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Cd11b+Ly6G+ neutrophil mobilization within the peripheral blood of LLC tumor-bearing animals (Fig. S7C). Mixture therapy of MEKi plus anti-VEGF significantly reduced tumor growth compared with anti-Ragweed or anti-VEGF monotherapy (Fig. S7A). Importantly, mixture therapy of MEKi plus antiVEGF or anti -CSF plus anti-VEGF resulted in marked reduction in angiogenesis (Fig. S7 D and E) as measured by CD31+ endothelial cell density relative to anti-Ragweed reated animals.PNAS | April 9, 2013 | vol. 110 | no. 15 |Medical SCIENCESATumor Volume mmLy6GG-CSFR+/+ RAG2-/- aRAG G-CSFR+/+ RAG2-/- aVEGF G-CSFR-/- RAG2-/- aRAG G-CSFR-/- RAG2-/- aVEGFBaRAGaVEGFaRAGaVEGF*0 4 8Ly6CG-CSFR+/+ RAG2-/CD11bG-CSFR-/- RAG2-/-Time (Days)C2000Tumor Volume mm1600 1400 1200 1000 800 600 400 200 0G-CSF (pg/ml)CD11b+ Ly6G+aRAG (n=10) MEKi (n=10) aVEGF (n=10) aG-CSF (n=10) MEKi + aG-CSF (n=10) MEKi + aVEGF (n=10) aG-CSF + aVEGF (n=10) * * * *5 7 11 14D1800 1600 1400 1200 1000 800 600 400 200E70 60 50 40 30 20 10* *** * * * **Time (Days)FGaRAG MEKiMicrovessel Density Coverage0 1 two three four five 6aRAGMEKiaVEGFaG-CSFaVEGF aG-CSF MEKi + aG-CSF MEKi + aVEGF* *MEKi + aG-CSFMEKi + aVEGFaVEGF + aG-CSFaVEGF + aG-CSFFig.Cefsulodin Purity 3. Targeting G-CSF in tumor is additive with anti-VEGF to cut down tumor angiogenesis and growth. (A) G-CSFR WT (G-CSFR+/+) or G-CSFR knockout (G-CSFR-/-) mice had been crossed with RAG2 knockout (RAG2-/-) mice to produce G-CSFR+/+ RAG2-/- and (G-CSFR-/-RAG2-/-. Mice were transplanted with KPP14388 PDAC cells (n = eight per group) and treated with anti-Ragweed (aRAG) manage or anti-VEGF (aVEGF). Starting three d following cell inoculation, tumor volumes were measured at numerous time points, as indicated, *P 1.PTCDA web 0 10-11.PMID:23439434 Error bars indicate SD. (B) Flow cytometry analysis of peripheral blood for the presence of CD11b+Ly6G+ neutrophils. Information are representative of each group as indicated. Myeloid cells had been gated for CD45+, followed by evaluation with an antibody that specifically recognizes Ly6G+ neutrophils. (C) KPP14388 PDAC tumor development in response to MEKi, antiVEGF, anti-G-CSF, or combination remedies. Nu/ Nu mice were transplanted with KPP14388 cells (n = ten per group). Three days immediately after tumor cell inoculation, diverse remedies have been initiated as indicated, *P 0.001. Error bars indicate SD. (D) GCSF levels in the plasma of KPP14388 PDAC tumorbearing mice; n = 10 per group, *P 0.001. Error bars indicate SD. (E) Flow cytometry analysis of peripheral blood of KPP14388 tumor-bearing mice have been monitored for CD11b+Ly6G+ neutrophils (n = 5 per group), *P 0.001. Error bars indicate SD. (F) KPP14388 PDAC tumor sections immunostained with anti-CD31 (red). Mice have been treated with antiRagweed (aRag), MEKi, anti-VEGF (aVEGF), anti CSF (aG-CSF), or combination treatment as indicated. (Scale bar, 100 m.) (G) Quantitative evaluation of tumor vascular surface area (microvessel density). Complete tumor cross-sections were stained with anti-CD31 and analyzed as described in SI Experimental Procedures (n = 4 per group). Significance compared with aRag-treated group *P 0.05. Error bars indicate SD.N ai v aR e A M G E aV Ki M E EK a GF aM i + G-C S aV EKi aG- F EG + CS F aVE F + aG GF -C SFCombined Inhibition of G-CSF or MEK with Anti-VEGF Therapy Is Efficacious inside a PDAC Allograft Model. We subsequent tested combina-tion therapies utilizing MEKi and anti-VEGF or anti -CSF and anti-VEGF in PDAC mouse models. Anti -CSF or MEKi alone considerably decreased CD11b+Ly6G+ neutrophil mobilization, but had tiny e.