Figure 4

Effect of PHY906 and/or Sorafenib (So) on the infiltration of macrophages in HepG2 tumors in NCR nude mice.

(A) Immunohistochemistry staining for F4/80 of HepG2 tumor section after treatment with Sorafenib (So) or So+PHY906 for 48 h and 96 h. Percentage of F4/80 stained cells per view of HepG2 tumor section after the treatments for 48 h (B) and 96 h(C). Each spot represents a mean of the number of F4/80 stained cells from 4 to 5 views of each tumor section against total live cells (PCNA-stained) in each treatment group. (D) Immunohistochemistry of hMCP1 of HepG2 tumor section tumor after PHY906 and/or Sorafenib (So) treatment for 96 h. (E) Quantification of immunohistochemistry staining of hMCP1 using imaging software. (Each spot represents a mean of the intensity of hMCP1 staining from 5 views of a tumor section; N = 14). (F) Heat map for mRNA expression of M1 and M2 macrophage markers in HepG2 tumor at 96 h following the drug treatment (Each bar represents a mean of two to three different qRT- PCR experiments (triplicate samples of each; N = 14). (G) Bayesian analysis for the probability of M1 phenotype. (H) Effect of clodronate liposomes treatment on macrophage infiltration with Sorafenib (So) or So+PHY906 for 96 h. Liposomes were given using i.p. injection at day -2), day 0 and day 2. (I) Effect of clodronate liposomes treatment on HepG2 tumor growth following treatment with Sorafenib (So) or So+PHY906 for 96 h. (J) Effect of clodronate liposomes treatment on apoptosis of HepG2 tumor following treatment with Sorafenib (So) or So+PHY906 for 96 h, N = 5. Sorafenib (30 mg/kg, b.i.d.) and PHY906 (500 mg/kg b.i.d.) were administered orally twice a day. Details of experimental procedures are given in Materials and Methods.