Fig 1.
Identification of lymphangiogenic and non-lymphangiogenic NSCLC cell lines.
(A) Representative images of lung tumor xenografts stained with an antibody against LYVE-1 (red) (B) Representative images of lung tumor xenografts stained with antibodies against podoplanin (green) and smooth muscle actin (SMA, red). (C) Graph showing intratumoral lymphatic vessel density for podoplanin and LYVE-1 positive lymphatic vessels in NSCLC xenografts grown in mice. We classified Calu-1, HCC827, HCC461, H1993, and H2122 cells as being lymphangiogenic and Calu-3, H1155, H1975, and H2073 as being non-lymphangiogenic. Graph shows mean ± SEM.
Fig 2.
Growth curves for lymphangiogenic and non-lymphangiogenic tumors.
(A) Graph showing the growth of lymphangiogenic (Calu-1, HCC827, HCC461, H1993, and H2122) tumors. (B) Graph showing the growth of non-lymphangiogenic (Calu-3, H1155, H1975, and H2073) tumors. Graph shows mean ± SEM.
Table 1.
Characteristics of lymphangiogenic and non-lymphangiogenic NSCLC cell lines.
Table 2.
Mutation status of lymphangiogenic and non-lymphangiogenic NSCLC cell lines.
Fig 3.
VEGF-C is differentially expressed between lymphangiogenic and non-lymphangiogenic NSCLC cell lines.
(A) Microarray results showing genes differentially expressed between lymphangiogenic (Calu-1, HCC827, HCC461, H1993, and H2122) and non-lymphangiogenic (Calu-3, H1155, H1975, and H2073) cells. (B) Quantitative PCR results showing that VEGF-C is expressed at a higher level in lymphangiogenic than in non-lymphangiogenic NSCLC cell lines. VEGF-C values are normalized to the housekeeping gene GAPDH. Values for the NSCLC cell lines are normalized to an immortalized human bronchial epithelial cell line (HBEC3KT).
Fig 4.
Forced expression of VEGF-C promotes tumor lymphangiogenesis.
(A) RT-PCR results showing that H1975-VEGFC cells, but not H1975-Ctrl cells, express VEGF-C. (B) Tumor growth curves for mice injected subcutaneously with either H1975-Ctrl or H1975-VEGFC cells. (C,D) Representative images of H1975-Ctrl and H1975-VEGFC tumor sections stained with an anti-endomucin antibody. (E) The number of blood vessels per microscopic field is not significantly different between H1975-Ctrl tumors (13.75 ± 1.263, n = 7) and H1975-VEGFC tumors (16.18 ± 1.998, n = 7). (F,G) Representative images of H1975-Ctrl and H1975-VEGFC tumor sections stained with an anti-LYVE-1 antibody. (H) There are significantly more lymphatic vessels per microscopic field in H1975-VEGFC tumors (11.83 ± 3.125, n = 7) than in H1975-Ctrl tumors (0.4286 ± 0.4286 N = 7). ** P < 0.01.
Fig 5.
Knockdown of VEGF-C inhibits tumor lymphangiogenesis.
(A) qPCR results showing that H1993-shVEGFC cells express a lower level of VEGF-C than H1993-Ctrl cells. (B) Tumor growth curves for mice injected subcutaneously with either H1993-Ctrl or H1993-shVEGFC cells. (C,D) Representative images of H1993-Ctrl and H1993-shVEGFC tumor sections stained with an anti-endomucin antibody. (E) The number of blood vessels per microscopic field is not significantly different between H1993-Ctrl tumors (11.17 ± 0.7817, n = 6) and H1993-shVEGFC tumors (10.42 ± 1.182, n = 7). (F,G) Representative images of H1993-Ctrl and H1993-shVEGFC tumor sections stained with an anti-LYVE-1 antibody. (H) The density of intratumoral lymphatic vessels is significantly lower in H1993-shVEGFC tumors (0.61 ± 0.400, n = 7) than H1993-Ctrl tumors (27.61 ± 1.391, n = 6). **** P < 0.0001.
Fig 6.
Nintedanib inhibits tumor lymphangiogenesis.
(A,B) Representative images of LYVE-1-stained sections of H1993 tumors from vehicle and nintedanib treated mice. (C) The density of lymphatic vessels in H1993 xenografts is significantly lower in nintedanib treated mice (5.254 ± 2.745, n = 5) than in vehicle treated mice (22.19 ± 2.536, n = 6). ** P < 0.01.
Fig 7.
VEGF-C copy number variation influences VEGF-C expression.
(A) Image showing VEGFC copy number variation for different lung cancer cell lines. Rows show data for individual lung cancer cell lines. Columns mark different positions along the VEGFC gene. Amplified positions are colored red, diploid positions are colored black, and deleted regions are colored green or white. (B) Graph showing log transformed VEGF-C mRNA levels from microarray data for cell lines that have more than 2 copies (range is between 3–5 copies of VEGFC), 2 copies, or less than 2 copies of the VEGFC gene. Graph shows mean ± SEM. **** P < 0.0001