Bioimaging Nucleic-Acid Aptamers with Different Specificities in Human Glioblastoma Tissues Highlights Tumoral Heterogeneity.

Nucleic-acid aptamers are of strong interest for diagnosis and therapy. Compared with antibodies, they are smaller, stable upon variations in temperature, easy to modify, and have higher tissue-penetration abilities. However, they have been little described as detection probes in histology studies of human tissue sections. In this study, we performed fluorescence imaging with two aptamers targeting cell-surface receptors EGFR and integrin α5ß1, both involved in the aggressiveness of glioblastoma. The aptamers' cell-binding specificities were confirmed using confocal imaging. The affinities of aptamers for glioblastoma cells expressing these receptors were in the 100-300 nM range. The two aptamers were then used to detect EGFR and integrin α5ß1 in human glioblastoma tissues and compared with antibody labeling. Our aptafluorescence assays proved to be able to very easily reveal, in a one-step process, not only inter-tumoral glioblastoma heterogeneity (differences observed at the population level) but also intra-tumoral heterogeneity (differences among cells within individual tumors) when aptamers with different specificities were used simultaneously in multiplexing labeling experiments. The discussion also addresses the strengths and limitations of nucleic-acid aptamers for biomarker detection in histology.

Past mastering of metal transformation enabled physicians to increase their therapeutic potential.

BACKGROUND: Metals are trace elements, vital in some instances or toxic in others. Due to this toxicity, they have been used since ancient time as antimicrobials, and prescribed when plant-only remedies were not efficient enough. These remedies could still contain secrets that may lead to the discovery of new therapeutically interesting combinations. The objective of this study was to give a proof of concept that such remedies combining metals and plants are worth studying again. METHODS: We exploited 4 medical formularies (aqrabadhin), from three Arab authors from the 9-12th century. We reproduced a remedy, and analyzed the role of each ingredient. We further looked for the minimum inhibitory concentration against three pathogenic bacteria, and we analyzed toxic and inflammatory effects of this remedy on macrophages. RESULTS: Even if plants were extensively used (almost 80 % of all ingredients), more than 36 different minerals have been found in these 4 aqrabadhin. When it came to remedies against infections that could be applied externally, the use of metals grew to 70 %. We focused on a remedy, containing mainly metals. We have been able to attribute a role for each ingredient, to show that this skin remedy helped to combat the infection and to resorb the wound, and to highlight the mastering of metal transformation by these physicians. CONCLUSIONS: With a very simple recipe, mainly composed of metals, these past physicians designed a complete and synergistic remedy to combat abscesses, while restricting the toxic effect of metals to the site of infection. It is a first example showing that different metal manufactures were evolved to improve their therapeutic potentials. The knowledge acquired by these physician should deserve more attention, and unexpected features, original organo-metallic compounds or therapeutic synergy could still be found from such research.

Preliminary Study of New Gallium-68 Radiolabeled Peptide Targeting NRP-1 to Detect Brain Metastases by Positron Emission Tomography.

Due to their very poor prognosis and a fatal outcome, secondary brain tumors are one of the biggest challenges in oncology today. From the point of view of the early diagnosis of these brain micro- and macro-tumors, the sensitivity and specificity of the diagnostic tools constitute an obstacle. Molecular imaging, such as Positron Emission Tomography (PET), is a promising technique but remains limited in the search for cerebral localizations, given the commercially available radiotracers. Indeed, the [18F]FDG PET remains constrained by the physiological fixation of the cerebral cortex, which hinders the visualization of cerebral metastases. Tumor angiogenesis is recognized as a crucial phenomenon in the progression of malignant tumors and is correlated with overexpression of the neuropilin-1 (NRP-1) receptor. Here, we describe the synthesis and the photophysical properties of the new gallium-68 radiolabeled peptide to target NRP-1. The KDKPPR peptide was coupled with gallium-68 anchored into a bifunctional NODAGA chelating agent, as well as Cy5 for fluorescence detection. The Cy5 absorbance spectra did not change, whereas the molar extinction coefficient (ε) decreased drastically. An enhancement of the fluorescence quantum yield (φF) could be observed due to the better water solubility of Cy5. [68Ga]Ga-NODAGA-K(Cy5)DKPPR was radiosynthesized efficiently, presented hydrophilic properties (log D = -1.86), and had high in vitro stability (>120 min). The molecular affinity and the cytotoxicity of this new chelated radiotracer were evaluated in vitro on endothelial cells (HUVEC) and MDA-MB-231 cancer cells (hormone-independent and triple-negative line) and in vivo on a brain model of metastasis in a nude rat using the MDA-MB-231 cell line. No in vitro toxicity has been observed. The in vivo preliminary experiments showed promising results, with a high contrast between the healthy brain and metastatic foci for [68Ga]Ga-NODAGA-K(Cy5)DKPPR.

Role of Endocytosis Proteins in Gefitinib-Mediated EGFR Internalisation in Glioma Cells.

EGFR (epidermal growth factor receptor), a member of the ErbB tyrosine kinase receptor family, is a clinical therapeutic target in numerous solid tumours. EGFR overexpression in glioblastoma (GBM) drives cell invasion and tumour progression. However, clinical trials were disappointing, and a molecular basis to explain these poor results is still missing. EGFR endocytosis and membrane trafficking, which tightly regulate EGFR oncosignaling, are often dysregulated in glioma. In a previous work, we showed that EGFR tyrosine kinase inhibitors, such as gefitinib, lead to enhanced EGFR endocytosis into fused early endosomes. Here, using pharmacological inhibitors, siRNA-mediated silencing, or expression of mutant proteins, we showed that dynamin 2 (DNM2), the small GTPase Rab5 and the endocytosis receptor LDL receptor-related protein 1 (LRP-1), contribute significantly to gefitinib-mediated EGFR endocytosis in glioma cells. Importantly, we showed that inhibition of DNM2 or LRP-1 also decreased glioma cell responsiveness to gefitinib during cell evasion from tumour spheroids. By highlighting the contribution of endocytosis proteins in the activity of gefitinib on glioma cells, this study suggests that endocytosis and membrane trafficking might be an attractive therapeutic target to improve GBM treatment.

Expression Analysis of α5 Integrin Subunit Reveals Its Upregulation as a Negative Prognostic Biomarker for Glioblastoma.

Integrin α5ß1 was suggested to be involved in glioblastoma (GBM) aggressiveness and treatment resistance through preclinical studies and genomic analysis in patients. However, further protein expression data are still required to confirm this hypothesis. In the present study, we investigated by immunofluorescence the expression of integrin α5 and its prognostic impact in a glioblastoma series of patients scheduled to undergo the Stupp protocol as first-line treatment for GBM. The integrin α5 protein expression level was estimated in each tumor by the mean fluorescence intensity (MFI) and allowed us to identify two subpopulations showing either a high or low expression level. The distribution of patients in both subpopulations was not significantly different according to age, gender, recursive partitioning analysis (RPA) prognostic score, molecular markers or surgical and medical treatment. A high integrin α5 protein expression level was associated with a high risk of recurrence (HR = 1.696, 95% CI 1.031-2.792, p = 0.0377) and reduced overall survival (OS), even more significant in patients who completed the Stupp protocol (median OS: 15.6 vs. 22.8 months; HR = 2.324; 95% CI 1.168-4.621, p = 0.0162). In multivariate analysis, a high integrin α5 protein expression level was confirmed as an independent prognostic factor in the subpopulation of patients who completed the temozolomide-based first-line treatment for predicting OS over age, extent of surgery, RPA score and O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation (p = 0.029). In summary, for the first time, our study validates that a high integrin α5 protein expression level is associated with poor prognosis in GBM and confirms its potential as a therapeutic target implicated in the Stupp protocol resistance.

A DNA Repair and Cell Cycle Gene Expression Signature in Pediatric High-Grade Gliomas: Prognostic and Therapeutic Value.

BACKGROUND: Pediatric high-grade gliomas (pHGGs) are the leading cause of mortality in pediatric neuro-oncology, displaying frequent resistance to standard therapies. Profiling DNA repair and cell cycle gene expression has recently been proposed as a strategy to classify adult glioblastomas. To improve our understanding of the DNA damage response pathways that operate in pHGGs and the vulnerabilities that these pathways might expose, we sought to identify and characterize a specific DNA repair and cell-cycle gene expression signature of pHGGs. METHODS: Transcriptomic analyses were performed to identify a DNA repair and cell-cycle gene expression signature able to discriminate pHGGs (n = 6) from low-grade gliomas (n = 10). This signature was compared to related signatures already established. We used the pHGG signature to explore already transcriptomic datasets of DIPGs and sus-tentorial pHGGs. Finally, we examined the expression of key proteins of the pHGG signature in 21 pHGG diagnostic samples and nine paired relapses. Functional inhibition of one DNA repair factor was carried out in four patients who derived H3.3 K27M mutant cell lines. RESULTS: We identified a 28-gene expression signature of DNA repair and cell cycle that clustered pHGGs cohorts, in particular sus-tentorial locations, in two groups. Differential protein expression levels of PARP1 and XRCC1 were associated to TP53 mutations and TOP2A amplification and linked significantly to the more radioresistant pHGGs displaying the worst outcome. Using patient-derived cell lines, we showed that the PARP-1/XRCC1 expression balance might be correlated with resistance to PARP1 inhibition. CONCLUSION: We provide evidence that PARP1 overexpression, associated to XRCC1 expression, TP53 mutations, and TOP2A amplification, is a new theranostic and potential therapeutic target.

A Systematic Review of Glioblastoma-Targeted Therapies in Phases II, III, IV Clinical Trials.

Glioblastoma (GBM), the most frequent and aggressive glial tumor, is currently treated as first line by the Stupp protocol, which combines, after surgery, radiotherapy and chemotherapy. For recurrent GBM, in absence of standard treatment or available clinical trials, various protocols including cytotoxic drugs and/or bevacizumab are currently applied. Despite these heavy treatments, the mean overall survival of patients is under 18 months. Many clinical studies are underway. Based on clinicaltrials.org and conducted up to 1 April 2020, this review lists, not only main, but all targeted therapies in phases II-IV of 257 clinical trials on adults with newly diagnosed or recurrent GBMs for the last twenty years. It does not involve targeted immunotherapies and therapies targeting tumor cell metabolism, that are well documented in other reviews. Without surprise, the most frequently reported drugs are those targeting (i) EGFR (40 clinical trials), and more generally tyrosine kinase receptors (85 clinical trials) and (ii) VEGF/VEGFR (75 clinical trials of which 53 involving bevacizumab). But many other targets and drugs are of interest. They are all listed and thoroughly described, on an one-on-one basis, in four sections related to targeting (i) GBM stem cells and stem cell pathways, (ii) the growth autonomy and migration, (iii) the cell cycle and the escape to cell death, (iv) and angiogenesis.

Gefitinib induces EGFR and α5ß1 integrin co-endocytosis in glioblastoma cells.

Overexpression of EGFR drives glioblastomas (GBM) cell invasion but these tumours remain resistant to EGFR-targeted therapies such as tyrosine kinase inhibitors (TKIs). Endocytosis, an important modulator of EGFR function, is often dysregulated in glioma cells and is associated with therapy resistance. However, the impact of TKIs on EGFR endocytosis has never been examined in GBM cells. In the present study, we showed that gefitinib and other tyrosine kinase inhibitors induced EGFR accumulation in early-endosomes as a result of an increased endocytosis. Moreover, TKIs trigger early-endosome re-localization of another membrane receptor, the fibronectin receptor alpha5beta1 integrin, a promising therapeutic target in GBM that regulates physiological EGFR endocytosis and recycling in cancer cells. Super-resolution dSTORM imaging showed a close-proximity between beta1 integrin and EGFR in intracellular membrane compartments of gefitinib-treated cells, suggesting their potential interaction. Interestingly, integrin depletion delayed gefitinib-mediated EGFR endocytosis. Co-endocytosis of EGFR and alpha5beta1 integrin may alter glioma cell response to gefitinib. Using an in vitro model of glioma cell dissemination from spheroid, we showed that alpha5 integrin-depleted cells were more sensitive to TKIs than alpha5-expressing cells. This work provides evidence for the first time that EGFR TKIs can trigger massive EGFR and alpha5beta1 integrin co-endocytosis, which may modulate glioma cell invasiveness under therapeutic treatment.

Biological Relevance of RGD-Integrin Subtype-Specific Ligands in Cancer.

Integrins are heterodimeric transmembrane proteins able to connect cells with the micro-environment. They represent a family of receptors involved in almost all the hallmarks of cancer. Integrins recognizing the Arg-Gly-Asp (RGD) peptide in their natural extracellular matrix ligands have been particularly investigated as tumoral therapeutic targets. In the last 30 years, intense research has been dedicated to designing specific RGD-like ligands able to discriminate selectively the different RGD-recognizing integrins. Chemists' efforts have led to the proposition of modified peptide or peptidomimetic libraries to be used for tumor targeting and/or tumor imaging. Here we review, from the biological point of view, the rationale underlying the need to clearly delineate each RGD-integrin subtype by selective tools. We describe the complex roles of RGD-integrins (mainly the most studied αvß3 and α5ß1 integrins) in tumors, the steps towards selective ligands and the current usefulness of such ligands. Although the impact of integrins in cancer is well acknowledged, the biological characteristics of each integrin subtype in a specific tumor are far from being completely resolved. Selective ligands might help us to reconsider integrins as therapeutic targets in specific clinical settings.

La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société.

Founded in 1919, the Society of Biology of Strasbourg (SBS) is a learned society whose purpose is the dissemination and promotion of scientific knowledge in biology. Subsidiary of the Society of Biology, the SBS celebrated its Centenary on Wednesday, the 16th of October 2019 on the Strasbourg University campus and at the Strasbourg City Hall. This day allowed retracing the various milestones of the SBS, through its main strengths, its difficulties and its permanent goal to meet scientific and societal challenges. The common thread of this day was the transmission of knowledge related to the past, the present, but also the future. At the start of the 21st century, the SBS must continue to reinvent itself to pursue its objective of transmitting scientific knowledge in biology and beyond. Scientific talks performed by senior scientists and former SBS thesis prizes awardees, a round table, and informal discussions reflected the history and the dynamism of the SBS association. All SBS Centennial participants have set the first milestone for the SBS Bicentennial.

TITLE: La Société de Biologie de Strasbourg : 100 ans au service de la science et de la société. ABSTRACT: Filiale de la Société de Biologie, la Société de Biologie de Strasbourg (SBS) est une société savante qui a pour objet la diffusion et la promotion du savoir scientifique en biologie et en médecine. Fondée en 1919, La SBS a célébré son Centenaire le mercredi 16 octobre 2019. Cette journée a permis de retracer les différents jalons de la SBS, à travers ses lignes de forces, ses difficultés et sa volonté permanente de mettre en exergue les défis scientifiques et sociétaux auxquels participent les recherches strasbourgeoises. Le fil rouge de cette journée a été la transmission d'un savoir en lien avec le passé, le présent, mais également le futur. En ce début du 21e siècle, la SBS se doit de continuer de se réinventer pour poursuivre son objectif de transmission des connaissances scientifiques en biologie et au-delà. L'ensemble des participants du Centenaire de la SBS a ainsi posé la première pierre du Bicentenaire de la SBS.

Multiscale Selectivity and in vivo Biodistribution of NRP-1-Targeted Theranostic AGuIX Nanoparticles for PDT of Glioblastoma.

BACKGROUND: Local recurrences of glioblastoma (GBM) after heavy standard treatments remain frequent and lead to a poor prognostic. Major challenges are the infiltrative part of the tumor tissue which is the ultimate cause of recurrence. The therapeutic arsenal faces the difficulty of eradicating this infiltrating part of the tumor tissue while increasing the targeting of tumor and endogenous stromal cells such as angiogenic endothelial cells. In this aim, neuropilin-1 (NRP-1), a transmembrane receptor mainly overexpressed by endothelial cells of the tumor vascular system and associated with malignancy, proliferation and migration of GBM, highlighted to be a relevant molecular target to promote the anti-vascular effect of photodynamic therapy (VTP). METHODS: The multiscale selectivity was investigated for KDKPPR peptide moiety targeting NRP-1 and a porphyrin molecule as photosensitizer (PS), both grafted onto original AGuIX design nanoparticle. AGuIX nanoparticle, currently in Phase II clinical trials for the treatment of brain metastases with radiotherapy, allows to achieve a real-time magnetic resonance imaging (MRI) and an accumulation in the tumor area by EPR (enhanced permeability and retention) effect. Using surface-plasmon resonance (SPR), we evaluated the affinities of KDKPPR and scramble free peptides, and also peptides-conjugated AGuIX nanoparticles to recombinant rat and human NRP-1 proteins. For in vivo selectivity, we used a cranial window model and parametric maps obtained from T2*-weighted perfusion MRI analysis. RESULTS: The photophysical characteristics of the PS and KDKPPR molecular affinity for recombinant human NRP-1 proteins were maintained after the functionalization of AGuIX nanoparticle with a dissociation constant of 4.7 µM determined by SPR assays. Cranial window model and parametric maps, both revealed a prolonged retention in the vascular system of human xenotransplanted GBM. Thanks to the fluorescence of porphyrin by non-invasive imaging and the concentration of gadolinium evaluated after extraction of organs, we checked the absence of nanoparticle in the brains of tumor-free animals and highlighted elimination by renal excretion and hepatic metabolism. CONCLUSION: Post-VTP follow-ups demonstrated promising tumor responses with a prolonged delay in tumor growth accompanied by a decrease in tumor metabolism.

Angiogenesis Inhibition by a Short 13 Amino Acid Peptide Sequence of Tetrastatin, the α4(IV) NC1 Domain of Collagen IV.

Angiogenesis is defined as the formation of new capillaries by sprouting from the pre-existing microvasculature. It occurs in physiological and pathological processes particularly in tumor growth and metastasis. α1, α2, α3, and α6 NC1 domains from type IV collagen were reported to inhibit tumor angiogenesis. We previously demonstrated that the α4 NC1 domain from type IV collagen, named Tetrastatin, inhibited tumor growth in a mouse melanoma model. The inhibitory activity was located in a 13 amino acid sequence named QS-13. In the present paper, we demonstrate that QS-13 decreases VEGF-induced-angiogenesis in vivo using the Matrigel plug model. Fluorescence molecular tomography allows the measurement of a 65% decrease in Matrigel plug angiogenesis following QS-13 administration. The results are confirmed by CD31 microvessel density analysis on Matrigel plug slices. QS-13 peptide decreases Human Umbilical Vein Endothelial Cells (HUVEC) migration and pseudotube formation in vitro. Relevant QS-13 conformations were obtained from molecular dynamics simulations and docking. A putative interaction of QS-13 with α5ß1 integrin was investigated. The interaction was confirmed by affinity chromatography, solid phase assay, and surface plasmon resonance. QS-13 binding site on α5ß1 integrin is located in close vicinity to the RGD binding site, as demonstrated by competition assays. Collectively, our results suggest that QS-13 exhibits a mighty anti-angiogenic activity that could be used in cancer treatment and other pathologies with excessive angiogenesis such as hemangioma, psoriasis or diabetes.

RNA Aptamers Targeting Integrin α5ß1 as Probes for Cyto- and Histofluorescence in Glioblastoma.

Nucleic acid aptamers are often referred to as chemical antibodies. Because they possess several advantages, like their smaller size, temperature stability, ease of chemical modification, lack of immunogenicity and toxicity, and lower cost of production, aptamers are promising tools for clinical applications. Aptamers against cell surface protein biomarkers are of particular interest for cancer diagnosis and targeted therapy. In this study, we identified and characterized RNA aptamers targeting cells expressing integrin α5ß1. This αß heterodimeric cell surface receptor is implicated in tumor angiogenesis and solid tumor aggressiveness. In glioblastoma, integrin α5ß1 expression is associated with an aggressive phenotype and a decrease in patient survival. We used a complex and original hybrid SELEX (selective evolution of ligands by exponential enrichment) strategy combining protein-SELEX cycles on the recombinant α5ß1 protein, surrounded by cell-SELEX cycles using two different cell lines. We identified aptamer H02, able to differentiate, in cyto- and histofluorescence assays, glioblastoma cell lines, and tissues from patient-derived tumor xenografts according to their α5 expression levels. Aptamer H02 is therefore an interesting tool for glioblastoma tumor characterization.

Role of Integrins in Resistance to Therapies Targeting Growth Factor Receptors in Cancer.

Integrins contribute to cancer progression and aggressiveness by activating intracellular signal transduction pathways and transducing mechanical tension forces. Remarkably, these adhesion receptors share common signaling networks with receptor tyrosine kinases (RTKs) and support their oncogenic activity, thereby promoting cancer cell proliferation, survival and invasion. During the last decade, preclinical studies have revealed that integrins play an important role in resistance to therapies targeting RTKs and their downstream pathways. A remarkable feature of integrins is their wide-ranging interconnection with RTKs, which helps cancer cells to adapt and better survive therapeutic treatments. In this context, we should consider not only the integrins expressed in cancer cells but also those expressed in stromal cells, since these can mechanically increase the rigidity of the tumor microenvironment and confer resistance to treatment. This review presents some of these mechanisms and outlines new treatment options for improving the efficacy of therapies targeting RTK signaling.

Selection of Nucleic Acid Aptamers Targeting Tumor Cell-Surface Protein Biomarkers.

Aptamers are nucleic acids referred to as chemical antibodies as they bind to their specific targets with high affinity and selectivity. They are selected via an iterative process known as 'selective evolution of ligands by exponential enrichment' (SELEX). Aptamers have been developed against numerous cancer targets and among them, many tumor cell-membrane protein biomarkers. The identification of aptamers targeting cell-surface proteins has mainly been performed by two different strategies: protein- and cell-based SELEX, when the targets used for selection were proteins and cells, respectively. This review aims to update the literature on aptamers targeting tumor cell surface protein biomarkers, highlighting potentials, pitfalls of protein- and cell-based selection processes and applications of such selected molecules. Aptamers as promising agents for diagnosis and therapeutic approaches in oncology are documented, as well as aptamers in clinical development.

Expression/activation of α5ß1 integrin is linked to the ß-catenin signaling pathway to drive migration in glioma cells.

The Wnt/beta catenin pathway has been highlighted as an important player of brain tumors aggressiveness and resistance to therapies. Increasing knowledges of the regulation of beta-catenin transactivation point out its hub position in different pathophysiological outcomes in glioma such as survival and migration. Crosstalks between integrins and beta-catenin pathways have been suggested in several tumor tissues. As we demonstrated earlier that α5ß1 integrin may be considered as a therapeutic target in high grade glioma through its contribution to glioma cell migration and resistance to chemotherapy, we addressed here the potential relationship between α5ß1 integrin and beta-catenin activation in glioma cells. We demonstrated that overexpression and activation by fibronectin of α5ß1 integrin allowed the transactivation of beta-catenin gene targets included in an EMT-like program that induced an increase in cell migration. Hampering of beta catenin activation and cell migration could be similarly achieved by a specific integrin antagonist. In addition we showed that α5ß1 integrin/AKT axis is mainly involved in these processes. However, blockade of beta-catenin by XAV939 (tankyrase inhibitor leading to beta-catenin degradation) did not synergize with p53 activation aiming to cell apoptosis as was the case with integrin antagonists. We therefore propose a dual implication of α5ß1 integrin/AKT axis in glioma cell resistance to therapies and migration each supported by different signaling pathways. Our data thus suggest that α5ß1 integrin may be added to the growing list of beta-catenin modulators and provide new evidences to assign this integrin as a valuable target to fight high grade glioma.

Glioma cell dispersion is driven by α5 integrin-mediated cell-matrix and cell-cell interactions.

Glioblastoma multiform (GBM) is the most common and most aggressive primary brain tumor. The fibronectin receptor, α5 integrin is a pertinent novel therapeutic target. Despite numerous data showing that α5 integrin support tumor cell migration and invasion, it has been reported that α5 integrin can also limit cell dispersion by increasing cell-cell interaction. In this study, we showed that α5 integrin was involved in cell-cell interaction and gliomasphere formation. α5-mediated cell-cell cohesion limited cell dispersion from spheroids in fibronectin-poor microenvironment. However, in fibronectin-rich microenvironment, α5 integrin promoted cell dispersion. Ligand-occupied α5 integrin and fibronectin were distributed in fibril-like pattern at cell-cell junction of evading cells, forming cell-cell fibrillar adhesions. Activated focal adhesion kinase was not present in these adhesions but was progressively relocalized with α5 integrin as cell migrates away from the spheroids. α5 integrin function in GBM appears to be more complex than previously suspected. As GBM overexpressed fibronectin, it is most likely that in vivo, α5-mediated dissemination from the tumor mass overrides α5-mediated tumor cell cohesion. In this respect, α5-integrin antagonists may be useful to limit GBM invasion in brain parenchyma.

Antibody Binding Selectivity: Alternative Sets of Antigen Residues Entail High-Affinity Recognition.

Understanding the relationship between protein sequence and molecular recognition selectivity remains a major challenge. The antibody fragment scFv1F4 recognizes with sub nM affinity a decapeptide (sequence 6TAMFQDPQER15) derived from the N-terminal end of human papilloma virus E6 oncoprotein. Using this decapeptide as antigen, we had previously shown that only the wild type amino-acid or conservative replacements were allowed at positions 9 to 12 and 15 of the peptide, indicating a strong binding selectivity. Nevertheless phenylalanine (F) was equally well tolerated as the wild type glutamine (Q) at position 13, while all other amino acids led to weaker scFv binding. The interfaces of complexes involving either Q or F are expected to diverge, due to the different physico-chemistry of these residues. This would imply that high-affinity binding can be achieved through distinct interfacial geometries. In order to investigate this point, we disrupted the scFv-peptide interface by modifying one or several peptide positions. We then analyzed the effect on binding of amino acid changes at the remaining positions, an altered susceptibility being indicative of an altered role in complex formation. The 23 starting variants analyzed contained replacements whose effects on scFv1F4 binding ranged from minor to drastic. A permutation analysis (effect of replacing each peptide position by all other amino acids except cysteine) was carried out on the 23 variants using the PEPperCHIP® Platform technology. A comparison of their permutation patterns with that of the wild type peptide indicated that starting replacements at position 11, 12 or 13 modified the tolerance to amino-acid changes at the other two positions. The interdependence between the three positions was confirmed by SPR (Biacore® technology). Our data demonstrate that binding selectivity does not preclude the existence of alternative high-affinity recognition modes.

Caveolin-1-negative head and neck squamous cell carcinoma primary tumors display increased epithelial to mesenchymal transition and prometastatic properties.

Distant metastases arise in 20-30% of patients with squamous cell carcinoma of the head and neck (HNSCC) in the 2 years following treatment. Therapeutic options are limited and the outcome of the patients is poor. The identification of predictive biomarkers of patient at risk for distant metastasis and therapies are urgently needed. We previously identified a clinical subgroup, called "R1" characterized by high propensity for rapid distant metastasis. Here, we showed that "R1" patients do not or at very low level express caveolin-1 (Cav1). Low or no expression of Cav1 is of bad prognosis. Disappearance of Cav1 enables cells to undergo epithelial-mesenchymal transition (EMT). EMT is associated with enhanced migration and invasion. Our study uncovered a new target, α5ß1 integrin. Targeting α5ß1 integrins might not only prevent metastasis of HNSCC but also delay the development of the primary tumor by reducing tumor cell viability. Cav1 detection might be taken into consideration in the future in the clinic not only to identify patients at high risk of metastasis but also to select patient who might benefit from an anti-integrin therapy.