Enriched Extracellular Vesicles Research Articles

B-356 Advancements in Commercial Viability of a Blood Test for the Early Detection of Breast Cancer

Abstract Background Mammography is the current diagnostic standard for breast cancer screening and monitoring. However, accessibility challenges, accuracy issues and patient discomfort all contribute to reduced patient compliance and utilization, resulting in a need for more effective diagnostic tools. A blood test to detect early-stage breast cancer is sought to increase the screening detection rate, provide more accurate monitoring tools, and improve outcomes for patients. We have previously reported a series of lipidomic studies and derived a lipid signature from plasma enriched extracellular vesicles (EVs) that effectively distinguished people with localized breast cancer from cancer-free controls. Here we report on a significant refinement to the test methodology allowing the assessment of the lipid signature directly from plasma samples and its performance, with the aim of advancing the commercial viability of the test as we move towards clinical application. Methods Lipids in EVs from fasted breast cancer and control blood samples (4 separate cohorts (n = 793)) were extracted and analysed by liquid chromatography-high resolution mass spectrometry (LC-HRAM-MS). Over 400 manually curated lipids were quantified. From these, an independent review and retrospective analysis of the data established a lipid signature. The lipid signature was modelled on each of the cohorts using leave-one-out internal cross-validation. Following variable selection, a lipid signature capable of distinguishing breast cancer samples from controls was derived. Furthermore, we investigated if the lipid signature can be assessed from plasma instead of EVs with the aim of further streamlining the process. We analysed the lipids in cancer and control plasma samples (n = 256) previously used for EV preparations, corresponding to patients from Cohorts 3 and 4, and applied the signature derived using EVs on plasma lipidomic data. NIST Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma was used for inter-laboratory quality control. Results Breast cancer subjects were differentiated from controls by the lipid signature with an area under the curve (AUC) of 0.77–0.89 across four cohorts. Assessing the signature directly from plasma, the test achieved a comparable AUC of 0.84. This improvement would make the test more clinically viable and easier to perform. Conclusion We derived a lipid signature, which shows high potential for distinguishing breast cancer samples from controls directly from plasma instead of EVs, reducing the test complexity. Ongoing studies will optimize the plasma lipidomic signature and prospectively compare the test against mammographic and pathological diagnoses.

Lipidomic signature from plasma to detect localised breast cancer.

565 Background: An effective and accurate blood test to detect localised breast cancer may increase the screening detection rate and improve patient outcomes. We have previously reported a series of lipidomic studies and derived a lipid signature from plasma enriched extracellular vesicles (EVs) that effectively distinguished people with localised breast cancer from cancer-free controls. here we report on a significant refinement to the test methodology allowing the assessment of the lipid signature directly from plasma samples and its performance, with the aim of advancing the commercial viability of the test as we move towards clinical application. Methods: Lipids were extracted from enriched EVs from plasma samples donated by fasted people with localised breast cancer and control samples (n=793) and analysed by high resolution accurate mass liquid chromatography-mass spectrometry (LC-MS). Over 400 manually curated lipids were quantified. Following variable selection, a lipid signature capable of distinguishing breast cancer samples from controls was derived. The lipid signature was modelled on each of the cohorts using leave-one-out internal cross-validation. Next, we analysed the lipids in cancer and control (n=256) plasma samples corresponding to patients from Cohorts 3 and 4 previously used for EV preparations, and applied the signature derived using EVs on plasma lipidomic data. Results: EV samples of people with breast cancer were distinguished from controls with an area under the curve (AUC) of 0.77-0.89 across 4 cohorts. When the lipid signature was assessed directly from plasma the test achieved a comparable AUC of 0.84. Assessing the markers directly from plasma samples would make the test more scalable, higher throughput and easier to perform. Conclusions: Our study demonstrated the high performance of a lipid biomarker signature derived from plasma enriched EVs for early detection of localised breast cancer. Our results suggest that that the lipidomic signature could potentially be assessed directly from plasma samples instead of EVs reducing the test complexity. Ongoing studies will optimise the plasma lipidomic signature and prospectively compare the test against mammographic and pathological diagnoses. [Table: see text]

MiR-210-3p enriched extracellular vesicles from hypoxic neuroblastoma cells stimulate migration and invasion of target cells

BackgroundTumor hypoxia stimulates release of extracellular vesicles (EVs) that facilitate short- and long-range intercellular communication and metastatization. Albeit hypoxia and EVs release are known features of Neuroblastoma (NB), a metastasis-prone childhood malignancy of the sympathetic nervous system, whether hypoxic EVs can facilitate NB dissemination is unclear.MethodsHere we isolated and characterized EVs from normoxic and hypoxic NB cell culture supernatants and performed microRNA (miRNA) cargo analysis to identify key mediators of EVs biological effects. We then validated if EVs promote pro-metastatic features both in vitro and in an in vivo zebrafish model.ResultsEVs from NB cells cultured at different oxygen tensions did not differ for type and abundance of surface markers nor for biophysical properties. However, EVs derived from hypoxic NB cells (hEVs) were more potent than their normoxic counterpart in inducing NB cells migration and colony formation. miR-210-3p was the most abundant miRNA in the cargo of hEVs; mechanistically, overexpression of miR-210-3p in normoxic EVs conferred them pro-metastatic features, whereas miR-210-3p silencing suppressed the metastatic ability of hypoxic EVs both in vitro and in vivo.ConclusionOur data identify a role for hypoxic EVs and their miR-210-3p cargo enrichment in the cellular and microenvironmental changes favoring NB dissemination.

Detection of early-stage breast cancer in women by plasma lipidomic profiling.

554 Background: Early detection of breast cancer provides the best opportunity for cure. Mammography is the benchmark for screening, but suffers technical, logistic, and diagnostic limitations. An effective and accurate blood test to detect early stages of the disease should increase the screening detection rate for breast cancer. We conducted a series of lipidomic studies in early-stage breast cancer patients and combined the datasets via a machine learning driven analysis to test if plasma lipidomic profiles can detect breast cancer. Methods: Blood samples were collected from women with stage 0-IV breast cancer (4 separate cohorts) with age and BMI matched breast cancer-free controls. Lipids from plasma enriched extracellular vesicles were extracted and analysed by high resolution accurate mass LC-MS. A commercially available software was used to annotate and quantify >400 manually curated lipid species. Following variable selection, a lipid signature was identified capable of distinguishing breast cancer samples from control. Results: Plasma samples from women with breast cancer were distinguished from controls with an average cross-validated accuracy of 0.81, and average AUC of 0.84 across 4 cohorts (Table 1). An optimised cross-cohort subset of early-stage IDC, DCIS and ILC were differentiated from controls with a cross-validated AUC of 0.90, sensitivity of 0.88 and specificity of 0.82 (201 early-stage breast cancer, 199 controls) (Table 1). For this optimised cohort our test achieved a sensitivity of 0.71 at a prescribed specificity of 0.90, or equivalently a sensitivity of 0.89 at a prescribed specificity of 0.80. Conclusions: Our study demonstrates the high sensitivity and specificity of a lipid biomarker signature with potential for early detection of breast cancer. Ongoing studies will prospectively compare the lipid-biomarker based test against mammographic and pathological diagnosis. [Table: see text]

DRAM1 increases the secretion of PKM2-enriched EVs from hepatocytes to promote macrophage activation and disease progression in ALD

DNA damage-regulated autophagy modulator 1 (DRAM1) could play important roles in inflammation and hepatic apoptosis, while its roles in alcohol-related liver disease (ALD), which is characterized by hepatic inflammation and apoptosis, are still unclear. In this study, we explored the expression, role, and mechanism of DRAM1 in ALD. Firstly, our results showed that DRAM1 was significantly increased in liver tissues of mice at the early stage of alcohol treatment. In addition, DRAM1 knockout reduced, and liver-specific overexpression of DRAM1 aggravated, alcohol-induced hepatic steatosis, injury, and expressions of M1 macrophage markers in mice. Furthermore, ethanol-induced DRAM1 of hepatic cells increased pyruvate kinase M2 (PKM2)-enriched extracellular vesicles (EVs), and ectosomes derived from hepatic cells with DRAM1 overexpression promoted macrophage activation. Mechanistic investigations showed that DRAM1 interacted with PKM2 and increased the PKM2 level in plasma membrane. At last, DRAM1 was significantly increased in liver tissues of ALD patients, and it was positively correlated with M1 macrophage markers. Taken together, this study revealed that ethanol-induced DRAM1 of hepatic cells could increase the PKM2-enriched EVs, promote macrophage activation, and aggravate the disease progression of ALD. These findings suggested that DRAM1 might be a potentially promising target for the therapy of ALD.

Polysialic Acid in Human Plasma Can Compensate the Cytotoxicity of Histones.

The innate immune system has numerous mechanisms to fight against pathogens, including the formation of neutrophil extracellular traps (NETs). By spreading out chromatin, antimicrobial peptides and enzymes, neutrophils efficiently trap pathogens like bacteria and facilitate their elimination. During this process, high concentrations of extracellular histones can be reached. Several researchers have demonstrated that the cytotoxic characteristics of these histones can trigger diseases like sepsis. Interestingly, the carbohydrate polysialic acid (polySia) can bind histones and reduce histone-mediated cytotoxicity in a chain length-dependent manner. In the present study, we examined the chain length of polySia in plasma and tested its ability to decrease the cytotoxic characteristics of extracellular histones. Remarkably, we detected polySia not only in the soluble fraction of plasma, but also on enriched extracellular vesicles (EVs). Chain length analysis revealed that polySia chains originating from human plasma can consists of more than 40 sialic acid residues and show a cytoprotective effect against extracellular histones. Intriguingly, polySia is not only present in human plasma but also in fish and other branches of vertebrates. Thus, polySia is a physiological element in plasma and may represent a natural buffer for extracellular histones.