The immunobiology of mushrooms

There has been enormous interest in the biologic activity of mushrooms and innumerable claims have been made that mushrooms have beneficial effects on immune function with subsequent implications for inhibition of tumor growth. The majority of these observations are anecdotal and often lack standardization. However, there remains considerable data on both in vitro and in vivo effects that reflect on the potential of mushroom compounds to influence human immunity. A number of these effects are beneficial but, unfortunately, many responses are still characterized based on phenomenology and there is more speculation than substance. With respect to tumor biology, although many neoplastic lesions are immunogenic, tumor antigens frequently are self antigens and induce tolerance and many patients with cancer exhibit suppressed immune responses, including defective antigen presentation. Therefore, if and when mushroom extracts are effective, they more likely function as a result of improved antigen presentation by dendritic cells than by a direct cytopathic effect. In this review we attempt to place these data in perspective, with a particular focus on dendritic cell populations and the ability of mushroom extracts to modulate immunity. There is, at present, no scientific basis for the use of either mushrooms or mushroom extracts in the treatment of human patients but there is significant potential for rigorous research to understand the potential of mushrooms in human disease and thence to focus on appropriate clinical trials to demonstrate effectiveness and/ or potential toxicity.     

Suppression of Tumor Growth by Pleurotus ferulae Ethanol Extract through Induction of Cell Apoptosis,and Inhibition of Cell Proliferation and Migration

Cancer is the second leading cause of death worldwide. Edible medicinal mushrooms have been used in traditional medicine as regimes for cancer patients. Recently anti-cancer bioactive components from some mushrooms have been isolated and their anti-cancer effects have been tested. Pleurotus ferulae, a typical edible medicinal mushroom in Xinjiang China, has also been used to treat cancer patients in folk medicine. However, little studies have been reported on the anti-cancer components of Pleurotus ferulae. This study aims to extract bioactive components from Pleurotus ferulae and to investigate the anti-cancer effects of the extracts. We used ethanol to extract anti-cancer bioactive components enriched with terpenoids from Pleurotus ferulae. We tested the anti-tumour effects of ethanol extracts on the melanoma cell line B16F10, the human gastric cancer cell line BGC 823 and the immortalized human gastric epithelial mucosa cell line GES-1 in vitro and a murine melanoma model in vivo. Cell toxicity and cell proliferation were measured by MTT assays. Cell cycle progression, apoptosis, caspase 3 activity, mitochondrial membrane potential (MMP), migration and gene expression were studied in vitro. PFEC suppressed tumor cell growth, inhibited cell proliferation, arrested cells at G0/G1 phases and was not toxic to non-cancer cells. PFEC also induced cell apoptosis and necrosis, increased caspase 3 activity, reduced the MMP, prevented cell invasion and changed the expression of genes associated with apoptosis and the cell cycle. PFEC delayed tumor formation and reduced tumor growth in vivo. In conclusion, ethanol extracted components from Pleurotus ferulae exert anti-cancer effects through direct suppression of tumor cell growth and invasion, demonstrating its therapeutic potential in cancer treatment.     

Mushrooms, tumors, and immunity.

Medicinal properties have been attributed to mushrooms for thousands of years. Mushroom extracts are widely sold as nutritional supplements and touted as beneficial for health. Yet, there has not been a critical review attempting to integrate their nutraceutical potential with basic science. Relatively few studies are available on the biologic effects of mushroom consumption, and those have been performed exclusively in murine models. In this paper, we review existing data on the mechanism of whole mushrooms and isolated mushroom compounds, in particular (1-->3)-beta-D-glucans, and the means by which they modulate the immune system and potentially exert tumor-inhibitory effects. We believe that the antitumor mechanisms of several species of whole mushrooms as well as of polysaccharides isolated from Lentinus edodes, Schizophyllum commune, Grifola frondosa, and Sclerotinia sclerotiorum are mediated largely by T cells and macrophages. Despite the structural and functional similarities of these glucans, they differ in their effectiveness against specific tumors and in their ability to elicit various cellular responses, particularly cytokine expression and production. Unfortunately, our data base on the involvement of these important mediators is still rather limited, as are studies concerning the molecular mechanisms of the interactions of glucans with their target cells. As long as it remains unclear what receptors are involved in, and what downstream events are triggered by, the binding of these glucans to their target cells, it will be difficult to make further progress in understanding not only their antitumor mechanisms but also their other biological activities.     

Mushrooms, tumors, and immunity: an update

There is significant interest in the use of mushrooms and/or mushroom extracts as dietary supplements based on theories that they enhance immune function and promote health. To some extent, select mushrooms have been shown to have stimulatory action on immune responsiveness, particularly when studied in vitro. However, despite their widespread use for potential health benefits, there is a surprising paucity of epidemiologic and experimental studies that address the biologic activities of mushrooms after oral administration to animals or humans. There have been a number of studies that have addressed the ability of mushrooms to modulate mononuclear cell activation and the phenotypic expression of cytokines and their cognate receptors. There have also been a number of attempts to determine antitumor activities of mushrooms. Such studies are important because many of the components of mushrooms do potentially have significant biologic activity. All data, however, should be tempered by the possibility that there are toxic levels of metals, including arsenic, lead, cadmium, and mercury as well as the presence of radioactive contamination with 137Cs. In this review, we will present the comparative biology with respect to both immunological and antitumor activities of mushroom extracts and also highlight the need for further evidence-based research.     

Electron microscopy reveals the presence of viruses in mouse embryonic fibroblasts but neither in human embryonic fibroblasts nor in human mesenchymal cells used for hESC maintenance: toward an implementation of microbiological quality assurance program in stem cell banks

Human embryonic stem cells (hESCs) are expected to open up new avenues in regenerative medicine by allowing the generation of transplantable cells to be used in future cell replacement therapies. Maintenance of hESCs in the presence of xenogenic compounds is likely to prevent their use in future therapeutic applications in humans. Recently, it has been claimed that human foreskin-derived human embryonic fibroblast (HEFs) and human adult marrow cells have the ability to support prolonged expansion of hESCs in culture similar to murine feeders. Here, to minimize the use of xenogenic components for hESC maintenance, we performed transmission electron microscopy-based microbiological studies in an attempt to implement a microbiological Quality Assurance Program in Stem Cell Banks by determining the potential presence of viral particles in MEFs compared with human HEFs and bone marrow-derived mesenchymal cells. We observed in three out of nine MEF samples (33.3%) viruses belonging to the Retroviridae family. Within the Retroviridae family, these viruses have a C morphology, which indicates they belong to the subfamily Orthoretroviridae. In contrast, no viral particles could be observed in either the HEF samples (n = 5) or the human BM-derived mesenchymal cells (n = 9) analyzed. Based on these experimental microbiological data, we recommend the implementation of microbiological Quality Assurance Programs by means of transmission electron microscopy as a routine technique to assess the potential presence of viral particles in any feeder cell used in stem cell banks and support the use of human cells rather than murine cells as feeders to maintain hESC cultures in an undifferentiated state.     

Using induced pluripotent stem cells as a tool for modelling carcinogenesis

Cancer is a highly heterogeneous group of diseases that despite improved treatments remain prevalent accounting for over 14 million new cases and 8.2 million deaths per year. Studies into the process of carcinogenesis are limited by lack of appropriate models for the development and pathogenesis of the disease based on human tissues. Primary culture of patient samples can help but is difficult to grow for a number of tissues. A potential opportunity to overcome these barriers is based on the landmark study by Yamanaka which demonstrated the ability of four factors;Oct4, Sox2, Klf4, and c-Myc to reprogram human somatic cells in to pluripotency. These cells were termed induced pluripotent stem cells(i PSCs) and display characteristic properties of embryonic stem cells. This technique has a wide range of potential uses including disease modelling, drug testing and transplantation studies. Interestingly i PSCs also share a number of characteristics with cancer cells including self-renewal and proliferation, expression of stem cell markers and altered metabolism. Recently, i PSCs have been generated from a number of human cancer cell lines and primary tumour samples from a range of cancers in an attempt to recapitulate the development of cancer and interrogate the underlying mechanisms involved. This review will outline the similarities between the reprogramming process and carcinogenesis, and how these similarities have been exploited to generate i PSC models for a number of cancers.     

The German cDNA Network: cDNAs,functional genomics and proteomics

Among the greatest challenges facing biology today is the exploitation of huge amounts of genomic data, and their conversion into functional information about the proteins encoded. For example, the large-scale cDNA sequencing project of the German cDNA Consortium is providing vast numbers of open reading frames (ORFs) encoding novel proteins of completely unknown function. As a first step towards their characterization we have tagged over 500 of these with the green fluorescent protein (GFP), and examined the subcellular localizations of these fusion proteins in living cells. These data have allowed us to classify the proteins into subcellular groups which determines the next step towards a detailed functional characterization. To make further use of these GFP-tagged constructs, a series of functional assays have been designed and implemented to assess the effect of these novel proteins on processes such as cell growth, cell death, and protein transport.Functional assays with such a large set of molecules is only possible by automation. Therefore, we have developed, and adapted, functional assays for use by robotic liquid handling stations and reading stations. A transport assay allows to identify proteins which localize to distinct organelles of the secretory pathway and have the potential to be new regulators in protein transport, a proliferation assay helps identifying proteins that stimulate or repress mitosis. Further assays to monitor the effects of the proteins in apoptosis and signal transduction pathways are in progress. Integrating the functional information that is generated in the assays with data from expression profiling and further functional genomics and proteomics approaches, will ultimately allow us to identify functional networks of proteins in a morphological context, and will greatly contribute to our understanding of cell function.     

Design, synthesis, biological evaluation and QSAR studies of novel bisepipodophyllotoxins as cytotoxic agents

Two moieties of epipodophyllotoxin have been linked at C4-position to provide novel bisepipodophyllotoxin analogues. These have been evaluated for their anticancer potential and DNA-topoisomerase II poisoning activity. Most of these analogues have exhibited promising in vitro anticancer activity against different human tumour cell lines and interestingly 4(')-O-methylated analogues have shown increased cytotoxic activity. Similarly, the DNA-topo II poisoning activity tested for these compounds has not only exhibited the DNA cleavage potential comparable to etoposide, but for some compounds this cleavage potential is superior to etoposide. Further, an interesting structure-activity relationship of these epipodophyllotoxin dimers have been generated on the basis of GI(50) values. The equations indicated that GI(50) activity is strongly dependent on structural and thermodynamic properties. These QSAR results are discussed in conjunction with conformational analysis from molecular modelling studies. QSAR models developed in these studies will be helpful in the future to design novel potent bispodophyllotoxin analogues by minor structural modifications.     

Cacospongionolide and scalaradial, two marine sesterterpenoids as potent apoptosis-inducing factors in human carcinoma cell lines

Apoptosis, a form of programmed cell death, is a critical defence mechanism against the formation and progression of cancer and acts by eliminating potentially deleterious cells without causing such adverse effects, as inflammatory response and ensuing scar formation. Therefore, targeting apoptotic pathways becomes an intriguing strategy for the development of chemotherapeutic agents. In last decades, marine natural products, such as sesterterpenoids, have played an important role in the discovery and development of new drugs. Interestingly, many of these compounds have a strong potential as anticancer drugs by inhibiting cell proliferation and/or inducing cell death. In the present study, we investigated the effects of scalaradial and cacospongionolide, two sesterterpenoids from Cacospongia scalaris and Fasciospongia cavernosa marine sponges, on the apoptotic signalling pathway in three different human tumoral cells. Results were obtained by using DNA fragmentation, comet and viability assays, quantification of the mitochondrial transmembrane potential and Western blot. The T47D (human breast carcinoma), A431 (human epidermoid carcinoma), HeLa (human cervix carcinoma) and HCT116 (human colon carcinoma) cells were incubated for 24 h with scalaradial or cacospongionolide. Treatment of T47D cells with scalaradial or cacospongionolide for 24 h brought about a significant increase in DNA migration as well as fragmentation. Moreover, incubation of HCT116 and HeLa cells with scalaradial or cacospongionolide for 24 h caused an increased expression of pro-apoptotic proteins. Furthermore, scalaradial or cacospongionolide, added to HCT116 and HeLa cells overnight, induced a significant and concentration-dependent loss of mitochondrial transmembrane potential, an early apoptosis signalling event. These effects paralleled with those achieved with p50 and p65, NF-κB subunits, nuclear level. In conclusion, scalaradial and cacospongionolide, by determining human cancer cell apoptosis, may represent new promising compounds to inhibit cancer cell proliferation.     

The scope of mycoplasma contamination within the biopharmaceutical industry

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants encountered within the manufacturing of biopharmaceuticals from the research phase to clinical development and production. The potential for mycoplasma contamination within cell culture systems was first identified by Robinson et al. in 1956 [1]. Presently, contamination rates in established cell cultures have been reported between 15 and 35% with considerably higher occurrence cited in certain selected populations [2]. In the last few years, there has been an expansion of diagnostic approaches for mycoplasma detection with the development and validation of rapid microbiological methods. The objective of this study was to determine current levels of mycoplasma infection of cell cultures, cell substrates and biologicals within a client based population. Retrospective comparison of 40,000 sample results was done to determine total contaminations rates amongst four (4) individual analytical assays. The establishment of reference data, such as existing contamination rates, becomes important in the critical appraisal of rapid microbiological methods for the detection of mycoplasma.     

A protocol for quantifying the birth, death and emigration rates of produce-associated bacteria, illustrated by its application to Salmonella enterica serovar Typhimurium on cultivated mushrooms

Colony counts, counts of immunostained cells, fluorescent assays for cell viability and titration of a superinfecting bacteriophage were incorporated into a protocol for studying the growth kinetics of produce-associated bacteria in vivo. A set of equations was assembled for measuring the true rates of birth, death and emigration of the bacteria within the frame of a "transit growth" model, thus allowing the independent measurement of the carrying capacity of the substrate and of the overall productivity of the system. Implementation of the protocol on two species of cultivated mushrooms inoculated with Salmonella enterica serovar Typhimurium showed that large bacterial populations developed on Agaricus bisporus A15 but emigration was not detected, whereas resident populations on Agrocybe aegerita FAR142 amounted to 79.7%, 65.1% and 80.7% of the cultivable, dead and total bacterial cells produced, owing to emigration.     

Characterization of amphiphilic secondary structures in neuropeptide Y through the design, synthesis, and study of model peptides

Neuropeptide Y (NPY) has the potential to form two amphiphilic secondary structures: a polyproline II-like helix in residues 1-8, and an alpha-helix in residues 13-32. NPY dimerizes in aqueous solution and forms stable monolayers at the air-water interface, suggesting that these amphiphilic conformations are stabilized at interfaces. Furthermore, the negative molar ellipticity of monomeric NPY at 222 nm (-8500 degree cm2/dmol), suggests that hydrophobic interactions with the NH2-terminal amphiphilic structure may stabilize the alpha-helix in residues 13-32 before it binds to cell surfaces, even at physiological concentrations. In order to investigate the role of these amphiphilic structures, five NPY models with multiple substitutions in positions 13-32 have been synthesized and studied. Our data demonstrate that the surfactant properties of NPY result from its potential to form amphiphilic secondary and tertiary structures and not from specific amino acid sequences in this region. However, specific residues on the hydrophilic face of the amphiphilic alpha-helix that have been substituted in the models appear to be required to reproduce the full potency of NPY in our pharmacological assays. A possible role for the amphiphilic structures in NPY in presenting such specific determinants to cell surface receptors in the correct conformation is suggested.     

Molecular modeling, design, synthesis, and biological evaluation of novel 3',4'-dicamphanoyl-(+)-cis-khellactone (DCK) analogs as potent anti-HIV agents

Our current studies aimed at developing new potential anti-AIDS drug candidates have focused on the design and synthesis of new DCK analogs with improved molecular water solubility. Based on the structures and biodata of previous DCK analogs, 3D-QSAR studies have been performed which resulted in two reliable computational models, CoMFA and CoMSIA, with r(2) values of 0.995 and 0.987, and q(2) values of 0.662 and 0.657, respectively. In accord with these 3D-QSAR models, 15 new DCK analogs with polar functional groups at the 3-position were subsequently designed, synthesized, and evaluated against HIV-1 replication in H9 and MT4 cell lines. New DCK analogs 3b, 3c, 4b, 4c, 6a, 7c, and 9a showed promising potency with EC(50) values ranging from 0.09 to 0.0002 microM in both assays. Meanwhile, these promising compounds also showed a wide range of predicted logP values from 0.90 to 5.19, which increased the probability of identifying anti-HIV drug candidates from this class of compounds for clinical trials. Furthermore, both experimental and predicted values matched well, corroborating the reliability of the established 3D-QSAR models.     

Neuroprotective properties of cannabinoids against oxidative stress: role of the cannabinoid receptor CB1

Neuroprotective effects have been described for many cannabinoids in several neurotoxicity models. However, the exact mechanisms have not been clearly understood yet. In the present study, antioxidant neuroprotective effects of cannabinoids and the involvement of the cannabinoid receptor 1 (CB1) were analysed in detail employing cell-free biochemical assays and cultured cells. As it was reported for oestrogens that the phenolic group is a lead structure for antioxidant neuroprotective effects, eight compounds were classified into three groups. Group A: phenolic compounds that do not bind to CB1. Group B: non-phenolic compounds that bind to CB1. Group C: phenolic compounds that bind to CB1. In the biochemical assays employed, a requirement of the phenolic lead structure for antioxidant activity was shown. The effects paralleled the protective potential of group A and C compounds against oxidative neuronal cell death using the mouse hippocampal HT22 cell line and rat primary cerebellar cell cultures. To elucidate the role of CB1 in neuroprotection, we established stably transfected HT22 cells containing CB1 and compared the protective potential of cannabinoids with that observed in the control transfected HT22 cell line. Furthermore, oxidative stress experiments were performed in cultured cerebellar granule cells, which were derived either from CB1 knock-out mice or from control wild-type littermates. The results strongly suggest that CB1 is not involved in the cellular antioxidant neuroprotective effects of cannabinoids.     

ECVAM prevalidation study on in vitro cell transformation assays: general outline and conclusions of the study

The potential for a compound to induce carcinogenicity is a key consideration when ascertaining hazard and risk assessment of chemicals. Among the in vitro alternatives that have been developed for predicting carcinogenicity, in vitro cell transformation assays (CTAs) have been shown to involve a multistage process that closely models important stages of in vivo carcinogenesis and have the potential to detect both genotoxic and non-genotoxic carcinogens. These assays have been in use for decades and a substantial amount of data demonstrating their performance is available in the literature. However, for the standardised use of these assays for regulatory purposes, a formal evaluation of the assays, in particular focusing on development of standardised transferable protocols and further information on assay reproducibility, was considered important to serve as a basis for the drafting of generally accepted OECD test guidelines. To address this issue, a prevalidation study of the CTAs using the BALB/c 3T3 cell line, SHE cells at pH 6.7, and SHE cells at pH 7.0 was coordinated by the European Centre for the Validation of Alternative Methods (ECVAM) and focused on issues of standardisation of protocols, test method transferability and within- and between-laboratory reproducibility. The study resulted in the availability of standardised protocols that had undergone prevalidation [1,2]. The results of the ECVAM study demonstrated that for the BALB/c 3T3 method, some modifications to the protocol were needed to obtain reproducible results between laboratories, while the SHE pH 6.7 and the SHE pH 7.0 protocols are transferable between laboratories, and results are reproducible within- and between-laboratories. It is recommended that the BALB/c 3T3 and SHE protocols as instituted in this prevalidation study should be used in future applications of these respective transformation assays. To support their harmonised use and regulatory application, the development of an OECD test guideline for the SHE CTAs, based on the protocol published in this issue, is recommended. The development of an OECD test guideline for the BALB/c 3T3 CTA should likewise be further pursued upon the availability of additional supportive data and improvement of the statistical analysis.     

Monitoring the Metabolic State of Fungal Hyphae and the Presence of Melanin by Nonlinear Spectral Imaging

Label-free nonlinear spectral imaging microscopy (NLSM) records two-photon-excited fluorescence emission spectra of endogenous fluorophores within the specimen. Here, NLSM is introduced as a novel, minimally invasive method to analyze the metabolic state of fungal hyphae by monitoring the autofluorescence of NAD(P)H and flavin adenine dinucleotide (FAD). Moreover, the presence of melanin was analyzed by NLSM. NAD(P)H, FAD, and melanin were used as biomarkers for freshness of mushrooms of Agaricus bisporus (white button mushroom) that had been stored at 4°C for 0 to 17 days. During this period, the mushrooms did not show changes in morphology or color detectable by eye. In contrast, FAD/NAD(P)H and melanin/NAD(P)H ratios increased over time. For instance, these ratios increased from 0.92 to 2.02 and from 0.76 to 1.53, respectively, at the surface of mushroom caps that had been harvested by cutting the stem. These ratios were lower under the skin than at the surface of fresh mushrooms (0.78 versus 0.92 and 0.41 versus 0.76, respectively), indicative of higher metabolism and lower pigment formation within the fruiting body. Signals were different not only between tissues of the mushroom but also between neighboring hyphae. These data show that NLSM can be used to determine the freshness of mushrooms and to monitor the postharvest browning process at an early stage. Moreover, these data demonstrate the potential of NLSM to address a broad range of fundamental and applied microbiological processes.     

Synthesis and biological evaluation of arylphosphonium-benzoxaborole conjugates as novel anticancer agents

Arylphosphonium-benzoxaborole conjugates have been synthesized as potential mitochondria targeting anticancer agents. The synthesized compounds have been tested for their effects on cell viability in various solid tumor cell lines including breast cancer 4T1 and MCF-7, pancreatic cancer MIAPaCa-2 and colorectal adenocarcinoma WiDr. Compound 6c is designated as a lead compound for further studies due to its enhanced effects on cell viability in the above-mentioned cell lines. Seahorse Xfe96 based metabolic assays reveal that the lead candidate 6c inhibits mitochondrial respiration in 4T1 and WiDr cell lines as evidenced by the reduction of mitochondrial ATP production and increase in proton leak. Epiflourescent microscopy experiments also illustrate that 6c causes significant mitochondrial fragmentation in 4T1 and WiDr cells, morphologically consistent with programmed cell death. Our current studies illustrate that arylphosphonium-benzoxaborole conjugates have potential to be further developed as anticancer agents.     

ALCAM regulates motility, invasiveness, and adherens junction formation in uveal melanoma cells

ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM's role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ?-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ?-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves.     

Allylmagnolol,a novel magnolol derivative as potent antioxidant

We reported the discovery of potent antioxidants based on magnolol, a naturally occurring biphenolic obtained from the bark of Magnolia officinalis. The allylmagnolols 3a,b were synthesized via O-alkylation of the biphenols followed by Claisen rearrangement. In-vitro using enhanced chemiluminescence (CL) and flow cytometric assays in whole cells revealed that both 3a and 3b displayed promising free radical scavenging effects in PMA- and LPS-stimulated models as compared with magnolol. Further DNA labeling analysis for cytotoxicity indicated that these analogues show no cytotoxic effects for the scavenging of the oxygen-derived free radicals under PMA-stimulated concentrations. The results from 3,3'-bisallylmagnolol (3b) suggested that the naturally occurring constituent was suitable to be a lead compound for the development of potential antioxidants for certain diseases.     

Serum-Induced Differentiation of Glioblastoma Neurospheres Leads to Enhanced Migration/Invasion Capacity That Is Associated with Increased MMP9

Glioblastoma (GBM) is a highly infiltrative brain tumor in which cells with properties of stem cells, called glioblastoma stem cells (GSCs), have been identified. In general, the dominant view is that GSCs are responsible for the initiation, progression, invasion and recurrence of this tumor. In this study, we addressed the question whether the differentiation status of GBM cells is associated with their invasive capacity. For this, several primary GBM cell lines were used, cultured either as neurospheres known to enrich for GSCs or in medium supplemented with 10% FCS that promotes differentiation. The differentiation state of the cells was confirmed by determining the expression of stem cell and differentiation markers. The migration/invasion potential of these cells was tested using in vitro assays and intracranial mouse models. Interestingly, we found that serum-induced differentiation enhanced the invasive potential of GBM cells, which was associated with enhanced MMP9 expression. Chemical inhibition of MMP9 significantly reduced the invasive potential of differentiated cells in vitro. Furthermore, the serum-differentiated cells could revert back to an undifferentiated/stem cell state that were able to form neurospheres, although with a reduced efficiency as compared to non-differentiated counterparts. We propose a model in which activation of the differentiation program in GBM cells enhances their infiltrative potential and that depending on microenvironmental cues a significant portion of these cells are able to revert back to an undifferentiated state with enhanced tumorigenic potential. Thus, effective therapy should target both GSCs and differentiated offspring and targeting of differentiation-associated pathways may offer therapeutic opportunities to reduce invasive growth of GBM.