Functional insight into the reciprocal paracrine crosstalk of stromal fibroblasts in the head and neck cancer microenvironment

Trabajo presentado en el 18th ASEICA International Congress, celebrado en Santiago de Compostela (España) del 16 al 18 de noviembre de 2022

A comprehensive systematic review of CSF proteins and peptides that defne Alzheimer’s disease

Background: During the last two decades, over 100 proteomics studies have identifed a variety of potential bio‑ markers in CSF of Alzheimer’s (AD) patients. Although several reviews have proposed specifc biomarkers, to date, the statistical relevance of these proteins has not been investigated and no peptidomic analyses have been generated on the basis of specifc up- or down- regulation. Herein, we perform an analysis of all unbiased explorative proteom‑ ics studies of CSF biomarkers in AD to critically evaluate whether proteins and peptides identifed in each study are consistent in distribution; direction change; and signifcance, which would strengthen their potential use in studies of AD pathology and progression. Methods: We generated a database containing all CSF proteins whose levels are known to be signifcantly altered in human AD from 47 independent, validated, proteomics studies. Using this database, which contains 2022 AD and 2562 control human samples, we examined whether each protein is consistently present on the basis of reliable statistical studies; and if so, whether it is over- or under-represented in AD. Additionally, we performed a direct analysis of available mass spectrometric data of these proteins to generate an AD CSF peptide database with 3221 peptides for further analysis. Results: Of the 162 proteins that were identifed in 2 or more studies, we investigated their enrichment or depletion in AD CSF. This allowed us to identify 23 proteins which were increased and 50 proteins which were decreased in AD, some of which have never been revealed as consistent AD biomarkers (i.e. SPRC or MUC18). Regarding the analysis of the tryptic peptide database, we identifed 87 peptides corresponding to 13 proteins as the most highly consistently altered peptides in AD. Analysis of tryptic peptide fngerprinting revealed specifc peptides encoded by CH3L1, VGF, SCG2, PCSK1N, FBLN3 and APOC2 with the highest probability of detection in AD. Conclusions: Our study reveals a panel of 27 proteins and 21 peptides highly altered in AD with consistent statistical signifcance; this panel constitutes a potent tool for the classifcation and diagnosis of AD

Biological Significance of the Protein Changes Occurring in the Cerebrospinal Fluid of Alzheimer’s Disease Patients: Getting Clues from Proteomic Studies

The fact that cerebrospinal fluid (CSF) deeply irrigates the brain together with the relative simplicity of sample extraction from patients make this biological fluid the best target for biomarker discovery in neurodegenerative diseases. During the last decade, biomarker discovery has been especially fruitful for the identification new proteins that appear in the CSF of Alzheimer’s disease (AD) patients together with amyloid-β (Aβ42), total tau (T-tau), and phosphorylated tau (P-tau). Thus, several proteins have been already stablished as important biomarkers, due to an increase (i.e., CHI3L1) or a decrease (i.e., VGF) in AD patients’ CSF. Notwithstanding this, only a deep analysis of a database generated with all the changes observed in CSF across multiple proteomic studies, and especially those using state-of-the-art methodologies, may expose those components or metabolic pathways disrupted at different levels in AD. Deep comparative analysis of all the up- and down-regulated proteins across these studies revealed that 66% of the most consistent protein changes in CSF correspond to intracellular proteins. Interestingly, processes such as those associated to glucose metabolism or RXR signaling appeared inversely represented in CSF from AD patients in a significant manner. Herein, we discuss whether certain cellular processes constitute accurate indicators of AD progression by examining CSF. Furthermore, we uncover new CSF AD markers, such as ITAM, PTPRZ or CXL16, identified by this study

Human amyloid-β enriched extracts: evaluation of in vitro and in vivo internalization and molecular characterization

[Background]: Intracerebral inoculation of extracts from post-mortem human Alzheimer’s disease brains into mice produces a prion-like spreading effect of amyloid-β. The differences observed between these extracts and the synthetic peptide, in terms of amyloid-β internalization and seed and cell-to-cell transmission of cytosolic protein aggregates, suggest that brain extracts contain key contributors that enhance the prion-like effect of amyloid-β. Nevertheless, these potential partners are still unknown due to the complexity of whole brain extracts.[Methods]: Herein, we established a method based on sequential detergent solubilization of post-mortem samples of human brains affected by Alzheimer’s disease that strongly enrich amyloid-β aggregates by eliminating 92% of the remaining proteins. Internalization of Aβ1–42 from the enriched AD extracts was evaluated in vitro, and internalization of fluorescent-labeled AD extracts was also investigated in vivo. Furthermore, we carried out a molecular characterization of the Aβ-enriched fraction using label-free proteomics, studying the distribution of representative components in the amygdala and the olfactory cortex of additional human AD brain samples by immunohistochemistry.[Results]: Aβ1–42 from the enriched AD extracts are internalized into endothelial cells in vitro after 48 h. Furthermore, accumulation of fluorescent-labeled Aβ-enriched extracts into mouse microglia was observed in vivo after 4 months of intracerebral inoculation. Label-free proteomics (FDR < 0.01) characterization of the amyloid-β-enriched fraction from different post-mortem samples allowed for the identification of more than 130 proteins, several of which were significantly overrepresented (i.e., ANXA5 and HIST1H2BK; p < 0.05) and underrepresented (i.e., COL6A or FN1; p < 0.05) in the samples with Alzheimer’s disease. We were also able to identify proteins exclusively observed in Alzheimer’s disease (i.e., RNF213) or only detected in samples not affected by the disease (i.e., CNTN1) after the enrichment process. Immunohistochemistry against these proteins in additional tissues revealed their particular distribution in the amygdala and the olfactory cortex in relation to the amyloid-β plaque.[Conclusions]: Identification and characterization of the unique features of these extracts, in terms of amyloid-β enrichment, identification of the components, in vitro and in vivo cell internalization, and tissue distribution, constitute the best initial tool to further investigate the seeding and transmissibility proposed in the prion-like hypothesis of Alzheimer’s disease.Sponsored by the Spanish Ministry of Economy and Competitiveness-FEDER (grant # SAF2016-75768-R) to AMM, MINECO-RETOS (AEI-FEDER) to MDP, and the Autonomous Government of Castilla-La Mancha/FEDER (grant no. SBPLY/ 17/180501/000430) to AMM and DSS.Peer reviewe

Bidirectional paracrine communication between head and neck cancer cells and stromal fibroblasts

Resumen del trabajo presentado en el European Association for Cancer Research (EACR) Congress, celebrado en Sevilla (España) del 20 al 23 de junio de 2023