Results of the Meso-ORIGINS feasibility study regarding collection of matched benign-mesothelioma tissue pairs by longitudinal surveillance.

OBJECTIVES: To assess key elements of the design for Meso-ORIGINS (Mesothelioma Observational study of RIsk prediction and Generation of paired benign-meso tissue samples, Including a Nested MRI Substudy), an ambitious, UK-wide, prospective study that will collect ≥63 matched benign-mesothelioma tissue pairs through longitudinal surveillance and repeat biopsy of patients with asbestos-associated pleural inflammation (AAPI). DESIGN: A multicentre, mixed-methods feasibility study, comprising a prospective observational element, evaluating recruitment feasibility, technical feasibility of repeat local anaesthetic thoracoscopy (LAT) and patient acceptability, and a retrospective cohort study focused on AAPI-mesothelioma evolution rate, informing sample size. SETTING: 4 UK pleural disease centres (February 2019-January 2020). PARTICIPANTS: Patients with AAPI (history or typical imaging plus appropriate pleural histology) were eligible for both elements. In August 2019, eligibility for the prospective element was broadened, including addition of radiological AAPI for technical feasibility and patient acceptability endpoints only. Retrospective cases required ≥2 years follow-up. OUTCOME MEASURES: A prospective recruitment target was set a priori at 27 histological AAPI cases (or 14 in any 6 months). Technical feasibility and patient acceptability were determined at 6-month follow-up by thoracic ultrasound surrogates and questionnaires, respectively. Retrospective malignant pleural mesothelioma evolution rate was defined by proportion (95% CI). Baseline predictors of evolution were identified using logistic regression. RESULTS: 296 patients with AAPI (39 prospective, 257 retrospective) were recruited/selected. 21/39 prospective recruits were histologically diagnosed (target n=27). Repeat LAT was technically feasible and acceptable in 13/28 (46%) and 24/36 (67%) cases with complete follow-up data. Mesothelioma evolution was confirmed histologically in 36/257 retrospective cases (14% (95% CI 10.3% to 18.8%)) and associated with malignant CT features (OR 4.78 (95% CI 2.36 to 9.86)) and age (OR 1.06 (95% CI 1.02 to 1.12)). CONCLUSIONS: Our initial eligibility criteria were too narrow. Meso-ORIGINS will recruit a broader cohort, including prevalent cases, any biopsy type and patients with malignant CT features. A range of rebiopsy techniques will be allowed, accounting for technical and patient factors. The sample size has been reduced to 500. TRIAL REGISTRATION NUMBER: ISRCTN12840870.

PTEN deficiency exposes a requirement for an ARF GTPase module for integrin-dependent invasion in ovarian cancer.

Dysregulation of the PI3K/AKT pathway is a common occurrence in high-grade serous ovarian carcinoma (HGSOC), with the loss of the tumour suppressor PTEN in HGSOC being associated with poor prognosis. The cellular mechanisms of how PTEN loss contributes to HGSOC are largely unknown. We here utilise time-lapse imaging of HGSOC spheroids coupled to a machine learning approach to classify the phenotype of PTEN loss. PTEN deficiency induces PI(3,4,5)P3 -rich and -dependent membrane protrusions into the extracellular matrix (ECM), resulting in a collective invasion phenotype. We identify the small GTPase ARF6 as a crucial vulnerability of HGSOC cells upon PTEN loss. Through a functional proteomic CRISPR screen of ARF6 interactors, we identify the ARF GTPase-activating protein (GAP) AGAP1 and the ECM receptor ß1-integrin (ITGB1) as key ARF6 interactors in HGSOC regulating PTEN loss-associated invasion. ARF6 functions to promote invasion by controlling the recycling of internalised, active ß1-integrin to maintain invasive activity into the ECM. The expression of the CYTH2-ARF6-AGAP1 complex in HGSOC patients is inversely associated with outcome, allowing the identification of patient groups with improved versus poor outcome. ARF6 may represent a therapeutic vulnerability in PTEN-depleted HGSOC.

Glioblastoma extracellular vesicles influence glial cell hyaluronic acid deposition to promote invasiveness.

Background: Infiltration of glioblastoma (GBM) throughout the brain leads to its inevitable recurrence following standard-of-care treatments, such as surgical resection, chemo-, and radiotherapy. A deeper understanding of the mechanisms invoked by GBM to infiltrate the brain is needed to develop approaches to contain the disease and reduce recurrence. The aim of this study was to discover mechanisms through which extracellular vesicles (EVs) released by GBM influence the brain microenvironment to facilitate infiltration, and to determine how altered extracellular matrix (ECM) deposition by glial cells might contribute to this. Methods: CRISPR was used to delete genes, previously established to drive carcinoma invasiveness and EV production, from patient-derived primary and GBM cell lines. We purified and characterized EVs released by these cells, assessed their capacity to foster pro-migratory microenvironments in mouse brain slices, and evaluated the contribution made by astrocyte-derived ECM to this. Finally, we determined how CRISPR-mediated deletion of genes, which we had found to control EV-mediated communication between GBM cells and astrocytes, influenced GBM infiltration when orthotopically injected into CD1-nude mice. Results: GBM cells expressing a p53 mutant (p53R273H) with established pro-invasive gain-of-function release EVs containing a sialomucin, podocalyxin (PODXL), which encourages astrocytes to deposit ECM with increased levels of hyaluronic acid (HA). This HA-rich ECM, in turn, promotes migration of GBM cells. Consistently, CRISPR-mediated deletion of PODXL opposes infiltration of GBM in vivo. Conclusions: This work describes several key components of an EV-mediated mechanism though which GBM cells educate astrocytes to support infiltration of the surrounding healthy brain tissue.

Phase II proof-of-concept study of atorvastatin in castration-resistant prostate cancer.

OBJECTIVES: To test for evidence of statin-mediated effects in patients with castration-resistant prostate cancer (CRPC) as post-diagnosis use of statins in patients with prostate cancer is associated with favourable survival outcome. PATIENTS AND METHODS: The SPECTRE trial was a 6-weeks-long proof-of-concept single-arm Phase II treatment trial, combining atorvastatin and androgen deprivation therapy in patients with CRPC (regardless of metastatic status), designed to test for evidence of statin-mediated effects in patients with CRPC. The primary study endpoint was the proportion of patients achieving a ≥50% drop from baseline in prostate-specific antigen (PSA) levels at any time over the 6-week period of atorvastatin medication (PSA response). Exploratory endpoints include PSA velocity and serum metabolites identified by mass spectrometry . RESULTS: At the scheduled interim analysis, one of 12 patients experienced a ≥50% drop in PSA levels (primary endpoint), with ≥2 patients satisfying the primary endpoint required for further recruitment. All 12 patients experienced substantial falls in serum cholesterol levels following statin treatment. While all patients had comparable pre-study PSA velocities, six of 12 patients showed decreased PSA velocities after statin treatment, suggestive of disease stabilization. Unbiased metabolomics analysis on serial weekly blood samples identified tryptophan to be the dominant metabolite associated with patient response to statin. CONCLUSIONS: Data from the SPECTRE study provide the first evidence of statin-mediated effects on CRPC and early sign of disease stabilization. Our data also highlight the possibility of altered tryptophan metabolism being associated with tumour response.

Traject3d allows label-free identification of distinct co-occurring phenotypes within 3D culture by live imaging.

Single cell profiling by genetic, proteomic and imaging methods has expanded the ability to identify programmes regulating distinct cell states. The 3-dimensional (3D) culture of cells or tissue fragments provides a system to study how such states contribute to multicellular morphogenesis. Whether cells plated into 3D cultures give rise to a singular phenotype or whether multiple biologically distinct phenotypes arise in parallel is largely unknown due to a lack of tools to detect such heterogeneity. Here we develop Traject3d (Trajectory identification in 3D), a method for identifying heterogeneous states in 3D culture and how these give rise to distinct phenotypes over time, from label-free multi-day time-lapse imaging. We use this to characterise the temporal landscape of morphological states of cancer cell lines, varying in metastatic potential and drug resistance, and use this information to identify drug combinations that inhibit such heterogeneity. Traject3d is therefore an important companion to other single-cell technologies by facilitating real-time identification via live imaging of how distinct states can lead to alternate phenotypes that occur in parallel in 3D culture.

Nuclear-capture of endosomes depletes nuclear G-actin to promote SRF/MRTF activation and cancer cell invasion.

Signals are relayed from receptor tyrosine kinases (RTKs) at the cell surface to effector systems in the cytoplasm and nucleus, and coordination of this process is important for the execution of migratory phenotypes, such as cell scattering and invasion. The endosomal system influences how RTK signalling is coded, but the ways in which it transmits these signals to the nucleus to influence gene expression are not yet clear. Here we show that hepatocyte growth factor, an activator of MET (an RTK), promotes Rab17- and clathrin-dependent endocytosis of EphA2, another RTK, followed by centripetal transport of EphA2-positive endosomes. EphA2 then mediates physical capture of endosomes on the outer surface of the nucleus; a process involving interaction between the nuclear import machinery and a nuclear localisation sequence in EphA2's cytodomain. Nuclear capture of EphA2 promotes RhoG-dependent phosphorylation of the actin-binding protein, cofilin to oppose nuclear import of G-actin. The resulting depletion of nuclear G-actin drives transcription of Myocardin-related transcription factor (MRTF)/serum-response factor (SRF)-target genes to implement cell scattering and the invasive behaviour of cancer cells.

Serum Proteomics and Plasma Fibulin-3 in Differentiation of Mesothelioma From Asbestos-Exposed Controls and Patients With Other Pleural Diseases.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is difficult to diagnose. An accurate blood biomarker could prompt specialist referral or be deployed in future screening. In earlier retrospective studies, SOMAscan proteomics (Somalogic, Boulder, CO) and fibulin-3 seemed highly accurate, but SOMAscan has not been validated prospectively and subsequent fibulin-3 data have been contradictory. METHODS: A multicenter prospective observational study was performed in 22 centers, generating a large intention-to-diagnose cohort. Blood sampling, processing, and diagnostic assessment were standardized, including a 1-year follow-up. Plasma fibulin-3 was measured using two enzyme-linked immunosorbent assays (CloudClone [used in previous studies] and BosterBio, Pleasanton, CA). Serum proteomics was measured using the SOMAscan assay. Diagnostic performance (sensitivity at 95% specificity, area under the curve [AUC]) was benchmarked against serum mesothelin (Mesomark, Fujirebio Diagnostics, Malvern, PA). Biomarkers were correlated against primary tumor volume, inflammatory markers, and asbestos exposure. RESULTS: A total of 638 patients with suspected pleural malignancy (SPM) and 110 asbestos-exposed controls (AECs) were recruited. SOMAscan reliably differentiated MPM from AECs (75% sensitivity, 88.2% specificity, validation cohort AUC 0.855) but was not useful in patients with differentiating non-MPM SPM. Fibulin-3 (by BosterBio after failed CloudClone validation) revealed 7.4% and 11.9% sensitivity at 95% specificity in MPM versus non-MPM SPM and AECs, respectively (associated AUCs 0.611 [0.557-0.664], p = 0.0015) and 0.516 [0.443-0.589], p = 0.671), both inferior to mesothelin. SOMAscan proteins correlated with inflammatory markers but not with asbestos exposure. Neither biomarker correlated with tumor volume. CONCLUSIONS: SOMAscan may prove useful as a future screening test for MPM in asbestos-exposed persons. Neither fibulin-3 nor SOMAscan should be used for diagnosis or pathway stratification.

Selection of established tumour cells through narrow diameter micropores enriches for elevated Ras/Raf/MEK/ERK MAPK signalling and enhanced tumour growth.

As normal cells become cancer cells, and progress towards malignancy, they become progressively softer. Advantages of this change are that tumour cells become more deformable, and better able to move through narrow constraints. We designed a positive selection strategy that enriched for cells which could move through narrow diameter micropores to identify cell phenotypes that enabled constrained migration. Using human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, we found that micropore selection favoured cells with relatively higher Ras/Raf/MEK/ERK mitogen-activated protein kinase (MAPK) signalling, which affected actin cytoskeleton organization, focal adhesion density and cell elasticity. In this follow-up study, we provide further evidence that selection through micropores enriched for cells with altered cell morphology and adhesion. Additional analysis of RNA sequencing data revealed a set of transcripts associated with small cell size that was independent of constrained migration. Gene set enrichment analysis identified the 'matrisome' as the most significantly altered gene set linked with small size. When grown as orthotopic xenograft tumours in immunocompromised mice, micropore selected cells grew significantly faster than Parent or Flow-Sorted cells. Using mathematical modelling, we determined that there is an interaction between 1) the cell to gap size ratio; 2) the bending rigidity of the cell, which enable movement through narrow gaps. These results extend our previous conclusion that Ras/Raf/MEK/ERK MAPK signalling has a significant role in regulating cell biomechanics by showing that the selective pressure of movement through narrow gaps also enriches for increased tumour growth in vivo.

Correction: BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

BRF1 accelerates prostate tumourigenesis and perturbs immune infiltration.

BRF1 is a rate-limiting factor for RNA Polymerase III-mediated transcription and is elevated in numerous cancers. Here, we report that elevated levels of BRF1 associate with poor prognosis in human prostate cancer. In vitro studies in human prostate cancer cell lines demonstrated that transient overexpression of BRF1 increased cell proliferation whereas the transient downregulation of BRF1 reduced proliferation and mediated cell cycle arrest. Consistent with our clinical observations, BRF1 overexpression in a Pten-deficient mouse (PtenΔ/Δ BRF1Tg) prostate cancer model accelerated prostate carcinogenesis and shortened survival. In PtenΔ/Δ BRF1Tg tumours, immune and inflammatory processes were altered, with reduced tumoral infiltration of neutrophils and CD4 positive T cells, which can be explained by decreased levels of complement factor D (CFD) and C7 components of the complement cascade, an innate immune pathway that influences the adaptive immune response. We tested if the secretome was involved in BRF1-driven tumorigenesis. Unbiased proteomic analysis on BRF1-overexpresing PC3 cells confirmed reduced levels of CFD in the secretome, implicating the complement system in prostate carcinogenesis. We further identify that expression of C7 significantly correlates with expression of CD4 and has the potential to alter clinical outcome in human prostate cancer, where low levels of C7 associate with poorer prognosis.

N-WASP Control of LPAR1 Trafficking Establishes Response to Self-Generated LPA Gradients to Promote Pancreatic Cancer Cell Metastasis.

Pancreatic ductal adenocarcinoma is one of the most invasive and metastatic cancers and has a dismal 5-year survival rate. We show that N-WASP drives pancreatic cancer metastasis, with roles in both chemotaxis and matrix remodeling. lysophosphatidic acid, a signaling lipid abundant in blood and ascites fluid, is both a mitogen and chemoattractant for cancer cells. Pancreatic cancer cells break lysophosphatidic acid down as they respond to it, setting up a self-generated gradient driving tumor egress. N-WASP-depleted cells do not recognize lysophosphatidic acid gradients, leading to altered RhoA activation, decreased contractility and traction forces, and reduced metastasis. We describe a signaling loop whereby N-WASP and the endocytic adapter SNX18 promote lysophosphatidic acid-induced RhoA-mediated contractility and force generation by controlling lysophosphatidic acid receptor recycling and preventing degradation. This chemotactic loop drives collagen remodeling, tumor invasion, and metastasis and could be an important target against pancreatic cancer spread.

Migration through physical constraints is enabled by MAPK-induced cell softening via actin cytoskeleton re-organization.

Cancer cells are softer than the normal cells, and metastatic cells are even softer. These changes in biomechanical properties contribute to cancer progression by facilitating cell movement through physically constraining environments. To identify properties that enabled passage through physical constraints, cells that were more efficient at moving through narrow membrane micropores were selected from established cell lines. By examining micropore-selected human MDA MB 231 breast cancer and MDA MB 435 melanoma cancer cells, membrane fluidity and nuclear elasticity were excluded as primary contributors. Instead, reduced actin cytoskeleton anisotropy, focal adhesion density and cell stiffness were characteristics associated with efficient passage through constraints. By comparing transcriptomic profiles between the parental and selected populations, increased Ras/MAPK signalling was linked with cytoskeleton rearrangements and cell softening. MEK inhibitor treatment reversed the transcriptional, cytoskeleton, focal adhesion and elasticity changes. Conversely, expression of oncogenic KRas in parental MDA MB 231 cells, or oncogenic BRaf in parental MDA MB 435 cells, significantly reduced cell stiffness. These results reveal that MAPK signalling, in addition to tumour cell proliferation, has a significant role in regulating cell biomechanics.This article has an associated First Person interview with the first author of the paper.

Radiation Exposure to the Sacroiliac Joint From Low-Dose CT Compared With Radiography.

OBJECTIVE: The primary objective of this study was to estimate the effective dose delivered to the sacroiliac joint (SIJ) from low-dose (LD) CT compared with that from radiography. Secondary objectives included evaluation of diagnostic quality of LD CT of the SIJ and development of a clinical protocol for LD CT of the SIJ. MATERIALS AND METHODS: Data from 36 patients (19 women, 17 men) undergoing LD CT for suspected renal colic were analyzed. Two effective dose estimates were calculated: one for the SIJ and another for an extended region from the iliac crest to 1 cm below the SIJ. Thirty-six anteroposterior pelvic and 36 SIJ view radiographs were age-, sex-, and body width-matched to CT scans. Effective dose from radiography was estimated using the method described in International Commission on Radiologic Protection Publication 60. RESULTS: Maximum effective dose to the SIJ from LD CT was less than 1 mSv in all cases, with a mean ± SD of 0.42 ± 0.18 mSv (range, 0.14-0.83 mSv), whereas mean dose to the extended region was 0.57 ± 0.24 mSv (range, 0.19-1.11 mSv). Mean dose from SIJ radiographs was 0.15 ± 0.10 mSv (range, 0.07-1.38 mSv), and mean dose from a single pelvic radiograph was 0.09 ± 0.06 mSv (range, 0.04-0.37 mSv). All CT studies were of diagnostic quality for assessment of the SIJ. CONCLUSION: LD CT of the SIJ can be consistently performed with an effective radiation dose of less than 1 mSv. Because reliability and sensitivity of radiography for sacroiliitis is poor, we recommend that LD CT replace radiography for dedicated evaluation of the SIJ.

Fam49/CYRI interacts with Rac1 and locally suppresses protrusions.

Actin-based protrusions are reinforced through positive feedback, but it is unclear what restricts their size, or limits positive signals when they retract or split. We identify an evolutionarily conserved regulator of actin-based protrusion: CYRI (CYFIP-related Rac interactor) also known as Fam49 (family of unknown function 49). CYRI binds activated Rac1 via a domain of unknown function (DUF1394) shared with CYFIP, defining DUF1394 as a Rac1-binding module. CYRI-depleted cells have broad lamellipodia enriched in Scar/WAVE, but reduced protrusion-retraction dynamics. Pseudopods induced by optogenetic Rac1 activation in CYRI-depleted cells are larger and longer lived. Conversely, CYRI overexpression suppresses recruitment of active Scar/WAVE to the cell edge, resulting in short-lived, unproductive protrusions. CYRI thus focuses protrusion signals and regulates pseudopod complexity by inhibiting Scar/WAVE-induced actin polymerization. It thus behaves like a 'local inhibitor' as predicted in widely accepted mathematical models, but not previously identified in cells. CYRI therefore regulates chemotaxis, cell migration and epithelial polarization by controlling the polarity and plasticity of protrusions.

A G-protein-coupled chemoattractant receptor recognizes lipopolysaccharide for bacterial phagocytosis.

Phagocytes locate microorganisms via chemotaxis and then consume them using phagocytosis. Dictyostelium amoebas are stereotypical phagocytes that prey on diverse bacteria using both processes. However, as typical phagocytic receptors, such as complement receptors or Fcγ receptors, have not been found in Dictyostelium, it remains mysterious how these cells recognize bacteria. Here, we show that a single G-protein-coupled receptor (GPCR), folic acid receptor 1 (fAR1), simultaneously recognizes the chemoattractant folate and the phagocytic cue lipopolysaccharide (LPS), a major component of bacterial surfaces. Cells lacking fAR1 or its cognate G-proteins are defective in chemotaxis toward folate and phagocytosis of Klebsiella aerogenes. Computational simulations combined with experiments show that responses associated with chemotaxis can also promote engulfment of particles coated with chemoattractants. Finally, the extracellular Venus-Flytrap (VFT) domain of fAR1 acts as the binding site for both folate and LPS. Thus, fAR1 represents a new member of the pattern recognition receptors (PRRs) and mediates signaling from both bacterial surfaces and diffusible chemoattractants to reorganize actin for chemotaxis and phagocytosis.

Histone chaperone HIRA deposits histone H3.3 onto foreign viral DNA and contributes to anti-viral intrinsic immunity.

The HIRA histone chaperone complex deposits histone H3.3 into nucleosomes in a DNA replication- and sequence-independent manner. As herpesvirus genomes enter the nucleus as naked DNA, we asked whether the HIRA chaperone complex affects herpesvirus infection. After infection of primary cells with HSV or CMV, or transient transfection with naked plasmid DNA, HIRA re-localizes to PML bodies, sites of cellular anti-viral activity. HIRA co-localizes with viral genomes, binds to incoming viral and plasmid DNAs and deposits histone H3.3 onto these. Anti-viral interferons (IFN) specifically induce HIRA/PML co-localization at PML nuclear bodies and HIRA recruitment to IFN target genes, although HIRA is not required for IFN-inducible expression of these genes. HIRA is, however, required for suppression of viral gene expression, virus replication and lytic infection and restricts murine CMV replication in vivo. We propose that the HIRA chaperone complex represses incoming naked viral DNAs through chromatinization as part of intrinsic cellular immunity.

The initiator methionine tRNA drives cell migration and invasion leading to increased metastatic potential in melanoma.

The cell's repertoire of transfer RNAs (tRNAs) has been linked to cancer. Recently, the level of the initiator methionine tRNA (tRNAiMet) in stromal fibroblasts has been shown to influence extracellular matrix (ECM) secretion to drive tumour growth and angiogenesis. Here we show that increased tRNAiMet within cancer cells does not influence tumour growth, but drives cell migration and invasion via a mechanism that is independent from ECM synthesis and dependent on α5ß1 integrin and levels of the translation initiation ternary complex. In vivo and ex vivo migration (but not proliferation) of melanoblasts is significantly enhanced in transgenic mice which express additional copies of the tRNAiMet gene. We show that increased tRNAiMet in melanoma drives migratory, invasive behaviour and metastatic potential without affecting cell proliferation and primary tumour growth, and that expression of RNA polymerase III-associated genes (which drive tRNA expression) are elevated in metastases by comparison with primary tumours. Thus, specific alterations to the cancer cell tRNA repertoire drive a migration/invasion programme that may lead to metastasis.