The Chemokine CXCL16 and Its Receptor, CXCR6, as Markers and Promoters of Inflammation-Associated Cancers

Clinical observations and mouse models have suggested that inflammation can be pro-tumorigenic. Since chemokines are critical in leukocyte trafficking, we hypothesized that chemokines play essential roles in inflammation-associated cancers. Screening for 37 chemokines in prostate cancer cell lines and xenografts revealed CXCL16, the ligand for the receptor CXCR6, as the most consistently expressed chemokine. Immunohistochemistry and/or immunofluorescence and confocal imaging of 121 human prostate specimens showed that CXCL16 and CXCR6 were co-expressed, both on prostate cancer cells and adjacent T cells. Expression levels of CXCL16 and CXCR6 on cancer cells correlated with poor prognostic features including high-stage and high-grade, and expression also correlated with post-inflammatory changes in the cancer stroma as revealed by loss of alpha-smooth muscle actin. Moreover, CXCL16 enhanced the growth of CXCR6-expressing cancer and primary CD4 T cells. We studied expression of CXCL16 in an additional 461 specimens covering 12 tumor types, and found that CXCL16 was expressed in multiple human cancers associated with inflammation. Our study is the first to describe the expression of CXCL16/CXCR6 on both cancer cells and adjacent T cells in humans, and to demonstrate correlations between CXCL16 and CXCR6 vs. poor both prognostic features and reactive changes in cancer stoma. Taken together, our data suggest that CXCL16 and CXCR6 may mark cancers arising in an inflammatory milieu and mediate pro-tumorigenic effects of inflammation through direct effects on cancer cell growth and by inducing the migration and proliferation of tumor-associated leukocytes

Fast chopper for single radio-frequency quadrupole bunch selection for neutron time-of-flight capabilities

A fast chopper system has been developed and tested for single bunch selection with the radio-frequency quadrupole (RFQ) accelerating element of the Soreq Applied Research Accelerator Facility (SARAF). The fast chopper consists of a high voltage (HV) deflector just before the RFQ, providing both positive and negative HV deflections and fast HV switching between polarities to enable momentary transmission of a single prebunch to the RFQ. Presently, the system enables single bunch selection for protons and deuterons at a repetition rate as determined by the user of up to 200 kHz, with bunch transmission of up to 50%, and with neighboring bunch contamination of less than 15%. Single bunch selection provides SARAF with fast neutron time-of-flight (TOF) capabilities. Measurements performed with liquid scintillation detectors show clear gamma and neutrons peaks, with TOF resolution of about 1 nanosecond FWHM. Beam dynamics simulations suggest possibilities for further improvements of the fast chopper and single bunch selection characteristics, with a significant lowering or elimination of the neighboring bunches, enhanced TOF resolution, and increased repetition rate to above 200 kHz. Fast neutron TOF capabilities, especially at phase II of SARAF, will provide exceptional opportunities for neutron induced reaction measurements for nuclear technology and fundamental research

Inflammatory stroma and cytokines induce CXCL16 and CXCR6 on normal and malignant prostate cells.

<p>(A) Staining for CXCL16 and CXCR6 in prostatectomy specimens using DAB (brown) for detection is shown for: chronic prostatitis, PIA and PIN. Panels are representative of over 80 prostate cancer cases examined. Each panel includes an H&E stain on the left. (B) CXCL16 secreted by primary normal epithelial cells (PrEC) and stromal cells (PrSC), and the DU145 and PC3 cell lines was measured 48 hours after no treatment (control), or treatment with 5 ng/ml IFN-γ and 0.5 µg/ml TNF-α alone and in combination. Bars show means±SEM from duplicate wells, of one representative experiment of three performed for each of three donors (D1–D3). *, p<0.05 and **, p<0.01 vs. untreated cells. (C) CXCL16 mRNA expression in PrEC from three donors and two prostate tumor cell lines (DU145 and PC3) treated with 5 ng/ml IFN-γ and 0.5 µg/ml TNF-α alone and in combination were analyzed using real-time RT-PCR. Values were normalized by setting the control sample with lowest expression equal to 1. Each bar represents the mean±SEM obtained from duplicate wells, from one representative experiment of three performed. **, p<0.01 vs. untreated cells. (D) CXCR6 mRNA expression in PrEC from three donors treated with 5 ng/ml IFN-γ and 0.5 µg/ml TNF-α alone and in combination were analyzed using real-time RT-PCR. Values were normalized by setting the sample with lowest expression equal to 1. **, p<0.01 vs. untreated cells. (E) Each row contains serial sections from the same regions of prostatectomy specimens stained using DAB (brown) for αSMA, CXCL16, and CXCR6 including an H&E stain on the left. Panels are representative of over 120 prostate specimens. (F) Separate slides of arrays containing prostate tissue from 40 patients were stained for αSMA, CXCL16 and CXCR6 and scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006695#s4" target="_blank">Material and Methods</a> for αSMA in stroma and CXCL16 and CXCR6 in cancer cells. Samples were grouped according to scores for CXCL16 or CXCR6 (n = samples in each group), and mean scores were calculated for αSMA in stroma. Data are displayed and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006695#pone-0006695-g001" target="_blank">Fig. 1E</a>.I. *, p<0.05.</p

CXCL16 and CXCR6 establish positive feedback loops contributing to the progression of inflammation-associated cancers.

<p>Secretion of CXCL16 from pre-cancer or cancer cells leads to CXCR6-mediated recruitment of leukocytes. Cytokines such as TNF-α and IFN-γ produced by these cells in turn induce expression of CXCL16 and CXCR6, creating a positive feedback loop that enhances tumorigenesis – directly, through effects on the growth of pre-cancer and cancer cells, and indirectly, by increasing the migration and proliferation of the inflammatory cells, together leading to the increased growth of inflammation-associated cancers.</p

Roles for CXCL16 and CXCR6 in prostate cancer stage, grade and cell growth.

<p>(A) Arrays containing prostate cancer tissue from 40 patients were stained for CXCL16 and CXCR6. Samples were scored for CXCL16 and CXCR6 expression as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006695#s4" target="_blank">Materials and Methods</a>. Samples were grouped according to stage or Gleason score (X-axes) as determined by the supplier, and mean scores for CXCL16 and CXCR6 expression were calculated. The number of samples (n) in each stage or Gleason score group is shown. Error bars show SEM. Cross-bars indicate comparisons that are significant. *, p<0.05 and ***, p<0.001. (B) PC3 cells were transfected with the CXCR6-YFP plasmid, cultured for 48 hours without chemokine (control, ctrl) or with 0.05–5 µg/ml CXCL16 and numbers of CXCR6-YFP⁺ (R6YFP⁺) vs. CXCR6YFP⁻ (R6YFP⁻) cells were counted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006695#s4" target="_blank">Materials and Methods</a>. Values were normalized to the control, untreated sample to yield the fold difference in cell number. Bars show means±SEM combined from three experiments. **, p<0.01 and ***, p<0.001 vs. control values. (C) Following transfection with the CXCR6-YFP plasmid, PC3 cells were cultured as in (B), without or with 5 µg/ml CXCL16. Left panel, gating is shown for separating untreated and CXCL16-treated cells into “low” (L) or “high” (H) for CXCR6-YFP. Percentages of cells in the high gates are shown. One representative experiment is shown out of three performed. Right panel, CXCR6-YFP⁺low (CXCR6+low) and CXCR6-YFP⁺high (CXCR6+high) cells from CXCL16-treated and control cultures were counted after 48 hours as in (B). Data were combined from three experiments. *, p<0.05 and ***, p<0.001 vs. untreated cells. (D) Confocal imaging displays anti-CXCL16 or anti-CXCR6 (488 tyramide) as green, anti-Ki67 (594 tyramide) as red, and nuclear staining with Hoechst 33258 as blue. Panel is representative of more than 15 cases.</p

CXCL16 can mediate the migration and proliferation of T cells expressing CXCR6.

<p>(A) CD4⁺ T cells were sorted into naïve (CD45RO-), total effector/memory (CD45RO+), CD45RO⁺CXCR6⁻ (CXCR6-) and CD45RO⁺CXCR6⁺ (CXCR6+) subsets and stimulated with PMA and ionomycin for 4 hours before measuring levels of CXCL16 mRNA by real-time RT-PCR. Values were normalized by setting the sample with lowest expression equal to 1. The data are means from duplicate samples from one representative experiment out of three performed. (B) Jurkat E6.1 T cells transfected with the CXCR6-YFP plasmid were analyzed for migration to CXCL16. Some samples were pre-incubated for two hours with 400 ng/ml pertussis toxin as indicated. Numbers of YFP+ and YFP- migrating cells were analyzed by flow cytometry. Each bar represents the mean±SEM obtained from triplicate wells, from one representative experiment of three performed. ***, p<0.001 vs. the corresponding control value. (C) Following transfection with the CXCR6-YFP plasmid, Jurkat E6.1 T cells were cultured for 48 hours without chemokine (control, ctrl) or with 1 µg/ml CXCL8 as an additional control, or with 0.05–5 µg/ml CXCL16 before counting numbers of CXCR6-YFP⁺ vs. CXCR6-YFP⁻ cells. Bars show means±SEM combined from three different experiments. *, p<0.05 and ***, p<0.001. (D) Transfected cells were treated as in (C) and stained with Annexin V and propidium iodide (PI) before counting numbers of CXCR6-YFP⁺ cells within the subgroups as noted. Bars show means±SEM combined from three experiments. **, p<0.01 and ***, p<0.001 vs. control untreated cells. (E) Following transfections with CXCR6-YFP plasmid, Jurkat E6.1 T cells were loaded with 2 µM Far Red DDAO dye and cultured without chemokine (control, ctrl) or with 0.05–5 µg/ml CXCL16 or 1 µg/ml CXCL8. Numbers of CXCR6YFP⁺ vs. CXCR6YFP⁺DDAO⁻ (proliferated) and CXCR6YFP⁺DDAO⁺ (non-proliferated) cells were determined after 48 hours using counting beads and flow cytometry. Bars show means±SEM from triplicate wells, from one representative experiment of three performed. ***, p<0.001 and *, p<0.05 vs. control untreated cells.</p

CXCL16 is highly expressed in multiple inflammation-associated cancers.

<p>(A) * The intensity of CXCL16 staining was scored as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006695#s4" target="_blank">Materials and Methods</a>. The % of cases staining high is shown for each cancer type. ** RCC, renal cell carcinoma; GBM, glioblastoma multiforme. (B) CXCL16 expression in arrays of multiple human cancers by immunohistochemistry using AEC (red) or DAB (brown) for detection. Panels are representative of the numbers of specimens of the various cancers as listed in (A).</p