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.

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.

Escherichia coli ribosomal protein S1 unfolds structured mRNAs onto the ribosome for active translation initiation.

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5' untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA-protein or mRNA-ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5' ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features.

Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5ß1 or αvß3/ß5 integrin antagonists in U87MG glioma cells.

BACKGROUND: Integrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5ß1 and αvß3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5ß1 and αvß3/ß5 purified integrins. METHODS: Small antagonists were either selective for α5ß1 integrin, for αvß3/ß5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5ß1 integrin in the biological assays. RESULTS: U87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5ß1 or αvß3/ß5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5ß1 integrin and was inhibited selectively by α5ß1 integrin antagonists but increased by αvß3/ß5 integrin antagonists. CONCLUSIONS: We provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions. GENERAL SIGNIFICANCE: Our data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5ß1 integrin-dependent cell migration.

Spot peptide arrays and SPR measurements: throughput and quantification in antibody selectivity studies.

Antibody selectivity represents a major issue in the development of efficient immuno-therapeutics and detection assays. Its description requires a comparison of the affinities of the antibody for a significant number of antigen variants. In the case of peptide antigens, this task can now be addressed to a significant level of details owing to improvements in spot peptide array technologies. They allow the high-throughput mutational analysis of peptides with, depending on assay design, an evaluation of binding stabilities. Here, we examine the cross-reactive capacity of an antibody fragment using the PEPperCHIP(®) technology platform (PEPperPRINT GmbH, Heidelberg, Germany; >8800 peptides per microarray) combined with the surface plasmon resonance characterization (Biacore(®) technology; GE-Healthcare Biacore, Uppsala, Sweden) of a subset of interactions. ScFv1F4 recognizes the N-terminal end of oncoprotein E6 of human papilloma virus 16. The spot permutation analysis (i.e. each position substituted by all amino acids except cysteine) of the wild type decapeptide (sequence (6)TAMFQDPQER(15)) and of 15 variants thereof defined the optimal epitope and provided a ranking for variant recognition. The SPR affinity measurements mostly validated the ranking of complex stabilities deduced from array data and defined the sensitivity of spot fluorescence intensities, bringing further insight into the conditions for cross-reactivity. Our data demonstrate the importance of throughput and quantification in the assessment of antibody selectivity.

An Arabidopsis dual-localized pentatricopeptide repeat protein interacts with nuclear proteins involved in gene expression regulation.

Following the endosymbiotic acquisition of mitochondria by eukaryotic cells, most of the genes in this organelle were transferred to the nucleus. To maintain mitochondrial biogenesis and function, nuclear and mitochondrial genomes require regulated and coordinated expression. In plant organelles, nuclear-encoded proteins targeted to the organelles control posttranscriptional and posttranslational mechanisms. Pentatricopeptide repeat (PPR) proteins are good candidates to play such regulatory roles. Here, we identify PNM1 (for PPR protein localized to the nucleus and mitochondria 1), a novel PPR protein that is dual localized to mitochondria and nuclei in Arabidopsis thaliana, as observed by green fluorescent protein fusions and immunodetection on subcellular fractions and on histological sections. Genetic complementation showed that loss of PNM1 function in mitochondria, but not in nuclei, is lethal for the embryo. In mitochondria, it is associated with polysomes and may play a role in translation. A genetic screen in yeast identified protein partners of PNM1. These partners, the nucleosome assembly protein NAP1, and the transcription factor TCP8 interact with PNM1 in the nucleus in planta. Furthermore, TCP8 can bind the promoter of PNM1. This suggests that PNM1 might be involved in the regulation of its own gene expression in the nucleus and could thus play a role in gene expression adjustments between mitochondria and the nucleus.

Chemical library screening using a SPR-based inhibition in solution assay: simulations and experimental validation.

We have developed a surface plasmon resonance (SPR)-based inhibition in solution assay (ISA) to search for inhibitors of the medium affinity (KD = 0.8 µM) interaction between an E6-derived peptide (E6peptide) immobilized on the sensor and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are widespread protein-protein interaction modules that recognize the C-terminus of various partners. Simulations indicated that relatively low compound concentrations (10 µM) and limited peptide densities (Rmax < 200 resonance units) should allow the detection of inhibitors with a target affinity close to 100 µM, which was then demonstrated experimentally. ISA screening, carried out on the Prestwick Chemical Library® (1120 compounds), identified 36 compounds that inhibited the interaction by more than 5%. Concentration-dependent ISA, carried out on a subset of 19 potential inhibitors, indicated that 13 of these indeed affected the interaction between MAGI-1 PDZ1 and the E6peptide. No effect was observed for 84 compounds randomly chosen among noninhibitors. One of the four best inhibitors was a peptide binder, and three were PDZ binders with KD in the 10-50 µM range. We propose that a medium (µM) affinity between the target and surface-bound partner is optimal for SPR-based ISA screening.

Deciphering complex protein interaction kinetics using Interaction Map.

Cellular receptor systems are expected to present complex ligand interaction patterns that cannot be evaluated assuming a simple one ligand:one receptor interaction model. We have previously evaluated heterogeneous interactions using an alternative method to regression analysis, called Interaction Map (IM). IM decomposes a time-resolved binding curve into its separate components. By replacing the reductionistic, scalar kinetic association rate constant k(a) and dissociation rate constant k(d) with a two-dimensional distribution of k(a) and k(d), it is possible to display heterogeneous data as a map where each peak corresponds to one of the components that contribute to the cumulative binding curve. Here we challenge the Interaction Map approach by artificially generating heterogeneous data from two known interactions, on either LigandTracer or Surface Plasmon Resonance devices. We prove the ability of IM to accurately decompose these man-made heterogeneous binding curves composed of two different interactions. We conclude that the Interaction Map approach is well suited for the analysis of complex binding data and forecast that it has a potential to resolve previously uninterpretable data, in particular those generated in cell-based assays.

Validation of surface plasmon resonance screening of a diverse chemical library for the discovery of protein tyrosine phosphatase 1b binders.

We investigated the suitability of surface plasmon resonance (SPR) for providing quantitative binding information from direct screening of a chemical library on protein tyrosine phosphatase 1b (PTP1B). The experimental design was established from simulations to detect binding with K(D) < 10⁻4 M. The 1120 compounds (cpds) were injected sequentially at concentrations [C(cpd)] of 0.5 or 10 µM over various target surfaces. An optimized evaluation procedure was applied. More than 90% of cpds showed no detectable signal in four screens. The 30 highest responders at C(cpd)=10 µM, of which 25 were selected in at least one of three screens at C(cpd)=0.5 µM, contained 22 promiscuous binders and 8 potential PTP1B-specific binders with K(D) ~10⁻5 M. Inhibition of PTP1B activity was assayed and confirmed for 6 of these, including sanguinarine, a known PTP1B inhibitor. C(cpd) dependence studies fully confirmed screening conclusions. The quantitative consistency of SPR data led us to propose a structure-activity relationship (SAR) model for developing selective PTP1B inhibitors based on the ranking of 10 arylbutylpiperidine analogs.

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.

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.

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.

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.

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.

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 peptide-based fluorescent ratiometric sensor for quantitative detection of proteins.

A ratiometric fluorescent sensor was obtained by solid-phase synthesis of a peptide singly labeled at its N-terminus with a 3-hydroxychromone (3HC) derivative, an environmentally sensitive fluorophore with a two-band emission. The construct contains the binding site recognized by an antibody fragment, scFv1F4(Q34S), with nanomolar (nM) affinity. The dye only marginally affected the kinetic and equilibrium binding parameters of the scFv-peptide interaction, as measured by surface plasmon resonance. On interaction with the antibody fragment, the sensor showed up to 47% change in the ratio of its two emission bands, indicating an enhanced screening of the 3 HC fluorophore from bulk water. Competition with two unlabeled peptides of different lengths led to a dynamic displacement of the construct governed by the relative binding constants. Calibration showed that the response is proportional to the ratio of scFv1F4(Q34S) to labeled peptide. The detection limit of scFv1F4(Q34S) was 15 nM. In a more complex medium (100 microg/ml bovine serum albumin), the scFv could be detected in the 50- to 100-nM range. This work demonstrates that, with the perspective of further improvements of the dye spectroscopic properties, fluorescent ratiometric sensing based on small synthetic peptides represents a promising tool for quantitative target detection.

Environmentally sensitive fluorescent sensors based on synthetic peptides.

Biosensors allow the direct detection of molecular analytes, by associating a biological receptor with a transducer able to convert the analyte-receptor recognition event into a measurable signal. We review recent work aimed at developing synthetic fluorescent molecular sensors for a variety of analytes, based on peptidic receptors labeled with environmentally sensitive fluorophores. Fluorescent indicators based on synthetic peptides are highly interesting alternatives to protein-based sensors, since they can be synthesized chemically, are stable, and can be easily modified in a site-specific manner for fluorophore coupling and for immobilization on solid supports.

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.

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.