Biochemical and cell biological tumor characteristics. A: Tumor cholesterol levels. Membranes were prepared from tumors and subjected to cholesterol extraction and analysis (see Materials and Methods). Data are plotted as cholesterol (mg)/g tumor tissue versus diet/ezetimibe (Z) group ± SE. Data were analyzed by analysis of variance, which indicated no significant interaction between diet and ezetimibe (P = 0.40) but highly significant main effects of both diet (P = 0.039) and ezetimibe (P < 0.0001) n = 9/group. B: Tumor cell apoptosis (TUNEL staining). Upper panel: representative TUNEL stained images. Left column shows TUNEL staining (fluorescein; green) of selected tumor sections; right column is the merged image of TUNEL and DAPI counterstaining. n = 40. Lower panel: quantitative evaluation of tumor cell apoptosis levels. Data are plotted as relative level of TUNEL staining versus diet/ezetimibe (Z) group ± SE. Data were analyzed by analysis of variance, which indicated no significant interaction between diet and ezetimibe (P = 0.85) but highly significant main effects of both diet (P < 0.0001) and ezetimibe (P < 0.0001). C: Tumor cell proliferation (Ki-67 staining). Upper panel: representative Ki-67 stained images. Left column, Ki-67 staining (red); Right column, merged image of Ki-67 (Cy3; red) and DAPI (blue; nuclei) counterstaining. n = 20. Lower panel: quantitative evaluation of tumor cell proliferation levels. Data are plotted as relative level of Ki-67 staining versus diet/ezetimibe (Z) group ± SE. The data were analyzed by two-way analysis of variance, which demonstrated that there is a significant ezetimibe by diet interaction (P = 0.027), implying that ezetimibe has a significant effect on lowering proliferation although the magnitude of this effect depends on the diet; LFNC: average 28.78 (95% CI; 25.42 to 32.14) w/ezetimibe: average 25.57 (95% CI; 22.21 to 28.93); HFHC: average 41.16 (CI; 37.80 to 44.52) w/ezetimibe: average 30.32 (95% CI; 26.96 to 33.68). Images were acquired and analyzed by AxioVision 4.0 software for quantification. In all cases data are considered significant at P < 0.05.

Characterization of the xenograft tumor microenvironment. A: Fibroblast analysis. Upper panel; Quantification of fibroblast staining. Data are plotted as relative level of fibroblast staining versus diet/ezetimibe (Z) group ± SE. Data were analyzed by two-way analysis of variance analysis, which indicated no statistically significant diet effect, however a statistically significant ezetimibe effect in the HFHC condition was detected (P = 0.03). n = 55/group. Lower panel; representative fibroblast staining. Sections were stained using a fibroblast specific mAb (see Materials and Methods) and nuclei were counterstained with DAPI. Left column; representative anti-fibroblast mAb staining (Alexa Fluor 488; green). Right column; representative fibroblast staining (Alexa Fluor 488; green) merged with DAPI staining (blue). B: Pericyte coverage (vessel quality). Pericyte coverage of microvessels was determined by SMA staining of peri-microvesicular regions of stained tumor sections (CD31 staining). Upper panel: quantitative evaluation of tumor section SMA levels. Data are plotted as relative level of SMA staining versus diet/ezetimibe (Z) group ± SE. Data were analyzed by mixed model analysis, which indicated no significant interaction between diet and ezetimibe (Z) (P = 0.062) but highly significant main effects of ezetimibe (P < 0.0001) and diet (P = 0.009). Lower panel: representative anti-SMA/anti-CD31 mAb stained images. Columns left to right: SMA staining (Alexa Fluor 568; Red); SMA (Alexa Fluor 568; Red) + CD31 (Alexa Fluor 488; Green) merged images; DAPI (blue), SMA (Alexa Fluor 568; Red), + CD31 (Alexa Fluor 488; Green) merged images. n = 34 to 36. C: TSP-1 levels in tumors. TSP-1 levels in tumor sections were determined by staining tumor sections with anti-TSP-1 and anti-CD31 mAbs. Left panel: quantitative evaluation of tumor section TSP-1 levels. Data are plotted as relative level of TSP-1 staining versus diet/ezetimibe (Z) group ± SE. Data were analyzed by mixed model analysis, which indicated highly significant main effects of ezetimibe (P < 0.0001) and diet (P < 0.0001). n = 34–39. Right panel; representative anti-TSP-1 (Alexa Fluor 568)/anti-CD31 (Alexa Fluor 488) mAb stained images. Columns top to bottom: DAPI (blue), TSP-1 (Alexa Fluor 568; Red), + CD31 (Alexa Fluor 488; Green) merged images; TSP-1 (Alexa Fluor 568; Red) + CD31 (Alexa Fluor 488; Green) merged images; TSP-1 staining (Alexa Fluor 568; Red). Tumors were fixed in OCT, and either 10 μm sections (part A) or 20 μm sections (parts B + C) were stained for the indicated markers. CD31 (PECAM) staining was performed as described.46 Fluorescent mages were acquired and analyzed by AxioVision 4.0 software for quantification. In all cases data are considered significant at P < 0.05.

Diet/ezetimibe effects on serum cholesterol levels and the growth of implanted prostate tumors in SCID mice. A: Serum cholesterol levels in diet/ezetimibe mouse cohorts. Animals were given various ezetimibe (Z)-diet combinations (see Figure) for 2 weeks after which the mice were bled by small tail vein incision, and cholesterol measured in the collected serum via Infinity colormetric assay. Data are plotted as cholesterol level (mg/dL) vs. group ± SE. n = 13 to 15/group. Two-way analysis of variance indicated significant effects of diet (F = 77.57, P < 0.001) and drug (F = 23.48, P < 0.001) on cholesterol levels, but no significant diet-by-drug interaction (F = 0.86, P = 0.36) suggesting that the effects are independent. B: Longitudinal volume measurements. SCID mice were fed various diet/ezetimibe (Z) combinations for 2 weeks before tumor implantation (see Materials and Methods). Tumors were measured daily by calipers starting at first appearance (day 1) and continued for 13 days. Data are plotted as tumor volume (mm³) per site versus time (days) ± SE. A mixed model analysis was used to calculate the significance of the both diet (P = 0.048) and ezetimibe (Z) (P = 0.035) on tumor growth. n = 52 to 60/group. C: Tumor wet weight. At sacrifice all tumors were removed and weighed. Data are plotted as average tumor mass (g) per site versus group ± SE. These data were statistically significant between LFNC + Z (0.63 ± 0.55 average grams/tumor site) vs. HFHC (0.88 ± 0.71 average grams/tumor site) groups (P = 0.021) and between the LFNC (0.67 ± 0.56 average grams/tumor site) vs. HFHC groups (P = 0.037). n = 52 to 60/group. In all cases data are considered significant at P < 0.05.

Angiogenesis in xenograft tumors. A: Tumor hemoglobin quantification. Tumors were subjected to mechanical disruption in PBS, followed by centrifugation (to remove debris) and the clarified supernatants were analyzed by optical density (absorbance at 530Å —absorbance at 650Å). Data are plotted as relative hemoglobin/mg tumor tissue versus group (mean value is indicated by line). All groups demonstrated statistical significance versus all other groups except for LFNC versus HFHC + Z (ezetimibe), and LFNC versus LFNC + Z, which were not statistically different. n = 20. B: MVD-CD31 analysis. Upper panel: quantitative evaluation of tumor section CD31 levels. Data are plotted as relative level of CD31 staining versus diet/ezetimibe (Z) group ± SE. Data were analyzed by mixed model analysis, which indicated no significant interaction between diet and ezetimibe (P = 0.199) but highly significant main effect of ezetimibe (P = 0.013) with larger effects when the HFHC was used (P = 0.01). Lower panel: representative anti-CD31 mAb stained images. Left column, CD31 staining (Alexa Fluor 488; Green); Right column, merged image of CD31 (Alexa Fluor 488; Green) and DAPI (blue; nuclei) counterstaining. n = 76 to 103. C: MVD-caveolin-1 analysis: Immunoblotting. Tumors were subjected to SDEM and raft fractions were then subjected to SDS-polyacrylamide gel electrophoresis and immunoblot analysis using anti-caveolin-1 mAb. Film exposures were analyzed by densitometry and the signal intensity data normalized for each film so that the largest signal had a value of 1. All signal ratios were then averaged to calculate the relative intensity of caveolin-1 for tumor samples from each diet/drug cohort. Data are plotted as average caveolin-1 signal (arbitrary units) vs. diet/ezetimibe (Z) group ± SE. Analysis of variance analysis indicated no statistically significant diet effect (P = 0.171) however a significant drug effect in lowering caveolin for both diet conditions (P = 0.027). n = 6/group. Immunofluorescence. Upper panel; Quantification of caveolin-1 staining. Data are plotted as relative level of caveolin-1 staining versus diet/ezetimibe (Z) group ± SE Data were analyzed by mixed model analysis, which indicated highly significant main effects of ezetimibe (P < 0.0001) and diet (P < 0.0001). n = 43/group. Lower panel; representative caveolin-1 staining. Sections were stained for caveolin-1 (see Materials and Methods) and nuclei were counterstained with DAPI. Left column; representative anti-caveolin-1 mAb staining (Alexa Fluor 488; green). Right column; representative caveolin-1 staining (Alexa Fluor 488; green) merged with DAPI staining (blue). Tumors were fixed in OCT, and 10 μm sections were stained for the indicated markers. CD31 (PECAM) staining was performed as described.46 Fluorescent mages were acquired and analyzed by AxioVision 4.0 software for quantification. In all cases data are considered significant at P < 0.05.