Publications by authors named "Boudewijn van der Sanden"

47 Publications

3D two-photon polymerization of smart cell gelatin - collagen matrixes with incorporated ruthenium complexes for the monitoring of local oxygen tensions.

Acta Biomater 2021 08 12;130:172-182. Epub 2021 Jun 12.

MoVe, Laboratoire interdisciplinaire de physique, CNRS UMR 5588, Université Grenoble Alpes, St-Martin d'Hères, France. Electronic address:

The extra cellular matrix plays a major role in the biomechanical properties of tissues that impact cell behavior and fate. It is therefore crucial to mimic these complex cell-matrix interactions in 3D cell cultures. Here, two-photon polymerization is applied to produce gelatin methacryloyl (GelMA) - collagen matrixes that further enable local pO measurement, when ruthenium complexes are used as photo-activators. The fluorescence intensity of these complexes has a direct and inverse relationship with the local pO. The 3D structures reached their maximum size in cell culture conditions after 3H with a swelling factor of ~1.5. Their shape and the ruthenium fluorescence intensity of the alveoli walls stayed constant for at least 2 weeks in the absence of cells. They were used in time series to monitor the local pO adjacent to cancer cells during their division, migration and the formation of a tumor tissue mass. At the presence of these cell activities that consume O, a significant ~3-fold increase of the ruthenium fluorescence intensity in the alveoli walls was observed. This study demonstrates that online monitoring of the local pO is possible. The ruthenium complexes provide the bio-optical sensors that are useful for further analysis of cancer and healthy cell energy metabolism in a 3D matrix that better mimics in vivo conditions and migration paths. Unraveling the cancer cell metabolic adaptations in a changing micro-environment will help the development of new therapeutic opportunities. STATEMENT OF SIGNIFICANCE: In 3D cell cultures, monitoring pericellular pO is as critical as controlling pH. This facility is currently missing. Here, we take advantage of the direct and inverse relationship between pO and the fluorescence intensity of ruthenium complexes to generate stable gelatin-collagen matrixes able to continuously monitoring the pO at the pericellular level. The ruthenium complexes, which are photo-activators in the two-photon polymerization of these matrixes, became covalently bind to the collagen fibers. Indeed, local O consumption by cancer cells during migration, mitosis and tumor mass formation caused a 3-fold increase of the ruthenium fluorescence. In the future, incorporating ruthenium complexes with other bio-optical sensors will create new drug screening platforms that monitor cell culture parameters at the pericellular level.
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http://dx.doi.org/10.1016/j.actbio.2021.06.021DOI Listing
August 2021

Vascular bifurcation mapping with photoacoustic microscopy.

Biomed Opt Express 2020 Mar 7;11(3):1298-1305. Epub 2020 Feb 7.

Université Grenoble Alpes, CNRS UMR 5588, Laboratoire interdisciplinaire de physique, St-Martin d'Hères, France.

The detection of in cancer diagnosis is needed in the clinic. A change in the vascular bifurcation density is a biomarker for the sprouting activity. Here, ptical-esolution hotocoustic icroscopy is used for quantitative vascular bifurcation mapping in 2D after the creation of rtual bes out of furcations. In stacks of OR-PAM images of the hemoglobin distribution, bifurcations become tubes and are selected by the 3D tubeness filter. These fast analyses will be compared to a classical approach and are easier to implement for functional analysis of the vascular bifurcation density in healthy and diseased tissues.
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http://dx.doi.org/10.1364/BOE.383583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7075599PMC
March 2020

Photo-acoustic tomography based on laser optical feedback imaging of surface displacements.

Appl Opt 2019 Sep;58(26):7195-7204

We present how a laser optical feedback imaging (LOFI) setup can be used for the optical detection of ultrasound in photo-acoustic tomography (PAT). A PAT image is reconstructed by an inversion algorithm using surface displacement measurements made at several locations with our LOFI setup and following the optical irradiation with a pulsed Nd:YAG laser of a sample with absorbing inclusions. The width of the reconstructed inclusions and the signal-to-noise ratio (SNR) of the reconstructed images are first studied on the numerical model of a sample with three absorbing inclusions (i.e., with three acoustic punctual sources). Finally, an experimental PAT image of a phantom composed of two polyamide tubes with an internal diameter of 800 μm filled with red ink and submerged at -3.5  mm depth in a tank filled with water is reconstructed. Experimentally, the water surface displacement measurements have been made with our LOFI vibrometer, which provides an amplitude sensitivity of 1 nm (for a single-shot measurement) in a detection bandwidth of roughly 1 MHz adapted to the detection of the polyamide tubes. Under our experimental conditions, the surface energy densities of the LOFI focalized beam for the detection and of the pulsed Nd:YAG laser used for the irradiation, are compatible with the maximum permissive exposure for future biomedical measurements. The SNR and the resolution of the reconstructed PAT images are in good agreement with the theoretical predictions.
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http://dx.doi.org/10.1364/AO.58.007195DOI Listing
September 2019

Multi-wavelength photo-acoustic microscopy in the frequency domain for simultaneous excitation and detection of dyes.

Biomed Opt Express 2019 Feb 29;10(2):932-943. Epub 2019 Jan 29.

Univ. Grenoble Alpes, CNRS, LiPhy, F-38000 Grenoble, France.

An optical-resolution photoacoustic microscope with modulated CW laser diodes allowing multi-channel imaging is presented that can be used for both imaging biological tissues and for targeted photo-dynamic therapy (PDT) varying the optical power and exposure time. The effects of this therapy are immediately monitored in order to optimize the time of irradiation. After the description of the experimental setup, and applications are presented on a synthetic sample and on the mouse ear using hemoglobin as endogenous and methylene blue as exogenous dye for imaging and PDT, respectively.
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http://dx.doi.org/10.1364/BOE.10.000932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6377889PMC
February 2019

General and Scalable Approach to Bright, Stable, and Functional AIE Fluorogen Colloidal Nanocrystals for in Vivo Imaging.

ACS Appl Mater Interfaces 2018 Aug 23;10(30):25154-25165. Epub 2018 Jul 23.

Université de Lyon , F-69003 Lyon , France.

Fluorescent nanoparticles built from aggregation-induced emission-active organic molecules (AIE-FONs) have emerged as powerful tools in life science research for in vivo bioimaging of organs, biosensing, and therapy. However, the practical use of such biotracers has been hindered owing to the difficulty of designing bright nanoparticles with controlled dimensions (typically below 200 nm), narrow size dispersity and long shelf stability. In this article, we present a very simple yet effective approach to produce monodisperse sub-200 nm AIE fluorescent organic solid dispersions with excellent redispersibility and colloidal stability in aqueous medium by combination of nanoprecipitation and freeze-drying procedures. By selecting polymer additives that simultaneously act as stabilizers, promoters of amorphous-crystalline transition, and functionalization/cross-linking platforms, we demonstrate a straightforward access to stable nanocrystalline FONs that exhibit significantly higher brightness than their amorphous precursors and constitute efficient probes for in vivo imaging of the normal and tumor vasculature. FONs design principles reported here are universal, applicable to a range of fluorophores with different chemical structures and crystallization abilities, and are suitable for high-throughput production and manufacturing of functional imaging probes.
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http://dx.doi.org/10.1021/acsami.8b07859DOI Listing
August 2018

Coumarin-containing thermoresponsive hyaluronic acid-based nanogels as delivery systems for anticancer chemotherapy.

Nanoscale 2017 Aug;9(33):12150-12162

Grenoble Alpes University, CERMAV-CNRS, Grenoble, France.

Multi-stimuli responsive nanogels based on biocompatible hydrophilic polymers have emerged as promising drug delivery systems to improve anticancer therapy with hydrophobic drugs, through increase of circulating-time in the bloodstream, tumor-targeting and reduction of systemic toxicity. This paper reports on the synthesis, characterization and biological perspectives of light- and thermoresponsive hyaluronic acid (HA)-based nanogels containing coumarin as the photocleavable group. Newly synthesized nanogels exhibited interesting features: formation by a temperature-triggered self-assembly process, successful incorporation of poorly water-soluble molecules, light-responsiveness as demonstrated by a significant shift in the critical aggregation temperature after light irradiation, efficient internalization by cancer cells overexpressing the CD44 receptor of HA, ability to circulate for a prolonged period of time in the bloodstream after intravenous injection in mice and considerable detection in tumor tissues. Our findings indicate that coumarin-containing HA-based nanogels may be promising delivery systems for anticancer chemotherapy.
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http://dx.doi.org/10.1039/c7nr03964fDOI Listing
August 2017

Keto-polymethines: a versatile class of dyes with outstanding spectroscopic properties for and two-photon microscopy imaging.

Chem Sci 2017 Jan 3;8(1):381-394. Epub 2016 Aug 3.

ENS Lyon , Université de Lyon 1 , CNRS Laboratoire de chimie de l'ENS Lyon , UMR 5182 CNRS, 46 allée d'Italie , 69364 Lyon , France . Email: ; Email:

The synthesis of keto-heptamethine derivatives has been expanded to various new symmetrical and asymmetrical structures, including an unprecedented di-anionic keto-polymethine. The spectroscopic behavior of these new dyes has been systematically and thoroughly investigated, revealing that the formation of hydrogen bond interactions with protic solvents is responsible for a dramatic enhancement of the fluorescence quantum yield in the far-red spectral region. The existence of these strong hydrogen-bond interactions was further confirmed by molecular dynamics simulations. These bis-dipolar polymethines exhibit large two-photon absorption (TPA) cross-sections ( in GM) in the near-infrared, making them ideal candidates for NIR-to-NIR two-photon microscopy imaging applications. We demonstrate that the molecular engineering of the hydrophilic/hydrophobic balance enables targeting of different cellular components, such as cytoplasm or cell membranes. Addition of appropriate substituents provides the molecule with high-water-solubility, affording efficient two-photon probes for angiography.
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http://dx.doi.org/10.1039/c6sc02488bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365052PMC
January 2017

Towards the Design of a Patient-Specific Virtual Tumour.

Comput Math Methods Med 2016 19;2016:7851789. Epub 2016 Dec 19.

Université Grenoble Alpes, CNRS, TIMC-IMAG/DyCTIM2, 38041 Grenoble, France.

The design of a patient-specific virtual tumour is an important step towards Personalized Medicine. However this requires to capture the description of many key events of tumour development, including angiogenesis, matrix remodelling, hypoxia, and cell state heterogeneity that will all influence the tumour growth kinetics and degree of tumour invasiveness. To that end, an integrated hybrid and multiscale approach has been developed based on data acquired on a preclinical mouse model as a proof of concept. Fluorescence imaging is exploited to build case-specific virtual tumours. Numerical simulations show that the virtual tumour matches the characteristics and spatiotemporal evolution of its real counterpart. We achieved this by combining image analysis and physiological modelling to accurately described the evolution of different tumour cases over a month. The development of such models is essential since a dedicated virtual tumour would be the perfect tool to identify the optimum therapeutic strategies that would make Personalized Medicine truly reachable and achievable.
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http://dx.doi.org/10.1155/2016/7851789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206790PMC
March 2017

Glioma resection and tumor recurrence: back to Semmelweis.

Neuro Oncol 2016 12 7;18(12):1688-1689. Epub 2016 Oct 7.

Clinatec, Centre de recherche biomédicale Edmond J. Safra, CEA-LETI 17 rue des Martyrs, 38054 Grenoble cedex, France (D.R.); INSERM U1205, bâtiment modulaire 40-23, CEA 17 rue des Martyrs, 38054 Grenoble cedex, France (B.v.d.S., D.W.).

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http://dx.doi.org/10.1093/neuonc/now201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744249PMC
December 2016

Microscopic DTI accurately identifies early glioma cell migration: correlation with multimodal imaging in a new glioma stem cell model.

NMR Biomed 2016 11 15;29(11):1553-1562. Epub 2016 Sep 15.

Inserm U1205, Grenoble, France.

Monitoring glioma cell infiltration in the brain is critical for diagnosis and therapy. Using a new glioma Glio6 mouse model derived from human stem cells we show how diffusion tensor imaging (DTI) may predict glioma cell migration/invasion. In vivo multiparametric MRI was performed at one, two and three months of Glio6 glioma growth (Glio6 (n = 6), sham (n = 3)). This longitudinal study reveals the existence of a time window to study glioma cell/migration/invasion selectively. Indeed, at two months only Glio6 cell invasion was detected, while tumor mass formation, edema, blood-brain barrier leakage and tumor angiogenesis were detected later, at three months. To robustly confirm the potential of DTI for detecting glioma cell migration/invasion, a microscopic 3D-DTI (80 μm isotropic spatial resolution) technique was developed and applied to fixed mouse brains (Glio6 (n = 6), sham (n = 3)). DTI changes were predominant in the corpus callosum (CC), a known path of cell migration. Fractional anisotropy (FA) and perpendicular diffusivity (D ) changes derived from ex vivo microscopic 3D-DTI were significant at two months of tumor growth. In the caudate putamen an FA increase of +38% (p < 0.001) was observed, while in the CC a - 28% decrease in FA (p < 0.005) and a + 95% increase in D (p < 0.005) were observed. In the CC, DTI changes and fluorescent Glio6 cell density obtained by two-photon microscopy in the same brains were correlated (p < 0.001, r = 0.69), validating FA and D as early quantitative biomarkers to detect glioma cell migration/invasion. The origin of DTI changes was assessed by electron microscopy of the same tract, showing axon bundle disorganization. During the first two months, Glio6 cells display a migratory phenotype without being associated with the constitution of a brain tumor mass. This offers a unique opportunity to apply microscopic 3D-DTI and to validate DTI parameters FA and D as biomarkers for glioma cell invasion.
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http://dx.doi.org/10.1002/nbm.3608DOI Listing
November 2016

Design of Hyaluronic Acid Hydrogels to Promote Neurite Outgrowth in Three Dimensions.

ACS Appl Mater Interfaces 2016 Sep 16;8(38):25051-9. Epub 2016 Sep 16.

Grenoble Alpes University , Centre de Recherches sur les Macromolécules Végétales (CERMAV-CNRS), 601, rue de la Chimie, BP 53, Grenoble 38041 Cedex 9, France.

A hyaluronic acid (HA)-based extracellular matrix (ECM) platform with independently tunable stiffness and density of cell-adhesive peptide (RGD, arginine-glycine-aspartic acid) that mimics key biochemical and mechanical features of brain matrix has been designed. We demonstrated here its utility in elucidating ECM regulation of neural progenitor cell behavior and neurite outgrowth. The analysis of neurite outgrowth in 3-D by two-photon microscopy showed several important results in the development of these hydrogels. First, the ability of neurites to extend deeply into these soft HA-based matrices even in the absence of cell-adhesive ligand further confirms the potential of HA hydrogels for central nervous system (CNS) regeneration. Second, the behavior of hippocampal neural progenitor cells differed markedly between the hydrogels with a storage modulus of 400 Pa and those with a modulus of 800 Pa. We observed an increased outgrowth and density of neurites in the softest hydrogels (G' = 400 Pa). Interestingly, cells seeded on the surface of the hydrogels functionalized with the RGD ligand experienced an optimum in neurite outgrowth as a function of ligand density. Surprinsingly, neurites preferentially progressed inside the gels in a vertical direction, suggesting that outgrowth is directed by the hydrogel structure. This work may provide design principles for the development of hydrogels to facilitate neuronal regeneration in the adult brain.
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http://dx.doi.org/10.1021/acsami.6b06446DOI Listing
September 2016

Glioma Recurrence following Surgery: Peritumoral or Perilesional?

Front Neurol 2016 31;7:52. Epub 2016 Mar 31.

INSERM UA01, Clinatec, Institut E.J. Safra, CEA , Grenoble , France.

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http://dx.doi.org/10.3389/fneur.2016.00052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4814895PMC
April 2016

The brain tissue response to surgical injury and its possible contribution to glioma recurrence.

J Neurooncol 2016 05 9;128(1):1-8. Epub 2016 Mar 9.

INSERM UA 01, Clinatec, Centre de recherche biomédicale Edmond J. Safra, CEA 17 rue des Martyrs, 38054, Grenoble cedex, France.

Surgery is the first line therapy for glioma. However, glioma recurs in 90 % of the patients in the resection margin. The impact of surgical brain injury (SBI) on glioma recurrence is largely overlooked. Herein, we review some of the mechanisms involved in tissue repair that may impact glioma recurrence at the resection margin. Many processes or molecules involved in tissue repair after brain injury are also critical for glioma growth. They include a wide array of secreted growth factors, cytokines and transcription factors including NFКB and STAT3 which in turn activate proliferative and anti-apoptotic genes and processes such as angiogenesis and inflammation. Because some residual glioma cells always remain in the tumor resection margin, there are now compelling arguments to suggest that some aspects of the brain tissue response to SBI can also participate to glioma recurrence at the resection margin. Brain tissue response to SBI recruits angiogenesis and inflammation that precede and then follow tumor recurrence at the resection margin. The healing response to SBI is double edged, as inflammation is involved in regeneration and healing, and has both pro- and anti-tumorigenic functions. A promising therapeutic approach is to normalize and re-educate the molecular and cellular responses at the resection margin to promote anti-tumorigenic processes involved in healing while inhibiting pro-tumorigenic activities. Manipulation of the inflammatory response to SBI to prevent local recurrence could also enhance the efficacy of other therapies such as immunotherapy. However, our current knowledge is far from sufficient to achieve this goal. Acknowledging, understanding and manipulating the double-edged role played by SBI in glioma recurrence is surely challenging, but it cannot be longer delayed.
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http://dx.doi.org/10.1007/s11060-016-2096-yDOI Listing
May 2016

Fluorescent Tobacco mosaic virus-Derived Bio-Nanoparticles for Intravital Two-Photon Imaging.

Front Plant Sci 2015 13;6:1244. Epub 2016 Jan 13.

Institut de Biologie Moléculaire des Plantes (IBMP-UPR2357), Centre National de la Recherche Scientifique Strasbourg, France.

Multi-photon intravital imaging has become a powerful tool to investigate the healthy and diseased brain vasculature in living animals. Although agents for multi-photon fluorescence microscopy of the microvasculature are available, issues related to stability, bioavailability, toxicity, cost or chemical adaptability remain to be solved. In particular, there is a need for highly fluorescent dyes linked to particles that do not cross the blood brain barrier (BBB) in brain diseases like tumor or stroke to estimate the functional blood supply. Plant virus particles possess a number of distinct advantages over other particles, the most important being the multi-valency of chemically addressable sites on the particle surface. This multi-valency, together with biological compatibility and inert nature, makes plant viruses ideal carriers for in vivo imaging agents. Here, we show that the well-known Tobacco mosaic virus is a suitable nanocarrier for two-photon dyes and for intravital imaging of the mouse brain vasculature.
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http://dx.doi.org/10.3389/fpls.2015.01244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4710741PMC
January 2016

Multiscale investigation of USPIO nanoparticles in atherosclerotic plaques and their catabolism and storage in vivo.

Nanomedicine 2016 Jan 12;12(1):191-200. Epub 2015 Sep 12.

Institut Lumière Matière, UMR 5306 Université Claude Bernard Lyon1-CNRS, Université de Lyon, Villeurbanne, Cedex, France. Electronic address:

The storage and catabolism of Ultrasmall SuperParamagnetic Iron Oxide (USPIO) nanoparticles were analyzed through a multiscale approach combining Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM) at different times after intravenous injection in an atherosclerotic ApoE(-/-) mouse model. The atherosclerotic plaque features and the USPIO heterogeneous biodistribution were revealed down from organ's scale to subcellular level. The biotransformation of the nanoparticle iron oxide (maghemite) core into ferritin, the non-toxic form of iron storage, was demonstrated for the first time ex vivo in atherosclerotic plaques as well as in spleen, the iron storage organ. These results rely on an innovative spatial and structural investigation of USPIO's catabolism in cellular phagolysosomes. This study showed that these nanoparticles were stored as non-toxic iron compounds: maghemite oxide or ferritin, which is promising for MRI detection of atherosclerotic plaques in clinics using these USPIOs. From the Clinical Editor: Advance in nanotechnology has brought new contrast agents for clinical imaging. In this article, the authors investigated the use and biotransformation of Ultrasmall Super-paramagnetic Iron Oxide (USPIO) nanoparticles for analysis of atherosclerotic plagues in Two Photon Laser Scanning Microscopy (TPLSM) and High-Resolution Transmission Electron Microscopy (HRTEM). The biophysical data generated from this study could enable the possible use of these nanoparticles for the benefits of clinical patients.
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http://dx.doi.org/10.1016/j.nano.2015.08.005DOI Listing
January 2016

Cancer research in need of a scientific revolution: Using 'paradigm shift' as a method of investigation.

J Biosci 2015 Sep;40(3):657-66

INSERM UA 01, Clinatec, Centre de Recherche Biomedicale Edmond J. Safra, CHU Michallon, Universite Joseph Fourier, CEA 17 rue des Martyrs, 38054, Grenoble Cedex, France,

Despite important human and financial resources and considerable accumulation of scientific publications, patents, and clinical trials, cancer research has been slow in achieving a therapeutic revolution similar to the one that occurred in the last century for infectious diseases. It has been proposed that science proceeds not only by accumulating data but also through paradigm shifts. Here, we propose to use the concept of 'paradigm shift' as a method of investigation when dominant paradigms fail to achieve their promises. The first step in using the 'paradigm shift' method in cancer research requires identifying its founding paradigms. In this review, two of these founding paradigms will be discussed: (i) the reification of cancer as a tumour mass and (ii) the translation of the concepts issued from infectious disease in cancer research. We show how these founding paradigms can generate biases that lead to over-diagnosis and over-treatment and also hamper the development of curative cancer therapies. We apply the 'paradigm shift' method to produce perspective reversals consistent with current experimental evidence. The 'paradigm shift' method enlightens the existence of a tumour physiologic-prophylactic-pathologic continuum. It integrates the target/antitarget concept and that cancer is also an extracellular disease. The 'paradigm shift' method has immediate implications for cancer prevention and therapy. It could be a general method of investigation for other diseases awaiting therapy.
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http://dx.doi.org/10.1007/s12038-015-9543-3DOI Listing
September 2015

Long-term in vivo clearance of gadolinium-based AGuIX nanoparticles and their biocompatibility after systemic injection.

ACS Nano 2015 Mar 26;9(3):2477-88. Epub 2015 Feb 26.

†Institut lumière matière, UMR5306, Université Claude Bernard Lyon1-CNRS, Université de Lyon 69622 Villeurbanne cedex, France.

We previously reported the synthesis of gadolinium-based nanoparticles (NPs) denoted AGuIX (activation and guiding of irradiation by X-ray) NPs and demonstrated their potential as an MRI contrast agent and their efficacy as radiosensitizing particles during X-ray cancer treatment. Here we focus on the elimination kinetics of AGuIX NPs from the subcellular to whole-organ scale using original and complementary methods such as laser-induced breakdown spectroscopy (LIBS), intravital two-photon microscopy, inductively coupled plasma optical emission spectrometry (ICP-OES), transmission electron microscopy (TEM), and electrospray ionization mass spectrometry (ESI-MS). This combination of techniques allows the exact mechanism of AGuIX NPs elimination to be elucidated, including their retention in proximal tubules and their excretion as degraded or native NPs. Finally, we demonstrated that systemic AGuIX NP administration induced moderate and transient effects on renal function. These results provide useful and promising preclinical information concerning the safety of theranostic AGuIX NPs.
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http://dx.doi.org/10.1021/acsnano.5b00552DOI Listing
March 2015

Thermoresponsive hyaluronic acid nanogels as hydrophobic drug carrier to macrophages.

Acta Biomater 2014 Nov 8;10(11):4750-4758. Epub 2014 Aug 8.

University Grenoble Alpes, CERMAV-CNRS, F-38000 Grenoble, France. Electronic address:

Delivery systems for macrophages are particularly attractive since these phagocytic cells play a important role in immunological and inflammatory responses, also acting as host cells for microorganisms that are involved in deadly infectious diseases, such as leishmaniasis. Hyaluronic acid (HA) is specifically recognized by macrophages that are known to express HA receptors. Therefore, in this study, we focused on HA-based nanogels as drug carriers for these cells. The drug delivery was validated in an in vivo study on mice using intravital two-photon laser scanning microscopy. HA derivatives were modified with a biocompatible oligo(ethylene glycol)-based thermoresponsive polymer to form nanogels. These HA conjugates were readily prepared by varying the molar mass of initial HA and the degree of substitution via radical-mediated thiol-ene chemistry in aqueous solution. The derivatives were shown to self-assemble into spherical gel particles with diameters ranging from 150 to 214 nm above 37 °C. A poorly water-soluble two-photon dye was successfully loaded into the nanogels during this self-assembly process. In vitro cellular uptake tests using a RAW 264.7 murine macrophage cell line showed successful intracellular delivery of the hydrophobic dye. After intravenous injection in mice, the nanogels circulated freely in the blood but were rapidly phagocytized within 13 min by circulating macrophages and stored in the liver and spleen, as observed by two-photon microscopy. Benefit can be thus expected in using such a delivery system for the liver and spleen macrophage-associated diseases.
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http://dx.doi.org/10.1016/j.actbio.2014.07.033DOI Listing
November 2014

Brain mesenchymal stem cells: The other stem cells of the brain?

World J Stem Cells 2014 Apr;6(2):134-43

Florence Appaix, Marie-France Nissou, Jean-Paul Issartel, Didier Wion, INSERM U836, Grenoble Institut des Neurosciences, Bâtiment Edmond J Safra, Université Joseph Fourier, CHU Michallon, 38042 Grenoble, France.

Multipotent mesenchymal stromal cells (MSC), have the potential to differentiate into cells of the mesenchymal lineage and have non-progenitor functions including immunomodulation. The demonstration that MSCs are perivascular cells found in almost all adult tissues raises fascinating perspectives on their role in tissue maintenance and repair. However, some controversies about the physiological role of the perivascular MSCs residing outside the bone marrow and on their therapeutic potential in regenerative medicine exist. In brain, perivascular MSCs like pericytes and adventitial cells, could constitute another stem cell population distinct to the neural stem cell pool. The demonstration of the neuronal potential of MSCs requires stringent criteria including morphological changes, the demonstration of neural biomarkers expression, electrophysiological recordings, and the absence of cell fusion. The recent finding that brain cancer stem cells can transdifferentiate into pericytes is another facet of the plasticity of these cells. It suggests that the perversion of the stem cell potential of pericytes might play an even unsuspected role in cancer formation and tumor progression.
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http://dx.doi.org/10.4252/wjsc.v6.i2.134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3999771PMC
April 2014

Rapid-Steady-State-T1 signal modeling during contrast agent extravasation: toward tumor blood volume quantification without requiring the arterial input function.

Magn Reson Med 2015 Mar 14;73(3):1005-14. Epub 2014 Apr 14.

Université Joseph Fourier, Grenoble Institut des Neurosciences, Institut National de la Santé et de la Recherche Médicale INSERM-U836, Bâtiment Edmond J. Safra, Chemin Fortuné Ferrini, Grenoble, France; CLINATEC, Commissariat à l'énergie atomique et aux énergies alternatives, MINATEC Campus, Grenoble, France; Université Saint Joseph-Faculté des sciences, Département de physique, campus des sciences et technologies, Mar Roukos, Mkallès, Lebanon.

Purpose: This study demonstrates how to quantify the tumor blood volume fraction (BVf) using the dynamic Rapid-Steady-State-T1 (RSST1 )-MRI method despite contrast agent (CA) leakage and without arterial input function (AIF) determination.

Methods: For vasculature impermeable to CAs, the BVf is directly quantified from the RSST1 signal amplitude. In case of CA extravasation, we propose a two-compartment model to describe the dynamic RSST1 signal increase. We applied the mathematical model in a pilot-study on a RG2-glioma model to compare extravasation of two Gd-based CAs. The BVf quantification using the mathematical model in a C6-glioma model (n = 8) with the clinical CA Gd-DOTA was validated using a ΔR2 *-steady-state MRI method with an USPIO and by immunohistochemical staining of perfused vessels labeled with Hoechst-33342 dye in the same rats.

Results: BVf in tumor and in healthy brain tissues (0.034 ± 0.005 and 0.026 ± 0.004, respectively) derived from the dynamic RSST1 signal were confirmed by ΔR2 *-steady-state MRI (0.036 ± 0.003 and 0.027 ± 0.002, respectively, correlation coefficient rS = 0.74) and by histology (0.036 ± 0.003 and 0.025 ± 0.004 respectively, rS = 0.87).

Conclusion: Straightforward tumor BVf quantification without AIF determination is demonstrated in presence of CA leakage. The method will facilitate angiogenesis assessment in longitudinal neuro-oncologic studies in particular when monitoring the response to antiangiogenic therapies.
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http://dx.doi.org/10.1002/mrm.25218DOI Listing
March 2015

Synergistic effect of cisplatin and synchrotron irradiation on F98 gliomas growing in nude mice.

J Synchrotron Radiat 2013 Sep 3;20(Pt 5):777-84. Epub 2013 Jul 3.

INSERM U836, Grenoble Institut des Neurosciences, Grenoble, France.

Among brain tumors, glioblastoma multiforme appears as one of the most aggressive forms of cancer with poor prognosis and no curative treatment available. Recently, a new kind of radio-chemotherapy has been developed using synchrotron irradiation for the photoactivation of molecules with high-Z elements such as cisplatin (PAT-Plat). This protocol showed a cure of 33% of rats bearing the F98 glioma but the efficiency of the treatment was only measured in terms of overall survival. Here, characterization of the effects of the PAT-Plat on tumor volume and tumor blood perfusion are proposed. Changes in these parameters may predict the overall survival. Firstly, changes in tumor growth of the F98 glioma implanted in the hindlimb of nude mice after the PAT-Plat treatment and its different modalities have been characterized. Secondly, the effects of the treatment on tumor blood perfusion have been observed by intravital two-photon microscopy. Cisplatin alone had no detectable effect on the tumor volume. A reduction of tumor growth was measured after a 15 Gy synchrotron irradiation, but the whole therapy (15 Gy irradiation + cisplatin) showed the largest decrease in tumor growth, indicating a synergistic effect of both synchrotron irradiation and cisplatin treatment. A high number of unperfused vessels (52%) were observed in the peritumoral area in comparison with untreated controls. In the PAT-Plat protocol the transient tumor growth reduction may be due to synergistic interactions of tumor-cell-killing effects and reduction of the tumor blood perfusion.
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http://dx.doi.org/10.1107/S0909049513016567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943558PMC
September 2013

Translation of the ecological trap concept to glioma therapy: the cancer cell trap concept.

Future Oncol 2013 Jun;9(6):817-24

INSERM U836, Grenoble Institut des Neurosciences, Université Joseph Fourier, CHU Michallon, Grenoble, France.

Viewing tumors as ecosystems offers the opportunity to consider how ecological concepts can be translated to novel therapeutic perspectives. The ecological trap concept emerged approximately half a century ago when it was observed that animals can prefer an environment of low quality for survival over other available environments of higher quality. The presence of such a trap can drive a local population to extinction. The cancer cell trap concept is the translation of the ecological trap into glioma therapy. It exploits and diverts the invasive potential of glioma cells by guiding their migration towards specific locations where a local therapy can be delivered efficiently. This illustrates how an ecological concept can change therapeutic obstacles into therapeutic tools.
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http://dx.doi.org/10.2217/fon.13.30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4788699PMC
June 2013

Hypoxia-induced expression of VE-cadherin and filamin B in glioma cell cultures and pseudopalisade structures.

J Neurooncol 2013 Jun 31;113(2):239-49. Epub 2013 Mar 31.

INSERM U836, Grenoble Institut des Neurosciences, Université Joseph Fourier, CHU Grenoble, Bâtiment E.J. Safra, 38042, Grenoble, France.

Most of our knowledge regarding glioma cell biology comes from cell culture experiments. For many years the standards for glioma cell culture were the use of cell lines cultured in the presence of serum and 20 % O2. However, in vivo, normoxia in many brain areas is in close to 3 % O2. Hence, in cell culture, the experimental value referred as the norm is hyperoxic compared to any brain physiological value. Likewise, cells in vivo are not usually exposed to serum, and low-passaged glioma neurosphere cultures maintained in serum-free medium is emerging as a new standard. A consequence of changing the experimental normoxic standard from 20 % O2 to the more brain physiological value of 3 % O2, is that a 3 % O2 normoxic reference point enabled a more rigorous characterization of the level of regulation of genes by hypoxia. Among the glioma hypoxia-regulated genes characterized using this approach we found VE-cadherin that is required for blood vessel formation, and filamin B a gene involved in endothelial cell motility. Both VE-cadherin and filamin B were found expressed in pseudopalisades, a glioblastoma pathognomonic structure made of hypoxic migrating cancer cells. These results provide additional clues on the role played by hypoxia in the acquisition of endothelial traits by glioma cells and on the functional links existing between pseudopalisades, hypoxia, and tumor progression.
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http://dx.doi.org/10.1007/s11060-013-1124-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4850216PMC
June 2013

On the importance of the submicrovascular network in a computational model of tumour growth.

Microvasc Res 2012 Sep 13;84(2):188-204. Epub 2012 Jun 13.

UJF-Grenoble 1, CNRS, Laboratory TIMC-IMAG UMR 5525, DyCTiM research team, Grenoble, F-38041, France.

A computational model is potentially a powerful tool to apprehend complex phenomena like solid tumour growth and to predict the outcome of therapies. To that end, the confrontation of the model with experiments is essential to validate this tool. In this study, we develop a computational model specifically dedicated to the interpretation of tumour growth as observed in a mouse model with a dorsal skinfold chamber. Observation of the skin vasculature at the dorsal window scale shows a sparse network of a few main vessels of several hundreds micrometers in diameter. However observation at a smaller scale reveals the presence of a dense and regular interconnected network of capillaries about ten times smaller. We conveniently designate this structure as the submicrovascular network (SMVN).(1) The question that we wish to answer concerns the necessity of explicitly taking into account the SMVN in the computational model to describe the tumour evolution observed in the dorsal chamber. For that, simulations of tumour growth realised with and without the SMVN are compared and lead to two distinct scenarios. Parameters that are known to strongly influence the tumour evolution are then tested in the two cases to determine to which extent those parameters can be used to compensate the observed differences between these scenarios. Explicit modelling of the smallest vessels appears mandatory although not necessarily under the form of a regular grid. A compromise between the two investigated cases can thus be reached.
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http://dx.doi.org/10.1016/j.mvr.2012.06.001DOI Listing
September 2012

Specific in vivo staining of astrocytes in the whole brain after intravenous injection of sulforhodamine dyes.

PLoS One 2012 11;7(4):e35169. Epub 2012 Apr 11.

Grenoble Institute of Neuroscience, Inserm U836, Grenoble, France.

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035169PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324425PMC
August 2012

How stereological analysis of vascular morphology can quantify the blood volume fraction as a marker for tumor vasculature: comparison with magnetic resonance imaging.

J Cereb Blood Flow Metab 2012 Mar 9;32(3):489-501. Epub 2011 Nov 9.

Université Joseph Fourier, Grenoble Institut des Neurosciences, Grenoble, France.

To assess angiogenesis noninvasively in a C6 rat brain tumor model, the rapid-steady-state-T(1) (RSST(1)) magnetic resonance imaging (MRI) method was used for microvascular blood volume fraction (BVf) quantification with a novel contrast agent gadolinium per (3,6 anhydro) α-cyclodextrin (Gd-ACX). In brain tissue contralateral to the tumor, equal BVfs were obtained with Gd-ACX and the clinically approved gadoterate meglumine (Gd-DOTA). Contrary to Gd-DOTA, which leaks out of the tumor vasculature, Gd-ACX was shown to remain vascular in the tumor tissue allowing quantification of the tumor BVf. We sought to confirm the obtained tumor BVf using an independent method: instead of using a 'standard' two-dimensional histologic method, we study here how vascular morphometry combined with a stereological technique can be used for three-dimensional assessment of the vascular volume fraction (V(V)). The V(V) is calculated from the vascular diameter and length density. First, the technique is evaluated on simulated data and the healthy rat brain vasculature and is then applied to the same C6 tumor vasculature previously quantified by RSST(1)-MRI with Gd-ACX. The mean perfused V(V) and the BVf obtained by MRI in tumor regions are practically equal and the technique confirms the spatial heterogeneity revealed by MRI.
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http://dx.doi.org/10.1038/jcbfm.2011.151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3293112PMC
March 2012

Deep in vivo two-photon imaging of blood vessels with a new dye encapsulated in pluronic nanomicelles.

J Biomed Opt 2011 Mar;16(3):036001

Laboratoire de Spectrométrie Physique, CNRS UMR 5588, Saint Martin d'Hères, France.

Our purpose is to test if Pluronic® fluorescent nanomicelles can be used for in vivo two-photon imaging of both the normal and the tumor vasculature. The nanomicelles were obtained after encapsulating a hydrophobic two-photon dye: di-stryl benzene derivative, in Pluronic block copolymers. Their performance with respect to imaging depth, blood plasma staining, and diffusion across the tumor vascular endothelium is compared to a classic blood pool dye Rhodamin B dextran (70 kDa) using two-photon microscopy. Pluronic nanomicelles show, like Rhodamin B dextran, a homogeneous blood plasma staining for at least 1 h after intravenous injection. Their two-photon imaging depth is similar in normal mouse brain, using 10 times less injected mass. In contrast with Rhodamin B dextran, no extravasation is observed in leaky tumor vessels due to their large size: 20-100 nm. In conclusion, Pluronic nanomicelles can be used as a blood pool dye, even in leaky tumor vessels. The use of Pluronic block copolymers is a valuable approach for encapsulating two-photon fluorescent dyes that are hydrophobic and not suitable for intravenous injection.
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http://dx.doi.org/10.1117/1.3548879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020796PMC
March 2011

Optimizing stem cell culture.

J Cell Biochem 2010 Nov;111(4):801-7

INSERM U836, Grenoble Institut des Neurosciences, Université Joseph Fourier, CHU Michallon, 38042 Grenoble, France.

Stem cells always balance between self-renewal and differentiation. Hence, stem cell culture parameters are critical and need to be continuously refined according to progress in our stem cell biology understanding and the latest technological developments. In the past few years, major efforts have been made to define more precisely the medium composition in which stem cells grow or differentiate. This led to the progressive replacement of ill-defined additives such as serum or feeder cell layers by recombinant cytokines or growth factors. Another example is the control of the oxygen pressure. For many years cell cultures have been done under atmospheric oxygen pressure which is much higher than the one experienced by stem cells in vivo. A consequence of cell metabolism is that cell culture conditions are constantly changing. Therefore, the development of high sensitive monitoring processes and control algorithms is required for ensuring cell culture medium homeostasis. Stem cells also sense the physical constraints of their microenvironment. Rigidity, stiffness, and geometry of the culture substrate influence stem cell fate. Hence, nanotopography is probably as important as medium formulation in the optimization of stem cell culture conditions. Recent advances include the development of synthetic bioinformative substrates designed at the micro- and nanoscale level. On going research in many different fields including stem cell biology, nanotechnology, and bioengineering suggest that our current way to culture cells in Petri dish or flasks will soon be outdated as flying across the Atlantic Ocean in the Lindbergh's plane.
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http://dx.doi.org/10.1002/jcb.22847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3348118PMC
November 2010

Tolerance of arteries to microplanar X-ray beams.

Int J Radiat Oncol Biol Phys 2010 Aug;77(5):1545-52

INSERM U836, Institute of Neuroscience Grenoble, France.

Purpose: The purpose is to evaluate effects of a new radiotherapy protocol, microbeam radiation therapy, on the artery wall. In previous studies on animal models, it was shown that capillaries recover well from hectogray doses of X-rays delivered in arrays of narrow (< or = 50 microm) beams with a minimum spacing of 200 microm. Here, short- and long-term effects of comparable microplanar beam configurations on the saphenous artery of the mouse hind leg were analyzed in situ by use of nonlinear optics and compared with histopathologic findings.

Methods And Materials: The left hind leg of normal mice including the saphenous artery was irradiated by an array of 26 microbeams of synchrotron X-rays (50 microm wide, spaced 400 microm on center) with peak entrance doses of 312 Gy and 2,000 Gy.

Results: The artery remained patent, but narrow arterial smooth muscle cell layer segments that were in the microplanar beam paths became atrophic and fibrotic in a dose-dependent pattern. The wide tunica media segments between those paths hypertrophied, as observed in situ by two-photon microscopy and histopathologically.

Conclusions: Clinical risks of long-delayed disruption or occlusion of nontargeted arteries from microbeam radiation therapy will prove less than corresponding risks from broad-beam radiosurgery, especially if peak doses are kept below 3 hectograys.
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http://dx.doi.org/10.1016/j.ijrobp.2010.02.019DOI Listing
August 2010
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