Fig 1: Schematic representation of the cross talk between tumor cells and lymphatic endothelial cells:(A) In autocrine regulation, VEGF-D produced by 468LN breast cancer cell line binds to α9β1 integrin cell surface receptor to induce the migration and invasion of cancer cell via Erk signaling. (B) In paracrine regulation, VEGF-D secreted by 468LN cells binds to VEGF-R3 on lymphatic endothelial cell (LEC) surface and induces migration and tube formation by the LEC via Erk signaling. (C) Binding of VEGF-D to α9β1 integrin located on the cancer cell membrane still leaves its binding site for VEGF-R3 located on the LEC membrane unoccupied. This leads to dual signaling by this ligand for both tumor and endothelial cells in a highly localised manner, defined as juxtacrine regulation.
Fig 2: Differential migratory, invasive, and VEGF-D producing capacity of 486LN cells, as compared to 468GFP cells:(A) Compared to 468GFP cells, 468LN cells were significantly more migratory and invasive. (B, C) 468LN cells expressed significantly higher level of VEGF-D mRNA measured with semi-quantitative RT-PCR (B) and total protein measured with western blot (C), compared to 468GFP cells. (MDA-MB-231 and MCF7-COX-2 cells served as positive controls for COX-2 and VEGF-C/D). (D) 468LN cells secreted significantly higher levels of VEGF-D but not VEGF-C, in comparison to 468GFP cells as measured by ELISA in cell supernatants; MDA-MB-231 cells served as positive controls for both VEGF-C and VEGF-D. (E) Exogenous rVEGF-D (2.5 ng/ml) increased both migration and invasion of 468LN, but not 468GFP cells. Migration/ invasion indices were normalized relative to 468GFP in SFM. Migration and invasion of 468GFP-SFM in Fig E was performed separately from the data in Fig A. All bars represent mean (n = 4) +/− S.E, *, P< 0.05; **, P< 0.01.
Fig 3: 468LN cell conditioned medium increases capillary-like tube formation by HMVEC-dLy cells:(A, E) HMVEC-dLy cells could hardly form any tube like structure with addition of conditioned medium (CM) from 468GFP cells at (A) 4 h and (E) 8 h. (B, F) HMVEC-dLy cells could form complete tubes as early as at (B) 4 h and (F) 8 h with 468LN CM. (C, G) VEGF-D knock down (KD) in 468LN cells, resulted in reduction in tube formation at both time points. (D, H) Again, addition of rVEGF-D in the CM of VEGF-D-KD 468LN cells stimulated tube formation. (I, J) Quantitative data for above observations are presented as (I) tube number index and (J) branching point index showing significant increase in tube and branch formation in presence of 468LN cell CM, but not 468GFP CM. Moreover VEGF-D KD in 468LN cells could block this function and HMVEC-dLy cells regained this function when rVEGF-D was added. Data represent mean (n = 3) ± SE. *,P<0.05; **,P<0.01.
Fig 4: Intra-tumoral lymphangiogenesis (LYVE-1 immunostatining-red) as well as angiogenesis (CD31 immunostaining-red) were abrogated by both α9 integrin and VEGF-D knock down in 468LN cells:(A) Direct measurements of lymphangiogenesis and angiogenesis respectively identified with immunofluorescent labelling for murine LYVE-1 and CD31 markers in serial frozen sections of tumors revealed significantly higher labelling for both markers in 468LN tumors compared to Δα9/468LN and ΔVEGF-D/468LN tumors. Nuclei were stained with DAPI (blue). (B) Fluorescence was quantified as corresponding “hot spot” scores. (n = 16, using the mean of 3 hot spots from each of the 16 tumors per group; Matrigel implants, n = 4) ± S.E, **p<0.001.
Fig 5: VEGF-D production by 468LN cells increased capillary-like tube formation of HMVEC-dLy cells:468GFP cells when co-cultured with HMVEC-dLy cells formed sparse and incomplete tube like structure at either 4 h (A) or 8 h (T), which were not different from HMVEC-dLy cells alone at both 4 h (B) and 8h (J) when seeded on growth factor reduced Matrigel in serum free medium (SFM). When 468LN cells were co-cultured with HMVEC-dLy cells, they showed enhanced formation of complete tubes as early as 4 h (C) and 8 h (K). (D, L) 468LN cells express GFP, so that it was evident from the images of tubes in co-culture, that both cancer cells and HMVEC-dLy cells aligned together in forming tubes, indicating cell-cell interaction or collaboration in aligned tube formation. (E, M) To test whether 468LN cell-derived VEGF-D contributed to tube formation by HMVEC-dLy cells, the same experiments were done after silencing the endogenous VEGF-D of 468LN cells. In this case, tube formation in co-cultured cells were reduced but not completely abolished. (G, O) Cells regained their tube forming capacity, when rVEGF-D (2.5 ng/ml) was added to the medium. (F, H, N, P) Again the GFP marker in 468LN cells confirms that these tubes were not exclusive to HMVEC-dLy. Quantitative data presented as (Q) tube number and (R) branching point indices. Data represent (n = 3) ± SEM. *,P<0.05; **,P<0.01.
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