Defining the relationship between cellular and extracellular vesicle (EV) content in breast cancer via an integrative multi-omic analysis.

Much recent research has been dedicated to exploring the utility of extracellular vesicles (EVs) as circulating disease biomarkers. Underpinning this work is the assumption that the molecular cargo of EVs directly reflects the originating cell. Few attempts have been made, however, to empirically validate this on the -omic level. To this end, we have performed an integrative multi-omic analysis of a panel of breast cancer cell lines and corresponding EVs. Whole transcriptome analysis validated that the cellular transcriptome remained stable when cultured cells are transitioned to low serum or serum-free medium for EV collection. Transcriptomic profiling of the isolated EVs indicated a positive correlation between transcript levels in cells and EVs, including disease-associated transcripts. Analysis of the EV proteome verified that HER2 protein is present in EVs, however neither the estrogen (ER) nor progesterone (PR) receptor proteins are detected regardless of cellular expression. Using multivariate analysis, we derived an EV protein signature to infer cellular patterns of ER and HER2 expression, though the ER protein could not be directly detected. Integrative analyses affirmed that the EV proteome and transcriptome captured key phenotypic hallmarks of the originating cells, supporting the potential of EVs for non-invasive monitoring of breast cancers.

Human proximal tubular epithelial cell-derived small extracellular vesicles mediate synchronized tubular ferroptosis in hypoxic kidney injury.

Hypoxia is the key pathobiological trigger of tubular oxidative stress and cell death that drives the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). The mitochondrial-rich proximal tubular epithelial cells (PTEC) are uniquely sensitive to hypoxia and thus, are pivotal in propagating the sustained tubular loss of AKI-to-CKD transition. Here, we examined the role of PTEC-derived small extracellular vesicles (sEV) in propagating the 'wave of tubular death'. Ex vivo patient-derived PTEC were cultured under normoxia (21 % O2) and hypoxia (1 % O2) on Transwell inserts for isolation and analysis of sEV secreted from apical versus basolateral PTEC surfaces. Increased numbers of sEV were secreted from the apical surface of hypoxic PTEC compared with normoxic PTEC. No differences in basolateral sEV numbers were observed between culture conditions. Biological pathway analysis of hypoxic-apical sEV cargo identified distinct miRNAs linked with cellular injury pathways. In functional assays, hypoxic-apical sEV selectively induced ferroptotic cell death (↓glutathione peroxidase-4, ↑lipid peroxidation) in autologous PTEC compared with normoxic-apical sEV. The addition of ferroptosis inhibitors, ferrostatin-1 and baicalein, attenuated PTEC ferroptosis. RNAse A pretreatment of hypoxic-apical sEV also abrogated PTEC ferroptosis, demonstrating a role for sEV RNA in ferroptotic 'wave of death' signalling. In line with these in vitro findings, in situ immunolabelling of diagnostic kidney biopsies from AKI patients with clinical progression to CKD (AKI-to-CKD transition) showed evidence of ferroptosis propagation (increased numbers of ACSL4+ PTEC), while urine-derived sEV (usEV) from these 'AKI-to-CKD transition' patients triggered PTEC ferroptosis (↑lipid peroxidation) in functional studies. Our data establish PTEC-derived apical sEV and their intravesicular RNA as mediators of tubular lipid peroxidation and ferroptosis in hypoxic kidney injury. This concept of how tubular pathology is propagated from the initiating insult into a 'wave of death' provides novel therapeutic check-points for targeting AKI-to-CKD transition.

Imetelstat-mediated alterations in fatty acid metabolism to induce ferroptosis as a therapeutic strategy for acute myeloid leukemia.

Telomerase enables replicative immortality in most cancers including acute myeloid leukemia (AML). Imetelstat is a first-in-class telomerase inhibitor with clinical efficacy in myelofibrosis and myelodysplastic syndromes. Here, we develop an AML patient-derived xenograft resource and perform integrated genomics, transcriptomics and lipidomics analyses combined with functional genetics to identify key mediators of imetelstat efficacy. In a randomized phase II-like preclinical trial in patient-derived xenografts, imetelstat effectively diminishes AML burden and preferentially targets subgroups containing mutant NRAS and oxidative stress-associated gene expression signatures. Unbiased, genome-wide CRISPR/Cas9 editing identifies ferroptosis regulators as key mediators of imetelstat efficacy. Imetelstat promotes the formation of polyunsaturated fatty acid-containing phospholipids, causing excessive levels of lipid peroxidation and oxidative stress. Pharmacological inhibition of ferroptosis diminishes imetelstat efficacy. We leverage these mechanistic insights to develop an optimized therapeutic strategy using oxidative stress-inducing chemotherapy to sensitize patient samples to imetelstat causing substantial disease control in AML.

Safety, Feasibility, and Acceptability of a Multisite Individualized Exercise Intervention for People with Multiple Myeloma.

INTRODUCTION: High rates of disease- and treatment-related symptoms, such as bone lesions, in people with multiple myeloma (MM) create uncertainty on the safety and feasibility of exercise. This study determined the safety, feasibility, and acceptability of an individualized exercise medicine program for people with MM at any disease stage. METHODS: A multisite, randomized waitlist-controlled trial was conducted of an individualized, high-intensity aerobic, resistance, and impact-loading exercise program. The exercise sessions were supervised twice weekly by accredited exercise physiologists, with one additional unsupervised session per week, for 12 wk. Safety was determined by number of adverse and serious adverse events. Feasibility outcome measures were study eligibility, recruitment, adherence, and attrition. Acceptability was determined by qualitative interviews and subjective levels of enjoyment. RESULTS: Of 203 people with MM screened, 88% were eligible, with 34% accepting participation (60 people) and 20% attrition for the between-group analysis, meeting a priori criteria (≥25% and <25%, respectively). No adverse or serious adverse events attributed to testing and/or exercise training were reported. Attendance at supervised exercise sessions was 98%, with 45% completion of the home-based exercise sessions. Adherence rates were 35%, 63%, and 34% for the aerobic, resistance, and impact-loading protocols, with 55%, 80%, and 37% of participants meeting a priori criteria (75% of protocol). Acceptability of the exercise program was high (mean, 82%; 95% confidence interval, 78%-87%) and highly supported by qualitative responses. CONCLUSIONS: An individualized, high-intensity aerobic, resistance, and impact-loading exercise medicine program is safe and acceptable, and feasible by some measures for people with MM. Adherence to the prescribed exercise protocols was limited by comorbidities and disease symptoms. Strategies to improve unsupervised exercise completion are warranted in this population.

Discovery and validation of serum glycoprotein biomarkers for high grade serous ovarian cancer.

PURPOSE: This study aimed to identify serum glycoprotein biomarkers for early detection of high-grade serous ovarian cancer (HGSOC), the most common and aggressive histotype of ovarian cancer. EXPERIMENTAL DESIGN: The glycoproteomics pipeline lectin magnetic bead array (LeMBA)-mass spectrometry (MS) was used in age-matched case-control serum samples. Clinical samples collected at diagnosis were divided into discovery (n = 30) and validation (n = 98) sets. We also analysed a set of preclinical sera (n = 30) collected prior to HGSOC diagnosis in the UK Collaborative Trial of Ovarian Cancer Screening. RESULTS: A 7-lectin LeMBA-MS/MS discovery screen shortlisted 59 candidate proteins and three lectins. Validation analysis using 3-lectin LeMBA-multiple reaction monitoring (MRM) confirmed elevated A1AT, AACT, CO9, HPT and ITIH3 and reduced A2MG, ALS, IBP3 and PON1 glycoforms in HGSOC. The best performing multimarker signature had 87.7% area under the receiver operating curve, 90.7% specificity and 70.4% sensitivity for distinguishing HGSOC from benign and healthy groups. In the preclinical set, CO9, ITIH3 and A2MG glycoforms were altered in samples collected 11.1 ± 5.1 months prior to HGSOC diagnosis, suggesting potential for early detection. CONCLUSIONS AND CLINICAL RELEVANCE: Our findings provide evidence of candidate early HGSOC serum glycoprotein biomarkers, laying the foundation for further study in larger cohorts.

Semi-Automated Lectin Magnetic Bead Array (LeMBA) for Translational Serum Glycoprotein Biomarker Discovery and Validation.

Aberrant protein glycosylation is a characteristic of diverse diseases which has been explored as biomarkers. To support translational serum glycoprotein biomarker discovery and validation, we developed a semi-automated workflow using individual lectin-coupled magnetic beads to conduct lectin pulldowns in a high-throughput format. Lectins are naturally occurring glycoprotein binding proteins widely used in glycobiology. While lectin-affinity isolation has been coupled to mass spectrometry-based proteomics, the lectin magnetic bead array (LeMBA) platform allows technically robust screening and measurement of clinical cohorts. This chapter describes detailed lectin-magnetic bead coupling, serum denaturation, lectin magnetic bead pulldown, and on-bead trypsin digest. The resulting tryptic peptides are analyzed by untargeted or targeted liquid chromatography-mass spectrometry (LC-MS), for biomarker discovery, or qualification/validation, respectively. LeMBA-MS generates quantitative data for glycoforms based on lectin affinity of the glycoprotein coupled with MS measurement of one or more prototypic peptides and has successfully been used to discover and validate novel serum cancer glycoprotein biomarkers. This chapter includes detailed protocols for two different liquid handlers, along with recommendations on quality control measures for clinical biomarker studies.

Extracellular vesicles for precision medicine in prostate cancer - Is it ready for clinical translation?

Biofluid-based biomarker tests hold great promise for precision medicine in prostate cancer (PCa) clinical practice. Extracellular vesicles (EV) are established as intercellular messengers in cancer development with EV cargos, including protein and nucleic acids, having the potential to serve as biofluid-based biomarkers. Recent clinical studies have begun to evaluate EV-based biomarkers for PCa diagnosis, prognosis, and disease/therapy resistance monitoring. Promising results have led to PCa EV biomarker validation studies which are currently underway with the next challenge being translation to robust clinical assays. However, EV research studies generally use low throughput EV isolation methods and costly molecular profiling technologies that are not suitable for clinical assays. Here, we consider the technical hurdles in translating EV biomarker research findings into precise and cost-effective clinical biomarker assays. Novel microfluidic devices coupling EV extraction with sensitive antibody-based biomarker detection are already being explored for point-of-care applications for rapid provision in personalised medicine approaches.

Safety, Feasibility, and Efficacy of Exercise Interventions for People With Multiple Myeloma: A Systematic Review.

Bone lesions and other disease- and treatment-related side effects commonly experienced by people with multiple myeloma (MM) may impede their ability to exercise. This systematic review evaluated the safety, feasibility, and efficacy of exercise program participation on the physiological and/or psychological health of people with MM. Literature searches were conducted through five electronic databases and appraised using the Delphi list of criteria. Controlled trials that assessed the safety and feasibility of an exercise intervention and its effects on disease- or treatment-related symptoms in people with MM were included. Seven studies of varying quality involving 563 participants were included. All studies concluded that exercise was safe, reporting zero serious and 4 adverse events attributable to exercise testing or training. Attendance ranged from 58% to 96%, however no study reported adherence to the exercise prescription. Compared to a control group, exercise did not appear to affect fatigue, depression, anxiety, body composition, quality of life, or sleep. Isolated studies identified between-group differences favoring exercise in lower limb strength (+8.4 kg, 95% CI 0.5, 16.3, P= .04), peak oxygen uptake (+1.2 mL/kg/min, 95% CI 0.3, 3.7, P= .02), physical activity (+6.5MET-hs/wk, P< .001), stem cell collection attempts (1.1 ± 0.2 vs. 1.5 ± 0.9, P< .01), and red blood cell (1.8 ± 2.2 vs. 2.4 ± 2.6, P< .05) and platelet transfusions (2.3 ± 1.6 vs. 3.5 ± 3.4, P < .05) during transplantation. Exercise interventions appear safe and well attended by people with MM. The lack of improvements in disease- and treatment-related symptoms requires further exploration to determine whether exercise is a sufficient stimulus to elicit benefits in this unique population.

Elevation of fatty acid desaturase 2 in esophageal adenocarcinoma increases polyunsaturated lipids and may exacerbate bile acid-induced DNA damage.

BACKGROUND: The risk of esophageal adenocarcinoma (EAC) is associated with gastro-esophageal reflux disease (GERD) and obesity. Lipid metabolism-targeted therapies decrease the risk of progressing from Barrett's esophagus (BE) to EAC, but the precise lipid metabolic changes and their roles in genotoxicity during EAC development are yet to be established. METHODS: Esophageal biopsies from the normal epithelium (NE), BE, and EAC, were analyzed using concurrent lipidomics and proteomics (n = 30) followed by orthogonal validation on independent samples using RNAseq transcriptomics (n = 22) and immunohistochemistry (IHC, n = 80). The EAC cell line FLO-1 was treated with FADS2 selective inhibitor SC26196, and/or bile acid cocktail, followed by immunofluorescence staining for γH2AX. RESULTS: Metabolism-focused Reactome analysis of the proteomics data revealed enrichment of fatty acid metabolism, ketone body metabolism, and biosynthesis of specialized pro-resolving mediators in EAC pathogenesis. Lipidomics revealed progressive alterations (NE-BE-EAC) in glycerophospholipid synthesis with decreasing triglycerides and increasing phosphatidylcholine and phosphatidylethanolamine, and sphingolipid synthesis with decreasing dihydroceramide and increasing ceramides. Furthermore, a progressive increase in lipids with C20 fatty acids and polyunsaturated lipids with ≥4 double bonds were also observed. Integration with transcriptome data identified candidate enzymes for IHC validation: Δ4-Desaturase, Sphingolipid 1 (DEGS1) which desaturates dihydroceramide to ceramide, and Δ5 and Δ6-Desaturases (fatty acid desaturases, FADS1 and FADS2), responsible for polyunsaturation. All three enzymes showed significant increases from BE through dysplasia to EAC, but transcript levels of DEGS1 were decreased suggesting post-translational regulation. Finally, the FADS2 selective inhibitor SC26196 significantly reduced polyunsaturated lipids with three and four double bonds and reduced bile acid-induced DNA double-strand breaks in FLO-1 cells in vitro. CONCLUSIONS: Integrated multiomics revealed sphingolipid and phospholipid metabolism rewiring during EAC development. FADS2 inhibition and reduction of the high polyunsaturated lipids effectively protected EAC cells from bile acid-induced DNA damage in vitro, potentially through reduced lipid peroxidation.

Skin Cancer-Associated S. aureus Strains Can Induce DNA Damage in Human Keratinocytes by Downregulating DNA Repair and Promoting Oxidative Stress.

Actinic keratosis (AK) is a premalignant lesion, common on severely photodamaged skin, that can progress over time to cutaneous squamous cell carcinoma (SCC). A high bacterial load of Staphylococcus aureus is associated with AK and SCC, but it is unknown whether this has a direct impact on skin cancer development. To determine whether S. aureus can have cancer-promoting effects on skin cells, we performed RNA sequencing and shotgun proteomics on primary human keratinocytes after challenge with sterile culture supernatant ('secretome') from four S. aureus clinical strains isolated from AK and SCC. Secretomes of two of the S. aureus strains induced keratinocytes to overexpress biomarkers associated with skin carcinogenesis and upregulated the expression of enzymes linked to reduced skin barrier function. Further, these strains induced oxidative stress markers and all secretomes downregulated DNA repair mechanisms. Subsequent experiments on an expanded set of lesion-associated S. aureus strains confirmed that exposure to their secretomes led to increased oxidative stress and DNA damage in primary human keratinocytes. A significant correlation between the concentration of S. aureus phenol soluble modulin toxins in secretome and the secretome-induced level of oxidative stress and genotoxicity in keratinocytes was observed. Taken together, these data demonstrate that secreted compounds from lesion-associated clinical isolates of S. aureus can have cancer-promoting effects in keratinocytes that may be relevant to skin oncogenesis.

C5b-9 Membrane Attack Complex Formation and Extracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett's Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

An Individualized Exercise Intervention for People with Multiple Myeloma-Study Protocol of a Randomized Waitlist-Controlled Trial.

People with multiple myeloma (MM) are second only to people with lung cancer for the poorest reported health-related quality of life (HRQoL) of all cancer types. Whether exercise can improve HRQoL in MM, where bone pain and lesions are common, requires investigation. This trial aims to evaluate the efficacy of an exercise intervention compared with control on HRQoL in people with MM. Following baseline testing, people with MM (n = 60) will be randomized to an exercise (EX) or waitlist control (WT) group. EX will complete 12-weeks of supervised (24 sessions) and unsupervised (12 sessions) individualized, modular multimodal exercise training. From weeks 12-52, EX continue unsupervised training thrice weekly, with one optional supervised group-based session weekly from weeks 12-24. The WT will be asked to maintain their current activity levels for the first 12-weeks, before completing the same protocol as EX for the following 52 weeks. Primary (patient-reported HRQoL) and secondary (bone health and pain, fatigue, cardiorespiratory fitness, muscle strength, body composition, disease response, and blood biomarkers) outcomes will be assessed at baseline, 12-, 24- and 52-weeks. Adverse events, attendance, and adherence will be recorded and cost-effectiveness analysis performed. The findings will inform whether exercise should be included as part of standard myeloma care to improve the health of this unique population.

Promoting exercise for patients with multiple myeloma: attitudes and practices of clinical haematologists.

PURPOSE: To explore the attitudes and practices of clinical haematologists towards promoting physical activity (PA) and exercise for patients with multiple myeloma (MM). METHODS: Using a quantitative cross-sectional survey, clinical haematologists reported on the perceived benefits and acceptability of PA and exercise and frequency, confidence and barriers to providing exercise advice. RESULTS: Clinical haematologists (n=34; 68% response rate), who cumulatively treated ~340 patients with MM each week, completed the survey. Almost all (97%) agreed that PA was important, with benefits for quality of life, activities of daily living, mental health and fatigue. Whilst 88% discussed PA at least occasionally with their patients, approximately two-thirds were not confident advising specific exercises (68%) or identifying PA resources (62%). Despite this, 44% never referred patients to exercise professionals, with 18% only doing so if the patient asked. Over half did not recommend exercise when patients had spine fractures or were physically unwell. No differences were observed in individual factors (age, gender, practice type and own PA participation) and promotion of PA. CONCLUSIONS: Clinical haematologists perceive PA as important, but lack confidence on what exercise/s to recommend and if exercise is appropriate for specific disease complications. They tend to not refer patients to exercise professionals. IMPLICATIONS FOR CANCER SURVIVORS: Patients with MM often suffer from symptoms and toxicities that may be alleviated through PA. However, PA participation rates are low. Support for clinical haematologists for when and how to discuss exercise, and clearer referral pathways to exercise professionals may improve PA uptake and hence ensure access to optimal care, thereby improving patient outcomes.

Potential Role of Exercise Induced Extracellular Vesicles in Prostate Cancer Suppression.

Physical exercise is increasingly recognized as a valuable treatment strategy in managing prostate cancer, not only enhancing supportive care but potentially influencing disease outcomes. However, there are limited studies investigating mechanisms of the tumor-suppressive effect of exercise. Recently, extracellular vesicles (EVs) have been recognized as a therapeutic target for cancer as tumor-derived EVs have the potential to promote metastatic capacity by transferring oncogenic proteins, integrins, and microRNAs to other cells and EVs are also involved in developing drug resistance. Skeletal muscle has been identified as an endocrine organ, releasing EVs into the circulation, and levels of EV-containing factors have been shown to increase in response to exercise. Moreover, preclinical studies have demonstrated the tumor-suppressive effect of protein and microRNA contents in skeletal muscle-derived EVs in various cancers, including prostate cancer. Here we review current knowledge of the tumor-derived EVs in prostate cancer progression and metastasis, the role of exercise in skeletal muscle-derived EVs circulating levels and the alteration of their contents, and the potential tumor-suppressive effect of skeletal muscle-derived EV contents in prostate cancer. In addition, we review the proposed mechanism of exercise in the uptake of skeletal muscle-derived EVs in prostate cancer.

An integrated mass spectrometry imaging and digital pathology workflow for objective detection of colorectal tumours by unique atomic signatures.

Tumours are abnormal growths of cells that reproduce by redirecting essential nutrients and resources from surrounding tissue. Changes to cell metabolism that trigger the growth of tumours are reflected in subtle differences between the chemical composition of healthy and malignant cells. We used LA-ICP-MS imaging to investigate whether these chemical differences can be used to spatially identify tumours and support detection of primary colorectal tumours in anatomical pathology. First, we generated quantitative LA-ICP-MS images of three colorectal surgical resections with case-matched normal intestinal wall tissue and used this data in a Monte Carlo optimisation experiment to develop an algorithm that can classify pixels as tumour positive or negative. Blinded testing and interrogation of LA-ICP-MS images with micrographs of haematoxylin and eosin stained and Ki67 immunolabelled sections revealed Monte Carlo optimisation accurately identified primary tumour cells, as well as returning false positive pixels in areas of high cell proliferation. We analysed an additional 11 surgical resections of primary colorectal tumours and re-developed our image processing method to include a random forest regression machine learning model to correctly identify heterogenous tumours and exclude false positive pixels in images of non-malignant tissue. Our final model used over 1.6 billion calculations to correctly discern healthy cells from various types and stages of invasive colorectal tumours. The imaging mass spectrometry and data analysis methods described, developed in partnership with clinical cancer researchers, have the potential to further support cancer detection as part of a comprehensive digital pathology approach to cancer care through validation of a new chemical biomarker of tumour cells.

Addressing Delicate and Variable Cancer Morphology in Spectral Histopathology Using Canine Visceral Hemangiosarcoma.

Spectral histopathology has shown promise for the classification and diagnosis of tumors with defined morphology, but application in tumors with variable or diffuse morphologies is yet to be investigated. To address this gap, we evaluated the application of Fourier transform infrared (FTIR) imaging as an accessory diagnostic tool for canine hemangiosarcoma (HSA), a vascular endothelial cell cancer that is difficult to diagnose. To preserve the delicate vascular tumor tissue structure, and potential classification of single endothelial cells, paraffin removal was not performed, and a partial least square discrimination analysis (PLSDA) and Random Forest (RF) models to classify different tissue types at individual pixel level were established using a calibration set (24 FTIR images from 13 spleen specimens). Next, the prediction capability of the PLSDA model was tested with an independent test set (n = 11), resulting in 74% correct classification of different tissue types at an individual pixel level. Finally, the performance of the FTIR spectropathology and chemometric algorithm for diagnosis of HSA was established in a blinded set of tissue samples (n = 24), with sensitivity and specificity of 80 and 81%, respectively. Taken together, these results show that FTIR imaging without paraffin removal can be applied to tumors with diffuse morphology, and this technique is a promising tool to assist in canine splenic HSA differential diagnosis.

Differential Regulation of Lacto-/Neolacto- Glycosphingolipid Biosynthesis Pathway Reveals Transcription Factors as Potential Candidates in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer with limited treatment options. Glycosylation has been implicated in cancer development, but TNBC-specific glycosylation pathways have not been examined. Here, we applied bioinformatic analyses on public datasets to discover TNBC-specific glycogenes and pathways, as well as their upstream regulatory mechanisms. Unsupervised clustering of 345 glycogene expressions in breast cancer datasets revealed a relative homogenous expression pattern in basal-like TNBC subtype. Differential expression analyses of the 345 glycogenes between basal-like TNBC (hereafter termed TNBC) and other BC subtypes, or normal controls, revealed 84 differential glycogenes in TNBC. Pathway enrichment showed two common TNBC-enriched pathways across all three datasets, cell cycle and lacto-/neolacto- glycosphingolipid (GSL) biosynthesis, while a total of four glycosylation-related pathways were significantly enriched in TNBC. We applied a selection criterion of the top 50% differential anabolic/catabolic glycogenes in the enriched pathways to define 34 TNBC-specific glycogenes. The lacto-/neolacto- GSL biosynthesis pathway was the most highly enriched, with seven glycogenes all up-regulated in TNBC. This data led us to investigate the hypothesis that a common upstream mechanism in TNBC up-regulates the lacto-/neolacto-GSL biosynthesis pathway. Using public multi-omic datasets, we excluded the involvement of copy-number alteration and DNA methylation, but identified three transcription factors (AR, GATA3 and ZNG622) that each target three candidate genes in the lacto-/neolacto- GSL biosynthesis pathway. Interestingly, a subset of TNBC has been reported to express AR and GATA3, and AR antagonists are being trialed for TNBC. Our findings suggest that AR and GATA3 may contribute to TNBC via GSL regulation, and provide a list of candidate glycogenes for further investigation.

Development of EndoScreen Chip, a Microfluidic Pre-Endoscopy Triage Test for Esophageal Adenocarcinoma.

The current endoscopy and biopsy diagnosis of esophageal adenocarcinoma (EAC) and its premalignant condition Barrett's esophagus (BE) is not cost-effective. To enable EAC screening and patient triaging for endoscopy, we developed a microfluidic lectin immunoassay, the EndoScreen Chip, which allows sensitive multiplex serum biomarker measurements. Here, we report the proof-of-concept deployment for the EAC biomarker Jacalin lectin binding complement C9 (JAC-C9), which we previously discovered and validated by mass spectrometry. A monoclonal C9 antibody (m26 3C9) was generated and validated in microplate ELISA, and then deployed for JAC-C9 measurement on EndoScreen Chip. Cohort evaluation (n = 46) confirmed the expected elevation of serum JAC-C9 in EAC, along with elevated total serum C9 level. Next, we asked if the small panel of serum biomarkers improves detection of EAC in this cohort when used in conjunction with patient risk factors (age, body mass index and heartburn history). Using logistic regression modeling, we found that serum C9 and JAC-C9 significantly improved EAC prediction from AUROC of 0.838 to 0.931, with JAC-C9 strongly predictive of EAC (vs. BE OR = 4.6, 95% CI: 1.6-15.6, p = 0.014; vs. Healthy OR = 4.1, 95% CI: 1.2-13.7, p = 0.024). This proof-of-concept study confirms the microfluidic EndoScreen Chip technology and supports the potential utility of blood biomarkers in improving triaging for diagnostic endoscopy. Future work will expand the number of markers on EndoScreen Chip from our list of validated EAC biomarkers.

Caveolin-1-driven membrane remodelling regulates hnRNPK-mediated exosomal microRNA sorting in cancer.

BACKGROUND: Caveolae proteins play diverse roles in cancer development and progression. In prostate cancer, non-caveolar caveolin-1 (CAV1) promotes metastasis, while CAVIN1 attenuates CAV1-induced metastasis. Here, we unveil a novel mechanism linking CAV1 to selective loading of exosomes with metastasis-promoting microRNAs. RESULTS: We identify hnRNPK as a CAV1-regulated microRNA binding protein. In the absence of CAVIN1, non-caveolar CAV1 drives localisation of hnRPNK to multi-vesicular bodies (MVBs), recruiting AsUGnA motif-containing miRNAs and causing their release within exosomes. This process is dependent on the lipid environment of membranes as shown by cholesterol depletion using methyl-ß-cyclodextrin or by treatment with n-3 polyunsaturated fatty acids. Consistent with a role in bone metastasis, knockdown of hnRNPK in prostate cancer PC3 cells abolished the ability of PC3 extracellular vesicles (EV) to induce osteoclastogenesis, and biofluid EV hnRNPK is elevated in metastatic prostate and colorectal cancer. CONCLUSIONS: Taken together, these results support a novel pan-cancer mechanism for CAV1-driven exosomal release of hnRNPK and associated miRNA in metastasis, which is modulated by the membrane lipid environment.