PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). separation of these distinct PTEN populations using RT-PCR analysis of mRNA expression (< 0.001; Physique ?Physique1A).1A). Cytokine profiling of these samples confirmed that low PTEN mRNA expression was correlative with increased 874819-74-6 IC50 CXCL8 mRNA (Spearman correlation: ?0.5088; = 0.0261) (Figure ?(Figure1B).1B). Conversely, subsequent 874819-74-6 IC50 profiling of these macro-dissected tumor samples confirmed that PTEN status did not correlate with significant changes in intrinsic expression of other cytokines including IL-6 (Spearman correlation: ?0.1091; = 0.6378) (Figure ?(Physique1C).1C). Other cytokines analyzed included CXCL1, CXCL2 and CXCL5 (data not shown). Physique 1 Comparative analysis of PTEN-status and cytokine expression in prostate cancer patient samples CXCL8 induces Rabbit Polyclonal to Cytochrome P450 51A1 chemotaxis of radioresistance-promoting THP1’s in a PTEN-deficient setting CXCL8 was initially characterized as a potent chemoattractant for leukocyte-derived immune cells [22]. Given the up-regulation of CXCL8 expression detected in PTEN-deficient tumors, further IHC analysis was performed to characterize the levels of CD68-positive macrophages detected within prostate patient samples (Physique ?(Figure2A).2A). Moderate to dense infiltration of CD68-positive macrophages was correlated with loss of PTEN protein expression across 70 analyzable cases (< 0.05). Lower levels of macrophage infiltration were detected within PTEN-positive tumors (Physique ?(Figure2B).2B). assays confirmed the function of CXCL8 in potentiating chemotactic migration of THP-1 cells, used in this context as a representative macrophage-like cell [243 66% above baseline migration towards serum-free medium; (Physique ?(Physique2C)].2C)]. Furthermore, our assays exhibited that the conditioned media (CM) harvested from irradiated PTEN-deficient Sh11.02 cells was capable of inducing THP-1 chemotaxis, and that this response could be partially inhibited following pre-treatment with a CXCL8 neutralizing antibody; however, this effect was not observed in PTEN-expressing NT01 cells (Physique ?(Figure2D).2D). Irradiating CaP cells can induce release of a multitude of cytokines (data not shown) and therefore total inhibition of cell migration may not be possible without considering this extensive signaling network. Physique 2 CXCL8 induces chemotaxis of radioresistance-promoting macrophages in a PTEN-deficient setting To establish how macrophage infiltration may affect therapeutic response, colony formation assays were used to characterize radiation sensitivity of PTEN-modulated DU145 cells, cultured in the presence or absence of THP-1 cells. Co-culture with THP-1 cells increased the resistance of both PTEN-positive NT01 and PTEN-deficient Sh11.02 cells to ionizing radiation (IR), with calculated dose enhancement factors (DEF) of 0.89 and 0.87, respectively. The equivalent degree of sensitization observed suggests that the mechanism of macrophage-afforded resistance was independent of the intrinsic PTEN status of the CaP cells (Physique 2E and 2F). These experiments were repeated in the PTEN-null PC3 cell line and although showing a pattern towards monocyte-driven radioresistance at the higher dose points, no significant difference in radiosensitivity was observed (Supplementary Physique 1A). This was also the case when PTEN expression was reconstituted under the control of a tetracycline-inducible promoter (Supplementary Physique 1B). IR induces secretion of TNF- from THP-1 cells but not from CaP cells Alongside CXCL8, the cytokine TNF- has recently been implicated with reduced overall survival and time to castration resistance in CaP patients [23]. Therefore the linkage of PTEN status to TNF- expression in malignant prostate cells was 874819-74-6 IC50 also decided. Analysis of DU145 NT01 and Sh11.02 cells indicated that loss of PTEN had no effect on TNF- secretion or mRNA levels in these isogenic lines (Supplementary Determine 2A and 2B). Furthermore, analysis of patient samples confirmed no correlation between PTEN mRNA levels and TNF- gene expression (Spearman correlation: ?0.1175; = 0.6318) (Physique ?(Figure3A).3A). Since we observed no intrinsic correlation of PTEN with TNF- expression, experimental results shown are derived from analysis of the PTEN-deficient Sh11.02 cell line while corresponding results 874819-74-6 IC50 derived from PTEN-positive NT01 cells presented in Supplementary Figures 2, 3 and 4. Physique 3 Impact of ionizing radiation 874819-74-6 IC50 and PTEN-status on TNF- signaling in prostate cancer and THP-1 cell lines The effect of radiation exposure upon TNF- secretion in CaP cells was also decided. Treatment of PTEN-deficient Sh11.02 cells with 3 Gy IR did not augment TNF- secretion levels compared to time-matched control samples, ranging from between 20C80 pg/ml/106 cells across a 24 h time course (Determine ?(Figure3B).3B). Ionizing radiation also had no effect on TNF- mRNA or protein expression (Supplementary Physique 2) or on expression of tumor necrosis factor receptor-1 (TNFR-1) in these cells (Physique ?(Physique3C).3C). Consistent with the secretion of TNF- and capacity of DU145 Sh11.02 cells and DU145 NT01 cells to respond in an autocrine manner, addition of 10 ng/ml.