Article

Evaluation of Poly (Glycerol-Adipate) Nanoparticle Uptake in an In Vitro 3-D Brain Tumor Co-Culture Model

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Abstract

Despite the inherent problems associated with in vivo animal models of tumor growth and metastases, many of the current in vitro brain tumor models also do not accurately mimic tumor-host brain interactions. Therefore, there is a need to develop such co-culture models to study tumor biology and, importantly, the efficacy of drug delivery systems targeting the brain. So far, few investigations of this nature have been published. In this paper we describe the development of a new model system and its application to drug delivery assessment. For our new model, a co-culture of DAOY cell brain tumor aggregates and organo-typic brain slices was developed. Initially, the DAOY aggregates attached to cerebellum slices and invaded as a unit. Single cells in the periphery of the aggregate detached from the DAOY aggregates and gradually replaced normal brain cells. This invasive behavior of DAOY cells toward organotypic cerebellum slices shows a similar pattern to that seen in vivo. After validation of the co-culture model using transmission electron microscopy, nanoparticle (NP) uptake was then evaluated. Confocal micrographs illustrated that DAOY cells in this co-culture model took up most of the NPs, but few NPs were distributed into brain cells. This finding corresponded with results of NP uptake in DAOY and brain aggregates reported elsewhere.

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... Employing cultures of both normal and tumour tissue provides information for the relative safety and efficacy of treatment and can be used to rank formulations according to their therapeutic safety ratio (Hickman, 2014). Our previous studies (Meng et al., 2007) have indicated that using the correct dimensionality plays a significant role in modelling the physiological response of in vitro cultures. Fluorescently-labelled biodegradable nanoparticles were taken up six times more in tumour spheroids made of DAOY medulloblastoma cells compared to normal tissue (rat brain slices) when cultured together in 3D (Meng, 2006). ...
... This is in agreement with the differential adhesion hypothesis postulated by Steinberg where spontaneous tissue segregation and sorting is thought to be guided by differential expression of cadherins, causing differences in surface tension between cell types (Foty and Steinberg, 2005). In previous work (Meng et al., 2007) we have seen invasion of DAOY medulloblastoma spheroids into organotypic cultures of rat neonatal cerebellum compared to cortical slices, which are not invaded significantly by DAOY spheroids suggesting that the cues for this cell behaviour can be very specific. Such interactions are reminiscent of the 'seeds and soils' hypothesis regarding the recognition between cancer and normal cells and the spread of metastasis to specific parts of the body (Langley and Fidler, 2011). ...
... Neurotoxic side effects in patients have been reported for other cytotoxics like cisplatin (Gregg et al., 1992), methotrexate (Bhojwani et al., 2014;Shuper et al., 2000) and cytarabine (Gállego Pérez-Larraya et al., 2011) but not for intrathecal etoposide (Fleischhack et al., 2001;Slavc et al., 2003;Van der Gaast et al., 1992) yet. Nevertheless, the findings in this study show that etoposide can damage proliferating cells regardless of their origin and suggest that more targeted approaches like nanoparticle encapsulation (Meng et al., 2007) should be considered to improve selectivity. Drug delivery strategies that limit normal tissue exposure and maximise the toxic effects to tumours are needed in order to prevent off-target toxicity. ...
Article
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Physiologically relevant in vitro models can serve as biological analytical platforms for testing novel treatments and drug delivery systems. We describe the first steps in the development of a 3D human brain tumour co-culture model that includes the interplay between normal and tumour tissue along with nutrient gradients, cell-cell and cell-matrix interactions. The human medulloblastoma cell line UW228-3 and human foetal brain tissue were marked with two supravital fluorescent dyes (CDCFDASE, Celltrace Violet) and cultured together in ultra-low attachment 96-well plates to form reproducible single co-culture spheroids (d = 600 μm, CV% = 10%). Spheroids were treated with model cytotoxic drug etoposide (0.3-100 μM) and the viability of normal and tumour tissue quantified separately using flow cytometry and multiphoton microscopy. Etoposide levels of 10 μM were found to maximise toxicity to tumours (6.5% viability) while stem cells maintained a surviving fraction of 40%. The flexible cell marking procedure and high-throughput compatible protocol make this platform highly transferable to other cell types, primary tissues and personalised screening programs. The model's key anticipated use is for screening and assessment of drug delivery strategies to target brain tumours, and is ready for further developments, e.g differentiation of stem cells to a range of cell types and more extensive biological validation
... Employing cultures of both normal and tumour tissue provides information for the relative safety and efficacy of treatment and can be used to rank formulations according to their therapeutic safety ratio (Hickman, 2014). Our previous studies (Meng et al., 2007) have indicated that using the correct dimensionality plays a significant role in modelling the physiological response of in vitro cultures. Fluorescently-labelled biodegradable nanoparticles were taken up six times more in tumour spheroids made of DAOY medulloblastoma cells compared to normal tissue (rat brain slices) when cultured together in 3D (Meng, 2006). ...
... This is in agreement with the differential adhesion hypothesis postulated by Steinberg where spontaneous tissue segregation and sorting is thought to be guided by differential expression of cadherins, causing differences in surface tension between cell types (Foty and Steinberg, 2005). In previous work (Meng et al., 2007) we have seen invasion of DAOY medulloblastoma spheroids into organotypic cultures of rat neonatal cerebellum compared to cortical slices, which are not invaded significantly by DAOY spheroids suggesting that the cues for this cell behaviour can be very specific. Such interactions are reminiscent of the 'seeds and soils' hypothesis regarding the recognition between cancer and normal cells and the spread of metastasis to specific parts of the body (Langley and Fidler, 2011). ...
... Neurotoxic side effects in patients have been reported for other cytotoxics like cisplatin (Gregg et al., 1992), methotrexate (Bhojwani et al., 2014;Shuper et al., 2000) and cytarabine (Gállego Pérez-Larraya et al., 2011) but not for intrathecal etoposide (Fleischhack et al., 2001;Slavc et al., 2003;Van der Gaast et al., 1992) yet. Nevertheless, the findings in this study show that etoposide can damage proliferating cells regardless of their origin and suggest that more targeted approaches like nanoparticle encapsulation (Meng et al., 2007) should be considered to improve selectivity. Drug delivery strategies that limit normal tissue exposure and maximise the toxic effects to tumours are needed in order to prevent off-target toxicity. ...
Conference Paper
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INTRODUCTION: Local interstitial or intra-cerebrospinal fluid (intra-CSF) delivery bypasses the blood-brain barrier, minimising systemic exposure and potentially reducing radiotherapy-induced neurotoxicity. Preclinical testing of enhanced local drug delivery systems can help refine these strategies maximising anti-tumour effect while minimising toxicity. AIMS: To develop a pre-clinical 3D co-culture system of medulloblastoma and human foetal stem cells in order to establish a therapeutic window of etoposide exposure during local drug delivery through high-throughput testing of efficacy and neurotoxicity. METHODS: We have developed a bio-representative 3D human spheroid co-culture in-vitro model of medulloblastoma using the cell line UW228-3 and human foetal brain stem cells marked with two supra-vital fluorescent dyes, cultured together in ultra-low attachment 96-well plates, forming reproducible single co-culture spheroids (d = 500 µm, CV% = 10%). Spheroids were exposed to concentrations of etoposide (0.3-100 µM), spanning CSF concentrations for systemic versus local delivery, based upon human pharmacokinetic data. Upon spheroid dissociation, flow cytometry quantified the absolute numbers of each cell population and determined their viability, separately. Multi-photon fluorescence microscopy was used to make qualitative assessment of tumour and foetal cell viability and distribution. RESULTS: Both the UW 228-3 tumour cells and normal foetal brain stem cells were easily distinguished permitting assessment of their numbers and viability. A concentration of etoposide of ∼ 10 µM was shown to maximise tumour cytotoxicity (5% viability), while sustaining a four-fold higher viability of human foetal brain cells. This concentration exceeds that achievable, clinically, by systemic administration by ∼10 fold and the AUC(-0-48) has been selected in the calculation of a ceiling concentration for a phase 1 trial of infusional intra-CSF etoposide, seeking to assess feasibility and safety of sustained intra-CSF drug delivery to leptomeningeal metastases. CONCLUSIONS: This proof-of-concept study offers the opportunity to perform relevant, pre-clinical assessment of novel drug delivery systems targeting brain tumours, with high clinical relevance.
... hydrophilic/hydrophobic balance of the grafted copolymers was fundamental in the encapsulation efficiency and drug release [37,38,]. To further remark on the versatility of the acylated PGA modification, Tchoryk et al. developed a revolutionary method to monitor NP penetration through 3D spheroid cell cultures [39], building on previous work in 3D models carried out by Meng et al. [40,41] Interestingly, NPs of around 100 nm, prepared from PGA-C18 end functionalized with a PEGylated chain, showed the same penetration as much smaller (50 nm) commercial model polystyrene NPs, suggesting the advantages of material flexibility shown by PGA. This flexibility effect, combined with the in vivo distribution of PGA NPs [7] and the exceptional ability of acyl-PGA alterations to encapsulate hydrophobic and hydrophilic drugs, opens up a wide range of possible applications in drug delivery [39]. ...
... In addition, the MTX nanoparticles showed 7 times higher toxicity to Saos-2 cells than MTX in 2D and 3D cell experiments (Scheme 1) [46]. These easily fabricated PGA-MTX conjugates, where the enzymatic degradability has replaced the need for To further remark on the versatility of the acylated PGA modification, Tchoryk et al. developed a revolutionary method to monitor NP penetration through 3D spheroid cell cultures [39], building on previous work in 3D models carried out by Meng et al. [40,41] Interestingly, NPs of around 100 nm, prepared from PGA-C 18 end functionalized with a PEGylated chain, showed the same penetration as much smaller (50 nm) commercial model polystyrene NPs, suggesting the advantages of material flexibility shown by PGA. This flexibility effect, combined with the in vivo distribution of PGA NPs [7] and the exceptional ability of acyl-PGA alterations to encapsulate hydrophobic and hydrophilic drugs, opens up a wide range of possible applications in drug delivery [39]. ...
Article
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The enzymatically synthesized poly (glycerol adipate) (PGA) has demonstrated all the desirable key properties required from a performing biomaterial to be considered a versatile "polymeric-tool" in the broad field of drug delivery. The step-growth polymerization pathway catalyzed by lipase generates a highly functionalizable platform while avoiding tedious steps of protection and deprotection. Synthesis requires only minor purification steps and uses cheap and readily available reagents. The final polymeric material is biodegradable, biocompatible and intrinsically amphiphilic, with a good propensity to self-assemble into nanoparticles (NPs). The free hydroxyl group lends itself to a variety of chemical derivatizations via simple reaction pathways which alter its physico-chemical properties with a possibility to generate an endless number of possible active macromolecules. The present work aims to summarize the available literature about PGA synthesis, architecture alterations, chemical modifications and its application in drug and gene delivery as a versatile carrier. Following on from this, the evolution of the concept of enzymatically-degradable PGA-drug conjugation has been explored, reporting recent examples in the literature.
... An increasing number of references relating to drug delivery have applied multicellular spheroid models for assessing various drug delivery systems. Our previous study 25 indicated selectivity of fluorescent dye loaded Poly(glyceroladipate) (PGA) NPs between tumour cells and brain cells in a 3-D in vitro co-culture brain tumour invasion model. However, it lacked evaluation of effect of ECM on cellular uptake and mobility of PGA NPs in various 3-D cell culture models. ...
... [29][30][31] The sections were subsequently incubated with fluorescein horse antimouse IgG(HþL) secondary antibody (1:1000 in Tris-HCl with 1%BSA) for 1 h at room temperature and were observed using fluorescence microscopy. 25 ...
Article
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Nanoparticle (NP) drug delivery systems may potentially enhance the efficacy of therapeutic agents. It is difficult to characterize many important properties of NPs in vivo and therefore attempts have been made to use realistic in vitro multicellular spheroids instead. In this paper, we have evaluated poly(glycerol-adipate) (PGA) NPs as a potential drug carrier for local brain cancer therapy. Various three-dimensional (3-D) cell culture models have been used to investigate the delivery properties of PGA NPs. Tumour cells in 3-D culture showed a much higher level of endocytic uptake of NPs than a mixed normal neonatal brain cell population. Differences in endocytic uptake of NPs in 2-D and 3-D models strongly suggest that it is very important to use in vitro 3-D cell culture models for evaluating this parameter. Tumour penetration of NPs is another important parameter which could be studied in 3-D cell models. The penetration of PGA NPs through 3-D cell culture varied between models, which will therefore require further study to develop useful and realistic in vitro models. Further use of 3-D cell culture models will be of benefit in the future development of new drug delivery systems, particularly for brain cancers which are more difficult to study in vivo.
... In her doctoral thesis Weina Meng was able to demonstrate that fluorescently labelled poly(glycerol adipate) nanoparticles had a 6-times higher uptake in tumour spheroids compared to normal rat brain [177]. The observed enhanced tumour endocytosis effect was strongly dependent on the dimensionality of culture and was only observed in physiologically relevant three-dimensional tissue models [178], [179]. At the same time, another member of the group, Sanyogitta Puri, was working on the incorporation of cytotoxic drugs into poly(glycerol adipate) nanoparticles and achieved reasonable drug loading with a selection of cytotoxic drugs [180]. ...
... The main purpose of the in vitro models was to be able to determine whether a drug delivery system offers selectivity of uptake and cytotoxicity towards medulloblastoma tumours compared to normal brain tissue. Conveniently, Meng et al. had already established some key in vitro models culturing medulloblastoma spheroids onto rat brain slices [177], [178]. These initial models, along with complementary research in the group [204]- [206], were instrumental in showing the importance of dimensionality not only in representing actual tissues rather than simple monolayers but also in altering the behaviour of cells and their propensity to take up nanoparticles. ...
Thesis
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This work describes the creation of a three-dimensional model of the children’s brain tumour medulloblastoma using primarily human cells. This in vitro cell culture model was created as a platform for testing novel drug delivery systems for local delivery in the brain. The aim of the local delivery strategy was to reduce radiotherapy through the use of nanoparticle-based chemotherapy. The nanoparticles would be delivered after surgery in the cavity left by the excised tumour tissue. The model was intended to evaluate the selective cytotoxicity of advanced drug delivery systems towards tumour tissue and the benefit of nanoparticle therapy compared to free drug. Normal tissue was modelled using human foetal brain tissue and tumour tissue was represented by a variety of medulloblastoma cell lines. Both were cultured as three-dimensional spheroids free of artificial matrix in ultra-low attachment plates. The tumour and normal cells could be cultured either separately or together and the viability for each cell population determined using a battery of methods. Co-cultures of both cell types had the additional benefit of mimicking the interaction between normal and tumour tissue. The use of physiologically relevant single and co-culture in vitro models could provide information on the relative safety and efficacy of novel brain tumour treatments. The high-throughput platforms used, the algorithms and the validation of a battery of tests in 3D may be extrapolated to other cancer models as well. Moreover the universal marking procedure employed can be employed to label, culture and analyse any two cell types, while preserving tissue heterogeneity and viability. The key benefit from this thesis is the framework for designing in vitro models of tumours that include normal tissue as an internal control. This is an important contribution that can substantiate IC50 values by putting them in the context of drug safety and efficacy. It also highlights the minimum checks and feasibility experiments that need to be done before an in vitro assay is accepted for 3D spheroids.
... Gliomas account for 80% of intracranial primary malignant tumors. The invasion process of malignant gliomas is complicated and consecutive (1,2). Glioblastoma multiforme cells are highly motile and not only often invade the normal brain parenchyma but can also migrate to distant sites along white matter tracts and blood vessels (3)(4)(5). ...
Article
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Although glioblastomas infiltrate diffusely into adjacent brain, it is difficult to unequivocally identify the solitary invading glioma cell. It is necessary to develop coculture models to study the motility of glioma cells, and to monitor the cellular morphology, movement direction, migration area and invasion rate. Cerebral slices were cultured on Millicell-CM membrane inserts in a petri dish. The neuronal viability and organizational structure of the brain sections were well maintained by experimental verification. C6 cell clones with persistent enhanced green fluorescent protein (EGFP) expression were established. EGFP-expressing glioma cells were cultured to form aggregates, which were implanted on the brain slices. The invasion area and migration rates of C6 cells on brain slices were measured. We evaluated the invasion area and depth after C6 cells were treated with the Rac1 inhibitor NSC23766. We successfully established the glioma cell-brain slice coculture model. In coculture, the average migration rate of C6 glioma cells within brain slices reached 11.36-15.27 μm/hour. The polarity of C6 glioma cells was parallel to the white matter tracts after 7 days. The invasive ability of C6 cells (depth: 105.3 ± 10.3 μm) treated with NSC23766 was weakened compared with the control group (depth: 198 ± 9.2 μm) within the white matter of brain slices (t = 16.26, p<0.05). We developed the model to analyze the invasion features of glioma cells. The significant suppression of glioma cell invasion by NSC23766 in brain slices indicates that anti-Rac1 treatment may represent an important future therapeutic strategy for glioblastoma.
... Applications of co-culture models are useful for investigating of the specificity of nanoparticle carriers. For example, Garnett and coworkers developed a brain tumor co-culture model consisting of cancerous cell aggregates and organotypic brain slices (50). Evaluation of the uptake of poly (glycerol-adipate) nanoparticles revealed that in 3-D co-cultures, nanoparticles were predominantly taken up by cancerous cells rather than normal cells. ...
Article
Nanoparticle carriers are attractive vehicles for a variety of drug delivery applications. In order to evaluate nanoparticle formulations for biological efficacy, monolayer cell cultures are typically used as in vitro testing platforms. However, these studies sometimes poorly predict the efficacy of the drug in vivo. The poor in vitro and in vivo correlation may be attributed in part to the inability of two-dimensional cultures to reproduce extracellular barriers, and may also be due to differences in cell phenotype between cells cultured as monolayers and cells in native tissue. In order to more accurately predict in vivo results, it is desirable to test nanoparticle therapeutics in cells cultured in three-dimensional (3-D) models that mimic in vivo conditions. In this review, we discuss some 3-D culture systems that have been used to assess nanoparticle delivery and highlight several implications for nanoparticle design garnered from studies using these systems. While our focus will be on nanoparticle drug formulations, many of the systems discussed here could, or have been, used for the assessment of small molecule or peptide/protein drugs. We also offer some examples of advancements in 3-D culture that could provide even more highly predictive data for designing nanoparticle therapeutics for in vivo applications.
... Of course, as the test tube never fully models the complex workings of in vivo biology, there are many circumstances where in vitro toxicity is not predictive of in vivo results. Three-dimensional cell culture (spheroids) may be better models than monolayer cells for predicting nanoparticle potency/toxicity in vivo, as they may provide conditions which better mimic in vivo tumor cell-nanoparticle interactions, 41 and coculture systems can be used to asses targeting/specificity and to include effects because of interactions with stromal cells. 42 ...
Article
Decreased toxicity via selective delivery of cancer therapeutics to tumors has become a hallmark achievement of nanotechnology. In order to be optimally efficacious, a systemically administered nanomedicine must reach cancer cells in sufficient quantities to elicit a response and assume its active form within the tumor microenvironment (e.g., be taken up by cancer cells and release a toxic component once within the cytosol or nuclei). Most nanomedicines achieve selective tumor accumulation via the enhanced permeability and retention (EPR) effect or a combination of the EPR effect and active targeting to cellular receptors. Here, we review how the fundamental physicochemical properties of a nanomedicine (its size, charge, hydrophobicity, etc.) can dramatically affect its distribution to cancerous tissue, transport across vascular walls, and retention in tumors. We also discuss how nanoparticle characteristics such as stability in the blood and tumor, cleavability of covalently bound components, cancer cell uptake, and cytotoxicity contribute to efficacy once the nanoparticle has reached the tumor's interstitial space. We elaborate on how tumor vascularization and receptor expression vary depending on cancer type, stage of disease, site of implantation, and host species, and review studies which have demonstrated that these variations affect tumor response to nanomedicines. Finally, we show how knowledge of these properties (both of the nanoparticle and the cancer/tumor under study) can be used to design meaningful in vivo tests to evaluate nanoparticle efficacy. WIREs Nanomed Nanobiotechnol 2010 2 99–112 For further resources related to this article, please visit the WIREs website.
... Interestingly, cocultures of lung epithelial cells with mast cells and macrophages gave a different cytokine profile than a similar coculture that also included an endothelial layer, suggesting that even amongst cocultures, the interplay between multiple cell types is evident, increasing the validity of coculture systems over conventional monoculture (Alfaro-Moreno et al., 2008). While the effects of nanoparticle exposure on coculture of pulmonary cells are the most prevalent, coculture models have also been adopted to determine the effect of nanomaterial exposure on other organ systems, such as the brain and intestinal tract, with similar congruent results to in vivo studies (Bouwmeester et al., 2011; Meng et al., 2007). Although there are currently no published coculture studies of MWCNT, the lack of association between results obtained in vitro and results obtained in vivo suggests that more relevant concentrations, nanoparticle dispersion, mechanisms, and methods of exposure must be incorporated into in vitro techniques to study MWCNT toxicity. ...
Article
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Nanotechnology is a rapidly expanding field with wide application for industrial and medical use; therefore, understanding the toxicity of engineered nanomaterials is critical for their commercialization. While short-term in vivo studies have been performed to understand the toxicity profile of various nanomaterials, there is a current effort to shift toxicological testing from in vivo observational models to predictive and high-throughput in vitro models. However, conventional monoculture results of nanoparticle exposure are often disparate and not predictive of in vivo toxic effects. A coculture system of multiple cell types allows for cross-talk between cells and better mimics the in vivo environment. This review proposes that advanced coculture models, combined with integrated analysis of genome-wide in vivo and in vitro toxicogenomic data, may lead to development of predictive multigene expression-based models to better determine toxicity profiles of nanomaterials and consequent potential human health risk due to exposure to these compounds.
... To facilitate co-culture, tumour spheres were generated after harvesting cells from monolayer cell culture. For DAOY cells, 0.5-1 × 10 6 cells were cultured in 10 ml complete media in 25 ml screw-top culture flasks and maintained at constant rotation of 70 rev/min on an orbital shaker, at 37°C until tumour spheres were obtained at 24 hr [24]. ICb1299 cells were cultured at 37°C in ultralow cluster 6-well plate (Costar) in Dulbecco's MEM (Invitrogen) supplemented with F12, EGF, FGF, B27 and penicillin-streptomycin until tumour spheres formed at 48 hr [25]. ...
Article
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Medulloblastoma is the most common intracranial childhood malignancy and a genetically heterogeneous disease. Despite recent advances, current therapeutic approaches are still associated with high morbidity and mortality. Recent molecular profiling has suggested the stratification of medulloblastoma from one single disease into four distinct subgroups namely: WNT Group (best prognosis), SHH Group (intermediate prognosis), Group 3 (worst prognosis) and Group 4 (intermediate prognosis). BMI1 is a Polycomb group repressor complex gene overexpressed across medulloblastoma subgroups but most significantly in Group 4 tumours. Bone morphogenetic proteins are morphogens belonging to TGF-beta superfamily of growth factors, known to inhibit medulloblastoma cell proliferation and induce apoptosis. Here we demonstrate that human medulloblastoma of Group 4 characterised by the greatest overexpression of BMI1, also display deregulation of cell adhesion molecules. We show that BMI1 controls intraparenchymal invasion in a novel xenograft model of human MB of Group 4, while in vitro assays highlight that cell adhesion and motility are controlled by BMI1 in a BMP dependent manner. BMI1 controls MB cell migration and invasion through repression of the BMP pathway, raising the possibility that BMI1 could be used as a biomarker to identify groups of patients who may benefit from a treatment with BMP agonists.
... On the other hand, the difficult reproduction of the disease in animal models demands intermediate stages to evaluate potentially useful therapeutics. In this framework, Meng and co-workers developed an in vitro 3D model of brain tumors [207,208]. The next challenge would be the development of an appropriate xenograft model. ...
Article
Nanotechnology has become a key tool to overcome the main (bio)pharmaceutical drawbacks of drugs and to enable their passive or active targeting to specific cells and tissues. Pediatric therapies usually rely on the previous clinical experience in adults. However, there exists scientific evidence that drug pharmacokinetics and pharmacodynamics in children differ from those in adults. For example, the interaction of specific drugs with their target receptors undergoes changes over the maturation of the different organs and systems. A similar phenomenon is observed for toxicity and adverse effects. Thus, it is clear that the treatment of disease in children cannot be simplified to the direct adjustment of the dose to the body weight/surface. In this context, the implementation of innovative technologies (e.g., nanotechnology) in the pediatric population becomes extremely challenging. The present article overviews the different attempts to use nanotechnology to treat diseases in the pediatric population. Due to the relevance, though limited available literature on the matter, we initially describe from preliminary in vitro studies to preclinical and clinical trials aiming to treat pediatric infectious diseases and pediatric solid tumors by means of nanotechnology. Then, the perspectives of pediatric nanomedicine are discussed.
... Another variation of 3D culture for nanomedicine application is the combination of cell cancer cell spheroids with explant cultures of healthy tissue. Meng et al. cultured medulloblastoma-derived cell spheroids on healthy rat cerebellum slices and found that these cells internalized poly(glycerol-adipate) nanoparticles while the healthy brain cells in the cerebellum slices did not [142]. ...
... Poly(glycerol adipate) (PGA) has recently been introduced in the pharmaceutical and biomedical areas due to its versatility, biocompatibility, enzymatic biodegradability and well-suited characteristics for human use. (Meng et al., 2006;Meng et al., 2007;Puri et al., 2008;Kallinteri et al., 2005) The structure of PGA can be modified using simple reactions due to an availability of free hydroxyl groups pendant along each repeating unit. The literature reported the modification of PGA to customize the physicochemical and mechanical properties of PGA, for instance, fatty acids Naolou et al., 2015;, N-acyl amino acids (Sagnelli et al., 2019;Swainson et al., 2019;, non-steroidal anti-inflammatory drugs (Wersig et al., 2017;Gordhan et al., 2020), methotrexate (Suksiriworapong et al., 2018), and fluorescent dyes Vestri et al., 2020). ...
Article
The study demonstrated the fabrication of new poly(glycerol adipate) (PGA) nanoparticles decorated with folic acid (FOL-PGA) and triphenylphonium (TPP-PGA) and the potential on the delivery of acetogenin-enriched Annona muricata Linn leaf extract to ovarian cancer cells. FOL-PGA and TPP-PGA were successfully synthesized and used to fabricate FOL-decorated nanoparticles (FOL-NPs) and FOL-/TPP- decorated nanoparticles (FOL/TPP-NPs) by blending two polymers at a mass ratio of 1:1. All nanoparticles had small size of around 100 nm, narrow size distribution and high negative surface charge about -30 mV. The stable FOL/TPP-NPs showed highest drug loading of 14.9±1.9% at 1:5 ratio of extract to polymer and reached to 35.8±2.1% at higher ratio. Both nanoparticles released the extract in a biphasic sustained release manner over 5 days. The toxicity of the extract to SKOV3 cells was potentiated by FOL-NPs and FOL/TPP-NPs by 2.0 – 2.6 fold through induction of cell apoptosis. FOL/TPP-NPs showed lower IC50 and higher cellular uptake as compared to FOL-NPs. FOL-NPs exhibited folate receptor-mediated endocytosis. FOL/TPP-NPs provided more advantages than FOL-NPs in terms of stability in physiological fluid, uptake efficiency and targeting ability to mitochondria and showed a promising potential PGA platform for targeted delivery of herbal cytotoxic extracts.
... Owing to the possibilities to control their physicochemical properties, these poly(glycerol adipate) microparticles have tremendous potential to behave as advanced materials for various biological applications. Meng et al. studied the uptake of functionalized poly(glycerol adipate) nanoparticles in a three-dimensional brain tumor cell culture model and observed the preferential uptake of nanoparticles into DAOY tumor spheroids as compared to similar mixed neonatal normal brain cells of the rat [80,81]. Stearic acid-functionalized poly(glycerol adipate) nanoparticles coated with N-(2-hydroxypropyl)methacrylamide via covalent and non-covalent interactions and double labelled with fluorescent dyes were studied for non-invasive tracking of nanoparticles by multi-spectral fluorescence imaging [82]. ...
Article
Amphiphilic polymeric nanocarriers have attained immense attention for transporting drugs and other bioactive species to the living systems owing to their high loading capacity and efficient internalization. To avoid the side-effects of undesired interactions within the biological system, the use of biocompatible building blocks is most crucial in preparing polymeric amphiphiles. The excellent biocompatibility and multivalency of both glycerol and triglycerol make them suitable monomers to construct macromolecular skeletons. Besides these, easy availability and remarkable aqueous solubility further enlarge their use in synthetic chemistry and give ample possibilities for creating fascinating entities for practical applications. The conversion of glycerol into azido-glycerol and azido-triglycerol further helped in differential functionalization of their polymers with various hydrophobic and hydrophilic groups in different ratios thereby assisting in tuning the amphiphilicity of resulting functionalized polymers. Herein, we review the enzymatic synthesis of glycerol, azido-glycerol and azido-triglycerol based amphiphilic polymeric architectures as nanocarriers for various bio-active species. Enzymatically synthesized linear copolymers synthesized by selective esterification of primary hydroxyl groups of glycerol and its derivatives with suitable diacids/diesters are explored in this review.
... Emerging in vitro cell co-culture system has, therefore, gradually attracted increasing attention within the fields of nanomedicine and toxicology because of the increasing demand for achieving more meaningful results that can better reflect the in vivo condition [16]. Indeed, co-culture systems have been proven to exhibit a realistic situation in mimicking healthy and diseased tissue states [17] and have been reliably utilized in NP cellular uptake and drug absorption studies [18][19][20][21]. Utilization of co-culture cancer cell and immune cell models generally offers a suitable platform for probing the uptake routes and mechanisms of these nanomaterials into cells, which may facilitate the design of nanocarriers that are better targeted to cancerous cells while simultaneously reducing NP phagocytosis. ...
Article
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Clinical translation of poly (lactic-co-glycolic acid) (PLGA)-based nanomedicine is limited, partly because of the poor delivery efficiency resulting from non-specific phagocytosis by phagocytes. Understanding the nanoparticle interplay between cancer cells and immune cells remains largely elusive. In this study, a quantitative investigation on cellular internalization of fluorescent PLGA particles (100 nm, 500 nm, and 1 µm) against laryngeal carcinoma cells with or without monocytes/macrophages in monoculture or co-culture systems was first performed. PLGA particles at concentrations of 5–20 µg/mL show superior biocompatibility except for 500 nm and 1 µm PLGA particles at 20 µg/mL slightly reduce cell viability. Microscopic observation has discovered all three sizes of particles are effectively ingested by both cancer cells and macrophages; however, quantitative fluorescence examination has disclosed that the uptake index of cancer cells (mean intracellular particle fluorescence per cancer cell normalized to that of per macrophage) is substantially declined for all PLGA particles in co-cultures compared to that in monocultures (1.35–1.05, 1.50–0.59, and 1.4–0.47 for 100 nm, 500 nm, and 1 µm particles, respectively). Quantitative analysis using flow cytometry further confirmed the reduced uptake index of cancer cells in co-cultures, but higher particle counts per macrophage. It has also been found that the formation of multinucleated giant cells via the fusion of macrophages increased after PLGA treatment, which could be further exploited as a potential approach for tumor drug delivery. Overall, these findings provide new insights into the interaction of nanoparticle-immune-cancer cells, which may facilitate the application of PLGA-based nanocarriers for the treatment of laryngeal carcinoma.
... They possess the in vivo-like architecture, mix of differentiated cells in the right spatial organisation and the native extracellular matrix of the brain. These advantages have stimulated their use in experiments modelling tumour invasion and metastasis in glioma (Aaberg-Jessen et al., 2013;Jung et al., 2002) as well as drug delivery in medulloblastoma (Meng et al., 2016(Meng et al., , 2007. They have also been utilised to study the effects of chemotherapy on the normal brain (Nørregaard et al., 2012). ...
Article
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The recently-defined four molecular subgroups of medulloblastoma have required updating of our understanding of in vitro models to include molecular classification and risk stratification features from clinical practice. This review seeks to build a more comprehensive picture of the in vitro systems available for modelling medulloblastoma. The subtype classification and molecular characterisation for over 40 medulloblastoma cell-lines has been compiled, making it possible to identify the strengths and weaknesses in current model systems. Less than half (18/44) of established medulloblastoma cell-lines have been subgrouped. The majority of the subgrouped cell-lines (11/18) are Group 3 with MYC-amplification. SHH cell-lines are the next most common (4/18), half of which exhibit TP53 mutation. WNT and Group 4 subgroups, accounting for 50% of patients, remain underrepresented with 1 and 2 cell-lines respectively. In vitro modelling relies not only on incorporating appropriate tumour cells, but also on using systems with the relevant tissue architecture and phenotype as well as normal tissues. Novel ways of improving the clinical relevance of in vitro models are reviewed, focusing on 3D cell culture, extracellular matrix, co-cultures with normal cells and organotypic slices. This paper champions the establishment of a collaborative online-database and linked cell-bank to catalyse preclinical medulloblastoma research.
... Ceux-ci sont résumés dans le Tableau I-7. ---- [249][250][251] Tableau I-7 : Différents systèmes 3D avantages et inconvénients [213,226,252] L'un des modèles les plus viables reste le vivant ou l'explant qui n'est autre qu'un morceau de tissu vivant remis en culture et maintenu, cependant ce genre de culture est très complexe à mettre en oeuvre et très difficile à se procurer surtout dans le travail chez l'humain comme il est question ici. ...
Thesis
L’objectif de cette thèse était de mettre au point une analyse critique de vecteurs polymères utilisés pour la thérapie photodynamique (PDT) et de faire le lien avec l’efficacité thérapeutique observée. Pour cela, une analyse complète des vecteurs a été réalisée par des techniques classiques comme la diffusion dynamique de la lumière ou la microscopie électronique, mais aussi grâce au fractionnement flux-force, technique peu utilisée jusqu’à présent dans le domaine des auto-assemblages polymères. Dans un deuxième temps, les auto-assemblages ont été utilisés comme vecteurs d’un photosensibilisateur, le Phéophorbide a, et l’efficacité thérapeutique évaluée en travaillant sur culture cellulaire 2D et 3D de lignées HCT116 (cancer du colon) ou FaDu (cancer tête et cou). Différents vecteurs polymères simples ont tout d’abord été examinés, à savoir des micelles ou des polymersomes à base de copolymères diblocs amphiphiles comme le poly(oxyde d’éthylène-b--caprolactone), le poly(oxyde d’éthylène-b-lactide) ou le poly(oxyde d’éthylène-b-styrène). Ceci a permis d’obtenir des vecteurs présentant des tailles et des morphologies variables. Les résultats en PDT ont montré des comportements différents et une meilleure efficacité en 3D pour les systèmes à base de PEO-PDLLA. La technique de fractionnement flux-force asymétrique (AsFlFFF) a particulièrement été utilisée pour ces vecteurs afin de démontrer la pureté des auto-assemblages. Les connaissances acquises dans cette première partie ont permis de caractériser des vecteurs faits à base de mélanges d’auto-assemblages micelles/vésicules. Ceux-ci ont révélé des phénomènes d’antagonisme ou de synergie dans l’efficacité en PDT, démontrant l’existence de processus complexes au niveau de la réponse cellulaire.Des auto-assemblages figés par réticulation ont aussi été développés, caractérisés et examinés en PDT. Ils se sont avérés extrêmement intéressants pour la PDT sur les cultures cellulaires en 3D, démontrant une efficacité accrue comparée aux systèmes simples. La comparaison de ces résultats avec ceux obtenus en culture 2D pour les mêmes objets a de plus permis de mettre en évidence la différence entre ces deux modèles biologiques. Enfin, des auto-assemblages à base de complexes poly-ioniques ont aussi été formés et caractérisés. Le fractionnement flux-force s’est là encore avéré efficace, mais a nécessité l’utilisation d’une injection spéciale par Frit-inlet. Leur efficacité en PDT s’est avérée faible.
... The cellular uptake of NPs (endosomal/lysosomal compartment) was dependent on the incubation time and the dose of NPs and it was found that the internalization of RBITC-labelled NPs was mostly mediated by an endocytic process. Furthermore, agreeing with results published elsewhere, tumor cells had higher rates of NP uptake than normal host cells [60,61]. After uptake, the release of RBITC from NPs within cells was about 30-40 times faster than the release in cell culture medium, suggesting a rapid breakdown of nanoparticles within the lysosomal compartment of cells. ...
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Investments in systems biology approaches by the pharmaceutical industry have not yet yielded the payoffs envisaged by many. In most cases, a plethora of novel drug targets arising from genomics led to many more failed projects in the pipeline, suggesting that target-based drug discovery may not be an optimal strategy for the industry. Before high-throughput “-omics” technologies and computer analysis became commonplace, most drug candidates were laboriously screened in animal systems to identify compounds that produced useful responses. Interestingly, the targets of many of the compounds that became drugs are still uncertain to this day. It is likely that drugs act on multiple targets in concert over time, the identification of which will require not only system level cataloguing and measurements, but next generation multiscale systems modelling. The concept of a “differentiated drug response” — elucidating and integrating responses composed of a range of effects on different tissues and, importantly, different time scales — may eventually prove to be the dominant paradigm of systems biology research. In this article, we explore key relevant concepts and technologies that we believe are critical for the future of systems biology and its place in pharmaceutical research.
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We investigate antitumor efficacy and 2D and 3D intratumoral distribution of 7-ethyl-10-hydroxycamptothecin (SN-38) from polymeric depots inside U-87MG xenograft tumor model in nude mice. Results showed that polymeric depots could be used to administer and controlled release of a large amount of SN-38 directly to the brain tumor model. SN-38 released from depots suppressed tumor growth, where the extent of suppression greatly depended on doses and the number of depot injections. Tumor suppression of SN-38 from depots was three-fold higher in animals which received double injections of depots at high dose (9.7 mg of SN-38) compared to single injection (2.2 mg). H&E staining of tumor sections showed that the area of tumor cell death/survival of the former group was two-fold higher than those of the latter group. Fluorescence imaging based on self-fluorescent property of SN-38 was used to evaluate the intratumoral distribution of this drug compared to histological results. The linear correlation between fluorescence intensity and the amount of SN-38 allowed quantitative determination of SN-38 in tumor tissues. Results clearly showed direct correlation between the amount of SN-38 in tumor sections and cancer cell death. Moreover, 3D reconstruction representing the distribution of SN-38 in tumors was obtained. Results from this study suggest the rationale for intratumoral drug administration and release of drugs inside tumor, which is necessary to design drug delivery systems with efficient antitumor activity. © 2015 by the Society for Experimental Biology and Medicine.
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The tumor microenvironment (TME) plays key roles in tumor development, progressive and metastasis. Recently, therapy strategies based on TME targeted modulation shows potential to improve the efficacy of therapeutics, which attracted much attention in pediatric brain cancer therapy. Moreover, the advanced technology on carrier design and drug formulation improved the penetrated efficiency and TME specific targeting delivery of nanoparticles for TME specific modulation. In this review, we briefly introduce the microenvironment substitutes in medulloblastoma (MB) including dendritic cells, macrophages, tumor vasculature and tumor-draining lymph nodes. In addition, we review various nano-based drug delivery strategies targeting these TME components and their applications in tumor therapy.
Chapter
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Investments in systems biology approaches by the pharmaceutical industry have not yet yielded the payoffs envisaged by many. In most cases, a plethora of novel drug targets arising from genomics led to many more failed projects in the pipeline, suggesting that target-based drug discovery may not be an optimal strategy for the industry. Before high-throughput '-omics' technologies and computer analysis became commonplace, most drug candidates were laboriously screened in animal systems to identify compounds that produced useful responses. Interestingly, the targets of many of the compounds that became drugs are still uncertain to this day. It is likely that drugs act on multiple targets in concert over time, the identification of which will require not only system level cataloguing and measurements, but next generation multiscale systems modelling. The concept of a 'differentiated drug response'- elucidating and integrating responses composed of a range of effects on different tissues and, importantly, different time scales - may eventually prove to be the dominant paradigm of systems biology research. In this article, we explore key relevant concepts and technologies that we believe are critical for the future of systems biology and its place in pharmaceutical research.
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Local invasion of the brain by neoplastic glial cells is a major obstacle to effective treatment of intrinsic brain tumors. Invasion is directly related to histologic malignancy, but occurs to some extent irrespective of tumor grade. Because the brain-to-tumor interface is not well demarcated, total surgical removal is rarely possible; moreover, as invading cells transiently arrest from cell division they are refractory to radiotherapeutic intervention. Invading cells may also be protected from the action of cytotoxic drugs by the presence of an intact blood-brain barrier. The invading cells, having migrated several millimeters or even centimeters from the main focus of the tumor, return to cycle phase under the control of some as yet unknown microenvironmental cue to form a recurrent tumor adjacent to the original site of presentation. Recent cellular and genetic information concerning factors underlying invasion may not only yield suitable targets for adaptation of existing therapies, but may also lead to novel approaches in glioma management.
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Little is known about the genetic regulation of medulloblastoma dissemination, but metastatic medulloblastoma is highly associated with poor outcome. We obtained expression profiles of 23 primary medulloblastomas clinically designated as either metastatic (M+) or non-metastatic (M0) and identified 85 genes whose expression differed significantly between classes. Using a class prediction algorithm based on these genes and a leave-one-out approach, we assigned sample class to these tumors (M+ or M0) with 72% accuracy and to four additional independent tumors with 100% accuracy. We also assigned the metastatic medulloblastoma cell line Daoy to the metastatic class. Notably, platelet-derived growth factor receptor alpha (PDGFRA) and members of the downstream RAS/mitogen-activated protein kinase (MAPK) signal transduction pathway are upregulated in M+ tumors. Immunohistochemical validation on an independent set of tumors shows significant overexpression of PDGFRA in M+ tumors compared to M0 tumors. Using in vitro assays, we show that platelet-derived growth factor alpha (PDGFA) enhances medulloblastoma migration and increases downstream MAP2K1 (MEK1), MAP2K2 (MEK2), MAPK1 (p42 MAPK) and MAPK3 (p44 MAPK) phosphorylation in a dose-dependent manner. Neutralizing antibodies to PDGFRA blocks MAP2K1, MAP2K2 and MAPK1/3 phosphorylation, whereas U0126, a highly specific inhibitor of MAP2K1 and MAP2K2, also blocks MAPK1/3. Both inhibit migration and prevent PDGFA-stimulated migration. These results provide the first insight into the genetic regulation of medulloblastoma metastasis and are the first to suggest a role for PDGFRA and the RAS/MAPK signaling pathway in medulloblastoma metastasis. Inhibitors of PDGFRA and RAS proteins should therefore be considered for investigation as possible novel therapeutic strategies against medulloblastoma.
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The process of metastasis consists of a series of linked, sequential steps that must be completed by tumour cells if a metastasis is to develop. Although some of the steps in this process contain stochastic elements, metastasis as a whole favors the survival and growth of a few subpopulations of tumour cells that pre-exist within the heterogeneous parent neoplasm. Metastases can have a clonal origin, and different metastases can originate from the proliferation of single cells. The outcome of metastasis depends on the interaction of metastatic cells with different organ environments. Organ-specific metastases have been demonstrated in a variety of experimental tumour systems, and tumour growth can be specific to a particular site within one organ. The factors that regulate metastasis are the intrinsic properties of some tumour cells and host factors involved in homeostasis. Understanding these factors should allow for the development of more effective of cancer metastasis.
Conference Paper
BACKGROUND: Conventional interpretation of methionine PET (Met-PET) scans in suspected brain tumors uses the ratio of the tracer uptake within the lesion to the corresponding contralateral area. The precise location at which the region of interest used to calculate the reference value is placed is vital, because local variations in methionine uptake may significantly alter the calculated ratio. Identifying a precise mirror region is complicated by the distorting effect of the tumor and the need for manual realignment of the image. METHOD: Patients with low-grade primary brain tumors or benignlesions were identified on the basis of a tissue diagnosis or surveillance neuroimaging that excluded a high-grade tumor. These conditions were selected because they primarily involve a single hemisphere, with methionine uptake in the unaffected hemisphere being essentially normal. A total of 180 Met-PET scans performed during 2003 – 2005 were identified from the database at the Max Planck Institute for Neurological Research, coded, and anonymized for analysis. Scans demonstrating midline lesions, significant mass effect, or evidence of substantial previous surgery were then excluded. A methionine template was prepared using data from patients who had undergone both FDG and Met-PET scans within eight weeks, with normalization to a previously developed FDG template. Methionine scans were coregistered to the template, after masking of any tumor, and the diseased hemispheres stripped. Mean uptake maps for each hemisphere were calculated on a voxel-by-voxel basis and merged to create the normal methionine uptake map. Scans unsuitable for inclusion into the normal map were reanalyzed using the contralateral hemisphere and the normal uptake map for reference values, allowing the methods to be compared. RESULTS: Good correlation was found between uptake ratios using reference values calculated by both methods. Reference values could be reliably calculated in tumors that were previously problematic to analyze, such as those that cross the midline. Coregistration of the normal map was impaired in some cases by loss of the normal architecture, but valid reference values were obtained despite this. CONCLUSION: Use of a normal uptake may facilitate calculation of PET uptake ratios in brain tumors. Further research is required to evaluate the correlation with histological findings and the accuracy of image coregistration in the presence of distorting tumors.
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Intrinsic tumours of the central nervous system (CNS) are set apart from solid, non-neural primary neoplasms in that, although they seldom metastasize to distant organs, they are generally characterised by a diffuse local invasive pattern. Indeed, it is this important biological characteristic which precludes successful therapeutic intervention in the majority of brain and spinal cord neoplasms. While tumours metastasising to the brain are generally well-circumscribed lesions, sub-populations of neoplastic cells from intrinsic, neuroectodermal tumours may migrate several millimetres away from the brain/tumour interface, resulting in a poor demarcation of the neoplasm. These migratory cells give rise to recurrent tumours following surgical and adjuvant chemo- and radio-therapeutic intervention. The mechanisms which facilitate such migration of neoplastic neural cells into the contiguous normal nervous tissue are poorly documented. However, migration in this context is likely to be a complex multifaceted phenomenon involving cell/cell and cell/extracellular matrix (ECM) adhesion, locomotion, angiogenesis- and enzymic degradation of the ECM. In particular, cell adhesion molecules, ganglio-sides, paracrine and autocrine growth and motility factors and matrix metalloproteinases (MMPs) and their inhibitors probably ail play important and inter-dependent roles in the migration of neoplastic neural cells.
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The plasminogen activation (PA) system plays an important role in tumor invasion by initiating pericellular proteolysis of the extracellular matrix (ECM) and inducing cell migration. Malignant brain tumors overexpress PA members and characteristically invade by migrating on ECM-producing white matter tracts and blood vessel walls. To determine whether urokinase-type plasminogen activator (uPA) and its receptor (uPAR) directly modulate the migration of brain tumor cells, we examined six human brain tumor cell lines, 2 astrocytomas (SW1088, SW1783), 2 medullobastomas (Daoy, D341Med), and 2 glioblastomas (U87MG, U118MG), for their surface uPAR expression, endogenous PA activity, and functional proteolytic activity by an ECM-degradation assay. Migration on Transwell membranes and invasion of Matrigel was then tested by pre-incubating the cells with increasing concentrations of either uPA, the proteolytically inactive amino-terminal fragment (ATF) of uPA, or the uPAR cleaving enzyme, phosphatidylinositol-specific phospholipase C (PI-PLC). All of the cell lines, except D341Med, express surface uPAR protein and uPA activity. High levels of uPAR and uPA activity correlated with cellular degradation of ECM, cell migration, and Matrigel invasion. Cell migration and invasion were enhanced by uPA or ATF in a dose dependent manner, while PI-PLC treatment abolished the uPA effect and inhibited migration and invasion. We conclude that ligation of uPAR by uPA directly induces brain tumor cell migration, independent of uPA-mediated proteolysis; and in concert with ECM degradation, markedly enhances invasion. Conversely, removing membrane bound uPAR from the surface of the cells studied inhibited their ability to migrate and invade even in the presence of proteolytically active uPA.
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Several model tumour systems are now known to display increased vascular permeability compared with normal tissues, permitting their selective targeting using macromolecular drugs. Preliminary clinical observations suggest that this pathology may be reflected in at least some types of human cancer, and this may have important implications in facilitating macromolecular drug treatments, including antibody targeting and delivery of DNA for gene therapy. The enhanced permeability of tumour vasculature is thought to be regulated by tumour-secreted growth factors, with vascular permeability facor (VPF), also known as vascular endothelial growth factor (VEGF), emerging as a particularly likely candidate. VPF/VEGF is known to be an important regulator of tumour-angiogenesis in vivo, and it exerts its endothelium-specific effects via its receptors KDR/Flk-1 and Flt-1 on the endothelial cell membrane. Although the precise mechanism of VEGF's permeabilising action is not yet understood, it is likely to contribute to the enhanced permeability and retention (EPR) effect in tumours which is thought to underlie the anticancer activity of macromolecular drugs.
Article
Glioblastoma multiforme is a malignant primary brain tumor associated with short patient survival in part because of the ability of individual cells to migrate significant distances into brain tissue. Invasion is a difficult process to model, because many such human tumors do not invade immunologically competent animal tissue, tumors grown in animals do not invade human tissue, and relevant human tissue substrates are not easily reproduced. We discuss models for examining invasion in vitro, and in particular review work using the tumor spheroid--fetal rat brain aggregate co-culture model, assessed with confocal microscopy and four-dimensional imaging. Quantitation of invasion in this model is discussed, as well as the invasion-inhibitory properties of tyrosine kinase (TK) inhibitors. The effects of receptor-specific tyrphostins strongly support a dominant role for Epidermal Growth Factor Receptor activation in this process and show that invasion can be effectively inhibited at much lower concentrations of TK inhibitors than is necessary for growth suppression. Inhibition of activation of the purported growth factor receptor second messenger phospholipase C- gamma 1, by pharmacological means and gene transfection, also profoundly inhibits the invasive properties of human glioblastoma and rat C6 glioma cells. We have assessed invasiveness in several human tumor specimens, which may provide information relative to prognosis and recurrence risk. Our data supports the concept of differential control of invasion and proliferation, and points to possible strategies for anti-invasive therapy for glioblastoma multiforme.
Article
Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium. They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator. No plasma clot or roller drum were used. This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization. Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage. Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques. After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced. Evidence for a sprouting response during the first days in culture or following sections is illustrated. This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture.
Article
The invasiveness of human intracranial tumours was studied in an organ culture system. Biopsies from six glioblastomas, four astrocytomas, two mixed gliomas, one ependymoma, four meningiomas and two carcinoma metastases were cut into fragments of 0.5 mm diameter, and placed in agar overlay tissue culture. The tumour specimens formed spheroids which were co-cultured with cell aggregates or fragments from fetal rat brain for up to 10 days in vitro. The invasiveness of the glioblastoma spheroids was characterised by a gradual destruction of normal brain tissue by tumour cells, followed by replacement of normal tissue by these cells. Co-cultures from two glioblastomas showed lesions in the normal brain tissue in areas removed from the tumour cells. Tumour spheroids from four glioblastomas totally destroyed the normal brain tissue without any change in the original tumour spheroid configuration. The low-grade gliomas were less invasive than the glioblastomas. The meningiomas and the metastases were non-invasive. This organ culture assay appeared to reflect the in situ invasive behaviour of the brain tumours examined. It is suggested that it may be used for evaluating the aggressiveness of individual brain tumours with the specific aim of correlating clinical data with the biological character of the tumour.
Article
Abnormal vascularization of malignant tumors is associated with the development of microregions of heterogeneous cells and environments. Experimental models such as multicell spheroids and a variety of new techniques are being used to determine the characteristics of these microregions and to study the interactions of the cells and microenvironments. The special cellular microecology of tumors influences responsiveness to therapeutic agents and has implications for future directions in cancer research.
Article
CEREBELLAR MEDULLOBLASTOMA is one of the most common types of primary intracranial tumors in children. The treatment of this tumor, unfortunately, is considered as “one of the darkest chapters in pediatric neurosurgery” (1). Cushing (2) reported an average survival of five and six-tenths months in 14 patients with medulloblastoma who received no postoperative radiotherapy. Ingraham and Matson (3) stated that “to our knowledge, no patient with cerebellar medulloblastoma has ever been cured.” Although medulloblastoma has long been known as the most sensitive primary brain tumor to ionizing radiation, the radiotherapy results for this tumor with respect to long-term survival is still very disappointing. In order to better evaluate the response of medulloblastoma to irradiation and to improve the survival rate, the operative records of 100 consecutive cases of tissue-proved medulloblastoma at the Neurological Institute, Columbia-Presbyterian Medical Center were arbitrarily taken in a period from 1940 to 1967 ...
Article
The cerebral and cerebellar cortices of man are richly provided with varicose noradrenaline nerve terminals, which are visualized by fluorescence histochemistry of brain smears obtained by a new technique. The density of such nerves in human cortices equals that of the rat. The method permits simple and rapid analysis of noradrenergic nerves of the human cortex during routine neurosurgical operations.
Article
The invasiveness of three medulloblastoma permanent cell lines (D-283, D-341, and DAOY), a human medulloblastoma biopsy, and in addition, a human rhabdomyosarcoma permanent cell line (TE-671), which previously had been regarded as a human medulloblastoma, was studied in an organ co-culture assay. All the four cell lines and the biopsy were co-cultured with normal rat brain cell aggregates for up to six days in vitro. The medulloblastoma biopsy, the D-283 and the D-341 cells invaded the brain tissue by diffuse single cell infiltration. The medulloblastoma cell line (DAOY) showed an invasive pattern similar to that observed earlier for most glioblastoma cell lines. This was characterized by massive cell replacement and destruction of normal brain tissue. The rhabdomyosarcoma cell line (TE-671) presented a solid invasive pattern with a fairly well defined border between normal brain and tumour tissue. Thus, the organ co-culture assay system in vitro seems to mimic several aspects of the in situ invasive behaviour of medulloblastomas. It may, therefore, provide new perspectives for pretreatment investigations with chemotherapy and radiotherapy of these malignancies.
Article
Tumor cells metastasize when they have successfully passed a number of invasion steps. We hypothesize that each step is a microecosystem, the basic elements of which are neoplastic cells, host cells, and extracellular matrix (ECM). We review here molecular pathways with a regulatory function in these microecosystems: homotypic cell-cell adhesion molecules counteracting invasion; complexes of lytic (pro-)enzymes, their receptors and inhibitors regulating focalized breakdown of the ECM; matrix components, their cellular receptors and motility factors governing cell migration; heterotypic cell-cell adhesion molecules initiating extravasation, encompassing in the vasculature the sequence: cell arrest, ECM lysis, cell migration; factors allowing survival and growth at ectopic sites. We conclude that delicate molecular balances within microecosystems are responsible for the temporary and repeated invasion events leading to metastasis.
Article
Slices of CNS tissue prepared from young rodents can be maintained in culture for many weeks to months. The basic requirements are simple: a stable substratum, culture medium, sufficient oxygenation and incubation at a temperature of about 36 degrees C. Under these conditions, nerve cells continue to differentiate and to develop a tissue organization that closely resembles that observed in situ. Several alternative culturing methods have been developed recently. Slices maintained in stationary culture with the interface method are ideally suited for questions requiring a three-dimensional structure, whereas slices cultured in roller-tubes remain the method of choice for experiments that require optimal optical conditions. In this report, three typical experiments are discussed that illustrate the potential of the slice-culture technique. The first example indicates that, due to their high neuronal connectivity, slice cultures provide a very useful tool for studying the properties of synaptic transmission between monosynaptically coupled cell pairs. The other two studies show how long-term application of substances to slice cultures can be used to examine the consequences of epileptic discharges in vitro, as well as the effects of slowly acting clostridial neurotoxins on synaptic transmission.
Article
Since it is difficult to study the dynamic biological aspects of brain tumour invasion using histological sections of surgical biopsy and autopsy tissues, various laboratory systems have been devised. Animal models are less than ideal as chemically-induced brain tumours suffer from the fact that they have a low incidence and a long latency, while transplanted tumours grow predominantly by expansion, due to high proliferative activity, and not by diffuse local invasion as in human brain tumours. Various in vitro assays have, therefore, been established for both migration and invasion. These include the simple scratch technique in a confluent cell monolayer, the use of cloning rings and the "Transwell" modified Boyden chamber technique. More complex, three-dimensional culture model systems have also been developed, using chick heart, optic nerve or reaggregated fetal brain as "targets" for the invasion of neoplastic glia. Each method has yielded important information on the mechanisms which underlie brain tumour invasion. Moreover, individual microenvironmental factors may be modulated in these laboratory systems to determine their influence on the migration/invasion process.
Article
The invasive cellular behavior of malignant gliomas is determined by receptor mediated cell-substratum contacts and cell-cell interaction as well as cellular locomotion. This study attempts to break down the complex phenomena of the invasive process into their components of attachment to neighboring cells, aggregate formation, adhesion to matrix substratum, migration and invasion into three-dimensional cellular aggregates separately analyzed in different in vitro assay systems. Using a panel of 13 glioma cell lines, adhesion to non-specifically or merosin coated surfaces was correlated to monolayer cell migration and dissemination of tumor cells from aggregates plated on these substrates. The formation kinetics of aggregates were determined and compared to the ability of these cells to rapidly attach and form mechanically stable cell-cell contacts. The motility rates in the different assay systems as well as cell-cell attachment was correlated to invasion of re-aggregated tumor cells into fetal rat brain. A tight positive correlation was found for substrate adhesion and monolayer migration. In contrast, cell-substratum contacts had little influence on dissemination of cells out of three-dimensional aggregates and no association between monolayer migration and migration of cells out of aggregates was detected. The ability of glioma cells to rapidly form aggregates was associated with enhanced migration out of aggregates. The capacity to invade fetal rat brain aggregates was correlated with the capacity to form stable intercellular adhesion as measured in a cell-cell adhesion assay. Invasion in this system was not found to be associated with migration in monolayer or with migration out of tumor aggregates. This study highlights that current in vitro assays for invasion only represent isolated aspects of the multi-cascade process which is involved in tumor cell invasion.
Article
Although it is known that malignant astrocytomas infiltrate diffusely into regions of normal brain, it is frequently difficult to identify unequivocally the solitary, invading astrocytoma cell in histopathological preparations or experimental astrocytoma models. The authors describe an experimental system that facilitates the tracking of astrocytoma cells by using nonneoplastic cerebral tissue as the substrate for invasion. Cerebral tissue was cut into 1-mm-thick slices and cultured in the upper chamber of a Transwell culture dish on top of a polyester membrane (0.4-mm pore size) that was bathed in medium supplied by the lower chamber. Two astrocytoma cell lines, U-87 MG (U87) and U343 MG-A (U343), were selected because of their differing basal cell motilities in monolayer cultures. The astrocytoma cells were stably transfected with vectors that expressed green fluorescent protein (GFP), either alone or as a fusion protein with the receptor for hyaluronic acid-mediated motility (RHAMM) in either sense or antisense orientations. Stably transfected clones that had high levels of GFP expression were selected using the direct visualization provided by fluorescence microscopy and fluorescence-activated cell-sorter analysis. The GFP-expressing astrocytoma cell clones were implanted into the center of the brain slice and the degree of astrocytoma invasion into brain tissue was measured at different time points by using the optical sectioning provided by the confocal laser microscope. The authors observed that GFP-expressing astrocytoma cells could be readily tracked and followed in this model system. Individual astrocytoma cells that exhibited green fluorescence could be readily identified following their migration through the brain slices. The GFP-labeled U87 astrocytoma cells migrated farther into the brain slice than the U343 astrocytoma cells. The RHAMM-transfected GFP-labeled astrocytoma cells also infiltrated farther than the GFP-labeled astrocytoma cells themselves. The expression of antisense RHAMM virtually abrogated the invasion of the brain slices by both astrocytoma cell lines. The authors believe that this organotypical culture system may be of considerable utility in studying the process of astrocytoma invasion, not only because it provides a better representation of the extracellular matrix molecules normally encountered by invading astrocytoma cells, but also because the GFP tag enables tracking of highly migratory and invasive astrocytoma cells under direct vision.
Article
The primary cause of local recurrence and therapeutic failure in the treatment of malignant gliomas is the invasion of tumor cells into the surrounding normal brain. While it is known that malignant gliomas infiltrate diffusely into regions of normal brain, it is frequently very difficult to unequivocally identify the solitary invading glioma cell in histopathological preparations, or in experimental glioma models. We have developed an experimental invasion assay system, which allows us to track the solitary invasive glioma cell, using human brain tissue obtained from routine craniotomies for seizures or trauma. This tissue is cut into 1-mm thick slices and cultured in the upper chamber of Transwell culture dishes on top of a 0.4- micro m pore size polyester membrane, which is fed on medium provided in the lower chamber. Glioma cells are stably transfected with vectors containing a green fluorescent protein (GFP) cDNA. Stable, high-level expression GFP transfectants were selected by direct visualization under fluorescence microscope. In addition, various tumor spheroids are stained with vital dye, DiI, to track the invading cells. GFP-expressing glioma cells or stained spheroids were then implanted on the center of the brain slice, and the degree of brain tumor invasion into the brain tissue was evaluated at different time points by optical sectioning using a confocal microscope. We observed that GFP-expressing glioma cells or stained spheroids could be readily tracked and followed with this model system. Individual tumor cells that exhibited green or red fluorescence could be identified and their migration path through the brain slices unequivocally followed. This experimental invasion system may be of considerable utility in studying the process of brain tumor invasion and in evaluating its invasiveness in individual brain tumor because it not only provides a better representation of extracellular matrix molecules normally encountered by invading glioma cells, but also provides the fluorescent tag applied to the tumor cells.
Article
Phospholipid spherules composed of lipid bilayer membranes entrapping a central aqueous core were first described more than 30 years ago (Bangham et al 1965). The term liposome was coined in 1968 (Sessa & Weissmann 1968) and the first suggestions that these vesicles might have potential as vehicles for targeted drug delivery for a range of diseases, including cancer, appeared shortly afterwards (Gregoriades et al 1974; Gregoriades 1976a, b). However, the process of turning this expectation into a clinical reality has suffered a number of setbacks and has taken more than a quarter of a century. In the process, new types of liposomes with favourable in-vivo pharmacokinetics and biodistribution patterns have been generated (Lasic & Papahadjopoulos 1995). Many of these preparations have been subjected to extensive examination and an increasing number of agents have entered clinical trials. In this review, we will trace the development of those liposomes that are currently undergoing (or are about to undergo) clinical evaluation.
Article
The invasion of neoplastic cells into healthy brain tissue is a pathologic hallmark of gliomas and contributes to the failure of current therapeutic modalities (surgery, radiation and chemotherapy). Transformed glial cells share the common attributes of the invasion process, including cell adhesion to extracellular matrix (ECM) components, cell locomotion, and the ability to remodel extracellular space. However, glioma cells have the ability to invade as single cells through the unique environment of the normal central nervous system (CNS). The brain parenchyma has a unique composition, mainly hyaluronan and is devoid of rigid protein barriers composed of collagen, fibronectin and laminin. The integrins and the hyaluronan receptor CD44 are specific adhesion receptors active in glioma-ECM adhesion. These adhesion molecules play a major role in glioma cell-matrix interactions because the neoplastic cells use these receptors to adhere to and migrate along the components of the brain ECM. They also interact with the proteases secreted during glioma progression that degrade ECM allowing tumor cells to spread and diffusely infiltrate the brain parenchyma. The plasminogen activators (PAs), matrix metalloproteinases (MMPs) and lysosomal cysteine peptidases called cathepsins are also induced during the invasive process. Understanding the mechanisms of tumor cell invasion is critical as it plays a central role in glioma progression and failure of current treatment due to tumor recurrence from micro-disseminated disease. This review will focus on the impact of microregional heterogeneity of the ECM on glioma invasion in the normal adult brain and its modifications in tumoral brain.
Article
We have prepared and screened a library of novel functionalized polymers for development of nanoparticle drug delivery systems. The polymer backbone consisting of two ester-linked, nontoxic, biological monomers, glycerol and adipic acid, was prepared using a hydrolytic enzyme. The specificity of the chosen enzyme yields a linear polymer with one free pendant hydroxyl group per repeat unit, which can be further functionalized. This protocol gives control over the backbone polymer molecular weight, together with the ability to incorporate various amounts of different fatty acyl substituents. These functionalized polymers are able to self-assemble into well-defined small particles of high homogeneity with a very low toxicity. They are able to incorporate a water soluble drug, dexamethasone phosphate, with a high efficiency and drug loading which varies with the polymer specification. The above characteristics strongly suggest that these polymers could be developed into useful nanoparticulate drug delivery systems.
Article
A useful route for the development of antitumour therapies is by creating improved methods for delivering therapeutic agents to tumour cells or subcellular compartments and increasing retention of drugs within target cells. In this study, we have characterized nanoparticle (NP) uptake and metabolism by DAOY cells, a human medulloblastoma cell line. NPs were formed from a novel polymer, poly (glycerol-adipate) (PGA), containing Rhodamine B Isothiocyanate (RBITC) as a fluorescent marker. It was observed that the cellular uptake of NPs depends on the incubation time and the concentration of NPs in the culture medium. The studies of retention and metabolism of NPs within cells indicated that 1) faster degradation of NPs within cells compared with that in cell culture medium in vitro; 2) a small fraction of NPs were recycled back to the outside of cell, whereas most NPs entered endosomes and lysosomes; and 3) recycled NPs were re-taken up in the following 2 h incubation time. These studies thus suggested that PGA NPs could be used for localising therapeutic agents into cells, and could provide prolonged drug effects because of their long sustained release in physiological conditions and their rapid release when taken up into cells.
A new in vitro 3-D brain tumour co-culture model used to study nanoparticle uptake and tumour invasion
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Meng W, Kallinteri P, Walker D, Garnett MC, Parker TL. A new in vitro 3-D brain tumour co-culture model used to study nanoparticle uptake and tumour invasion. Neuro Oncol 8:293–372, 2006.
World Health Organisation Classification of Tumours: Pathology and Genetics of Tumours of the Nervous System Lyon
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Kleihues P, Cavenee WK. World Health Organisation Classification of Tumours: Pathology and Genetics of Tumours of the Nervous System Lyon: IARC Press, 2000.