Article Figures & Data
Figures
- Figure 1.
Flt1 is a miR-200 target gene. A, Kaplan–Meier analysis of 2 cohorts of lung denocarcinoma patients on the basis of VEGFR1 expression (top quartile vs. lower 3 quartiles) in resected primary tumor specimens. The number of patients analyzed (n; top right corner) and the results of statistical tests (P; bottom left corner) are indicated for each cohort. B, miR-200 targets sequences in the Flt1 gene 3′-UTR. Reporter constructs containing wild-type and mutant Flt1 3′UTR are illustrated diagrammatically. 344SQ cells were transiently cotransfected with pre-miRs (10 nmol/L) and reporter plasmids (500 ng) that express luciferase alone (control) or are linked to the full-length 3′-UTR of Zeb1 (Zeb1) or Flt1 that is wild-type (Flt1) or mutated at the 5′ (mut1) or 3′ (mut2) putative miR-200 binding site. Results were normalized on the basis of renilla luciferase and expressed as the mean values of triplicate wells. *, P < 0.05. C, miR-200 suppresses VEGFR1 expression. Western blot analysis (left) of 344SQ cells and murine lung endothelial cells (MEC) as a positive control. Quantitative RT-PCR analysis (bar graphs) of 344SQ cells stably transfected with miR-200 (200b) or empty (control) lentiviral vectors normalized on the basis of L32 ribosomal protein mRNA levels and expressed as mean values of triplicate cultures relative to control transfectants, which were set at 1.0. Western blot analysis (right) of VEGFR1 in the same transfectants, which was quantified densitometrically, normalized on the basis of actin, and expressed relative to control, which was set at 1.0.
- Figure 2.
Characterization of LFs and CAFs. A, quantitative RT-PCR analysis of LFs. Values normalized on the basis of L32 ribosomal protein mRNA levels. B, Detection of fibroblast markers in LFs and CAFs by immunocytochemistry imaged by fluorescent or light microscopic analysis (20× magnification) of identical fields. Bar indicates 200 μm. C, morphology of primary lung fibroblasts (light microscopic images at 20× magnification). Bar indicates 100 μm. D, distinct patterns of stress fibers in LFs and CAFs. Immunofluorescent staining and laser scanning confocal imaging of α-smooth muscle actin in 3D matrices produced by fibroblasts after extraction of cellular debris. Bar indicates 100 μm.
- Figure 3.
Fibroblast-induced tumor cell invasion. 344SQ cells were monocultured (−) or cocultured with LFs or CAFs. Invasive tumor cells were photographed (images) and counted in at least 5 separate microscopic fields per well, which were averaged, and the mean values per well ± SD were calculated from replicate wells (bar graphs).
- Figure 4.
VEGF mediates CAF-induced lung adenocarcinoma cell invasion. A, 344SQ cells were cocultured with CAFs in the presence (+) or absence (−) of anti-VEGF-A neutralizing antibodies. Invasive tumor cells were photographed (images) and counted in at least 5 separate microscopic fields per well, which were averaged, and the mean values per well ± SD were calculated from replicate wells (bar graphs). B, miR-200 suppresses CAF-induced tumor cell invasion. Transwell invasion assays of 344SQ cells that stably express miR-200b (200b) or empty (control) lentiviral vectors in coculture with CAFs. Invaded cells were stained, photographed (image), and counted in at least 5 separate microscopic fields per well, which were averaged, and the mean values per well ± SD were calculated from replicate wells (bar graph). C, The indicated tumor cell lines derived from KrasLA1/+; p53R172HΔG/+ mice were monocultured in the upper chamber of a dual-chamber well, and recombinant VEGF-A was added (+) or not added (−) to the lower chamber. Invasive tumor cells were photographed (images) and counted in at least 5 separate microscopic fields per well, which were averaged, and the mean values per well ± SD were calculated from replicate wells (bar graphs).
- Figure 5.
Flt1 regulates multiple biologic properties of lung adenocarcinoma cells. A, Flt1/VEGFR1 detected in 344SQ syngeneic tumor. Immunofluorescent staining of a 344SQ syngeneic tumor with anti-VEGFR1 antibody (green) and 4′,6-diamidino-2-phenylindole (DAPI, blue) to detect nuclei. Adjacent tissue section stained with hematoxylin and eosin. B, suppression of Flt1 expression by Flt1 shRNA. Quantification of Flt1 in 2 independent clones of 344SQ transfected with Flt1 shRNA (#2–3, #4–1) or scrambled shRNA (SCR) by quantitative RT-PCR of triplicate RNA samples (bar graph) and Western blot analysis of protein (gels); bands were quantified densitometrically. Flt1 RNA and protein levels were normalized on the basis of L32 ribosomal protein mRNA and actin levels, respectively, and expressed relative to controls, which were set at 1.0. C, Flt1 knockdown inhibits 344SQ proliferation. Transfectants were seeded in monolayer cultures, counted, and expressed as mean values (± SD) of quadruplicate samples. D, Flt1 knockdown inhibits 344SQ anchorage-independent growth. Transfectants were seeded in soft agar, and visible colonies were photographed (images) and counted (bar graph) at 21 days. Results expressed as mean values ± SD of triplicate samples. E, Flt1 knockdown inhibits CAF-induced tumor cell invasion. Transfectants were cocultured with CAF; invasive tumor cells were photographed (images) and counted in 5 separate microscopic fields per well, averaged, and mean values per well ± SD were calculated from replicate wells (bar graphs). F, Flt1 knockdown inhibits tumor metastasis. Syngeneic mice were injected subcutaneously with tumor cells and necropsied at 6 weeks to measure primary tumor size and count lung metastases (scatter plots). Mean values, long horizontal lines; standard deviations, short horizontal lines.
Tables
- Table 1.
Protein concentrations in conditioned media from monocultures and cocultures
Proteins 344SQ LF CAF LF/344SQ CAF/344SQ MCP-1 9.11 282.46 2,524.16 1,182.22a 2,711.63a MIP-1α 21.29 21.67 59.85 33.2 177.97a MIP-1β 0.75 2.71 60.64 14.16a 142.34a RANTES 0.5 1.22 19.65 9.40a 29.24 Eotaxin 76.12 36.8 149.24 86.01 196.3 KC 486.38 52.69 1,191.99 809.42 2,370.50a MIP-2 2.24 3.9 118.74 31.07a 1,119.31a MIG 2.51 1.58 31.82 10.08a 69.44a bFGF 1.94 2.07 12.57 5.28a 19.08a PDGF-β 38.86 22.36 17.41 13.67 13.58 VEGF-A 61.94 8.3 409.68 85.75a 712.91a VEGF-D 0.92 6.33 3.82 0.5 11.75 IL-1α 0.2 0.33 1.71 0.43 1.56 IL-1β 6.87 3.36 23.35 10.15a 34.51a IL-2 0.75 0.7 1.55 0.69 1.64 IL-3 0.03 0.18 0.24 0.09 0.29 IL-4 0.04 1.67 0.21 0.04 0.27 IL-5 0.41 0.6 1.28 0.49 1.66 IL-6 1.29 30.2 1,838.95 128.90a 675 IL-9 5.15 2.35 23.67 4.42 24.96 IL-10 0.87 0.48 2.08 0.85 2.95a IL-12(p40) 0.43 0.32 1.65 0.54a 2.21a IL-12(p70) 22.89 22.64 39.19 28.82a 41.69a IL-13 34.08 27.36 53.69 30.29 61.19 IL-17 0.23 0.34 0.97 0.25 1.22 IL-15 2.38 1.78 40.17 12.2a 76.29a IL-18 16.02 18.35 55.4 19.44 78.42* TGF-β1 852.39 110.68 1,875.37 903.56a 2,687.85a G-CSF 10.09 3.67 47.43 32.76a 122.32a GM-CSF 16.37 3.93 12.22 16.21 27.56a M-CSF 13.29 32.54 106.95 59.80a 132.21a IFN-γ 3.24 2 8.58 3.48 10.85 TNF-α 7.67 5.82 15.7 8.61 20.92a LIF 30.62 7.74 103.94 36.23 171.74a NOTE: Protein concentrations are pg/mL. Comparisons (coculture vs. either of 2 monocultures) were based on Dunnett's test.
↵aProtein concentrations that were significantly different (P < 0.05) in coculture relative to that of either of the monocultures.
Supplementary Data
Files in this Data Supplement:
- Supplementary Figure 1 - PDF file - 49K
- Supplementary Figure Legend - PDF file - 34K
- Supplementary Tables 1-2 - PDF file - 57K
Volume 9, Issue 1
