EGCG treatment inhibits growth and lymphangiogenic potential of tumors derived from ALDH-positive cells in mice.

<p>(A) Female NOD/SCID mice were implanted with SUM-149 ALDH-positive cells in the 4^th inguinal mammary fat pad (MFP injection) and separated into two groups (n = 6/group) once tumors became palpable (Tumors detected). Mice were treated by intraperitoneal injection with 16.5 mg/kg EGCG or Control 1X PBS (Treatment start). Tumor size was measured with calipers twice a week and tumor volumes calculated. Values shown are averages per group ± SE. *, p-value <0.05; **, p-value <0.005. (B) Following sacrifice, tumors were dissected out and weighed. Values shown are averages per group ± SE. *, p-value <0.05. (C–D) Tumor RNA was isolated and analyzed by RT-PCR using primers specific for human <i>VEGF-D</i> and for <i>18S</i> rRNA (C). Levels of h<i>VEGF-D</i> normalized to <i>18S</i> rRNA for each mouse per group are presented. Solid line represents median of the group (D). **, p-value <0.005. (E–F) Tumor proteins were extracted and separated by gel electrophoresis. Proteins were stained with Ponceau red and subjected to Western blotting for VEGF-D (E). Values of VEGF-D normalized to total Ponceau staining per sample are presented for each mouse per group. Solid line represents median of the group (F). *, p-value <0.05. (G–H) Tumor sections from Control or EGCG-treated mice were stained with an anti-podoplanin antibody. Representative images at 400× are shown (G). P-peritumoral area; T-tumor. Immunostained sections were analyzed and lymphatic vessel density quantified as described in Materials and Methods (H). Lymphatic vessel density for the Control and EGCG-treated groups is given as the average number of vessels in 3 hot spots in each of 4 podoplanin stained tumor sections per mouse. Solid line represents median of the group. **, p-value <0.005.