 33342 to analyze the side population. A representative side population subset (SP) with its percentage in H1299 lung cancer cells (C) and proportions of five cancer cells in the side population (D) are shown. The cell proportion is reported as the mean percentage ± standard error per group (n = 3). Asterisks indicate significant differences compared with the control vehicle.

Article Snippet: Using an ultra-low-attachment 96-well plate, 100 A549 cells were seeded and cultured in 100 μl of serum-free DMEM–F-12 containing 10 ng/ml human recombinant epidermal growth factor (EGF) (R&D Systems) and 10 ng/ml human recombinant basic fibroblast growth factor (bFGF) (R&D Systems) with 50 ng/ml recombinant human FGF10 (rFGF10) or an equal volume of vehicle (PBS) on day 1.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Functional Assay, Recombinant, Cell Culture, Staining

Journal: Molecular and Cellular Biology

Article Title: Human Marrow Stromal Cells Downsize the Stem Cell Fraction of Lung Cancers by Fibroblast Growth Factor 10

doi: 10.1128/MCB.00871-13

Figure Lengend Snippet: MSCs expressing FGF10 accumulate in lung tumors. (A and B) Specimen from a 59-year-old woman with squamous lung cancer. (C and D) Specimen from an 81-year-old man with lung adenocarcinoma. (E and F) Specimen from a 59-year-old woman with squamous lung cancer. Lung specimens from lung cancer patients were stained with FITC- and PE-conjugated antibodies specific for CD90 (an essential marker for MSCs) and FGF10, respectively. Nuclei were counterstained with DAPI. Scale bars, 100 μm. The lung cancer regions and the normal lung regions are shown in panels A, C, and E and panels B, D, and F, respectively. In all panels, controls included the isotype-matched control antibodies (Ctrl Ab).

Article Snippet: Using an ultra-low-attachment 96-well plate, 100 A549 cells were seeded and cultured in 100 μl of serum-free DMEM–F-12 containing 10 ng/ml human recombinant epidermal growth factor (EGF) (R&D Systems) and 10 ng/ml human recombinant basic fibroblast growth factor (bFGF) (R&D Systems) with 50 ng/ml recombinant human FGF10 (rFGF10) or an equal volume of vehicle (PBS) on day 1.

Techniques: Expressing, Staining, Marker

Journal: Molecular and Cellular Biology

Article Title: Human Marrow Stromal Cells Downsize the Stem Cell Fraction of Lung Cancers by Fibroblast Growth Factor 10

doi: 10.1128/MCB.00871-13

Figure Lengend Snippet: MSCs produce FGF10 affecting lung cancer stem cells. (A) Western blot analysis of FGF10 in transwell-cocultured MSCs with H1299 or A549 lung cancer cells. Controls included MSCs cultured without lung cancer cells. MSCs were from three unrelated donors, and β-actin production (ACTB) was used as a control. (B and C) Functional analysis of FGF10 production. The side population was analyzed in cancer cells stained with Hoechst 33342 after the 48-h culture with conditioned medium of MSCs in the presence of anti-FGF10 neutralizing antibody or control antibody. A representative side population subset (SP) with the percentage in H1299 lung cancer cells (B) and proportions of five cancer cells in the side population (C) are shown. The cell proportion is reported as the mean percentage ± standard error per group (n = 3). Asterisks indicate significant differences compared with the control antibody.

Article Snippet: Using an ultra-low-attachment 96-well plate, 100 A549 cells were seeded and cultured in 100 μl of serum-free DMEM–F-12 containing 10 ng/ml human recombinant epidermal growth factor (EGF) (R&D Systems) and 10 ng/ml human recombinant basic fibroblast growth factor (bFGF) (R&D Systems) with 50 ng/ml recombinant human FGF10 (rFGF10) or an equal volume of vehicle (PBS) on day 1.

Techniques: Western Blot, Cell Culture, Functional Assay, Staining

Journal: Molecular and Cellular Biology

Article Title: Human Marrow Stromal Cells Downsize the Stem Cell Fraction of Lung Cancers by Fibroblast Growth Factor 10

doi: 10.1128/MCB.00871-13

Figure Lengend Snippet: FGF10 suppresses reproliferation after an anticancer treatment. A549 lung cancer cells were treated with the anticancer drug cisplatin (CDDP) in the presence of recombinant FGF10 or vehicle (PBS [control]) for 2 days. (A) Time course of reproliferation after the anticancer treatment. The cell density relative to that at start of the experiment (day 0, indicated by the dotted horizontal line) was monitored by a colorimetric assay using MTS. (B) OCT3/4, SOX2, and LGR4 gene expression in A549 cells on day 4. The level of expression relative to that on day 0 was analyzed by quantitative RT-PCR. Data are presented as the mean ± standard error per group (n = 3). Asterisks indicate significant differences compared with the control vehicle.

Article Snippet: Using an ultra-low-attachment 96-well plate, 100 A549 cells were seeded and cultured in 100 μl of serum-free DMEM–F-12 containing 10 ng/ml human recombinant epidermal growth factor (EGF) (R&D Systems) and 10 ng/ml human recombinant basic fibroblast growth factor (bFGF) (R&D Systems) with 50 ng/ml recombinant human FGF10 (rFGF10) or an equal volume of vehicle (PBS) on day 1.

Techniques: Recombinant, Colorimetric Assay, Expressing, Quantitative RT-PCR