Article Figures & Data
Figures
- Figure 1.
RISH analysis revealed differential mRNA expression of IL1β, IL6, IL8, and IL10 in prostatectomy tissues. Modest IL1β expression was observed primarily in stromal compartment cells consistent with immune/inflammatory cells (arrows), or at times in atrophic epithelial cells (arrowhead). IL6 expression was largely confined to the stromal compartment in endothelial cell lining vessels (arrowheads) and on immune cells (arrows). IL8 was the most highly expressed cytokine assessed, observed both in benign and tumor regions. IL8 expression was predominant in atrophic glands (red arrowheads) but was also seen on immune cells (red arrows) and potentially tumor cells (blue arrowhead). Limited IL10 expression was observed on immune cells (arrows). Cytokine expression was dramatically increased in highly inflamed regions.
- Figure 2.
RISH analysis demonstrated IL8 mRNA expression in prostate tissues from men with prostate cancer. IL8 expression was predominant in atrophic glands particularly (A) in regions of intense inflammation, (B) atrophic glands involving basal cell proliferation, and (C) atrophic glands encapsulating corpora amylacea. IL8 expression was markedly increased in (D) urethral tissue in most cases. There was considerable IL8 expression observed in (E and F) tumor regions on immune infiltrates and in tumor cells (arrowheads). G and H, Concurrent IL8 RISH (brown) and CK8 IHC (red) analysis confirms that IL8 mRNA is expressed within prostate epithelial cells (arrows) in tumor regions; (I) CD68 (red)-positive macrophages do not express IL8, but some (J) CD66 (red)-positive neutrophils do (black arrows). There were also some IL8− neutrophils (red arrow).
- Figure 3.
Quantification of RISH IL8 expression in TMAs showed (A) significantly increased expression in tumor regions compared with benign regions. B, There was elevated IL8 expression in both benign and tumor regions in higher-grade cases. C, There was no apparent difference in expression between races, (D) but a modest increase was observed in higher-grade tumors compared with lower-grade tumors from AA and EA men, but only significant for EA men. E, There was significantly elevated IL8 expression in atrophic glands compared with the remaining regions of the prostate. F, IHC analysis of CD66 showed comparable neutrophils in benign and tumor regions. Each data point represents the median of up to 4 TMA spots except for E where all TMA spots are shown. Center line shows the median for each group (*, P < 0.05; **, P < 0.005; ***, P < 0.0005; and ****, P < 0.0001).
- Figure 4.
RISH analysis of a TMA set constructed from metastatic tumors obtained via autopsy showed that (A) IL8 mRNA is differentially expressed in every site assessed. B, Metastatic tissues from liver had the most intense signal compared with prostate (P < 0.0001). Each data point represents a TMA spot, and centerline shows the median for each group. The number of patients from which each site was sampled is noted (n = x). C, IL8 RISH analysis revealed IL8 mRNA expression in AR-null cell lines compared with AR-expressing cell lines (**, P < 0.005 and ****, P < 0.0001).
- Figure 5.
A, AR-null metastatic tissues have modestly elevated IL8 mRNA in epithelial cells compared with in AR-positive metastases (IL8 CISH left, AR IHC right). B, Regions of prostatic atrophy with dramatically elevated IL8 expression (red arrowheads) were more likely to have AR downregulation in radical prostatectomy tissues.
- Figure 6.
IL8 RISH analysis of 42 LuCaP PDX TMA set revealed (A and C) comparable IL8 mRNA expression between PDX and castration-resistant PDX lines, but (B and C) elevated IL8 expression in AR− PDX compared with AR+ PDX lines. Each data point in A and B represents the mean of 3 TMA spots, and the centerline shows the mean of each group. ***, P < 0.0001.
- Figure 7.
A, ChIP-seq analysis shows AR binding at the IL8 promoter (blue arrow) in LNCaP and C4-2b cell lines. B, LPS treatment induced IL8 protein expression in AR-negative PC3 cells but not in AR-positive LNCaP, LAPC4, or CWR22rv1 cells, even with AR antagonist (enzalutamide) treatment. C, AR-targeted shRNA (AR3717, AR3718, and AR26211) knockdown in LNCaP cells resulted in modest IL8 mRNA expression.
Additional Files
Supplementary Data
- Supplementary Figure S1 - Supplementary Figure S1 shows positive and negative controls for IL-1B, IL-6, IL-8, and IL-10 RISH and a comparison of IL-8 IHC versus IL-8 RISH.
- Supplementary Figure S2 - Supplementary Figure S2 shows quantification of IL-8 expression by RISH in whole tissue sections and TMAs by tumor grade and race.
- Supplementary Figure S3 - Supplementary Figure S3 shows quantification of CD66+ neutrophils in TMAs.
- Supplementary Figure S4 - Supplementary Figure S4 shows examples of liver metastases stained for IL-8 by RISH and CD66 by IHC as well as quantification of IL-8 and AR in metastatic tissues of AA versus EA men.
- Supplementary Figure S5 - Supplementary Figure S5 shows CCLE data on IL-8 and AR mRNA co-expression as well as examples of IL-8 expression in AR null metastases.
- Supplementary Figure S6 - Supplementary Figure S6 shows cBioPortal data for IL-8 and AR expression in primary prostate cancer, metastatic, and neuroendocrine tumors as well as CXCR1, CXCR2, and AR co-expression.
- Supplementary Figure S7 - Supplementary Figure S7 shows IL-8 western blot and RISH on LPS treated PC3 and DU145 cells and LNCaP cells with or without enzalutamide treatment.
- Supplementary Table S1 - Patient characteristics for whole tissue sections, 120 case TMA set, and autopsy TMA set.
Volume 18, Issue 1
