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Cataracts and glaucoma account for a high percentage of vision loss and blindness worldwide. Small extracellular vesicles (sEVs) are released into different body fluids, including the eye’s aqueous humor. Information about their proteome content and characterization in ocular pathologies is not yet well established. In this study, aqueous humor sEVs from healthy individuals, cataracts, and glaucoma patients were studied, and their specific protein profiles were characterized. Moreover, the potential of identified proteins as diagnostic glaucoma biomarkers was evaluated. The protein content of sEVs from patients’ aqueous humor with cataracts and glaucoma compared to healthy individuals was analyzed by quantitative proteomics. Validation was performed by western blot (WB) and ELISA. A total of 828 peptides and 192 proteins were identified and quantified. After data analysis with the R program, 8 significantly dysregulated proteins from aqueous humor sEVs in cataracts and 16 in glaucoma showed an expression ratio ≥ 1.5. By WB and ELISA using directly aqueous humor samples, the dysregulation of 9 proteins was mostly confirmed. Importantly, GAS6 and SPP1 showed high diagnostic ability of glaucoma, which in combination allowed for discriminating glaucoma patients from control individuals with an area under the curve of 76.1% and a sensitivity of 65.6% and a specificity of 87.7%.

Abstract Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

Transthyretin (TTR) amyloid cardiomyopathy (ATTR-CM) is a life-threatening disease caused by the abnormal production of misfolded TTR protein by liver cells, which is then released systemically. Its amyloid deposition in the heart is linked to cardiac toxicity and progression toward heart failure. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) from a patient suffering familial transthyretin amyloid cardiomyopathy carrying a c.128G>A (p.Ser43Asn) mutation in the TTR gene. This iPSC line offers a useful resource to study the disease pathophysiology and a cell-based model for therapeutic discovery.

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.

SPRY domain-containing protein 7 (SPRYD7) is a barely known protein identified via spatial proteomics as being upregulated in highly metastatic-to-liver KM12SM colorectal cancer (CRC) cells in comparison to its isogenic poorly metastatic KM12C CRC cells. Here, we aimed to analyze SPRYD7’s role in CRC via functional proteomics. Through immunohistochemistry, the overexpression of SPRYD7 was observed to be associated with the poor survival of CRC patients and with an aggressive and metastatic phenotype. Stable SPRYD7 overexpression was performed in KM12C and SW480 poorly metastatic CRC cells and in their isogenic highly metastatic-to-liver-KM12SM-and-to-lymph-nodes SW620 CRC cells, respectively. Upon upregulation of SPRYD7, in vitro and in vivo functional assays confirmed a key role of SPRYD7 in the invasion and migration of CRC cells and in liver homing and tumor growth. Additionally, transient siRNA SPRYD7 silencing allowed us to confirm in vitro functional results. Furthermore, SPRYD7 was observed as an inductor of angiogenesis. In addition, the dysregulated SPRYD7-associated proteome and SPRYD7 interactors were elucidated via 10-plex TMT quantitative proteins, immunoproteomics, and bioinformatics. After WB validation, the biological pathways associated with the stable overexpression of SPRYD7 were visualized. In conclusion, it was demonstrated here that SPRYD7 is a novel protein associated with CRC progression and metastasis. Thus, SPRYD7 and its interactors might be of relevance in identifying novel therapeutic targets for advanced CRC.

The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = −45 and CV = −60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.

Although blood autoantibodies were initially associated with autoimmune diseases, multiple evidence have been accumulated showing their presence in many types of cancer. This has opened their use in clinics, since cancer autoantibodies might be useful for early detection, prognosis, and monitoring of cancer patients. In this review, we discuss the different techniques available for their discovery and validation. Additionally, we discuss here in detail those autoantibody panels verified in at least two different reports that should be more likely to be specificof eachof the four most incident cancers. We also report the recent developed kits for breast and lung cancer detectionmostly based on autoantibodies and the identification of novel therapeutic targets because of the screening of the cancer humoral immune response. Finally, we discuss unsolved issues that still need to be addressed for the implementation of cancer autoantibodies in clinical routine for cancer diagnosis, prognosis, and/or monitoring. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. The five-year survival rate of CRC patients depends on the stage at diagnosis, being higher than 80% when CRC is diagnosed in the early stages but lower than 10% when CRC is diagnosed in advanced stages. Autoantibodies against specific CRC autoantigens (tumor-associated antigens (TAAs)) in the sera of patients have been widely demonstrated to aid in early diagnosis. Thus, we herein aim to identify autoantigens target of autoantibodies specific to CRC that possess a significant ability to discriminate between CRC patients and healthy individuals by means of liquid biopsy. To that end, we examined the protein content of the exosomes released by five CRC cell lines and tissue samples from CRC patients by means of immunoprecipitation coupled with mass spectrometry analysis. A total of 103 proteins were identified as potential autoantigens specific to CRC. After bioinformatics and meta-analysis, we selected 15 proteins that are more likely to be actual CRC autoantigens in order to evaluate their role in CRC prognosis by Western blot (WB) and immunohistochemistry (IHC). We found dysregulation at the protein level for 11 of these proteins in both tissue and plasma exosome samples from patients, along with an association of nine of these proteins with CRC prognosis. After validation, all but one showed a statistically significant high diagnostic ability to distinguish CRC patients and individuals with premalignant lesions from healthy individuals, either by luminescence Halotag-based beads, or by a multiplexed biosensing platform involving the use of magnetic microcarriers as solid support modified with covalently immobilized Halotag fusion proteins constructed for CRC detection. Taken together, our results highlight the usefulness of the approach defined here to identify the TAAs specific to chronic diseases; they also demonstrate that the measurement of autoantibody levels in plasma against the TAAs identified here could be integrated into a point-of-care (POC) device for CRC detection with high diagnostic ability.

The great opportunities represented by exosomes in liquid biopsy diagnostics and the relevance of CD147 protein as diagnostic and prognostic cancer biomarker led us to develop the first bio-electroanalytical platform for the determination of exosomal CD147 (exoCD147) by exploiting micro-sized magnetic beads coated with specific anti-CD147 antibodies. The captured exosomal target protein was sandwiched by specific biotin functionalized detector antibodies followed by attaching streptavidin-HRP conjugate to perform the amperometric reading using screen-printed carbon electrodes (SPCEs) as electrode transducers in the presence of hydroquinone (HQ) and H2O2. The analytical and operational characteristics achieved by implementing this simple methodology allowed the sensitive (LOD 29 pg mL−1) and selective determination of CD147 and the analysis of exoCD147 in different but inter-related real clinical scenarios including lysed and entire exosomes previously isolated from CRC cell lines with different metastatic potential. The obtained results, in agreement with those provided by ELISA and WB, proved the reliability of the developed immunosensor and its potential to isolate or identify specific subpopulations of exosomes based on the differential expression of characteristic surface biomarkers.

Approximately 25% of colorectal cancer (CRC) patients experience systemic metastases, with the most frequent target organs being the liver and lung. Metabolic reprogramming has been recognized as one of the hallmarks of cancer. Here, metabolic and functional differences between two CRC cells with different metastatic organotropisms (metastatic KM12SM CRC cells to the liver and KM12L4a to the lung when injected in the spleen and in the tail vein of mice) were analysed in comparison to their parental non-metastatic isogenic KM12C cells, for a subsequent investigation of identified metabolic targets in CRC patients. Meta-analysis from proteomic and transcriptomic data deposited in databases, qPCR, WB, in vitro cell-based assays, and in vivo experiments were used to survey for metabolic alterations contributing to their different organotropism and for the subsequent analysis of identified metabolic markers in CRC patients. Although no changes in cell proliferation were observed between metastatic cells, KM12SM cells were highly dependent on oxidative phosphorylation at mitochondria, whereas KM12L4a cells were characterized by being more energetically efficient with lower basal respiration levels and a better redox management. Lipid metabolism-related targets were found altered in both cell lines, including LDLR, CD36, FABP4, SCD, AGPAT1, and FASN, which were also associated with the prognosis of CRC patients. Moreover, CD36 association with lung metastatic tropism of CRC cells was validated in vivo. Altogether, our results suggest that LDLR, CD36, FABP4, SCD, FASN, LPL, and APOA1 metabolic targets are associated with CRC metastatic tropism to the liver or lung. These features exemplify specific metabolic adaptations for invasive cancer cells which stem at the primary tumour.

Colorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer-related death worldwide. The detection in plasma samples of autoantibodies against specific tumor-associated antigens has been demonstrated to be useful for the early diagnosis of CRC by liquid biopsy. However, new studies related to the humoral immune response in cancer are needed to enable blood-based diagnosis of the disease. Here, our aim was to characterize the humoral immune response associated with the different p53 and p63 proteoforms derived from alternative splicing and previously described as aberrantly expressed in CRC. Thus, here we investigated the diagnostic ability of the twelve p53 proteoforms and the eight p63 proteoforms described to date, and their specific N-terminal and C-terminal end peptides, by means of luminescence HaloTag beads immunoassays. Full-length proteoforms or specific peptides were cloned as HaloTag fusion proteins and their seroreactivity analyzed using plasma from CRC patients at stages I-IV (n = 31), individuals with premalignant lesions (n = 31), and healthy individuals (n = 48). p53γ, Δ40p53β, Δ40p53γ, Δ133p53γ, Δ160p53γ, TAp63α, TAp63δ, ΔNp63α, and ΔNp63δ, together with the specific C-terminal end α and δ p63 peptides, were found to be more seroreactive against plasma from CRC patients and/or individuals with premalignant lesions than from healthy individuals. In addition, ROC (receiver operating characteristic) curves revealed a high diagnostic ability of those p53 and p63 proteoforms to detect CRC and premalignant individuals (AUC higher than 85%). Finally, electrochemical biosensing platforms were employed in POC-like devices to investigate their usefulness for CRC detection using selected p53 and p63 proteoforms. Our results demonstrate not only the potential of these biosensors for the simultaneous analysis of proteoforms’ seroreactivity, but also their convenience and versatility for the clinical detection of CRC by liquid biopsy. In conclusion, we here show that p53 and p63 proteoforms possess differential seroreactivity in CRC patients in comparison to controls, distinctive from canonical proteins, which should improve the diagnostic panels for obtaining a blood-based biomarker signature for CRC detection.