Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages <i>In Vivo</i> in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Cardiff University

Stromal fibroblasts become altered in response to solid cancers, to exhibit myofibroblastic characteristics, with disease promoting influence. Infiltrating mesenchymal stem cells (MSC) may contribute towards these changes, but the factors secreted by cancer cells that impact MSC differentiation are poorly understood. We investigated the role of nano-metre sized vesicles (exosomes), secreted by prostate cancer cells, on the differentiation of bone-marrow MSC (BM-MSC), and the subsequent functional consequences of such changes. Purified exosomes impaired classical adipogenic differentiation, skewing differentiation towards alpha-smooth muscle actin (αSMA) positive myofibroblastic cells. A single exosomes treatment generated myofibroblasts secreting high levels of VEGF-A, HGF and matrix regulating factors (MMP-1, -3 and -13). Differentiated MSC had pro-angiogenic functions and enhanced tumour proliferation and invasivity assessed in a 3D co-culture model. Differentiation was dependent on exosomal-TGFβ, but soluble TGFβ at matched dose could not generate the same phenotype. Exosomes present in the cancer cell secretome were the principal factors driving this phenotype. Prostate cancer exosomes dominantly dictate a programme of MSC differentiation generating myofibroblasts with functional properties consistent with disease promotion.

Cancer exosomes trigger mesenchymal stem cell differentiation into pro-angiogenic and pro-invasive myofibroblasts.

Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVβ5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios.

Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array.

Exosomes are a distinct population of extracellular vesicles of endocytic origin with a protein repertoire similar to the parent cell. Although tumour-derived exosomes harbour immunosuppressive characteristics, they also carry tumour antigens and thus potentially contribute to immune activation. The aim of this study was to examine the impact of prostate cancer exosomes on tumour antigen cross-presentation. DU145 cells, transduced with shRNA to knockdown Rab27a (DU145) that inhibits exosome secretion, triggered significantly stronger tumour-antigen-specific T cell responses when loaded onto dendritic cells (DC) than control DU145 cells. Enhanced T cell response was prevented by adding purified exogenous DU145 exosomes to DU145 cells, demonstrating that the dominant effect of tumour exosomes is immunosuppression and not antigen delivery. CD8 T cell responses were impaired via exosomal regulation of DC function; exosomes triggered the expression of CD73, an ecto-5-nucleotidase responsible for AMP to adenosine hydrolysis, on DC. CD73 induction on DC that constitutively express CD39 resulted in an ATP-dependent inhibition of TNFα- and IL-12-production. We identified exosomal prostaglandin E (PGE) as a potential driver of CD73 induction, as inhibition of PGE receptors significantly reduced exosome-dependent CD73 induction. The results reveal a hitherto unknown suppression of DC function via exosomal PGE, adding a new element to tumour exosome-immune cell cross-talk. : AMP: adenosine monophosphate; ATP: adenosine triphosphate; BLCL: B lymphoblastoid cell line; CME: exosomes enriched from cell line conditioned media; DC: dendritic cell; DMSO: dimethyl-sulfoxide; DU145: DU145 cells with irrelevant knockdown control; DU145: DU145 cells with Rab27a knockdown; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; GM-CSF: granulocyte-monocyte colony stimulating factor; HLA: human lymphocyte antigen; IL: interleukin; LPS: lipopolysaccharide; mfi: mean fluorescence intensity; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffer solution; PGE: prostaglandin E; TRF: time-resolved fluorescence.

Dominant immunosuppression of dendritic cell function by prostate-cancer-derived exosomes.

Loss of latexin (LXN) expression negatively correlates with the prognosis of several human cancers. Despite association with numerous processes including haematopoietic stem cell (HSC) fate, inflammation and tumour suppression, a clearly defined biological role for LXN is still lacking. Therefore, we sought to understand LXN expression and function in the normal and malignant prostate to assess its potential as a therapeutic target. Our data demonstrate that LXN is highly expressed in normal prostate luminal cells but downregulated in high Gleason grade cancers. LXN protein is both cytosolic and secreted by prostate cells and expression is directly and potently upregulated by all-trans retinoic acid (atRA). Whilst overexpression of LXN in prostate epithelial basal cells did not affect cell fate, LXN overexpression in the luminal cancer line LNCaP reduced plating efficiency. Transcriptome analysis revealed that LXN overexpression had no direct effects on gene expression but had significant indirect effects on important genes involved in both retinoid metabolism and IFN-associated inflammatory responses. These data highlight a potential role for LXN in retinoid signaling and inflammatory pathways. Investigating the effects of LXN on immune cell function in the tumour microenvironment (TME) may reveal how observed intratumoural loss of LXN affects the prognosis of many adenocarcinomas.

The putative tumour suppressor protein Latexin is secreted by prostate luminal cells and is downregulated in malignancy.

Immunocompromised patients are highly susceptible to invasive aspergillosis. Herein, we identified a homozygous deletion mutation (507 del C) resulting in a frameshift (N170I) and early stop codon in the fungal binding Dectin-2 receptor, in an immunocompromised patient. The mutated form of Dectin-2 was weakly expressed, did not form clusters at/near the cell surface and was functionally defective. Peripheral blood mononuclear cells from this patient were unable to mount a cytokine (tumor necrosis factor, interleukin 6) response to Aspergillus fumigatus, and this first identified Dectin-2-deficient patient died of complications of invasive aspergillosis.

A Human Dectin-2 Deficiency Associated With Invasive Aspergillosis.

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.

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

Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial β-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin β-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by β-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial β-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.

Mark Gurney

Systems Immunity Research Institute,
Division of Infection and Immunity,
Systems Immunity Research Institute, Division of Infection and Immunity

Cardiff University

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice

Mark Gurney

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Cardiff University

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue-Resident Macrophages In Vivo in Mice

Systems Immunity Research Institute,
Division of Infection and Immunity,
Systems Immunity Research Institute, Division of Infection and Immunity