Figure 4. PrSC or HFD conditions stimulate PCa metastasis and invasiveness in vivo.

For in vivo metastatic tumor studies, PC-3M-luc-C6 cells were intraperitoneallyinjected into mice randomly assigned to four groups (5 mice per group): control (Ctrl), PrSC, HFD, and HFD+BMS. For the PrSC group, PrSC cells were injected with the tumor cells. Mice in the HFD and HFD+BMS groups were fed HFD, and mice in the Ctrl and PrSC groups were fed a control diet. Mice in the HFD+BMS group were administrated BMS309403 by dissolution in drinking water. (A and B) Increased luciferase activity by PrSC or HFD. At 14 and 28 days inoculation, bioluminescence was used to detect intraperitoneal tumor growth and metastases by the intraperitoneal injection of luciferin. (C) Increased tumor metastasis in the PrSC and HFD groups. At 29 days after the injection of cells, mice were sacrificed, and the proportion of metastatic tumor burden in peritoneal organs, peritoneum, intestine, stomach, liver, and diaphragm were evaluated. (D, E and F) Increased invasive capacity in the PrSC and HFD groups by the increased adipocyte infiltration, stromal fibroblast activation, and upregulation of FABP4 and MMPs. (D) Slides of mouse tumor tissues were subjected to hematoxylin and eosin staining (H & E), and immunohistochemistry to detect the expression of FABP4 and αSMA. (E and F) mRNA expressions of FABP4 and MMP2 and 9 in tumors from each group were analyzed by quantitative RT-PCR. The mRNA expression levels of FABP4 and MMP2 and 9 was normalized to the levels of beta-actin, and the relative values were compared with the level of the Ctrl group. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001. (G) HFD stimulated FABP4 expression. The serum FABP4 concentration was measured by a mouse FABP4 ELISA kit. The serum FABP4 level was significantly higher in the HFD and HFD+BMS groups compared with the Ctrl group. ^*P < 0.05. (H) Increased serum IL-8 levels in the PrSC and HFD groups. The serum cytokine concentrations were measured by a Cytometric bead array kit. The serum level of IL-8 was significantly higher in the PrSC and HFD groups compared with the Ctrl group, and in the HFD group compared with the HFD+BMS group. ^*P < 0.05, ^**P < 0.01. (I and J) Stimulation of PC-3 cell invasiveness by mouse serum containing higher concentrations of FABP4 and IL-8. In vitro cell invasion is shown (I), and in the presence of 30 μM BMS309403 or 10 μg ml^-1 IL-8 blocking antibody (J). All invading cells were counted and compared with control cells. Mean ± S.D., ^*P < 0.05, ^**P < 0.01.