Article Figures & Data
Figures
- Figure 1.
IL6 mRNA expression in benign and malignant prostate tissues as assessed by qRT-PCR. RNA extracts were prepared from matched tumor and benign tissues from radical prostatectomy specimens. IL6 mRNA expression was then determined by qRT-PCR. A, log2 IL6 mRNA expression in tumor relative to matched benign tissue in 10 prostatectomy specimens. IL6 mRNA expression levels were normalized to GAPDH, and then tumor was compared with benign using the 2−ΔΔCT method followed by log2 transformation. B, log2 relative IL6 mRNA expression in benign samples versus tumor samples.
- Figure 2.
Validation of IL6 CISH assay. A, IL6-transfected (positive control) or nontransfected (negative control) FFPE MCF7 cells assayed by IL6 CISH. B, IL6 qRT-PCR on 11 prostate cell lines. IL6 mRNA expression levels were normalized to GAPDH and then to PrEC cell expression level using the 2−ΔΔCT method. C, IL6 CISH was performed on the same 11 prostate cell lines, and representative examples are shown. Brown staining (arrowheads) represents positive IL6 mRNA expression. Single dots in the nucleus of cells are interpreted as the genomic copy of the gene (PC3 cells, arrow). Both qRT-PCR and CISH analyses indicated that three of the prostate cultured cells or cell lines (PrSC, RWPE-1, DU145) were positive for IL6 mRNA, indicating complete concordance between the two assays.
- Figure 3.
IL6 mRNA is not detected in prostate adenocarcinoma cells in primary tumors. Twenty-one prostatectomy specimens were selected for IL6 CISH (Table 1). PPIB (housekeeping gene) is used as a positive control for the CISH assay. A, representative examples of low-grade (Gleason pattern 3) and higher-grade (Gleason patterns 4 and 3) prostate cancer with positive staining for PPIB (positive control) and negative staining for IL6. B, IL6-positive cells in endothelium (arrow) in an area of cancer.
- Figure 4.
IL6 mRNA expression is nearly exclusively restricted to the prostate stromal compartment. A and B, IL6 mRNA-expressing cells (brown staining) are highly enriched in the stroma in areas of acute inflammation as indicated by the presence of neutrophils in glandular lumens (red arrows). C, IL6-positive cells in the stroma (arrows) and endothelium (arrowheads) surrounding prostate atrophy. D, positive epithelial staining was rare and restricted to prostatic atrophy (arrowheads).
- Figure 5.
IL6 mRNA is not detected in metastatic prostate cancer cells. In this study, metastatic prostate cancer in lymph node and liver biopsy samples and autopsy samples (liver, lung, bone, and lymph node) were assayed by IL6 CISH. No IL6-positive mRNA expression was observed in prostate cancer cells in any of the metastatic tissues. A, representative examples of lymph node biopsy and autopsy liver, lung, and bone metastases with positive staining for PPIB (positive control) and negative staining for IL6. B, example of IL6 mRNA-positive blood vessels (arrowheads) and IL6-negative tumor cells (arrows) in bone metastases.
- Figure 6.
A requirement for protein transport inhibition for IL6 IHC. A, IL6 CISH performed on the IL6-positive cell line NCI-H460. B and C, IL6 IHC performed on (B) PC3 cells (IL6 negative as assessed by qRT-PCR and CISH) and (C) NCI-H460 cells. D, marked difference in IHC results when NCI-H460 cells are treated with monensin prior to fixation. Arrows point to IL6-positive cells. E and F, the same prostatectomy case containing acute inflammation assayed for IL6 by CISH (E) and IHC (F). G and H, example of IL6 IHC on primary prostate cancer (G) and prostate cancer bone metastasis (H). All tumor cells were negative in all samples analyzed. Positive staining observed in extracellular spaces (arrows in H) was considered to be nonspecific.
Tables
- Table 1.
Assessment of levels and distribution of IL6 mRNA expression in prostatectomy tissues
Patient IL6 epithelium IL6 stroma Patient # Gleason score Clinical stage Tumor Normal Atrophy Noninflamed Inflamed Acute inflammation present? 1 3+3 = 6 T2N0MX − − ++ ++ ++++ Yes 2 3+4 = 7 T2N0MX − − − − +++ Yes 3 4+5 = 9 T2N0MX − − + − − No 4 3+4 = 7 T2N0MX − − + − + No 5 3+3 = 6 T2N0MX − − − − + No 6 3+4 = 7a T2N0MX − − − − ++ No 7 3+3 = 6b T2N0MX − − ++ − ++ No 8 3+4 = 7 T2N0MX − − − − ++++ Yes 9 5+4 = 9 T2N0MX − − − − ++ No 10 3+3 = 6b T2N0MX − − − − − No 11 3+4 = 7a T2N0MX − − − − − No 12 3+4 = 7 T2N0MX − − + − ++ No 13 3+4 = 7 T3AN0MX − − − − − No 14 3+4 = 7 T2N0MX − − ++ − ++ No 15 3+3 = 6 T2N0MX − − + − ++ No 16 3+4 = 7 T2N0MX − − +++ ++ ++++ Yes 17 3+3 = 6 T2N0MX − − − − +++ Yes 18 4+3 = 7a T3AN0MX − − + − + No 19 3+4 = 7 T3AN0MX − − − − − No 20 4+3 = 7 T3BN0MX − − ++ − + No 21 3+4 = 7a T2N0MX − − + − ++ No
Supplementary Data
- Supplementary Figure S1 - <b>IL-6 mRNA expression in macrophages and endothelial cells.</b> RNAscope 2-plex assay for IL-6 (red) and CD68 (green) <b>(A)</b> or IL-6 (red) and CD31 (green) <b>(B)</b> indicated that some IL-6 positive cells in the stroma are macrophages or endothelial cells, respectively (arrows).
- Supplementary Figure S2 - <b>IL-6 mRNA positive blood vessels in prostate cancer bone metastases.</b> Example of IL-6 positive endothelial cells and IL-6 negative metastatic tumor cells in prostate cancer bone metastases.
- Supplementary Figure S3 - <b>Western blot on NCI-H460 cells.</b> Western blot on protein lysates from NCI-H460 cells grown without (1) or with (2) protein transport inhibition with monensin (Golgi-Stop{trade mark, serif}). (3) 300 ng recombinant IL-6, positive control.
- Supplementary Figure S4 - <b>IL-6 CISH assay is more sensitive than IL-6 IHC. (A)</b> Standard curve generated using estimated copies of IL-6 cDNA clone expression vector (Origene, SC125236) used to determine IL-6 mRNA copy number in DU145 and PC3 cells. <b>(B) </b>IL-6 CISH on corresponding DU145 and PC3 cells. Note DU145 cells with many copies of IL-6 mRNA (arrow) and cells where lower copy numbers (5-10 dots per cell, arrowhead) can be visualized. Side-by-side IL-6 IHC is negative.
- Supplementary Figure Legends - Supplementary Figure Legends
- Supplementary Tables S1-S3 - Supplementary Table S1. Clinical Characteristics of Patient Samples Used in the Study. Supplementary Table S2. Results of Oncomine Database Query for IL-6 Expression. Supplementary Table S3. Assessment of Levels and Distribution of IL-6 mRNA Expression in Metastatic Tissue.
Volume 3, Issue 10
