Knock down of α9 integrin and VEGF-D in 468LN cells reduced primary tumor growth in nude mice:

<p>(<b>A</b>) Representative images of tumors and Matrigel alone as well as the draining lymph nodes on day 20 (a scale in mm shown in the background). (<b>B</b>) Tumor growth rates determined by volume measured externally were dramatically reduced in both α9 integrin (Δα9/468LN) and VEGF-D knock down (ΔVEGF-D/468LN) tumors (indistinguishable from Matrigel alone). (<b>C</b>) Tumors were excised and mean weights of tumors and Matrigel were retrieved on day 20. Weights of 468LN tumors were significantly higher than those of Matrigel alone or Δα9/468LN and ΔVEGF-D/468LN tumors. Data represented as means (n = 16 for tumors and 4 for Matrigel) ± S.E. *p<0.05, **p<0.001.</p

Metastatic spread of tumor cells to the draining lymph nodes is abrogated by α9 integrin and VEGF-D knock down in 468LN cells:

<p>(<b>A</b>) Metastasis to lymph nodes were validated by the visual localisation of many cancer cells in 468LN tumor draining nodes identified by the GFP-marker and their absence in lymph nodes draining α9 integrin and VEGF-D knock down tumor implants. Nuclei were stained blue by DAPI. (<b>B, C</b>) Above conclusions were further validated by measuring the human mitochondrial-specific marker MTCO2 at the mRNA level with qRT-PCR (<b>B</b>) and protein level with western blot (<b>C</b>) was high in lymph nodes draining 468LN tumors. This process was nearly completely abrogated in both Δα9/468LN and ΔVEGF-D/468LN tumor implants. Data were derived from 16 lymph nodes. Four lymph nodes draining into an individual tumor in a single mouse were pooled to extract RNA and proteins, showing the data in four mice; Matrigel was retrieved from four implants. Data represents mean (n = 4) ± S.E, *p<0.05, *p<0.001.</p

VEGF-D induced migration of HMVEC-dLy cells is Erk dependent:

<p>(<b>A</b>) Cells were serum starved for 24 h before stimulation with r-VEGF-D (20 ng/ml) for varying lengths of time (0, 15, 30 and 60 minutes) or pre-treated with varying concentrations of Erk inhibitor U0126 (0, 5, 10 and 15 µM) for one hour and further treated with 20 ng/ml r-VEGF-D for 30 minutes. Cell lysates were prepared and analyzed by western blot for Erk1/2 activity. VEGF-D stimulated Erk activation in HMVEC-dLy cells and this stimulation was prevented by pre-treatment with U126. (<b>B</b>) Cell migration was quantified by transwell migration assays; VEGF-D stimulated migration was inhibited with U126 (5–15 µM). 2% FBS served as a positive control. Migration data were represented as a mean ± S.E. for three independent experiments (n = 3). For recombinant VEGF-D stimulation, data were normalised to SFM and for Erk inhibition data were normalised to r-VEGF-D stimulation.</p

VEGF-D production by 468LN cells increased capillary-like tube formation of HMVEC-dLy cells:

<p>468GFP cells when co-cultured with HMVEC-dLy cells formed sparse and incomplete tube like structure at either 4 h (<b>A</b>) or 8 h (<b>T</b>), which were not different from HMVEC-dLy cells alone at both 4 h (<b>B</b>) and 8h (<b>J</b>) 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 (<b>C</b>) and 8 h (<b>K</b>). (<b>D, L</b>) 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. (<b>E, M</b>) 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. (<b>G, O</b>) Cells regained their tube forming capacity, when rVEGF-D (2.5 ng/ml) was added to the medium. (<b>F, H, N, P</b>) Again the GFP marker in 468LN cells confirms that these tubes were not exclusive to HMVEC-dLy. Quantitative data presented as (<b>Q</b>) tube number and (<b>R</b>) branching point indices. Data represent (n = 3) ± SEM. *,P<0.05; **,P<0.01.</p

VEGF-D knock down reduced lymphatic ingrowths into the tumor:

<p>(<b>A</b>) Digital camera captured picture at 5 minutes show the Evans blue dye injection sites and the dye stained lymphatics going towards the 468LN tumor (T). (<b>B</b>) Images were captured at 15 minutes using a dissection microscope. These images show lymphatics (arrows) going into the 468LN tumors as well as tumor draining axillary lymph nodes (LN). (<b>C</b>) Image of same tumor at a higher magnification (see inset) captured at 20 minutes showing blue dye inside the tumor (T), and (<b>D</b>) inside the lymph node (LN) at a higher magnification (see inset). (<b>E</b>) In contrast, VEGF-D knock down (ΔVEGF-D/468LN) tumor implants showed very few lymphatics traceable from the injection site captured with a digital camera. (<b>F</b>) Picture taken at 15 minutes showing lymphatics alone but very little dye in the tumor. (<b>G</b>) Even at 25 minutes no dye-stained lymphatics was visible on the surface or inside the tumor at a higher magnification (see inset).</p

Reduction of migration and invasion of 468LN cells by knocking down VEGF-D production:

<p>(<b>A–C</b>) Levels of VEGF-D knock down in 468LN cells was confirmed by three different methods, <b>(A)</b> qRT-PCR, (<b>B</b>) western blot and (<b>C</b>) ELISA. After VEGF-D knock down (KD) in 468LN cells, (<b>D</b>) migration, invasion and (<b>E</b>) proliferation significantly dropped as compared to scrambled knock down (SCR-KD) cells. (<b>F</b>) Addition of exogenous rVEGF-D or FBS increased both migration and invasion of VEGF-D knocked down 468LN cells. Bars represent mean (n = 4 in all cases except <b>C</b>, n = 6) ± SE, *,P<0.05; **,P<0.01.</p

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:

<p>(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.</p

Ability to induce intra-tumoral lymphangiogenesis was abrogated by both VEGF-D and α9 integrin knock down in 468LN cells:

<p>(<b>A, B</b>) Intra-tumoral lymphangiogenesis was measured indirectly from the expression of murine lymphatic endothelial marker LYVE-1 in tumors at the mRNA level with qRT-PCR (<b>A</b>) and protein level with western blot (<b>B</b>). Murine LYVE-1 expression was very high in 468LN tumors and this expression was nearly completely abolished in both Δα9/468LN and ΔVEGF-D/468LN tumor implants. Data were derived from 16 tumors. Four tumors in a single mouse were pooled to extract RNA and proteins, showing the data in four mice; Matrigel was retrieved from four implants. Data represents mean (n = 4) ± S.E, *p<0.05, *p<0.001.</p

Expression of VEGF-D-binding receptors by 468LN cells:

<p>(<b>A</b>) Expression of VEGF-R2 and R3 mRNA examined with semi quantitative RT-PCR, and (<b>B</b>) α9 integrin mRNA measured with qRT-PCR. (<b>C</b>) Confirmation of VEGF-R2, VEGF-R3, and α9 integrin expression at protein levels by western blot. MDA-MB-231 and MCF7-COX-2 cells were used as positive controls primarily for VEGF-R2 and VEGF-R3 expression. Differentially high α9 integrin expression by 468 LN cells was evident. Bars represent mean (n = 4) ± SE, *,P<0.05.</p

Blockade of α9β1/VEGF-D interaction hinders autocrine migratory/invasive functions of 468LN cells and α9-associated signaling:

<p>(<b>A</b>) A strong inhibition of both migration and invasion indices of 468LN cells, noted with α9β1 blocking antibody (5 µg/ml). This inhibition could not be reversed by addition of rVEGF-D. Data were normalized to control IgG for α9β1 blocking antibody (ab) and to α9β1 blocking antibody treated cells for rVEGF-D. Knock down of α9 was validated with qRT-PCR (<b>B</b>) and western blot (<b>C</b>). (<b>D</b>) Knock down of α9 integrin (α9-KD) significantly compromised both migration and invasion of 468LN cells compared to scrambled knock down (SCR-KD) cells. (E) Following α9-KD, but not SCR-KD, migratory and invasive functions could be stimulated with FBS, but not with rVEGF-D. Data presented as the mean (n = 4 except invasion data in D, n = 6) ± SE. *, P<0.05; **,P<0.01. (F) Phospho Erk1/2 was measured using western blots in wild type and α9-silenced, serum starved (24 h) 468LN cells in the presence or absence of rVEGF-D (3 µg/ml) at 0–60 min as compared to total Erk1/2. Significant constitutive Erk1/2 activity noted in the absence of rVEGF-D was stimulated further in the presence of rVEGF-D at 15–45 min (peak shown at 45 min). Knock down of α9 integrin resulted in suppressed Erk1/2 activity in both cases.</p