6 research outputs found
Selective isolation of extracellular vesicles from minimally processed human plasma as a translational strategy for liquid biopsies
Background: Intercellular communication is mediated by extracellular vesicles (EVs), as they enclose selectively
packaged biomolecules that can be horizontally transferred from donor to recipient cells. Because all cells constantly
generate and recycle EVs, they provide accurate timed snapshots of individual pathophysiological status. Since blood
plasma circulates through the whole body, it is often the biofluid of choice for biomarker detection in EVs. Blood
collection is easy and minimally invasive, yet reproducible procedures to obtain pure EV samples from circulating
biofluids are still lacking. Here, we addressed central aspects of EV immunoaffinity isolation from simple and complex
matrices, such as plasma.
Methods: Cell-generated EV spike-in models were isolated and purified by size-exclusion chromatography, stained
with cellular dyes and characterized by nano flow cytometry. Fluorescently-labelled spike-in EVs emerged as reliable,
high-throughput and easily measurable readouts, which were employed to optimize our EV immunoprecipitation
strategy and evaluate its performance. Plasma-derived EVs were captured and detected using this straightforward
protocol, sequentially combining isolation and staining of specific surface markers, such as CD9 or CD41. Multiplexed
digital transcript detection data was generated using the Nanostring nCounter platform and evaluated through a
dedicated bioinformatics pipeline.
Results: Beads with covalently-conjugated antibodies on their surface outperformed streptavidin-conjugated beads,
coated with biotinylated antibodies, in EV immunoprecipitation. Fluorescent EV spike recovery evidenced that target
EV subpopulations can be efficiently retrieved from plasma, and that their enrichment is dependent not only on
complex matrix composition, but also on the EV surface phenotype. Finally, mRNA profiling experiments proved that
distinct EV subpopulations can be captured by directly targeting different surface markers. Furthermore, EVs isolated
with anti-CD61 beads enclosed mRNA expression patterns that might be associated to early-stage lung cancer, in
contrast with EVs captured through CD9, CD63 or CD81. The differential clinical value carried within each distinct EV
subset highlights the advantages of selective isolation.
Conclusions: This EV isolation protocol facilitated the extraction of clinically useful information from plasma. Compatible
with common downstream analytics, it is a readily implementable research tool, tailored to provide a truly
translational solution in routine clinical workflows, fostering the inclusion of EVs in novel liquid biopsy settings.European Commission 765492
95218
Multiplex Analysis of CircRNAs from Plasma Extracellular Vesicle-Enriched Samples for the Detection of Early-Stage Non-Small Cell Lung Cancer
Background: The analysis of liquid biopsies brings new opportunities in the precision
oncology field. Under this context, extracellular vesicle circular RNAs (EV-circRNAs) have gained
interest as biomarkers for lung cancer (LC) detection. However, standardized and robust protocols
need to be developed to boost their potential in the clinical setting. Although nCounter has been
used for the analysis of other liquid biopsy substrates and biomarkers, it has never been employed
for EV-circRNA analysis of LC patients. Methods: EVs were isolated from early-stage LC patients
(n = 36) and controls (n = 30). Different volumes of plasma, together with different number of preamplification
cycles, were tested to reach the best nCounter outcome. Differential expression analysis
of circRNAs was performed, along with the testing of different machine learning (ML) methods for
the development of a prognostic signature for LC. Results: A combination of 500 L of plasma input
with 10 cycles of pre-amplification was selected for the rest of the study. Eight circRNAs were found
upregulated in LC. Further ML analysis selected a 10-circRNA signature able to discriminate LC from
controls with AUC ROC of 0.86. Conclusions: This study validates the use of the nCounter platform
for multiplexed EV-circRNA expression studies in LC patient samples, allowing the development of
prognostic signatures.European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant 76549
Combinatorial Blood Platelets-Derived circRNA and mRNA Signature for Early-Stage Lung Cancer Detection
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24054881/s1.Despite the diversity of liquid biopsy transcriptomic repertoire, numerous studies often
exploit only a single RNA type signature for diagnostic biomarker potential. This frequently results in
insufficient sensitivity and specificity necessary to reach diagnostic utility. Combinatorial biomarker
approaches may offer a more reliable diagnosis. Here, we investigated the synergistic contributions
of circRNA and mRNA signatures derived from blood platelets as biomarkers for lung cancer
detection. We developed a comprehensive bioinformatics pipeline permitting an analysis of platelet-
circRNA and mRNA derived from non-cancer individuals and lung cancer patients. An optimal
selected signature is then used to generate the predictive classification model using machine learning
algorithm. Using an individual signature of 21 circRNA and 28 mRNA, the predictive models
reached an area under the curve (AUC) of 0.88 and 0.81, respectively. Importantly, combinatorial
analysis including both types of RNAs resulted in an 8-target signature (6 mRNA and 2 circRNA),
enhancing the differentiation of lung cancer from controls (AUC of 0.92). Additionally, we identified
five biomarkers potentially specific for early-stage detection of lung cancer. Our proof-of-concept
study presents the first multi-analyte-based approach for the analysis of platelets-derived biomarkers,
providing a potential combinatorial diagnostic signature for lung cancer detection.European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie 765492
Assessing the complementary information from an increased number of biologically relevant features in liquid biopsy-derived RNA-Seq data
Liquid biopsy-derived RNA sequencing (lbRNA-seq) exhibits significant promise for clinicoriented
cancer diagnostics due to its non-invasiveness and ease of repeatability. Despite substantial
advancements, obstacles like technical artefacts and process standardisation impede
seamless clinical integration. Alongside addressing technical aspects such as normalising fluctuating
low-input material and establishing a standardised clinical workflow, the lack of result
validation using independent datasets remains a critical factor contributing to the often low
reproducibility of liquid biopsy-detected biomarkers.
Considering the outlined drawbacks, our objective was to establish a workflow/methodology
characterised by: 1. Harness the rich diversity of biological features accessible through lbRNA-seq
data, encompassing a holistic range of molecular and functional attributes. These components are
seamlessly integrated via a Machine Learning-based Ensemble Classification framework, enabling
a unified and comprehensive analysis of the intricate information encoded within the data. 2.
Implementing and rigorously benchmarking intra-sample normalisation methods to heighten
their relevance within clinical settings. 3. Thoroughly assessing its efficacy across independent
test sets to ascertain its robustness and potential utility.
Using ten datasets from several studies comprising three different sources of biological material,
we first show that while the best-performing normalisation methods depend strongly on the
dataset and coupled Machine Learning method, the rather simple Counts Per Million method is
generally very robust, showing comparable performance to cross-sample methods. Subsequently, we demonstrate that the innovative biofeature types introduced in this study, such as the Fraction
of Canonical Transcript, harbour complementary information. Consequently, their inclusion
consistently enhances prediction power compared to models relying solely on gene expressionbased
biofeatures. Finally, we demonstrate that the workflow is robust on completely independent
datasets, generally from different labs and/or different protocols. Taken together, the
workflow presented here outperforms generally employed methods in prediction accuracy and
may hold potential for clinical diagnostics application due to its specific design.European Union’s Horizon 2020 research and innovation program under the Marie
Skłodowska-Curie grant agreement ELBA No 76549
Digital multiplexed analysis of circular RNAs in FFPE and fresh non-small cell lung cancer specimens
We would like to thank Stephanie Davis for her language editing assistance. The investigators also wish to thank the patients for kindly agreeing to donate samples to this study. We thank all the physicians who collaborated by providing clinical information. Graphical Abstract, Figs 1A, 8A and Fig. S1 were created with Biorender.com. This project has received funding from a European Union's Horizon 2020 research and innovation program under the Marie SklodowskaCurie grant agreement ELBA No 765492.Although many studies highlight the implication of circular RNAs (circRNAs)
in carcinogenesis and tumor progression, their potential as cancer
biomarkers has not yet been fully explored in the clinic due to the limitations
of current quantification methods. Here, we report the use of the
nCounter platform as a valid technology for the analysis of circRNA
expression patterns in non-small cell lung cancer (NSCLC) specimens.
Under this context, our custom-made circRNA panel was able to detect
circRNA expression both in NSCLC cells and formalin-fixed paraffinembedded
(FFPE) tissues. CircFUT8 was overexpressed in NSCLC, contrasting
with circEPB41L2, circBNC2, and circSOX13 downregulation even
at the early stages of the disease. Machine learning (ML) approaches from
different paradigms allowed discrimination of NSCLC from nontumor controls
(NTCs) with an 8-circRNA signature. An additional 4-circRNA signature
was able to classify early-stage NSCLC samples from NTC,
reaching a maximum area under the ROC curve (AUC) of 0.981. Our
results not only present two circRNA signatures with diagnosis potential
but also introduce nCounter processing following ML as a feasible protocol
for the study and development of circRNA signatures for NSCLC.European Commission 76549
sRNAbench and sRNAtoolbox 2022 update: accurate miRNA and sncRNA profiling for model and non-model organisms
European Union [765492 to M.H.]; Spanish Government [AGL2017-88702-C2-2-R to M.H.]; Chair 'Doctors Galera-Requena in cancer stem cell research' (to J.A.M.); Tromsoforskningsstiftelse (TFS) [20 SG BF 'MIRevolution' to B.F.]; Stichting Cancer Center Amsterdam [CCA2021-9-77 to C.G]; TKI-Health Holland ['AQrate' project to C.G. and M.P.]. This publication is part of a project that has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 765492.The NCBI Sequence Read Archive currently hosts
microRNA sequencing data for over 800 different
species, evidencing the existence of a broad taxonomic
distribution in the field of small RNA research.
Simultaneously, the number of samples per
miRNA-seq study continues to increase resulting in
a vast amount of data that requires accurate, fast
and user-friendly analysis methods. Since the previous
release of sRNAtoolbox in 2019, 55 000 sRNAbench
jobs have been submitted which has motivated
many improvements in its usability and the
scope of the underlying annotation database. With
this update, users can upload an unlimited number
of samples or import them from Google Drive,
Dropbox or URLs. Micro- and small RNA profiling
can now be carried out using high-confidence Metazoan
and plant specific databases, MirGeneDB and
PmiREN respectively, together with genome assemblies
and libraries from 441 Ensembl species. The
new results page includes straightforward sample
annotation to allow downstream differential expression
analysis with sRNAde. Unassigned reads can
also be explored by means of a new tool that performsmapping
to microbial references, which can reveal
contamination events or biologically meaningful
findings as we describe in the example. sRNAtoolbox
is available at: https://arn.ugr.es/srnatoolbox/.European Commission 765492Spanish GovernmentEuropean Commission AGL2017-88702-C2-2-RChair 'Doctors Galera-Requena in cancer stem cell research'Stichting Cancer Center Amsterdam CCA2021-9-77Tromsoforskningsstiftelse (TFS) ['MIRevolution'] 20 SG BFTKI-Health Holland ['AQrate' project