Peptide-based biosensing approaches for targeting breast cancer-derived exosomes.

Exosomes are nanovesicles present in all the biological fluids, making them attractive as non-invasive biomarkers for diseases like cancer, among many others. However, exosomes are complex to separate and detect, requiring comprehensive molecular characterization for their routine use in diagnostics. This study explores the use of peptides as cost-effective and stable alternatives to antibodies for exosome binding. To achieve that, phage display technology was employed to select peptides with high specificity for target molecules in exosomes. Specifically, a selected peptide was evaluated for its ability to selectively bind breast cancer-derived exosomes. Proteomic analysis identified 38 protein candidates targeted by the peptide on exosome membranes. The binding of the peptide to breast cancer-derived exosomes was successfully demonstrated by flow cytometry and magneto-actuated immunoassays. Furthermore, an electrochemical biosensor was also tested for breast cancer-derived exosome detection and quantification. The peptide demonstrated effective binding to exosomes from aggressive cancer cell lines, offering promising results in terms of specificity and recovery. This research shows potential for developing rapid, accessible diagnostic tools for breast cancer, especially in low-resource healthcare settings.

Effect of Pharmaceutical Intervention in Pharmacologically Treated Hypertensive Patients-A Cluster-Randomized Clinical Trial: AFPRES-CLM Study.

BACKGROUND: Evaluate the effect of a community pharmaceutical intervention on the control of blood pressure in hypertensive patients treated pharmacologically. METHODS: A cluster-randomized clinical trial of 6 months was carried out. It was conducted in the Autonomous Community of Castilla-La Mancha (Spain). Sixty-three community pharmacies and 347 patients completed the study. Intervention patients received the community pharmaceutical intervention based on a protocol that addresses the individual needs of each patient related to the control of their blood pressure, which included Health Education, Pharmacotherapy Follow-up and 24 h Ambulatory Blood Pressure Measurement. Control patients received usual care in the community pharmacy. RESULTS: The pharmaceutical intervention resulted in better control of blood pressure (85.8% vs. 66.3% p < 0.001), lower use of emergencies (p = 0.002) and improvement trends in the physical components of quality of life, measured by SF-36 questionnaire, after 6 months of pharmaceutical intervention. No significant changes were observed for any of these variables in the control group. There were also detected 354 negative medication-related outcomes that were satisfactorily resolved in a 74.9% of the cases and 330 healthcare education interventions and 29 Ambulatory Blood Pressure Monitorings were performed in order to increase adherence to pharmacological treatment and minimize Negative Outcomes associated with Medication and prevent medication-related problems. CONCLUSIONS: Community pharmaceutical intervention can increase hypertensive patients with controlled blood pressure, after 6 months, compared with usual care.

Analysis of tumor infiltrating CD4+ and CD8+ CDR3 sequences reveals shared features putatively associated to the anti-tumor immune response.

Introduction: Tumor-infiltrating lymphocytes (TILs) have predictive and prognostic value in breast cancer (BC) and exert a protective function against tumor growth, indicating that it is susceptible to treatment using adoptive cell transfer of TILs or T cell receptor (TCR)-based therapies. TCR can be used to identify naturally tumor-reactive T cells, but little is known about the differences in the TCR repertoires of CD4+ and CD8+ TILs. Methods: TCR high-throughput sequencing was performed using TILs derived from the initial cultures of 11 BC biopsies and expanded and sorted CD4+ and CD8+ TILs as well as using PBMCs from healthy donors expanded and sorted using the same methodology. Results: Physicochemical TCR differences between T cell subsets were observed, as CD4+ TILs presented larger N(D)Nnt TRB sequences and with a higher usage of positively charged residues, although only the latest was also observed in peripheral T cells from healthy individuals. Moreover, in CD4+ TILs, a more restricted TCR repertoire with a higher abundance of similar sequences containing certain amino acid motifs was observed. Discussion: Some differences between CD4+ and CD8+ TCRs were intrinsic to T cell subsets as can also be observed in peripheral T cells from healthy individuals, while other were only found in TILs samples and therefore may be tumor-driven. Notably, the higher similarity among CD4+ TCRs suggests a higher TCR promiscuity in this subset.

Construction and validation of a gene expression classifier to predict immunotherapy response in primary triple-negative breast cancer.

BACKGROUND: Immune checkpoint inhibitors (ICI) improve clinical outcomes in triple-negative breast cancer (TNBC) patients. However, a subset of patients does not respond to treatment. Biomarkers that show ICI predictive potential in other solid tumors, such as levels of PD-L1 and the tumor mutational burden, among others, show a modest predictive performance in patients with TNBC. METHODS: We built machine learning models based on pre-ICI treatment gene expression profiles to construct gene expression classifiers to identify primary TNBC ICI-responder patients. This study involved 188 ICI-naïve and 721 specimens treated with ICI plus chemotherapy, including TNBC tumors, HR+/HER2- breast tumors, and other solid non-breast tumors. RESULTS: The 37-gene TNBC ICI predictive (TNBC-ICI) classifier performs well in predicting pathological complete response (pCR) to ICI plus chemotherapy on an independent TNBC validation cohort (AUC = 0.86). The TNBC-ICI classifier shows better performance than other molecular signatures, including PD-1 (PDCD1) and PD-L1 (CD274) gene expression (AUC = 0.67). Integrating TNBC-ICI with molecular signatures does not improve the efficiency of the classifier (AUC = 0.75). TNBC-ICI displays a modest accuracy in predicting ICI response in two different cohorts of patients with HR + /HER2- breast cancer (AUC = 0.72 to pembrolizumab and AUC = 0.75 to durvalumab). Evaluation of six cohorts of patients with non-breast solid tumors treated with ICI plus chemotherapy shows overall poor performance (median AUC = 0.67). CONCLUSION: TNBC-ICI predicts pCR to ICI plus chemotherapy in patients with primary TNBC. The study provides a guide to implementing the TNBC-ICI classifier in clinical studies. Further validations will consolidate a novel predictive panel to improve the treatment decision-making for patients with TNBC.

Triple-Negative Breast Cancer (TNBC) is an aggressive type of breast cancer, responsible for a substantial burden of breast cancer-related deaths. In recent years, immunotherapy, a therapy that triggers the patient's immune system to attack the tumor, has arisen as a promising treatment in various cancers, including TNBC. However, a subset of patients with TNBC does not respond to this treatment. Here, we employed advanced computational techniques to predict response to immunotherapy plus chemotherapy in patients with primary TNBC. Our method is more accurate than using other existing markers, such as PD-L1, but is not very accurate in patients with non-TNBC breast cancers or non-breast cancers. This method could potentially be used to better select patients for immunotherapy, upfront, avoiding the side effects and costs of treating patients in which immunotherapy might not work.

Evaluation of triple negative breast cancer with heterogeneous immune infiltration.

Introduction: Tumor infiltrating lymphocytes (TILs) are known to be a prognostic and predictive biomarker in breast cancer, particularly in triple negative breast cancer (TNBC) patients. International guidelines have been proposed to evaluate them in the clinical setting as a continuous variable, without a clear defined cut-off. However, there are scenarios where the immune infiltration is heterogeneous that some areas of the patient's tumour have high numbers of TILs while other areas completely lack them. This spontaneous presentation of a heterogeneous immune infiltration could be a great opportunity to study why some tumours present TILs at diagnosis but others do not, while eliminating inter patient's differences. Methods: In this study, we have identified five TNBC patients that showed great TIL heterogeneity, with areas of low (≤5%) and high (≥50%) numbers of TILs in their surgical specimens. To evaluate immune infiltration heterogeneity, we performed and analyzed bulk RNA-sequencing in three independent triplicates from the high and low TIL areas of each patient. Results: Gene expression was homogeneous within the triplicates in each area but was remarkable different between TILs regions. These differences were not only due to the presence of TILs as there were other non-inflammatory genes and pathways differentially expressed between the two areas. Discussion: This highlights the importance of intratumour heterogeneity driving the immune infiltration, and not patient's characteristics like the HLA phenotype, germline DNA or immune repertoire.

Magnetic Separation of Cell-Secreted Vesicles with Tailored Magnetic Particles and Downstream Applications.

The analysis of the receptors on the surface of the cell-secreted vesicles provides valuable information of the cell signature and may also offer diagnosis and/or prognosis of a wide range of diseases, including cancer.Due to their low concentration, conventional procedures for extracellular vesicle (EV) detection usually require relatively large sample volumes, involving preliminary purification or preconcentration steps from complex specimens. Here, we describe the separation and preconcentration in magnetic particles of extracellular vesicles obtained from cell culture supernatants from MCF7, MDA-MB-231, and SKBR3 breast cancer cell lines, human fetal osteoblastic cell line (hFOB), and human neuroblastoma SH-SY5Y cell line, as well as exosomes from human serum. The first approach involves the covalent immobilization for the exosomes directly on micro (4.5 µm)-sized magnetic particles. The second approach is based on tailored magnetic particles modified with antibodies for further immunomagnetic separation of the exosomes. In these instances, micro (4.5 µm)-sized magnetic particles are modified with different commercial antibodies against selected receptors, including the general tetraspanins CD9, CD63, and CD81 and the specific receptors (CD24, CD44, CD54, CD326, CD340, and CD171). The magnetic separation can be easily coupled with downstream characterization and quantification methods, including molecular biology techniques such as immunoassays, confocal microscopy, or flow cytometry.

Plasma extracellular vesicles reveal early molecular differences in amyloid positive patients with early-onset mild cognitive impairment.

In the clinical course of Alzheimer's disease (AD) development, the dementia phase is commonly preceded by a prodromal AD phase, which is mainly characterized by reaching the highest levels of Aß and p-tau-mediated neuronal injury and a mild cognitive impairment (MCI) clinical status. Because of that, most AD cases are diagnosed when neuronal damage is already established and irreversible. Therefore, a differential diagnosis of MCI causes in these prodromal stages is one of the greatest challenges for clinicians. Blood biomarkers are emerging as desirable tools for pre-screening purposes, but the current results are still being analyzed and much more data is needed to be implemented in clinical practice. Because of that, plasma extracellular vesicles (pEVs) are gaining popularity as a new source of biomarkers for the early stages of AD development. To identify an exosome proteomics signature linked to prodromal AD, we performed a cross-sectional study in a cohort of early-onset MCI (EOMCI) patients in which 184 biomarkers were measured in pEVs, cerebrospinal fluid (CSF), and plasma samples using multiplex PEA technology of Olink© proteomics. The obtained results showed that proteins measured in pEVs from EOMCI patients with established amyloidosis correlated with CSF p-tau181 levels, brain ventricle volume changes, brain hyperintensities, and MMSE scores. In addition, the correlations of pEVs proteins with different parameters distinguished between EOMCI Aß( +) and Aß(-) patients, whereas the CSF or plasma proteome did not. In conclusion, our findings suggest that pEVs may be able to provide information regarding the initial amyloidotic changes of AD. Circulating exosomes may acquire a pathological protein signature of AD before raw plasma, becoming potential biomarkers for identifying subjects at the earliest stages of AD development.

Extracellular vesicles, the emerging mirrors of brain physiopathology.

Extracellular vesicles are secreted by a wide variety of cells, and their primary functions include intercellular communication, immune responses, human reproduction, and synaptic plasticity. Their molecular cargo reflects the physiological processes that their cells of origin are undergoing. Thus, many studies have suggested that extracellular vesicles could be a promising biomarker tool for many diseases, mainly due to their biological relevance and easy accessibility to a broad range of body fluids. Moreover, since their biological composition leads them to cross the blood-brain barrier bidirectionally, growing evidence points to extracellular vesicles as emerging mirrors of brain diseases processes. In this regard, this review explores the biogenesis and biological functions of extracellular vesicles, their role in different physiological and pathological processes, their potential in clinical practice, and the recent outstanding studies about the role of exosomes in major human brain diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), or brain tumors.

Advances in exosome analysis.

There is growing demand for novel biomarkers that detect early stage disease as well as monitor clinical management and therapeutic strategies. Exosome analysis could provide the next advance in attaining that goal. Exosomes are membrane encapsulated biologic nanometric-sized particles of endocytic origin which are released by all cell types. Unfortunately, exosomes are exceptionally challenging to characterize with current technologies. Exosomes are between 30 and 200nm in diameter, a size that makes them out of the sensitivity range to most cell-oriented sorting or analysis platforms, i.e., traditional flow cytometers. The most common methods for targeting exosomes to date typically involve purification followed by the characterization and the specific determination of their cargo. The whole procedure is time consuming, requiring thus skilled personnel as well as laboratory facilities and benchtop instrumentation. The most relevant methodology for exosome isolation, characterization and quantification is addressed in this chapter, including the most up-to-date approaches to explore the potential usefulness of exosomes as biomarkers in liquid biopsies and in advanced nanomedicine.

Electrochemical Genosensing of Overexpressed GAPDH Transcripts in Breast Cancer Exosomes.

Exosomes are receiving highlighted attention as new biomarkers for the detection of cancer since they are profusely released by tumor cells in different biological fluids. In this paper, the exosomes are preconcentrated from the serum by immunomagnetic separation (IMS) based on a CD326 receptor as a specific epithelial cancer-related biomarker and detected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts. Following the lysis of the captured exosomes, the released GAPDH transcripts are amplified by reverse transcription polymerase chain reaction (RT-PCR) with a double-tagging set of primers on poly(dT)-modified-MPs to increase the sensitivity. The double-tagged amplicon is then quantified by electrochemical genosensing. The IMS/double-tagging RT-PCR/electrochemical genosensing approach is first demonstrated for the sensitive detection of exosomes derived from MCF7 breast cancer cells and compared with CTCs in terms of the analytical performance, showing an LOD of 4 × 102 exosomes µL-1. The genosensor was applied to human samples by immunocapturing the exosomes directly from serum from breast cancer patients and showed a higher electrochemical signal (3.3-fold, p < 0.05), when compared with healthy controls, suggesting an overexpression of GAPDH on serum-derived exosomes from breast cancer patients. The detection of GAPDH transcripts is performed from only 1.0 mL of human serum using specific magnetic particles, improving the analytical simplification and avoiding ultracentrifugation steps, demonstrating to be a promising strategy for minimal invasive liquid biopsy.

Corrigendum: Ligand-based CAR T-cell: Different strategies to drive T-cells in future new treatments.

[This corrects the article DOI: 10.3389/fimmu.2022.932559.].

Ligand-based CAR-T cell: Different strategies to drive T cells in future new treatments.

Chimeric antigen receptor (CAR)-based therapies are presented as innovative treatments for multiple malignancies. Despite their clinical success, there is scientific evidence of the limitations of these therapies mainly due to immunogenicity issues, toxicities associated with the infusion of the product, and relapses of the tumor. As a result, novel approaches are appearing aiming to solve and/or mitigate the harmful effects of CAR-T therapies. These include strategies based on the use of ligands as binding moieties or ligand-based CAR-T cells. Several proposals are currently under development, with some undergoing clinical trials to assess their potential benefits. In addition to these, therapies such as chimeric autoantibody receptor (CAAR), B-cell receptor antigen for reverse targeting (BAR), and even chimeric human leukocyte antigen (HLA) antibody receptor (CHAR) have emerged, benefiting from the advantages of antigenic ligands as antibody-binding motifs. This review focuses on the potential role that ligands can play in current and future antitumor treatments and in other types of diseases, such as autoimmune diseases or problems associated with transplantation.

Evaluation of the TCR Repertoire as a Predictive and Prognostic Biomarker in Cancer: Diversity or Clonality?

T cells play a vital role in the anti-tumoural response, and the presence of tumour-infiltrating lymphocytes has shown to be directly correlated with a good prognosis in several cancer types. Nevertheless, some patients presenting tumour-infiltrating lymphocytes do not have favourable outcomes. The TCR determines the specificities of T cells, so the analysis of the TCR repertoire has been recently considered to be a potential biomarker for patients' progression and response to therapies with immune checkpoint inhibitors. The TCR repertoire is one of the multiple elements comprising the immune system and is conditioned by several factors, including tissue type, tumour mutational burden, and patients' immunogenetics. Its study is crucial to understanding the anti-tumoural response, how to beneficially modulate the immune response with current or new treatments, and how to better predict the prognosis. Here, we present a critical review including essential studies on TCR repertoire conducted in patients with cancer with the aim to draw the current conclusions and try to elucidate whether it is better to encounter higher clonality with few TCRs at higher frequencies, or higher diversity with many different TCRs at lower frequencies.

The activity of alkaline phosphatase in breast cancer exosomes simplifies the biosensing design.

This work addresses a biosensor combining the immunomagnetic separation and the electrochemical biosensing based on the intrinsic ALP activity of the exosomes. This approach explores for the first time two different types of biomarkers on exosomes, in a unique biosensing device combining two different biorecognition reaction: immunological and enzymatic. Besides, the intrinsic activity of alkaline phosphatase (ALP) in exosomes as a potential biomarker of carcinogenesis as well as osseous metastatic invasion is also explored. To achieve that, as an in vitro model, exosomes from human fetal osteoblasts are used. It is demonstrated that the electrochemical biosensor improves the analytical performance of the gold standard colorimetric assay for the detection of ALP activity in exosomes, providing a limit of detection of 4.39 mU L-1, equivalent to 105 exosomes µL-1. Furthermore, this approach is used to detect and quantify exosomes derived from serum samples of breast cancer patients. The electrochemical biosensor shows reliable results for the differentiation of healthy donors and breast cancer individuals based on the immunomagnetic separation using specific epithelial biomarkers CD326 (EpCAM) combined with the intrinsic ALP activity electrochemical readout.

Epstein-Barr Virus+ B Cells in Breast Cancer Immune Response: A Case Report.

EBV-specific T cells have been recently described to be involved in fatal encephalitis and myocarditis in cancer patients after immune checkpoint therapies. Here, we report the study of a human triple-negative breast cancer tumor (TNBC) and EBV-transformed B cells obtained from a patient-derived xenograft (PDX) that progressed into a lymphocytic neoplasm named xenograft-associated B-cell lymphoma (XABCL). T-cell receptor (TCR) high-throughput sequencing was performed to monitor the T-cell clonotypes present in the different samples. Forty-three T-cell clonotypes were found infiltrating the XABCL tissue after three passes in mice along 6 months. Eighteen of these (42%) were also found in the TNBC biopsy. TCR infiltrating the XABCL tissue showed a very restricted T-cell repertoire as compared with the biopsy-infiltrating T cells. Consequently, T cells derived from the TNBC biopsy were expanded in the presence of the B-cell line obtained from the XABCL (XABCL-LCL), after which the TCR repertoire obtained was again very restricted, i.e., only certain clonotypes were selected by the B cells. A number of these TCRs had previously been reported as sequences involved in infection, cancer, and/or autoimmunity. We then analyzed the immunopeptidome from the XABCL-LCL, to identify putative B-cell-associated peptides that might have been expanding these T cells. The HLA class I and class II-associated peptides from XABCL-LCL were then compared with published repertoires from LCL of different HLA typing. Proteins from the antigen processing and presentation pathway remained significantly enriched in the XABCL-LCL repertoire. Interestingly, some class II-presented peptides were derived from cancer-related proteins. These results suggest that bystander tumor-infiltrating EBV+ B cells acting as APC may be able to interact with tumor-infiltrating T cells and influence the TCR repertoire in the tumor site.

Comparative Study of Gold and Carbon Nanoparticles in Nucleic Acid Lateral Flow Assay.

A lateral flow assay (LFA) is a paper-based, point-of-need test designed to detect a specific analyte in complex samples in low-resource settings. Although LFA has been successfully used in different applications, its use is still limited when high sensitivity is required, especially in the diagnosis of an early-stage condition. The limit of detection (LOD) is clearly related to the signal-generating system used to achieve the visual readout, in many cases involving nanoparticles coupled to a biomolecule, which, when combined, provides sensitivity and specificity, respectively. While colloidal gold is currently the most-used label, other detection systems are being developed. Carbon nanoparticles (CNPs) demonstrate outstanding features to improve the sensitivity of this technology by producing an increased contrast in the paper background. Based on the necessity of sensitivity improvement, the aim of this work is a comparative study, in terms of analytical performance, between commercial streptavidin gold nanoparticles (streptAv-AuNPs) and avidin carbon nanoparticles (Av-CNPs) in a nucleic acid lateral flow assay. The visual LOD of the method was calculated by serial dilution of the DNA template, ranging from 0.0 to 7 pg µL-1/1.5 × 104 CFU mL-1). The LFA achieved visual detection of as low as 2.2 × 10-2 pg µL-1 using Av-CNPs and 8.4 × 10-2 pg µL-1 using streptAv-AuNPs. These LODs could be obtained without the assistance of any instrumentation. The results demonstrate that CNPs showed an increased sensitivity, achieving the nanomolar range even by visual inspection. Furthermore, CNPs are the cheapest labels, and the suspensions are very stable and easy to modify.

Immune analysis of lymph nodes in relation to the presence or absence of tumor infiltrating lymphocytes in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with unmet medical needs. Several studies have proved that high levels of tumor infiltrating lymphocytes (TILs) at diagnosis of TNBC confer better prognosis and patients respond better to specific chemotherapies. Nonetheless, current evidence suggests that only 15% of TNBC patients have very high levels of TILs, and another 15% lacks TILs. One possible reason to explain why patients have low TILs at diagnosis is that lymphocytes might be deactivated by an immune checkpoint in local lymph nodes, provoking their retention in there as they are unresponsive to other immune stimuli. We have identified 15 high TILs (≥50%) and 20 low TILs (≤5%) TNBC patients with localised tumour (T1c-T2N0M0) and compared the protein expression of five immune checkpoints in lymph nodes. We have also performed a customised 50-immune gene NanoString expression panel, the NanoString 360 Breast Cancer panel, and whole exome sequencing for mutation and neoantigen load analyses. In low TILs, we observed higher expression of CTLA-4 in local lymph nodes, which could explain why lymphocytes get retained in there and do not migrate to tumour. These patients have also higher neoantigen load and higher expression of B7.H3 and B7.H4 in the tumour. In high TILs, we observed more PD-L1+ tumour cells and more expanded humoral response. These results could provide a strategy to revert low tumour immune infiltration at diagnosis of TNBC, improving their prognosis.

Osteoblastic exosomes. A non-destructive quantitative approach of alkaline phosphatase to assess osteoconductive nanomaterials.

Alkaline phosphatase (ALP) is an essential biomarker of osteoblastic activity. Currently, ALP activity has been used to study bone mineralization mechanisms and osteoactive biomaterials among others. The ALP quantification is usually performed by destructive methods either on growing cells or cells lysate in which the osteoconductive biomaterial is being assessed. This work addresses the evaluation of a non-destructive colorimetric approach for the determination of ALP activity on osteoblast-derived exosomes from culture supernatants. The efficiency of the method was evaluated on osteoconductive electrospun scaffolds of PCL compounded with ZnO as a reference biomaterial. The results demonstrated that the osteoblast cell line mineralization induced by osteoconductive scaffolds can be monitorized over time by the non-destructive measurement of ALP activity on osteoblast derived exosomes. Consequently, this non-destructive approach suggested to be a reliable alternative technique for the quantification of biomaterials osteoconductivity or even evaluation of osteoblastic response at stem cells.

Multiplex detection and characterization of breast cancer exosomes by magneto-actuated immunoassay.

The exosomes are emerging as biomarkers for the detection of cancer in early stages, as well as for the follow-up of the patients under treatment. This paper describes the characterization of exosomes derived from three different breast cancer cell lines (MCF7, MDA-MB-231 and SKBR3), and the quantification based on a magneto-actuated immunoassay. The exosomes are separated and preconcentrated on magnetic particles by immunomagnetic separation and labelled with a second antibody conjugated with an enzyme for the optical readout performed with a standard microplate reader. Several molecular biomarkers, including the general tetraspanin CD9, CD63 and CD81, and the receptors related with cancer (CD24, CD44, CD54, CD326 and CD340) were studied either for the immunomagnetic separation or the labelling, in different formats. After a rational selection of the biomarkers, this immunoassay is able to detect 105 exosomes µL-1 directly in human serum without any treatment, such as ultracentrifugation. The interference from free receptors in the samples could easily be prevented by performing the immunomagnetic separation with antiCD81 modified magnetic particles and the labeling based on either CD24 or CD340. Furthermore, the differentiation of healthy donors and breast cancer individuals was also demonstrated. This approach is a highly suitable alternative method for flow cytometry, providing a sensitive method for the multiplex detection but using instrumentation widely available in resource-constrained laboratories and requiring low-maintenance, as is the case of a microplate reader operated by filters.

Matrix Effect in the Isolation of Breast Cancer-Derived Nanovesicles by Immunomagnetic Separation and Electrochemical Immunosensing-A Comparative Study.

Exosomes are cell-derived nanovesicles released into biological fluids, which are involved in cell-to-cell communication. The analysis of the content and the surface of the exosomes allow conclusions about the cells they are originating from and the underlying condition, pathology or disease. Therefore, the exosomes are currently considered good candidates as biomarkers to improve the current methods for clinical diagnosis, including cancer. However, due to their low concentration, conventional procedures for exosome detection including biosensing usually require relatively large sample volumes and involve preliminary purification and preconcentration steps by ultracentrifugation. In this paper, the immunomagnetic separation is presented as an alternative method for the specific isolation of exosomes in serum. To achieve that, a rational study of the surface proteins in exosomes, which can be recognized by magnetic particles, is presented. The characterization was performed in exosomes obtained from cell culture supernatants of MCF7, MDA-MB-231 and SKBR3 breast cancer cell lines, including TEM and nanoparticle tracking analysis (NTA). For the specific characterization by flow cytometry and confocal microscopy, different commercial antibodies against selected receptors were used, including the general tetraspanins CD9, CD63 and CD81, and cancer-related receptors (CD24, CD44, CD54, CD326 and CD340). The effect of the serum matrix on the immunomagnetic separation was then carefully evaluated by spiking the exosomes in depleted human serum. Based on this study, the exosomes were preconcentrated by immunomagnetic separation on antiCD81-modified magnetic particles in order to achieve further magnetic actuation on the surface of the electrode for the electrochemical readout. The performance of this approach is discussed and compared with classical characterization methods.