In vitro expansion of human glioblastoma cells at non-physiological oxygen tension irreversibly alters subsequent in vivo aggresiveness and AC 133 expression

Among markers of glioblastoma initiating cells, AC133 has been shown to be associated with glioblastoma resistance and malignancy. Recently, it was demonstrated that increasing oxygen tension (pO(2)) down-regulated AC133 expression in glioblastoma cells in vitro. In order to better understand extrinsic factor regulation of AC133, this work aimed to investigate the relationship between cell culture pO(2), AC133 expression, and tumor development and phenotype. Using treatments with CoCl(2) and HIF-1α shRNA knockdowns on non-sorted human primary glioblastoma cells cultured at low (3%) versus high (21%) oxygen tension, we established a responsibility for low pO(2) in the maintenance of high levels of AC133 expression, with a major but non-exclusive role for HIF-1α. We also demonstrated that human glioblastoma cells previously cultured under high oxygen tension can lose part of their aggressiveness when orthotopically engrafted in SCID mice or lead to tumors with distinct phenotypes and no re-expression of AC133. These observations showed that the specific pO(2) microenvironment irreversibly impacts glioblastoma cell phenotypes, highlighting the pertinence of culture conditions when extrapolating data from xenogenic models to human cells in their source environment. They also raised AC133 as a marker of non-exposure to oxygenated areas rather than a marker of aggressiveness or low pO(2) niches

The Importance of the Stem Cell Marker Prominin-1/CD133 in the Uptake of Transferrin and in Iron Metabolism in Human Colon Cancer Caco-2 Cells

As the pentaspan stem cell marker CD133 was shown to bind cholesterol and to localize in plasma membrane protrusions, we investigated a possible function for CD133 in endocytosis. Using the CD133 siRNA knockdown strategy and non-differentiated human colon cancer Caco-2 cells that constitutively over-expressed CD133, we provide for the first time direct evidence for a role of CD133 in the intracellular accumulation of fluorescently labeled extracellular compounds. Assessed using AC133 monoclonal antibody, CD133 knockdown was shown to improve Alexa488-transferrin (Tf) uptake in Caco-2 cells but had no impact on FITC-dextran or FITC-cholera-toxin. Absence of effect of the CD133 knockdown on Tf recycling established a role for CD133 in inhibiting Tf endocytosis rather than in stimulating Tf exocytosis. Use of previously identified inhibitors of known endocytic pathways and the positive impact of CD133 knockdown on cellular uptake of clathrin-endocytosed synthetic lipid nanocapsules supported that CD133 impact on endocytosis was primarily ascribed to the clathrin pathway. Also, cholesterol extraction with methyl-β-cyclodextrine up regulated Tf uptake at greater intensity in the CD133high situation than in the CD133low situation, thus suggesting a role for cholesterol in the inhibitory effect of CD133 on endocytosis. Interestingly, cell treatment with the AC133 antibody down regulated Tf uptake, thus demonstrating that direct extracellular binding to CD133 could affect endocytosis. Moreover, flow cytometry and confocal microscopy established that down regulation of CD133 improved the accessibility to the TfR from the extracellular space, providing a mechanism by which CD133 inhibited Tf uptake. As Tf is involved in supplying iron to the cell, effects of iron supplementation and deprivation on CD133/AC133 expression were investigated. Both demonstrated a dose-dependent down regulation here discussed to the light of transcriptional and post-transciptional effects. Taken together, these data extend our knowledge of the function of CD133 and underline the interest of further exploring the CD133-Tf-iron network

" Locked Nucleic Acid " nanovectorisés pour la répression de l'activité de microARN impliqués dans la radiorésistance des cellules de glioblastome

Glioblastoma is the primary malignant tumour of the most common and most aggressive brain in humans. Its conventional treatment is only palliative and the appearance of recurrences is systematic. In order to develop innovative therapies based on targeting new tumour entities, two targets were investigated: the marker of radioresistance AC133/1 and oncomicroRNAs. Using glioblastoma cells from patients, we demonstrated that their in vitro expansion to a nonphysiological pO2 (21%) impairs in vivo their tumour aggressiveness and original expression of AC133/1, unlike physiological pO2 (3%) highlighting qu'AC133/1 is an early marker of non- exposure to high pO2. We also identify a role for AC133/1 in endocytosis of transferrin receptor and its partnership with iron metabolism. Finally we have developed and characterized immunonanoparticules able to convey chemotherapy or radiopharmaceuticals to this functional epitope. In parallel, in a second search axis, we establish the human miRnome in response to the action of radiation. Biomimetic lipid nanocapsules having on their surface and papillomavirus peptides capable of complexing with antagonists microRNAs have been developed and evaluated and have demonstrated their interest with synergy radiotherapy. Collectively and upstream of in vivo experiments in progress, these results underscore the relevance of applying novel targeted nanomedicine for circumvention of radioresistance in glioblastoma.Le glioblastome est la tumeur maligne primaire du cerveau la plus courante et la plus agressive chez l'homme. Son traitement conventionnel est palliatif et l'apparition de récidives est systématique. Dans le but de développer des thérapies innovantes basées sur le ciblage de nouvelles entités tumorales à l'aide de nanovecteur de médicaments, deux cibles ont été investiguées : le marqueur de radiorésistance AC133/1 et les onco-microARN. En utilisant des cellules de glioblastomes issues de patients, nous avons démontré que leur expansion in vitro à une pO2 non-physiologique (21%) altère leur agressivité tumorale in vivo et l'expression originelle d'AC133/1, au contraire d'une pO2 physiologique (3%) soulignant qu'AC133/1 est un marqueur précoce de non exposition à des pO2 élevées. Nous identifions par ailleurs un rôle pour AC133/1 dans l'endocytose du récepteur de la transferrine et son partenariat avec le métabolisme du fer. Enfin nous avons développé et caractérisé des immunonanoparticules capables de véhiculer des chimiothérapies ou des radiopharmaceutiques vers cet épitope fonctionnel. Dans un second axe de recherche, en parallèle d'établir le miRnome humain en réponse à l'action d'une radiothérapie, des nanocapsules lipidiques biomimétiques présentant à leur surface des peptides de papillomavirus et capables de se complexer avec des acides nucléiques antagonistes de microARN ont été développées et évaluées, démontrant leur intérêt en synergie d'une radiothérapie. Collectivement et en amont d'expérimentations in vivo en cours, ces résultats soulignent la pertinence d'appliquer de nouvelles nanomédecines ciblées pour le contournement de la radiorésistance dans le glioblastome

Cell penetrating peptide (CPP) gold( iii ) – complex – bioconjugates: from chemical design to interaction with cancer cells for nanomedicine applications

International audienceInnovative synthesis of a nanotheragnostic scaffold capable of targeting and destroying pancreatic cancer cells (PDAC) using the Biotinylated NFL-TBS.40-63 peptide (BIOT-NFL)

Hypoxia induces fructose accumulation in human U87 brain tumor cells: A 13C NMR study

International audienc

NFL-TBS.40-63 Peptide Gold Complex Nanovector: A Novel Therapeutic Approach to Increase Anticancer Activity by Breakdown of Microtubules in Pancreatic Adenocarcinoma (PDAC)

International audienc

Silencing of miR-21 and miR-210 by LNA-lipid nanocapsules complexes sensitize human glioblastoma cells to radiation-induced cell death

National audienc

MicroRNA-based therapeutics and nanomedicine for brain tumour treatment

Shubaash ANTHIYA and Audrey GRIVEAU contributed equally to this work.miRNAs are key cell regulatory elements code by the genome. Although their signature in cancer is incompletely known and not yet an element of diagnosis, the role of oncomiRs become increasingly documented. Alongside cancer intrinsic signals in which they exert essential functions, miRNAs are also responding to environmental cues. Developing novel approaches to target miRNA-pathways represents a pivotal issue in research and may result in a major breakthrough for human health. Due to their submicron size and versatile physicochemical properties the emergence of innovative nanomedicines may be here largely advantageous for the development of targeted clinical strategies. With a special focus on glioblastoma, the present review addresses cancer functions of miRNAs and raises the question of an adapted nanomedicine

CELL PENETRATING PEPTIDE DECORATED-MAGNETIC POROUS SILICON NANORODS FOR GLIOBLASTOMA IMAGING AND THERAPY

Glioblastoma multiforme (GBM) is the most aggressive type of cancer that begins within the brain. Despite advances in treatment, it remains largely untreatable , in part due to the low permeability of chemotherapeutic drugs to cross the blood-brain barrier (BBB) which significantly compromises their effectiveness. To circumvent the lack of drugs efficiency, we designed multifunctional nanoparticles based on porous silicon nanorods (pSiNRs). A Neurofilament light subunit derived 24 amino acid tubulin binding site peptide called NFL-peptide was reported to preferentially targeted human GBM cells compared to the healthy human cells.1, 2 Motivated by this approach, we investigated the use of magnetic-pSiNRs decorated with the NFL-TBS.40-63 peptide (NFL-peptide) to facilitate the targeting and enhance the internalization into human GBM cells. Additionally, we showed that the uptake of NFL-peptide coated magnetic-pSiNRs was preferential towards human GBM cells