Stroma-derived extracellular vesicle mRNA signatures inform histological nature of prostate cancer.

Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non-invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell-derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor-matched pairs of adjacent-normal versus disease-associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease-discriminating mRNA. A set of transcripts including Caveolin-1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression-free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV-associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in-silico model was produced, showcasing the superiority of this multi-modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy.

Prostaglandin E2-mediated adenosinergic effects on CD14+ cells: Self-amplifying immunosuppression in cancer.

CD39 and CD73 are surface-expressed ectonucleotidases that hydrolyze ATP in a highly regulated, serial manner into ADP, AMP and adenosine. The end product, adenosine, has both tumor-promoting and immunosuppressive effects. The aim of this study was to determine CD73 expression on immune cells in pleural effusion (PE) in order to have a better understanding of the immune environment in mesothelioma. PE- or blood-derived CD14+ cells of mesothelioma patients and healthy donors were analyzed by flow cytometry for the expression of CD39 and CD73. CD73-induction was studied by exposure of CD14+ cells to the soluble fraction of PE (sPE), while the signaling mechanism, responsible for CD73 induction, by phosphoflow cytometry and receptor-inhibition studies. We observed CD73 expression on CD14+ cells in PE but not peripheral blood of mesothelioma patients or healthy donors. CD73 expression was inducible on CD14+ cells with sPE, cyclic-AMP (cAMP)-inducers (forskolin and prostaglandin-E2 (PGE2)) and adenosine. Inhibition of PGE2 receptors or adenosine A2 receptors blocked CD73-induction by sPE. sPE treatment triggered protein kinase A and p38 activation. However, signal-transducer and activator of transcription 3 (STAT3)-blocking led to enhanced CD73 expression, demonstrating a hitherto unknown negative control of purinergic signaling by STAT3 in CD14+ cells. TNFα production by CD73+ CD14+ cells was significantly impaired in the presence of AMP, confirming immunosuppressive function. Taken together, CD73 expression can be induced by PGE2, cAMP or adenosine on human CD14+ cells. We suggest that targeting this autocrine loop is a valid therapeutic approach in mesothelioma that may also enhance immunotherapy.

Prostate stromal cell proteomics analysis discriminates normal from tumour reactive stromal phenotypes.

Changes within interstitial stromal compartments often accompany carcinogenesis, and this is true of prostate cancer. Typically, the tissue becomes populated by myofibroblasts that can promote progression. Not all myofibroblasts exhibit the same negative influence, however, and identifying the aggressive form of myofibroblast may provide useful information at diagnosis. A means of molecularly defining such myofibroblasts is unknown. We compared protein profiles of normal and diseased stroma isolated from prostate cancer patients to identify discriminating hallmarks of disease-associated stroma. We included the stimulation of normal stromal cells with known myofibroblast inducers namely soluble TGFß and exosome-associated-TGFß and compared the function and protein profiles arising. In all 6-patients examined, diseased stroma exhibited a pro-angiogenic influence on endothelial cells, generating large multicellular vessel-like structures. Identical structures were apparent following stimulation of normal stroma with exosomes (5/6 patients), but TGFß-stimulation generated a non-angiogenic stroma. Proteomics highlighted disease-related cytoskeleton alterations such as elevated Transgelin (TAGLN). Many of these were also changed following TGFß or exosome stimulation and did not well discriminate the nature of the stimulus. Soluble TGFß, however triggered differential expression of proteins related to mitochondrial function including voltage dependent ion channels VDAC1 and 2, and this was not found in the other stromal types studied. Surprisingly, Aldehyde Dehydrogenase (ALDH1A1), a stem-cell associated protein was detected in normal stromal cells and found to decrease in disease. In summary, we have discovered a set of proteins that contribute to defining disease-associated myofibroblasts, and emphasise the similarity between exosome-generated myofibroblasts and those naturally arising in situ.

Cross-Presentation of the Oncofetal Tumor Antigen 5T4 from Irradiated Prostate Cancer Cells--A Key Role for Heat-Shock Protein 70 and Receptor CD91.

Immune responses contribute to the success of radiotherapy of solid tumors; however, the mechanism of triggering CD8(+) T-cell responses is poorly understood. Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8(+) T-cell stimulation. We established a cross-presentation model in which DCs present a naturally expressed oncofetal tumor antigen (5T4) from irradiated DU145 prostate cancer cells to 5T4-specific T cells. The aim was to establish which immunogenic signals are important in radiation-induced cross-presentation. Radiation (12 Gy) caused G2-M cell-cycle arrest and cell death, increased cellular 5T4 levels, high-mobility protein group-B1 (HMGB1) release, and surface calreticulin and heat-shock protein-70 (Hsp70) expression in DU145 cells. DCs phagocytosed irradiated tumor cells efficiently, followed by upregulation of CD86 on phagocytic DCs. CD8(+) 5T4-specific T cells, stimulated with these DCs, proliferated and produced IFNγ. Inhibition of HMGB1 or the TRIF/MyD88 pathway only had a partial effect on T-cell stimulation. Unlike previous investigators, we found no evidence that DCs carrying Asp299Gly Toll-like receptor-4 (TLR4) single-nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, pretreatment of tumor cells with Hsp70 inhibitors resulted in a highly statistically significant and robust prevention of antigen cross-presentation and CD86 upregulation on DCs cocultured with irradiated tumor cells. Blocking the Hsp70 receptor CD91 also abolished cross-presentation. Together, the results from our study demonstrate that irradiation induces immunologically relevant changes in tumor cells, which can trigger CD8(+) T-cell responses via a predominantly Hsp70-dependent antigen cross-presentation process.

Enhancement of T cell responses as a result of synergy between lower doses of radiation and T cell stimulation.

As a side effect of cancer radiotherapy, immune cells receive varying doses of radiation. Whereas high doses of radiation (>10 Gy) can lead to lymphopenia, lower radiation doses (2-4 Gy) represent a valid treatment option in some hematological cancers, triggering clinically relevant immunological changes. Based on our earlier observations, we hypothesized that lower radiation doses have a direct positive effect on T cells. In this study, we show that 0.6-2.4 Gy radiation enhances proliferation and IFN-γ production of PBMC or purified T cells induced by stimulation via the TCR. Radiation with 1.2 Gy also lowered T cell activation threshold and broadened the Th1 cytokine profile. Although radiation alone did not activate T cells, when followed by TCR stimulation, ERK1/2 and Akt phosphorylation increased above that induced by stimulation alone. These changes were followed by an early increase in glucose uptake. Naive (CD45RA(+)) or memory (CD45RA(-)) T cell responses to stimulation were boosted at similar rates by radiation. Whereas increased Ag-specific cytotoxic activity of a CD8(+) T cell line manifested in a 4-h assay (10-20% increase), highly significant (5- to 10-fold) differences in cytokine production were detected in 6-d Ag-stimulation assays of PBMC, probably as a net outcome of death of nonstimulated and enhanced response of Ag-stimulated T cells. T cells from patients receiving pelvic radiation (2.2-2.75 Gy) also displayed increased cytokine production when stimulated in vitro. We report in this study enhanced T cell function induced by synergistic radiation treatment, with potential physiological significance in a wide range of T cell responses.

Tumor stroma-derived factors skew monocyte to dendritic cell differentiation toward a suppressive CD14+ PD-L1+ phenotype in prostate cancer.

Tumor-associated stromal myofibroblasts are essential for the progression and metastatic spread of solid tumors. Corresponding myeloid cell infiltration into primary tumors is a negative prognostic factor in some malignancies. The aim of this study was to define the exact role of stromal myofibroblasts and stromal factors in early prostate carcinoma (PCa) regulating monocyte infiltration and differentiation into dendritic cells (DCs). Epithelial and stromal primary cultures were generated from PCa biopsies and their purity confirmed. Stromal cells produced significantly more of the (C-C) motif chemokine ligand 2 (CCL2), interleukin 6 (IL-6) and transforming growth factor ß (TGFß) than epithelial cells. Monocyte chemoattraction was predominantly due to stromal-derived factors, mainly CCL2. DCs generated in the presence of stromal (but not epithelial) factors upregulated CD209, but failed to downregulate the monocyte marker CD14 in a signal transducer and activator of transcription 3 (STAT3)-dependent manner. Monocytes exposed to stromal factors did not produce detectable amounts of IL-10, however, upon lipopolysaccharide stimulation, stromal factor generated dendritic cells (sDC) produced significantly more IL-10 and less IL-12 than their conventional DC counterparts. sDC failed to cross-present tumor-antigen to CD8+ T cells and suppressed T-cell proliferation. Most importantly, sDC expressed significantly elevated levels of programmed cell death ligand-1 (PD-L1) in a primarily STAT3 and IL-6-dependent manner. In parallel with our findings in vitro, tumor-infiltrating CD14+ cells in situ were found to express both PD-L1 and CD209, and a higher percentage of tumor-associated CD3+ T cells expressed programmed cell death-1 (PD-1) molecules compared to T cells in blood. These results demonstrate a hitherto undescribed, fundamental contribution of tumor-associated stromal myofibroblasts to the development of an immunosuppressive microenvironment in early PCa.

Resistance of CD45RA- T cells to apoptosis and functional impairment, and activation of tumor-antigen specific T cells during radiation therapy of prostate cancer.

The effect of radiation therapy (RT) to the pelvis on circulating T cells was studied in prostate cancer (PCa) patients to provide a baseline for a more informed design of combination radioimmunotherapy. Peripheral blood samples taken from 12 PCa patients with locally advanced tumor before, during, and after hypofractionated RT were analyzed for T cell phenotype and function. There was significantly more loss of naive and early memory compared with more differentiated T cells during RT. The proportions of annexin-V(+) and Fas-expressing T cells were elevated in patients during RT and in PBMC irradiated in vitro (< or = 5.0 Gy), with preferential increases in CD45RA(+) T cells. The baseline level of apoptosis of CD45RA(-) T cells increased > 2-fold in the presence of an IkappaB-kinase inhibitor, indicating a protective effect via this pathway. T cell proliferation was impaired during RT with IL-2-dependent recovery post-RT. Recall T cell responses to common viral Ags, measured by IFN-gamma production, were little affected by RT. In vitro irradiation of healthy donor PBMCs resulted in a significantly increased frequency of responding T cells, due at least partly to the preferential elimination of CD45RA(+) T cells. Most importantly, antitumor CD4(+) and CD8(+) T cell responses were detectable after, but not before or during RT. The results indicate that generating tumor-specific T cell responses before RT and boosting their activity post-RT are ways likely to amplify the frequency and function of antitumor T cells, with implications for scheduling immunotherapy in PCa.