Fig 2: Erythropoietin blockade using soluble EPOR or anti-EPO mab suppress tumor angiogenesis and delay growth.(A–C) Representative images of window chambers implanted with R3230-GFP cells co-injected with (A) control buffer or protein, (B) sEPOR, or (C) anti-EPO mab. Fluorescent and transmitted light images were acquired serially on postoperative days 2, 4, 6, and 8. Scale bar = 2.5 mm. (D) Quantification of tumor neovascularization revealed significantly decreased angiogenesis in sEPOR and mab-treated chambers compared to controls. Control buffer was PBS (n = 3) and control protein was mouse IgG1 (n = 4), *P<0.001; **P<0.01, (n = 7 animals/group). (E) Quantification of tumor growth revealed significantly decreased tumor area in sEPOR and mab-treated chambers compared to controls, *P<0.001; †P<0.05, (n = 7 animals/group).

Fig 3: Increased EPO-induced phosphorylation of ERK1/2 and JNK in mammary carcinoma cells expressing EPOR-R129C.R3230-GFP cells transfected with empty pCR3.1 vector or EPOR-R129C were either left untreated as controls (C) or incubated with the indicated concentration of rEPO for 10 minutes. Whole cell lysates were analyzed by Western blotting using antibodies against phosphorylated or total proteins to detect (A) JAK2, JAK1 and STAT-5, (B) ERK1/2, and (C) JNK. Representative blots are shown. The phosphorylated (P) and total (T) proteins are indicated by the arrows. Comparable sample loading and protein integrity were confirmed by stripping the blots and hybridizing to respective antibodies to detect total protein amounts in the samples. The relative positions of the molecular weight markers are indicated in each blot. Quantitative representation of phosphorylated proteins using densitometry normalized to total protein levels are shown below the blots for ERK1/2 and JNK (n = 3 independent experiments using 3 different single cell clones, (*P<0.05).

Fig 4: Stimulation of tumor angiogenesis and growth in response to tumor cell EPOR-R129C expression.Representative images of dorsal skin-fold window chambers implanted with R3230-GFP cells transfected with (A) empty pCR3.1 vector control, or (B) EPOR-R129C expression vector are shown. Scale bar = 2.5 mm. Two independent single cell clones each of empty pCR3.1 vector and EPOR-R129C-transfected cells were tested for a total of 7 animals in each group. (C) Quantification of tumor neovascularization in window chambers implanted with EPOR-R129C or empty vector-transfected R3230-GFP cells revealed significantly increased VLD in EPOR-R129C expressing group compared to vector controls, *P<0.001. (D) Quantification of tumor growth in window chambers implanted with EPOR-R129C or vector transfected R3230-GFP cells revealed increased tumor area in EPOR-R129C expressing group compared to vector controls, *P<0.001; **P<0.01 (n = 7 animals/group).

Fig 5: In vivo tumor growth of R3230 mammary carcinoma cells in the mammary fat pad of nude mice.Stably transfected R3230-GFP cells were injected into the mammary fat pad of mice, tumor volumes were measured and expressed as fold-increase of palpable tumor size at day 7. Three independent single cell clones of each cell line were analyzed and empty vector-transfected cells (pCR3.1 and pcDNA3.1) served as negative controls. Expression of EPOR-R129C was associated with a significant increase in tumor growth rate compared to pCR3.1 vector controls (*P<0.001, n = 19 tumors in EPOR-R129C and n = 16 in pCR3.1 group). No significant tumor growth was observed in animals injected with cells expressing R103A-EPO antagonist (n = 14) compared to pcDNA3.1 vector transfected cells (**P<0.001, n = 15 tumors in pcDNA3.1 group).