American Society of Hematology

Figure 4

$Figure 4. Netrin-4–overexpressing MCF7 tumors have more lymphatic vessels and are more metastatic. (A) Human breast cancer MCF7 cells (WT) stably transduced with a Netrin-4 (Net4), VEGF-C or empty vector (control). Protein expression was determined in the cell supernatant (SUP), the fraction secreted and bound to the cytoplasmic membranes (Mb) or in the total cell lysate (CL) by Western blot using an anti–Netrin-4 or an anti–VEGF-C antibody. (rNet4; Recombinant Netrin-4). All control, Netrin-4, and VEGF-C MCF7 tumor cells also express at similar levels both GFP and luciferase (B) and proliferate in vitro at an identical rate (C; Ctrl; control empty vector, Net4; Netrin-4; scale bars in panel B: 100 μm and 1 cm). (D) Control, Netrin-4, and VEGF-C–overexpressing MCF7 cells injected subcutaneously into the midline of the back of NOS/SCID mice. Tumors were removed after 12 weeks, sectioned and costained with an anti–VEGFR-3/Flt-4 and anti–LYVE-1 antibody (scale bar: 100 μm). (E) VEGFR-3+/LYVE-1+ costaining density was quantified using ImageJ and represented as fold increase over the control-empty vector tumor condition. (F) GFP-positive tumor cells in the lumen of tumor lymphatic vessels stained with LYVE-1 antibody in the Netrin-4–overexpressing tumors (scale bars: 100 and 20 μm). (G) Luciferase-positive tumor cells metastasized into the auxillary lymph nodes of the Netrin-4 and VEGF-C tumor-bearing mice. (H) luciferase-positive lymph nodes contain GFP-positive tumor cells and are surrounded by enlarged LYVE-1 stained vessels in mice bearing Netrin-4 and VEGF-C–overexpressing tumors, but not in control condition (scale bar: 200 μm). 66C14 murine mammary carcinoma line metastasizing via the lymphatic system to lungs were stably transfected with a Netrin-4 encoding or empty-vector and injected into the exposed inguinal right mammary fat pad of Balb/C mice. Netrin-4–overexpressing 66C14 tumor bearing mice die faster than controls (I) and present more metastatic nodules per lung (J; scale bar: 5 mm). Pictures were taken on an Olympus IX71 microscope, at 100×, 200×, and 400× magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus software. The number of metastatic nodules per lung was quantified by visual inspection (K). *P < .05.$

Netrin-4–overexpressing MCF7 tumors have more lymphatic vessels and are more metastatic. (A) Human breast cancer MCF7 cells (WT) stably transduced with a Netrin-4 (Net4), VEGF-C or empty vector (control). Protein expression was determined in the cell supernatant (SUP), the fraction secreted and bound to the cytoplasmic membranes (Mb) or in the total cell lysate (CL) by Western blot using an anti–Netrin-4 or an anti–VEGF-C antibody. (rNet4; Recombinant Netrin-4). All control, Netrin-4, and VEGF-C MCF7 tumor cells also express at similar levels both GFP and luciferase (B) and proliferate in vitro at an identical rate (C; Ctrl; control empty vector, Net4; Netrin-4; scale bars in panel B: 100 μm and 1 cm). (D) Control, Netrin-4, and VEGF-C–overexpressing MCF7 cells injected subcutaneously into the midline of the back of NOS/SCID mice. Tumors were removed after 12 weeks, sectioned and costained with an anti–VEGFR-3/Flt-4 and anti–LYVE-1 antibody (scale bar: 100 μm). (E) VEGFR-3⁺/LYVE-1⁺ costaining density was quantified using ImageJ and represented as fold increase over the control-empty vector tumor condition. (F) GFP-positive tumor cells in the lumen of tumor lymphatic vessels stained with LYVE-1 antibody in the Netrin-4–overexpressing tumors (scale bars: 100 and 20 μm). (G) Luciferase-positive tumor cells metastasized into the auxillary lymph nodes of the Netrin-4 and VEGF-C tumor-bearing mice. (H) luciferase-positive lymph nodes contain GFP-positive tumor cells and are surrounded by enlarged LYVE-1 stained vessels in mice bearing Netrin-4 and VEGF-C–overexpressing tumors, but not in control condition (scale bar: 200 μm). 66C14 murine mammary carcinoma line metastasizing via the lymphatic system to lungs were stably transfected with a Netrin-4 encoding or empty-vector and injected into the exposed inguinal right mammary fat pad of Balb/C mice. Netrin-4–overexpressing 66C14 tumor bearing mice die faster than controls (I) and present more metastatic nodules per lung (J; scale bar: 5 mm). Pictures were taken on an Olympus IX71 microscope, at 100×, 200×, and 400× magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus software. The number of metastatic nodules per lung was quantified by visual inspection (K). *P < .05.

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