Stroma NCL inhibition increases pericyte coverage of tumor blood vessels.

mPDAC orthopic model characterization.

N6L and angiogenic molecules regulation.

N6L inhibits PDAC proliferation and migration

NCL blocking antibody and Ncl deletion affects PDAC cell proliferation.

Proteome array analysis of angiogenic molecules secreted by ECs.

L’index Ki67 dépend de l’hétérogénéité tumorale, de la qualité de l’immunomarquage et de l’interprétation du marquage par le pathologiste. L’utilisation des logiciels d’analyse d’image quantitative a pour but de rendre cette interprétation moins sujette aux variations intra- et inter-observateurs. L’objectif de ce travail était d’analyser une série de lames d’un test qualité AFAQAP avec la plateforme digitale d’IMSTAR et de comparer les valeurs obtenues avec celles des pathologistes experts relecteurs. Les 55 lames blanches comparables d’un carcinome mammaire ont été immunomarquées par 55 laboratoires participant au test de comparaison inter-laboratoire AFAQAP 2021, avec leur propre protocole de routine d’IHC Ki67. L’index a été obtenu sur chaque lame selon deux méthodes de lecture centralisée : visuelle par les deux pathologistes relecteurs et automatisée avec le logiciel, selon les recommandations du Cancéropôle de l’Inter-Région Grand-Est 2014 ( www.afaqap.fr ). La plateforme de scanning digitale et d’analyse IMSTAR PathfinderTM a utilisé le microscope Olympus BX63 motorisé (moyen-débit, 80-160 lames), le scanner Olympus VS200 (haut-débit, 210 lames) et le logiciel d’analyse PathoScan T-M Ki67. L‘index Ki67, évalué par les experts relecteurs sur les 55 lames émanant des participants auxquelles s’ajoutent 3 lames de référence AFAQAP, pouvait être réparti en 3 classes : 12–15 % (36 lames), 8–12 % (15 lames) et 5–8 % (7 lames). Les index calculés par le logiciel dans les zones tumorales sélectionnées par les experts relecteurs concordent à 92 % avec les index évalués visuellement par ces experts. Ils se situent dans l’intervalle défini pour 53 lames et ont été surévalués pour 5 lames. L’enjeu de l’analyse de Ki67 est de maitriser la partie technique du marquage IHC et l’interprétation de cet immunomarquage par le pathologiste. La maitrise technique relève de la formation et des contrôles qualité techniques. La maitrise de l’interprétation relève de la formation et du respect des recommandations. Elle se heurte néanmoins aux difficultés de l’évaluation à l’œil sujette à variabilité. C’est dans ce domaine que l’analyse d’image est d’un apport important. Le logiciel a été en phase avec l’évaluation des experts aguerris dans 92 % des cas. C’est d’autant plus remarquable que les techniques d’IHC émanaient de 55 laboratoires. Le logiciel a ainsi su s’adapter aux variations des préparations quels que soient les réactifs utilisés. La surévaluation constatée dans 8 % des cas avec le logiciel a pu être expliquée par la présence d’artéfacts de coloration ou d’un bruit de fond d’intensité comparable à une faible expression du Ki67. L’outil apporte une aide significative à la démarche qualité de l’AFAQAP visant à proposer une standardisation de l’évaluation Ki67, notamment pour les tumeurs présentant des index proches des seuils d’utilité clinique.

Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is highly fibrotic and hypoxic, with poor immune cell infiltration. Recently, we showed that nucleolin (NCL) inhibition normalizes tumour vessels and impairs PDAC growth. Methods: Immunocompetent mouse models of PDAC were treated by the pseudopeptide N6L, which selectively inhibits NCL. Tumour-infiltrating immune cells and changes in the tumour microenvironment were analysed. Results: N6L reduced the proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and increased tumour-infiltrated T lymphocytes (TILs) with an activated phenotype. Low-dose anti-VEGFR2 treatment normalized PDAC vessels but did not modulate the immune suppressive microenvironment. RNAseq analysis of N6L-treated PDAC tumours revealed a reduction of cancer-associated fibroblast (CAF) expansion in vivo and in vitro. Notably, N6L treatment decreased IL-6 levels both in tumour tissues and in serum. Treating mPDAC by an antibody blocking IL-6 reduced the proportion of Tregs and MDSCs and increased the amount of TILs, thus mimicking the effects of N6L. Conclusions: These results demonstrate that NCL inhibition blocks the amplification of lymphoid and myeloid immunosuppressive cells and promotes T cell activation in PDAC through a new mechanism of action dependent on the direct inhibition of the tumoral stroma.

Hypothesis: We describe the possibility of using the same block copolymer carriers prepared by PISA for in situ drug encapsulation or drug conjugation.Experiments: Block copolymers containing poly((ethylene glycol) methacrylate)-co-poly (pentafluorophenyl methacrylate)-b-poly(hydroxypropyl methacrylate) (P((PEGMA-co-PFBMA)-b- PHPMA)) were synthesized at 10 wt% using PISA. The first approach involved in situ Doxorubicin (DOX) loading during PISA, while the second exhibited surface functionalization of PISA-made vesicles with dual drug therapies, N-acetyl cysteine (NAC) and DOX using para-fluoro-thiol reaction (PFTR) and carbodiimide chemistry, respectively. Cytotoxicity, cell uptake, and cell apoptosis were assessed on MDA-MB-231 cell lines.Findings: P((PEGMA-co-PFBMA)-b-PHPMA) nanocarriers were prepared, showing size and shape transformations from spheres, cylinders to raspberry-forming vesicles. DOX was readily loaded into NPs during PISA with relatively high encapsulation efficiency of 70 %, whereas the plain PISA-made vesicles could be functionalized with NAC and DOX at high yields. DOX-free NPs showed biocompatibility, whilst DOXconjugated NPs imparted a concentration-dependent cytotoxicity, as well as an enhanced cell uptake compared to free DOX. The results demonstrated that the same PISA-derived self-assemblies enabled either in situ drug encapsulation, or post-polymerization surface engineering with useful functionalities upon tuning the macro-CTA block, thus holding promises for future drug delivery and biomedical applications.(c) 2022 Elsevier Inc. All rights reserved.

In the original publication [...].

The stroma is a relevant player in driving and supporting the progression of pancreatic ductal adenocarcinoma (PDAC), and a large body of evidence highlights its role in hindering the efficacy of current therapies. In fact, the dense extracellular matrix (ECM) characterizing this tumor acts as a natural physical barrier, impairing drug penetration. Consequently, all of the approaches combining stroma-targeting and anticancer therapy constitute an appealing option for improving drug penetration. Several strategies have been adopted in order to target the PDAC stroma, such as the depletion of ECM components and the targeting of cancer-associated fibroblasts (CAFs), which are responsible for the increased matrix deposition in cancer. Additionally, the leaky and collapsing blood vessels characterizing the tumor might be normalized, thus restoring blood perfusion and allowing drug penetration. Even though many stroma-targeting strategies have reported disappointing results in clinical trials, the ECM offers a wide range of potential therapeutic targets that are now being investigated. The dense ECM might be bypassed by implementing nanoparticle-based systems or by using mesenchymal stem cells as drug carriers. The present review aims to provide an overview of the principal mechanisms involved in the ECM remodeling and of new promising therapeutic strategies for PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact with β-catenin. We found that the Wnt/β-catenin pathway is activated in PDAC and is necessary for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited Wnt pathway activation by preventing β-catenin stabilization in PDAC cells. N6L also inhibited the growth and the activation of the Wnt/β-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased β-catenin stabilization. In conclusion, in this study, we identified β-catenin as a new nucleolin interactor and suggest that the Wnt/β-catenin pathway could be a new target of the nucleolin antagonist N6L in PDAC.

Pancreatic cancer is a growing cause of concern, leading to an increasing number of deaths worldwide. Most of the antitumor drugs for pancreatic cancer are given by systemic administration. However, even if this is at therapeutic doses, its tends to lead to severe side-effects because of nonspecific uptake by healthy tissues. In this context, we aim to develop an innovative drug delivery approach for the treatment of pancreatic cancer based on tailor-made nanomaterials with presenting controlled size, chemistry and stiffness. In this study, we have designed tailor-made N6L-nanomaterials, via the combination of both a templating method and a self-assembly process onto colloidal gold nanoparticles (diameters 20 nm and 60 nm) using a core template. The removal of the core leads to hollow particles, also called capsules. We judiciously functionalised the outer layer of the nanomaterial with N6L, which corresponds to a synthetic agent that is non-toxic to normal mammalian cells, targets surface nucleolin and induces tumour cell death by apoptosis. Our data demonstrated, via quartz crystal microbalance measurement, the possibility of directly using N6L in a self-assembly process through electrostatic interactions. In addition, as shown by measuring zeta potential values, we showed that electrostatic interactions between the protonated amine groups of poly (allylamine) macromolecules (+40 mV), the sulphate groups of dextran derivatives (-20 mV) and the protonated amine groups of N6L (+20 mV) are the driving attraction force during multilayer onto colloidal gold. Transmission electron microscopy was used to confirm that the gold nanoparticles were coated and the nanocapsules were formed. Finally, we successfully showed that these N6L-based nanostructures target cancer cells and inhibited, in a dose dependent manner, cell survival in pancreatic cancer model. This result clearly highlights the potential antitumor activity of the developed nanostructures.

Retinal vascular diseases (RVD) have been identified as a major cause of blindness worldwide. These pathologies, including the wet form of age-related macular degeneration, retinopathy of prematurity, and diabetic retinopathy are currently treated by intravitreal delivery of anti-vascular endothelial growth factor (VEGF) agents. However, repeated intravitreal injections can lead to ocular complications and resistance to these treatments. Thus, there is a need to find new targeted therapies. Nucleolin regulates the endothelial cell (EC) activation and angiogenesis. In previous studies, we designed a pseudopeptide, N6L, that binds the nucleolin and blocks the tumor angiogenesis. In this study, the effect of N6L was investigated in two experimental models of retinopathies including oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV). We found that in mouse OIR, intraperitoneal injection of N6L is delivered to activated ECs and induced a 50% reduction of pathological neovascularization. The anti-angiogenic effect of N6L has been tested in CNV model in which the systemic injection of N6L induced a 33% reduction of angiogenesis. This effect is comparable to those obtained with VEGF-trap, a standard of care drug for RVD. Interestingly, with preventive and curative treatments, neoangiogenesis is inhibited by 59%. Our results have potential interest in the development of new therapies targeting other molecules than VEGF for RVD.

Objective Weibel-Palade bodies (WPBs) are endothelial cell (EC)-specific organelles formed by vWF (von Willebrand factor) polymerization and that contain the proangiogenic factor Ang-2 (angiopoietin-2). WPB exocytosis has been shown to be implicated for vascular repair and inflammatory responses. Here, we investigate the role of WPBs during angiogenesis and vessel stabilization. Approach and Results WPB density in ECs decreased at the angiogenic front of retinal vascular network during development and neovascularization compared with stable vessels. In vitro, VEGF (vascular endothelial growth factor) induced a VEGFR-2 (vascular endothelial growth factor receptor-2)-dependent exocytosis of WPBs that contain Ang-2 and consequently the secretion of vWF and Ang-2. Blocking VEGF-dependant WPB exocytosis and Ang-2 secretion promoted pericyte migration toward ECs. Pericyte migration was inhibited by adding recombinant Ang-2 or by silencing Ang-1 (angiopoietin-1) or Tie2 (angiopoietin-1 receptor) in pericytes. Consistently, in vivo anti-VEGF treatment induced accumulation of WPBs in retinal vessels because of the inhibition of WPB exocytosis and promoted the increase of pericyte coverage of retinal vessels during angiogenesis. In tumor angiogenesis, depletion of WPBs in vWF knockout tumor-bearing mice promoted an increase of tumor angiogenesis and a decrease of pericyte coverage of tumor vessels. By another approach, normalized tumor vessels had higher WPB density. Conclusions We demonstrate that WPB exocytosis and Ang-2 secretion are regulated during angiogenesis to limit pericyte coverage of remodeling vessels by disrupting Ang-1/Tie2 autocrine signaling in pericytes.

A major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components.

In this study, a novel anticancer reagent based on polyplexes nanoparticles was developed. These nanoparticles are obtained by mixing negatively charged polyelectrolytes with the antitumour cationically-charged pseudopeptide N6L. Using two in vivo experimental tumor pancreatic models based upon PANC-1 and mPDAC cells, we found that the antitumour activity of N6L is significantly raised via its incorporation in polyplexed nanoparticles. Study of the mechanism of action using affinity isolation and si-RNA experiments indicated that N6L-polyplexes are internalized through their interaction with nucleolin. In addition, using a very aggressive model of pancreatic cancer in which gemcitabine, a standard of care for this type of cancer, has a weak effect on tumour growth, we observed that N6L-polyplexes administration has a stronger efficacy than gemcitabine. Biodistribution studies carried out in tumour-bearing mice indicated that N6L-polyplexes localises in tumour tissue, in agreement with its antitumour effect. These results support the idea that N6L nanoparticles could develop into a promising strategy for the treatment of cancer, especially hard-to-treat pancreatic cancers.

Abstract Pancreatic cancer is a highly aggressive tumor, mostly resistant to the standard treatments. Nucleolin is overexpressed in cancers and its inhibition impairs tumor growth. Herein, we showed that nucleolin was overexpressed in human specimens of pancreatic ductal adenocarcinoma (PDAC) and that the overall survival significantly increased in patients with low levels of nucleolin. The nucleolin antagonist N6L strongly impaired the growth of primary tumors and liver metastasis in an orthotopic mouse model of PDAC (mPDAC). Similar antitumor effect of N6L has been observed in a highly angiogenic mouse model of pancreatic neuroendocrine tumor RIP-Tag2. N6L significantly inhibited both human and mouse pancreatic cell proliferation and invasion. Notably, the analysis of tumor vasculature revealed a strong increase of pericyte coverage and vessel perfusion both in mPDAC and RIP-Tag2 tumors, in parallel to an inhibition of tumor hypoxia. Nucleolin inhibition directly affected endothelial cell (EC) activation and changed a proangiogenic signature. Among the vascular activators, nucleolin inhibition significantly decreased angiopoietin-2 (Ang-2) secretion and expression in ECs, in the tumor and in the plasma of mPDAC mice. As a consequence of the observed N6L-induced tumor vessel normalization, pre-treatment with N6L efficiently improved chemotherapeutic drug delivery and increased the antitumor properties of gemcitabine in PDAC mice. In conclusion, nucleolin inhibition is a new anti-pancreatic cancer therapeutic strategy that dually blocks tumor progression and normalizes tumor vasculature, improving the delivery and efficacy of chemotherapeutic drugs. Moreover, we unveiled Ang-2 as a potential target and suitable response biomarker for N6L treatment in pancreatic cancer. Cancer Res; 76(24); 7181–93. ©2016 AACR.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor, mostly resistant to the standard treatments. NCL is overexpressed in cancers and its inhibition impairs tumor growth. Herein we described, that NCL was overexpressed in human specimens of PDAC, and low NCL staining patients had increased overall survival. Previously, we described a family of multivalent pseudopeptides binding to NCL and inhibiting tumour growth. Here, NCL antagonist N6L, strongly impaired tumor growth, liver metastasis formation and angiogenesis in an orthothopic mouse model of PDAC. N6L inhibited both human and mouse tumor cell proliferation and invasion. Proteome analysis of endothelial cell secreted proteins showed that NCL inhibition decreased Ang-2 levels and switched a pro-angiogenic signature. Importantly, Ang-2 levels were decreased in plasma of N6L-treated PDAC mice. The analysis of tumor vasculature revealed a strong increase of pericyte coverage and vessel perfusion in parallel to an inhibition of tumor hypoxia. As consequence of N6L-induced tumor vessel normalization, pre-treatment with N6L efficiently improved chemotherapeutic drug delivery and increased the anti-tumor properties of gemcitabine in PDAC mice. In conclusion, NCL inhibition is a new anti-tumor therapeutic strategy that dually blocks tumor progression and normalizes tumor vessels improving the delivery and efficacy of chemotherapeutic drugs in PDAC cancers. Moreover, we identified Ang-2 as a potential target and suitable response biomarker for N6L treatment in PDAC. Citation Format: Maud-Emmanuelle Gilles, Federica Maione, Mélissande Cossutta, Gilles Carpentier, Laure Caruana, Silvia Di Maria, Damien Destouches, Ksenya Shchors, Christopher Prochasson, Anne Couvelard, José Courty, Enrico Giraudo, Ilaria Cascone. NCL targeting impairs the progression of pancreatic ductal adenocarcinoma and promotes tumor vessel normalization through Ang-2 inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3366.