(A) Spontaneous LLC metastasis is increased in Tie2-CYP2C8-Tr and sEH-null mice relative to WT 10 days after primary tumor removal (LLC resection). Images show representative lung metastasis in transgenic and WT mice. n = 5 mice/group; *P = 0.011, **P = 0.004, ***P ≤ 0.001 versus WT. The experiment was performed 3 times, with similar results. Scale bars: 1 cm. (B) Spontaneous LLC metastasis is decreased in Tie2-sEH-Tr relative to WT 17 days after primary tumor removal. n = 6 mice/group; *P = 0.001, **P = 0.002. (C) Systemic administration of 14,15-EET (15 μg/kg/d) via osmotic minipump increases spontaneous LLC lung metastasis and distant axillary and inguinal lymph node metastasis 12 days after LLC resection. Dashed circles indicates metastatic and normal lung; arrows show inguinal lymph node metastasis (white arrow, 14,15-EET; black arrow, control); scale bars: 1 cm. GFP-labeled LLC tumor cells (green) demonstrate bilateral axillary and inguinal lymph node metastasis in mice treated with 14,15-EET 17 days after LLC-GFP tumor resection; scale bars: 500 μm (for GFP staining). n = 10 mice/group; *P = 0.001, **P ≤ 0.001. (D) 14,15-EET (15 μg/kg/d) reduces survival and stimulates multiorgan lung, liver, and kidney metastasis of B16F10-GFP melanoma cells injected intravenously. Dashed circles indicate (top to bottom) representative lung, liver, and kidney metastasis 19 days after injection; scale bar: 1 cm. GFP-labeled melanoma tumor cells (green) confirm lung, liver, and kidney metastasis in mice treated with 14,15-EET; scale bar: 500 μm (for GFP staining). n = 7–9 mice/group; *P = 0.021. (E) 14,15-EET (15 μg/kg/d) stimulates liver metastasis in an orthotopic human prostate cancer (PC3M-LN4) model. H&E-stained section of liver metastasis of mice treated with 14,15-EET; arrows indicate nodules of tumor within hepatic parenchyma; scale bar: 100 μm. Black circles indicate representative orthotopic prostate tumors; scale bars: 1 cm. n = 5–6 mice/group.

(A) EC migration is decreased in Tie2-sEH-Tr mice (left panel) but increased in Tie2-CYP2J2-Tr and Tie2-CYP2C8-Tr relative to WT mice (middle). tAUCB and TUPS stimulate VEGF-mediated endothelial migration (right). n = 3–4/group; *P = 0.0139, **P = 0.029, and ***P < 0.001 versus WT; ^†P < 0.05 versus VEGF alone. (B) 14,15-EEZE inhibits VEGF-induced EC (left panel) but not tumor cell (LLC) migration (right). n = 3–4/group; *P = 0.038, **P = 0.012, ***P < 0.001 versus control. (C) VEGF ELISA of plasma of Tie2-CYP2C8-Tr and sEH-null mice 17 days after B16F10 resection shows an increase in VEGF levels. n = 5/group (left panel). Systemic administration of 14,15-EET regulates Vegfr2 mRNA levels in primary LLC tumors in mice compared with size matched control tumors, but has no effect on Vegfr1 mRNA levels (right). n = 5/group. *P < 0.05 versus WT; **P = 0.0095 versus control. (D) VEGF depletion with Ad-sFlt suppresses B16F10 tumor growth in Tie2-CYP2J2-Tr and sEH-null but not in WT mice. Plasma levels of sFlt1 on day 10 were 35,999 ± 10,225 pg/ml (Tie2-CYP2J2-Tr) and 36,680 ± 1,308 pg/ml (sEH-null). n = 5 mice/group; *P = 0.016, **P = 0.023. (E) tAUCB does not promote spontaneous LLC metastasis in mice depleted of VEGF with Ad-sFlt (tAUCB + Ad-sFlt). n = 5 mice/group; *P = 0.004, **P = 0.002 versus tAUCB + Ad-null. (F) The angiogenesis inhibitor TSP1 is downregulated in plasma of Tie2-CYP2C8-Tr, sEH-null, and Tie2-CYP2J2-Tr relative to WT mice on day 13 after LLC injection. rTSP1, recombinant TSP1. (G) 14,15-EEZE (0.21 mg/mouse) does not significantly inhibit primary LLC tumor growth in TSP1 null mice. n = 5 mice/group.

(A) Growth of B16F10 melanoma and T241 fibrosarcoma primary tumors in sEH-null, Tie2-CYP2C8-Tr, and WT mice. n = 10–14 mice/group; *P = 0.042, **P = 0.024, ^#P = 0.008, ^§P < 0.001 versus WT; unpaired Student’s t test is used throughout. (B) Primary B16F10 melanoma and T241 fibrosarcoma tumor growth is inhibited in Tie2-sEH-Tr mice on days 22–28. n = 5–6 mice/group; *P = 0.001, **P = 0.006 versus WT. (C) Systemic administration of 14,15-EET (15 μg/kg/d) via osmotic minipump stimulates primary LLC tumor growth. When 1 × 10⁴ or 1 × 10³ LLC cells were injected, 5 of 9 control mice and 9 of 9 control mice, respectively, did not exhibit visible tumor growth up to 209 days after injection. Images show representative mice on day 29 after injection with 1 × 10⁴ LLC cells. n = 6–9 mice/group; *P ≤ 0.02, **P ≤ 0.001, ^#P ≤ 0.006 versus WT. The experiment was performed 3 times with similar results. Rulers show centimeters. (D) 14,15-EET (15 μg/kg/d) stimulates tumor growth in a genetically engineered model of cancer (TRAMP). Ruler shows centimeters. At 90 days of treatment, H&E-stained section of prostate of control TRAMP mice reveals prostatic intraepithelial neoplasia (PIN) surrounded by tumor cells, while H&E-stained section of prostate of TRAMP mice treated with 14,15-EET shows glandular parenchyma surrounded by tumor cells. n = 6 mice/group; *P = 0.002 versus control. Scale bars: 100 μm. (E) 14,15-EET accelerates escape in a tumor dormancy model of human liposarcoma cells (clone 4). After treatment with 14,15-EET for 100 days, large palpable liposarcoma tumors are visible. No tumor is visible in the control mice. Ruler shows centimeters. H&E-stained section of tumors of mice treated with 14,15-EET reveal sheets of lipoblasts. n = 5–6 mice/group; *P = 0.008 versus control. Scale bar: 100 μm.

(A) Systemic administration of an sEH inhibitor (tAUCB) stimulates primary LLC-GFP tumor growth. Images show representative tumors after 13 days of treatment. n = 6 mice/group; *P = 0.007. Scale bar: 1 cm. (B) Immunofluorescence double staining for VEGF and GFP (tumor cells) shows increased tumor cell expression of VEGF in LLC-GFP tumor cells from tAUCB- versus vehicle-treated mice. Green, GFP-stained tumor cells; red, VEGF-containing cells. Colocalization of red and green fluorescence (yellow) indicates tumor cells expressing VEGF (arrows). tAUCB-treated tumors have an increase in MECA-32–positive ECs (green). Scale bars: 20 μm. (C) Systemic administration of tAUCB and TUPS (10 mg/kg/d each) increases spontaneous B16F10 axillary lymph node metastasis 21 days after B16F10 resection. Representative axillary lymph nodes after 21 days of treatment are shown. Scale bars: 1 cm. n = 6 mice/group; *P = 0.029 versus control. (D) The EET antagonist 14,15-EEZE (0.21 mg/mouse) inhibits primary LLC growth (left panel), prolongs survival (middle panel), and reduces plasma VEGF levels (right panel) in a spontaneous LLC lung metastasis model. n = 5 mice/group; *P = 0.017, **P = 0.035, ***P = 0.044. (E) The EET antagonist 14,15-EEZE-mSI (0.21 mg/mouse) inhibits 14,15-EET–induced (15 μg/kg/d) spontaneous LLC metastasis. The stable EET metabolite 14,15-DHET (15 μg/kg/d) does not stimulate metastasis. Representative photographs on day 12 after LLC resection are shown. Scale bars: 1 cm. n = 5 mice/group; *P < 0.001, **P = 0.003 versus 14,15-EET.

(A) LLC tumors in VEGF-LacZ-Tr mice treated with 14,15-EET (15 μg/kg/d) show β-galactosidase staining (marker of VEGF production) in tumor endothelium and stromal fibroblasts (arrows). Scale bars: 20 μm. (B) Expression of sEH, but not CYP2J and CYP2C, is downregulated in tumor (TEC) versus normal (NEC) ECs (two left panels). Control tissue: mouse liver. Expression of sEH, but not CYP2J and CYP2C, is downregulated in tumor lysates from larger LLC tumors (>5 cm³) versus smaller LLC tumors (<1 cm³) (third panel). sEH expression (brown staining) is also downregulated in B16F10 melanoma liver metastasis compared with normal adjacent liver (far right panel). Scale bar: 100 μm.