Journal: Cancer Research

Article Title: Macrophage-Derived Cholesterol Contributes to Therapeutic Resistance in Prostate Cancer

doi: 10.1158/0008-5472.can-20-4028

Figure Lengend Snippet: Figure 1. Development of syngeneic mouse models representing CRPC. A, Mouse model scheme. B, Tamoxifen induced GFP and Ki67 expression in prostate glands of a Ptenpce/Trp53pce/TMPRSS2-ERGpceþ mouse after 2 months of tamoxifen administration; n ¼ 1. C, Detection of prostate tumors and overall survival in Ptenpce/Trp53pce/ and Ptenpce/Trp53pce/TMPRSS2-ERGpceþ mice. Days reflect time post tamoxifen administration. Tumor incidence and size were monitored by monthly MRI starting at 5 to 6 months. n ¼ 17–19 mice, pooled from two independent cohorts. Significance between the two groups was determined by log-rank. D, H&E and Masson trichrome staining in Ptenpce/Trp53pce/TMPRSS2-ERGpceþ mice from C. Control prostate glands were harvested from mice at 6 and 12 months of age. E, Representative T2-weighted image by MRI of tumor lesion–bearing Ptenpce/Trp53pce/ mice treated with vehicle or 0.6 mg Lupron subcutaneously every 28 days for two cycles, starting 4 months after tamoxifen administration. n ¼ 3–5 mice per group; data from a one cohort of mice. F, Expression of Krt5, Krt8, Krt14, Krt18 in the different cell lines generated from either Ptenpce/Trp53pce/ (PT) or Ptenpce/Trp53pce/TMPRSS2-ERGpceþ (PTE) mice at end-stage. Data shown as the mean SEM. TRAMP-C2 cell line was used as a comparison. Right, representative confocal microscopy immunofluorescent images of the PTE-82 cell line stained for cytokeratin 14 and cytokeratin 18. Similar results were obtained for the PT-09, PT-25, and PTE-24 cell lines. Images represent one of two independent experiments. G, Expression of Ar in the different cell lines. Data shown as the mean SEM. H, Confocal microscopy immunofluorescent images of the PTE-82 cell line either treated with 10 nmol/L DHT or left untreated in CSS. AR (red) and DAPI (blue). Images are representative of one of three independent experiments. I, Dose–response curve of enzalutamide in five different prostate cancer cell lines. Phase contrast images were acquired at 8-hour intervals using Incucyte, with confluence per well calculated per cell line. n ¼ 3; data shown as the mean SEM from one of at least three independent experiments. , P < 0.01.

Article Snippet: Mice were then randomly assigned to experimental groups and treated with 0.6 mg Lupron Depot (AbbVie Inc.) by subcutaneous injection or 20 mg/kg enzalutamide (Selleck Chemicals) by oral administration.

Techniques: Expressing, Staining, Control, Generated, Comparison, Confocal Microscopy

Journal: Cancer Research

Article Title: Macrophage-Derived Cholesterol Contributes to Therapeutic Resistance in Prostate Cancer

doi: 10.1158/0008-5472.can-20-4028

Figure Lengend Snippet: Figure 3. Macrophages directly regulate AR nuclear translocation. A, Representative immunohistochemistry for tumor macrophages (F4/80) and AR within orthotopic prostate tumors from mice treated with IgG (control), aCSF or Lupron. B, Quantification of the percent of F4/80þ cells and ARhi nuclei in serial sections. n ¼ 5–6 mice per group from one of two independent experiments. Four to five regions of interest per each slide were selected in nonnecrotic areas of the tumor. Significance determined by unpaired t test and is shown as , P < 0.001. C, Experimental schematic for cancer cell and BMDM coculture and quantification of nuclear AR by confocal microscopy. D, Kernel density estimation of the AR nuclear to cytoplasmic ratio on a cell-by-cell basis following incubation of PTE-82, PTE-24, and PTE-09 cancer cell lines alone, or in coculture with BMDMs under androgen-deprived conditions (i.e., CSS) for 48 hours. Percent of ARhi cells was computed per individual images using the third quartile of the nucleus/cytoplasm AR intensity ratio as a threshold (third quartile of PTE-82, PTE-24, or PTE-09 þ BMDM ¼ 3, 3, 2.4, respectively). n ¼ 3; data from one of at least three independent experiments. Significance determined by Mann–Whitney. E, Representative confocal microscopy images of the GFPþ PTE-24 cell line. AR (red), F4/80 (white), and DAPI (blue) fluorescence is shown. F, Impact of BMDMs on the proliferation of PTE-24 cells in the presence of serial concentrations of enzalutamide. Cell proliferation was monitored using live imaging with phase contrast images acquired every 6 hours. Data shown as the mean SEM and reflects one of two independent experiments. Significance determined by two-way ANOVA.

Article Snippet: Mice were then randomly assigned to experimental groups and treated with 0.6 mg Lupron Depot (AbbVie Inc.) by subcutaneous injection or 20 mg/kg enzalutamide (Selleck Chemicals) by oral administration.

Techniques: Translocation Assay, Immunohistochemistry, Control, Confocal Microscopy, Incubation, MANN-WHITNEY, Imaging