Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer’s disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (− 0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories.Depto. de Química AnalíticaFac. de Ciencias QuímicasTRUESpanish Ministerio de Ciencia e InnovaciónAES-ISCIIIComunidad de Madridpu

Angiogenesis inhibitor or aggressiveness marker? The function of endostatin in cancer through electrochemical biosensing

This work reports the first electrochemical bioplatform developed for the determination of human endostatin (HE), a biomarker with recognized antiangiogenic potential whose elevated circulating levels have also been associated with the development of aggressive cancers. The developed electroanalytical biotool combines the benefits of using magnetic microparticles for the implementation of sandwich immunoassays and amperometric transduction on disposable carbon electrodes. A limit of detection (LOD) of 34.1 pg mL-1 for HE standards and a selectivity suitable for its foray into the clinical oncology area, are demonstrated. The determination of HE in clinical samples such as lysates and secretomes of colorectal cancer (CRC) cells, plasma, and tissue samples from patients with CRC in different stages, has been faced with satisfactory results showing the ability for discriminating the metastatic capabilities of cells and for identifying and staging CRC patients. The developed bioplatform allows precise quantitative determinations, requiring minimal pre-treatments and sample amounts in only 75 min. In addition, due to the instrumentation and the type of substrates used in the detection step, the biotool is compatible with implementation in multiplexed and/or point-of-need devices, features in which this bioplatform is advantageous with respect to the enzyme linked immunosorbent assay (ELISA) or immunoblotting technologies.The financial support of PID2019-103899RB-I00 (Spanish Ministerio de Ciencia e Innovación) Research Project and PI20CIII/00019 Grant from the AES-ISCIII Program co-founded by FEDER funds and the TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349) are gratefully acknowledged. M.G-A. acknowledges the postdoctoral contract Margarita Salas for the requalification of the Spanish University System. A.M-C. was supported by a FPU predoctoral contract supported by the Spanish Ministerio de Educación, Cultura y Deporte. S.T.M. acknowledges a predoctoral contract from the Spanish Ministerio de Ciencia e Innovación (PRE2020-092859).S

First bioelectronic immunoplatform for quantitative secretomic analysis of total and metastasis-driven glycosylated haptoglobin

The glycosylation status of proteins is increasingly used as biomarker to improve the reliability in the diagnosis and prognosis of diseases as relevant as cancer. This feeds the need for tools that allow its simple and reliable analysis and are compatible with applicability in the clinic. With this objective in mind, this work reports the first bioelectronic immunoplatforms described to date for the determination of glycosylated haptoglobin (Hp) and the simultaneous determination of total and glycosylated Hp. The bioelectronic immunoplatform is based on the implementation of non-competitive bioassays using two different antibodies or an antibody and a lectin on the surface of commercial magnetic microcarriers. The resulting bioconjugates are labeled with the horseradish peroxidase (HRP) enzyme, and after their magnetic capture on disposable electroplatforms, the amperometric transduction using the H2O2/hydroquinone (HQ) system allows the single or multiple detection. The developed immunoplatform achieves limits of detection (LODs) of 0.07 and 0.46 ng mL-1 for total and glycosylated Hp in buffer solution, respectively. The immunoplatform allows accurate determination using simple and relatively short protocols (approx. 75 min) of total and glycosylated Hp in the secretomes of in vitro-cultured colorectal cancer (CRC) cells with different metastatic potentials, which is not feasible, due to lack of sensitivity, by means of some commercial ELISA kits and Western blot methodology.Funding Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Spanish Ministerio de Ciencia e Innovación (PID2019-103899RB-I00 and RTI2018-095756-B-I00), AES-ISCIII Program co-founded by FEDER funds (PI17CIII/00045 and PI20CIII/00019 grants), TRANSNANOAVANSENS-CM Program from the Comunidad de Madrid (Grant S2018/NMT-4349).S

Electrochemical sensor for rapid determination of fibroblast growth factor receptor 4 in raw cancer cell lysates

The first electrochemical immunosensor for the determination of fibroblast growth factor receptor 4 (FGFR4) biomarker is reported in this work. The biosensor involves a sandwich configuration with covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic microcarriers (HOOC-MBs) and amperometric detection at disposable carbon screen-printed electrodes (SPCEs). The biosensor exhibits a great analytical performance regarding selectivity for the target protein and a low LOD of 48.2 pg mL-1. The electrochemical platform was successfully applied for the determination of FGFR4 in different cancer cell lysates without any apparent matrix effect after a simple sample dilution and using only 2.5 μg of the raw lysate. Comparison of the results with those provided by a commercial ELISA kit shows competitive advantages by using the developed immunosensor in terms of simplicity, analysis time, and portability and cost-affordability of the required instrumentation for the accurate determination of FGFR4 in cell lysates

The specific seroreactivity to ∆Np73 isoforms shows higher diagnostic ability in colorectal cancer patients than the canonical p73 protein

The p53-family is tightly regulated at transcriptional level. Due to alternative splicing, up to 40 different theoretical proteoforms have been described for p73 and at least 20 and 10 for p53 and p63, respectively. However, only the canonical proteins have been evaluated as autoantibody targets in cancer patients for diagnosis. In this study, we have cloned and expressed in vitro the most upregulated proteoforms of p73, ΔNp73α and ΔNp73β, for the analysis of their seroreactivity by a developed luminescence based immunoassay test using 145 individual plasma from colorectal cancer, premalignant individuals and healthy controls. ∆Np73α seroreactivity showed the highest diagnostic ability to discriminate between groups. The combination of ∆Np73α, ∆Np73β and p73 proteoforms seroreactivity were able to improve their individual diagnostic ability. Competitive inhibition experiments further demonstrated the presence of unique specific epitopes in ΔNp73 isoforms not present in p73, with several colorectal patients showing unique and specific seroreactivity to the ΔNp73 proteoforms. Overall, we have increased the complexity of the humoral immune response to the p53-family in cancer patients, showing that the proteoforms derived from the alternative splicing of p73 possess a higher diagnostic ability than the canonical protein, which might be extensive for p53 and p63 proteins.This work was supported by the Ramon y Cajal programme of the MINECO and the financial support of the PI17CIII/00045 grant from the AES-ISCIII program to R.B., cofounded by FEDER funds. G.D. acknowledges the financial support of PI15/00246 grant of the FIS and Cátedra UAM-Roche en Medicina de Innovación. M.G-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. We thank the excellent technical support of Maricruz Sánchez. A.M-C. is a recipient of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte.S

Tumor-derived pericytes driven by egfr mutations govern the vascular and immune microenvironment of gliomas

The extraordinary plasticity of glioma cells allows them to contribute to different cellular compartments in tumor vessels, reinforcing the vascular architecture. It was recently revealed that targeting glioma-derived pericytes, which represent a big percentage of the mural cell population in aggressive tumors, increases the permeability of the vessels and improves the efficiency of chemotherapy. However, the molecular determinants of this transdifferentiation process have not been elucidated. Here we show that mutations in EGFR stimulate the capacity of glioma cells to function as pericytes in a BMX- (bone marrow and X-linked) and SOX9-dependent manner. Subsequent activation of platelet-derived growth factor receptor beta in the vessel walls of EGFR-mutant gliomas stabilized the vasculature and facilitated the recruitment of immune cells. These changes in the tumor microenvironment conferred a growth advantage to the tumors but also rendered them sensitive to pericyte-targeting molecules such as ibrutinib or sunitinib. In the absence of EGFR mutations, high-grade gliomas were enriched in blood vessels, but showed a highly disrupted blood–brain barrier due to the decreased BMX/SOX9 activation and pericyte coverage, which led to poor oxygenation, necrosis, and hypoxia. Overall, these findings identify EGFR mutations as key regulators of the glioma-to-pericyte transdifferentiation, highlighting the intricate relationship between the tumor cells and their vascular and immune milieu. Our results lay the foundations for a vascular-dependent stratification of gliomas and suggest different therapeutic vulnerabilities determined by the genetic status of EGFR.This work was supported by FONDECYT grant (1140697 to V. Palma), CONICYT Fellowship (to B.S. Casas), by Ministerio de Economía y Competitividad and FEDER funds (PI13/01258 to A. Hernandez-Laín; PI17/01621 to J.M. Sepulveda-S anchez; and PI16/01580 and DTS18/00181 to A. Matheu), by Young Employment Initiative (Comunidad de Madrid) to M. Garranzo-Asensio, by “Asociacion Espanola contra ~ el Cancer” (AECC) grants (INVES192GARG to R. Gargini; GCTRA16015SEDA to J.M. Sepulveda-S anchez); and by Ministerio de Ciencia, Innovacion y Universidades and FEDER funds (RTI2018-093596 to P. Sanchez-Gomez).Peer reviewe

In-depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Alterations in proteins of the p53-family are a common event in CRC. ΔNp73, a p53-family member, shows oncogenic properties and its effectors are largely unknown. We performed an in-depth proteomics characterization of transcriptional control by ∆Np73 of the secretome of human colon cancer cells and validated its clinical potential. The secretome was analyzed using high-density antibody microarrays and stable isotopic metabolic labeling. Validation was performed by semiquantitative PCR, ELISA, dot-blot and western blot analysis. Evaluation of selected effectors was carried out using 60 plasma samples from CRC patients, individuals carrying premalignant colorectal lesions and colonoscopy-negative controls. In total, 51 dysregulated proteins were observed showing at least 1.5-foldchange in expression. We found an important association between the overexpression of ∆Np73 and effectors related to lymphangiogenesis, vasculogenesis and metastasis, such as brain-derived neurotrophic factor (BDNF) and the putative aminoacyl tRNA synthase complex-interacting multifunctional protein 1 (EMAP-II)-vascular endothelial growth factor C-vascular endothelial growth factor receptor 3 axis. We further demonstrated the usefulness of BDNF as a potential CRC biomarker able to discriminate between CRC patients and premalignant individuals from controls with high sensitivity and specificity.This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project “PI18/00473” and co-funded by the European Union (FEDER funds) and Cátedra UAM-Roche en Medicina de Innovación to GD, and the Ramón y Cajal Programme of the MINECO, PI17CIII/00045 and PI20CIII/00019 research projects from AES-ISCIII to RB. MG-A and JR-C were supported by contracts of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. AM-C FPU predoctoral contract is supported by the MECD. GS-F is a recipient of a predoctoral contract (grant num-ber 1193818N) supported by The Flanders Research Foundation (FWO).S

Revealing prevalent cancers by interrogating glycoproteins with sustainable immunoelectrochemical tools

Trabajo presentado en el 4th European Biosensor Symposium, celebrado en Aquisgrán (Alemania), del 27 al 30 de agosto de 2023Introduction. The worldwide incidence and death toll of colorectal and pancreatic cancers (CRC and PDAC) have increased considerably since 1990. For this reason, both early detection and regular follow-up are considered key factors in improving patient prognosis. In this sense, the determination of the total content of specific proteins and their aberrantly glycosylated fraction in oncologic processes could help to achieve the proposed goals. Results and Discussion. In this work, two simple but highly competitive electrochemical immunoplatforms for the determination of total and glycosylated post-translational modified haptoglobin (Hp) [1], and CA19-9 [2] (candidate biomarkers associated with colorectal and pancreatic cancer, respectively) are presented. As seen in Figure 1, these biotools are uplifted in the use of magnetic immunocaptors and another antibody or a lectin as detector elements lastly labeled with HRP, which enables subsequent amperometric detection. The presented bioplatforms exhibit attractive characteristics in terms of simplicity, affordability, and point-of-care application compared to the conventional available methodologies, highlighting low detection limits (0.07 and 0.46 ng mL¿1 for total and glycosylated Hp, respectively, and 1.5 U mL¿1 for CA19-9), and short assay times (< 2 h). The workability of these quantitative bioplatforms for the analysis of secretomes from cultured CRC cells with the distinct potential to metastasize (Hp) or serum samples from healthy and PDAC-diagnosed subjects (CA19-9) was assessed to definitely confirm full exploitation of all the above exposed enticing attributes. Conclusions. Our findings clearly revealed the unquestionable ability of these modern electrochemical immunoplatforms to discriminate between healthy and cancer-diagnosed subjects, as well as to assess disease progression, positioning these simple but effective methodologies as advanced electroanalytical tools with proven real biomedical applications, and the hope of aiding in the accurate diagnosis of prevalent and high mortality cancers

In-depth proteomics characterization of ∆Np73 effectors identifies key proteins with diagnostic potential implicated in lymphangiogenesis, vasculogenesis and metastasis in colorectal cancer

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death worldwide. Alterations in proteins of the p53-family are a common event in CRC. ΔNp73, a p53-family member, shows oncogenic properties and its effectors are largely unknown. We performed an in-depth proteomics characterization of transcriptional control by ∆Np73 of the secretome of human colon cancer cells and validated its clinical potential. The secretome was analyzed using high-density antibody microarrays and stable isotopic metabolic labeling. Validation was performed by semiquantitative PCR, ELISA, dot-blot and western blot analysis. Evaluation of selected effectors was carried out using 60 plasma samples from CRC patients, individuals carrying premalignant colorectal lesions and colonoscopy-negative controls. In total, 51 dysregulated proteins were observed showing at least 1.5-foldchange in expression. We found an important association between the overexpression of ∆Np73 and effectors related to lymphangiogenesis, vasculogenesis and metastasis, such as brain-derived neurotrophic factor (BDNF) and the putative aminoacyl tRNA synthase complex-interacting multifunctional protein 1 (EMAP-II)–vascular endothelial growth factor C–vascular endothelial growth factor receptor 3 axis. We further demonstrated the usefulness of BDNF as a potential CRC biomarker able to discriminate between CRC patients and premalignant individuals from controls with high sensitivity and specificity.This study has been funded by Instituto de Salud Carlos III (ISCIII) through the project “PI18/00473” and co-funded by the European Union (FEDER funds) and Catedra UAM-Roche en Medicina de Innovacion to GD, and the Ramon y Cajal Programme of the MINECO, PI17CIII/00045 and PI20CIII/00019 research projects from AES-ISCIII to RB. MG-A and JR-C were supported by contracts of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejerıa de Educacion, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo. AM-C FPU predoctoral contract is supported by the MECD. GSF is a recipient of a predoctoral contract (grant number 1193818N) supported by The Flanders Research Foundation (FWO).Peer reviewe