Search

An introduction is presented which discusses various topics within the issue including extracellular vesicles (EV) biology in the tumour microenvironment, EV heterogeneity and role of lipids and EV in tumour progress.

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 PGE2receptors or adenosine A2receptors 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. [ABSTRACT FROM AUTHOR]

Autoimmune conditions, such as rheumatoid arthritis, are characterised by a loss of immune tolerance, whereby the immune cells attack self-antigens causing pain and inflammation. These conditions can be brought into remission using pharmaceutical treatments, but often have adverse side effects and some patients do not respond favourably to them. Human umbilical cord mesenchymal stromal cells (UCMSCs) present a promising alternative therapeutic due to their innate anti-inflammatory properties which can be strengthened using pro-inflammatory conditions. Their therapeutic mechanism of action has been attributed to paracrine signalling, by which nanosized acellular particles called 'extracellular vesicles' (EVs) are one of the essential components. Therefore, this research analysed the anti-inflammatory properties of UCMSC-EVs 'primed' with pro-inflammatory cytokines and at baseline with no inflammatory cytokines (control). Both control and primed EVs were co-cultured with un-pooled peripheral blood mononuclear cells (PBMCs; n = 6) from healthy donors. Neither control nor primed EVs exerted a pro-inflammatory effect on PBMCs. Instead, the primed EVs showed the immunosuppressive potential by increasing the expression of the anti-inflammatory protein FoxP3 in PBMCs. This may be attributed to the upregulated miRNAs identified in primed EVs in comparison to control EVs (miR-139-5p, miR-140-5p, miR-214-5p). These findings aid in understanding how UCMSC-EVs mediate immunosuppression and support their potential use in treating autoimmune conditions. [ABSTRACT FROM AUTHOR]

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 β (TGFβ) 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 findingsin vitro, tumor-infiltrating CD14+cellsin situwere 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. [ABSTRACT FROM AUTHOR]

The prostate gland is a complex and heterogeneous organ composed of epithelium and stroma. Whilst many studies into prostate cancer focus on epithelium, the stroma is known to play a key role in disease with the emergence of a cancer-associated fibroblasts (CAF) phenotype associated upon disease progression. In this work, we studied the metabolic rewiring of stromal fibroblasts following differentiation to a cancer-associated, myofibroblast-like, phenotype. We determined that CAFs were metabolically more active compared to normal fibroblasts. This corresponded with a heightened lipogenic metabolism, as both reservoir species and building block compounds. Interestingly, lipid metabolism affects mitochondria functioning yet the mechanisms of lipid-mediated functions are unclear. Data showing oxidised fatty acids and glutathione system are elevated in CAFs, compared to normal fibroblasts, strengthens the hypothesis that increased metabolic activity is related to mitochondrial activity. This manuscript describes mechanisms responsible for the altered metabolic flux and shows that prostate cancer-derived extracellular vesicles can increase basal respiration in normal fibroblasts, mirroring that of the disease-like phenotype. This indicates that extracellular vesicles derived from prostate cancer cells may drive an altered oxygen-dependent metabolism associated to mitochondria in CAFs. [Display omitted] • Lipogenic metabolic pathways are more prevalent in CAFs. • Energy producing pathways alternative to glycolysis support CAFs metabolic activity. • Mitochondrial metabolism is altered in fibroblast differentiation towards myofibroblasts. • Extracellular vesicles provoke a higher basal respiration associated to mitochondria in normal fibroblasts. [ABSTRACT FROM AUTHOR]

Acoustofluidic devices becomes one of the emerging and versatile tools for many biomedical applications. Most of the previous acoustofluidic devices are used for cells manipulation, and the few devices for cell phenotyping with a limitation in throughput. In this study, an enhanced tilted-angle (ETA) acoustofluidic device is developed and applied for mechanophenotyping of live cells. The ETA Device consists of an interdigital transducer which is positioned along a microfluidic channel. An inclination angle of 5° is introduced between the interdigital transducer and the liquid flow direction. The pressure nodes formed inside the acoustofluidic field in the channel deflect the biological cells from their original course in accordance with their mechanical properties, including volume, compressibility, and density. The threshold power for fully converging the cells to the pressure node is used to calculate the acoustic contrast factor. To demonstrate the ETA device in cell mechanophenotyping, and distinguishing between different cell types, further experimentation is carried out by using A549 (lung cancer cells), MDB-MA-231 (breast cancer cells), and leukocytes. The resulting acoustic contrast factors for the lung and breast cancer cells are different from that of the leukocytes by 27.9% and 21.5%, respectively. These results suggest this methodology can successfully distinguish and phenotype different cell types based on the acoustic contrast factor. [Display omitted] • An acoustic tweezer is developed to distinguish different cell types based on the acoustic contrast factor. • Reference trajectories combined with new algorithms simplify equipment requirements. • The acoustic contrast factor and compressibility of cells are measured by cell reference trajectories. • The acoustic contrast factor and compressibility of different cells show significant differences. [ABSTRACT FROM AUTHOR]

Cytopathology, crucial in disease diagnosis, commonly uses microscopic slides to scrutinize cellular abnormalities. However, processing high volumes of samples often results in numerous negative diagnoses, consuming significant time and resources in healthcare. To address this challenge, a surface acoustic wave-enhanced multi-view acoustofluidic rotation cytometry (MARC) technique is developed for pre-cytopathological screening. MARC enhances cellular morphology analysis through comprehensive and multi-angle observations and amplifies subtle cell differences, particularly in the nuclear-to-cytoplasmic ratio, across various cell types and between cancerous and normal tissue cells. By prioritizing MARC-screened positive cases, this approach can potentially streamline traditional cytopathology, reducing the workload and resources spent on negative diagnoses. This significant advancement enhances overall diagnostic efficiency, offering a transformative vision for cytopathological screening., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Extracellular vesicles (EVs) are secreted nanostructures that play various roles in critical cancer processes. They operate as an intercellular communication system, transferring complex sets of biomolecules from cell to cell. The concentration of EVs is difficult to decipher, and there is an unmet technological need for improved (faster, simpler, and gentler) approaches to isolate EVs from complex matrices. Herein, an acoustofluidic concentration of extracellular vesicles (ACEV) is presented, based on a thin-film printed circuit board with interdigital electrodes mounted on a piezoelectric substrate. An angle of 120° is identified between the electrodes and the reference flat of the piezoelectric substrate for simultaneous generation of Rayleigh and shear horizontal waves. The dual waves create a complex acoustic field in a droplet, resulting in effective concentration of nanoparticles and EVs. The ACEV is able to concentrate 20 nm nanospheres within 105 s and four EV dilutions derived from the human prostate cancer (Du145) cell line in approximately 30 s. Cryo-electron microscopy confirmed the preservation of EV integrity. The ACEV device holds great potential to revolutionize investigations of EVs. Its faster, simpler, and gentler approach to EV isolation and concentration can save time and effort in phenotypic and functional studies of EVs., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Extracellular vesicles (EVs) are increasingly recognized as important mediators of intercellular communication. They have important roles in numerous physiological and pathological processes, and show considerable promise as novel biomarkers of disease, as therapeutic agents and as drug delivery vehicles. Intriguingly, however, understanding of the cellular and molecular mechanisms that govern the many observed functions of EVs remains far from comprehensive, at least partly due to technical challenges in working with these small messengers. Here, we highlight areas of consensus as well as contentious issues in our understanding of the intracellular and intercellular journey of EVs: from biogenesis, release and dynamics in the extracellular space, to interaction with and uptake by recipient cells. We define knowledge gaps, identify key questions and challenges, and make recommendations on how to address these., (© 2022. Springer Nature Limited.)

Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5., (© 2019 The Authors.)

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin and inherent intercellular biomolecule delivery capability. An in-depth knowledge of their in vivo biodistribution is therefore essential. This work aimed to develop a novel, reliable and universal method to radiolabel exosomes to study their in vivo biodistribution. Methods : Melanoma (B16F10) cells were cultured in bioreactor flasks to increase exosome yield. B16F10-derived exosomes (Exo B16 ) were isolated using ultracentrfugation onto a single sucrose cushion, and were characterised for size, yield, purity, exosomal markers and morphology using Nanoparticle Tracking Analysis (NTA), protein measurements, flow cytometry and electron microscopy. Exo B16 were radiolabelled using 2 different approaches - intraluminal labelling (entrapment of 111 Indium via tropolone shuttling); and membrane labelling (chelation of 111 Indium via covalently attached bifunctional chelator DTPA-anhydride). Labelling efficiency and stability was assessed using gel filtration and thin layer chromatography. Melanoma-bearing immunocompetent (C57BL/6) and immunodeficient (NSG) mice were injected intravenously with radiolabelled Exo B16 (1x10 11 particles/mouse) followed by metabolic cages study, whole body SPECT-CT imaging and ex vivo gamma counting at 1, 4 and 24 h post-injection. Results : Membrane-labelled Exo B16 showed superior radiolabelling efficiency and radiochemical stability (19.2 ± 4.53 % and 80.4 ± 1.6 % respectively) compared to the intraluminal-labelled exosomes (4.73 ± 0.39 % and 14.21 ± 2.76 % respectively). Using the membrane-labelling approach, the in vivo biodistribution of Exo B16 in melanoma-bearing C57Bl/6 mice was carried out, and was found to accumulate primarily in the liver and spleen (~56% and ~38% ID/gT respectively), followed by the kidneys (~3% ID/gT). Exo B16 showed minimal tumour i.e. self-tissue accumulation (~0.7% ID/gT). The membrane-labelling approach was also used to study Exo B16 biodistribution in melanoma-bearing immunocompromised (NSG) mice, to compare with that in the immunocompetent C57Bl/6 mice. Similar biodistribution profile was observed in both C57BL/6 and NSG mice, where prominent accumulation was seen in liver and spleen, apart from the significantly lower tumour accumulation observed in the NSG mice (~0.3% ID/gT). Conclusion : Membrane radiolabelling of exosomes is a reliable approach that allows for accurate live imaging and quantitative biodistribution studies to be performed on potentially all exosome types without engineering parent cells., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.

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-E 2 (PGE 2 )) and adenosine. Inhibition of PGE 2 receptors or adenosine A 2 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 PGE 2 , 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.

The UK Extracellular Vesicles (UKEV) Forum meetings were born of the realization that there were a number of UK laboratories studying extracellular vesicle biology and using similar techniques but without a regular national meeting dedicated to EVs at which to share their findings. This was compounded by the fact that many of these labs were working in different fields and thus networking and sharing of ideas and best practice was sometimes difficult. The first workshop was organized in 2013 by Dr Charlotte Lawson, under the auspices of the Society for Endocrinology, led to the founding of the UKEV Forum and the organization of a British Heart Foundation sponsored 1-day conference held in London in December 2014. Although growing in size every year, the central aims of these workshops have remained the same: to provide a forum for discussion and exchange of ideas, to allow young scientists to present their data in the form of short talks and poster presentations and to discuss their work with more established scientists in the field. Here we include the presented abstracts for the 2015 1-day conference hosted by Cardiff University. This meeting was attended by approximately 130 delegates throughout the United Kingdom, but also attended by delegates from Belgium, Netherlands, France, Ireland and other nations. The day composed of plenary presentations from Prof Matthias Belting, Lund University, Sweden and Dr Guillaume van Niel, Institut Curie, Paris together with 10 short presentations from submitted abstracts. The topics covered were broad, with sessions on Mechanisms of EV production, EVs in Infection, EVs in Cancer and in Blood and Characterizing EVs in Biological fluids. This hopefully gives a reflection of the range of EV-related studies being conducted currently in the UK. There were also 33 poster presentations equally broad in subject matter. The organizers are grateful to the Life Science Research Network Wales - a Welsh government-funding scheme that part-sponsored the conference. We are also grateful to commercial sponsors, and 3 paid-presentations are included in the abstracts. The UK EV Forum is expected to become an established annual event held at different Universities across the UK and continue to attract increasing delegate numbers and abstract submissions. We look forward to the next planned conference, which will be hosted by David Carter and his colleagues at Oxford Brookes University on 13th December 2016.

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging., Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research., Availability and Implementation: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

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.

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 β (TGFβ) 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.