RETRACTED: IP1867B suppresses the insulin-like growth factor 1 receptor (IGF1R) ablating epidermal growth factor receptor inhibitor resistance in adult high grade gliomas.

This article has been retracted at the request of the Editor-in-Chief due to concerns regarding the legitimacy of images and data presented in the paper. Though a corrigendum (Can. Lett. Vol. 469, 2020, pages 524-535) was previously published to address some of these concerns, this corrigendum has also been found to contain errors and therefore cannot stand. Specific concerns are listed below. The Editor and Publisher received a letter from the University of Portsmouth alerting us to an investigation into alleged research misconduct. The University concluded their investigation with external experts and determined that misconduct did take place in relation to the research involved in this paper. Upon our separate investigation, it has been determined that the paper headline relies on showing that there was considerable reduction of IGF1R, IL6R and EGFR post treatment in all cell lines. During review, it was determined that this cannot be concluded from the presented data. For example, in SEBTA-003 the EGFR levels go up and there is no difference in IGFR1. It is apparent from Fig 4d that in the SEBTA-003 cell line the EGFR level does not go down, which is stated in the Results section on page 32, it is rather going up. The data for IGFR1 are inconclusive and there are concerns regarding the blot. The general implications would be that the effects of the drug IP1867B does not seem to be the same for all tested cell lines, and this should have been discussed in detail by the authors. Additionally, in subsequent experiments (Fig. 4g and h) the SEBTA-003 cell line (no reduction of EGFR, rather increased expression) and the other 3 cell lines (reduction of EGFR) show similar responses. This is particularly evident in Fig. 4g: Two cell lines are compared, SEBTA-003 (increased EGFR expression) and UP-029 (decreased EGFR expression), both behave similarly after exposure to drugs. The corrigendum (https://doi.org/10.1016/j.canlet.2019.10.002) issue is with respect to the Supplemental Figure 6i EGFR, particularly panel IP1867B. The Corrigendum states that the left part is a cut out of the very right part. If so, the bands for IP1867B should show the same staining pattern - but they do not. Also, in the Corrigendum, there are incorrect mentions between day 14 in the Figure and day 19 in the Figure legend. All authors were informed of the retraction in advance. Drs. Pritchard and Duckworth agreed to the retraction. The corresponding author, Dr Hill, did not agree to the retraction. No response had been received from Drs. Mihajluk, Simms, Reay, Madureira, Howarth, Murray, Nasser and Pilkinton at the time of the retraction being published.

CD44 mediates human glioma cell adhesion and invasion in vitro.

Human gliomas are characterized by their invasion of normal brain structures irrespective of their grade of malignancy. Factors involved in the control of this invasive behavior are poorly documented. Human gliomas have also been found to express CD44 adhesion molecules. Expression of splice variants of CD44 has been correlated to metastasis in nonglial solid tumors. In this study, 8-microns porosity polycarbonate filters incorporated in modified Boyden chambers and coated with the extracellular matrix composite Matrigel were used to investigate the role of CD44 in invasion of eight human glioma cell lines in vitro. Invasion of Matrigel was found to be inhibited to different extents by a CD44 monoclonal antibody. Moreover, this invasion was highly inhibited in two cell lines and completely arrested in five other glioma cell lines by a CD44-specific antisense oligonucleotide which inhibited CD44 expression. In addition, adhesion of glioma cells to fibronectin, laminin, vitronectin, and collagen I was inhibited by the CD44 monoclonal antibody. These results strongly suggest that CD44 is involved in human glioma cell invasion in vitro, probably through its role in cell interactions with extracellular matrix proteins. Interference with glioma invasion, by targeting CD44 expression, may be envisaged in animal models.

Axl-EGFR receptor tyrosine kinase hetero-interaction provides EGFR with access to pro-invasive signalling in cancer cells.

Acquired resistance to conventional and targeted therapies is becoming a major hindrance in cancer management. It is increasingly clear that cancer cells are able to evolve and rewire canonical signalling pathways to their advantage, thus evading cell death and promoting cell invasion. The Axl receptor tyrosine kinase (RTK) has been shown to modulate acquired resistance to EGFR-targeted therapies in both breast and lung cancers. Glioblastoma multiforme (GBM) is a highly infiltrative and invasive form of brain tumour with little response to therapy. Both Axl and EGFR have been identified as major players in gliomagenesis and invasiveness. However, the mechanisms underlying a potential signalling crosstalk between EGFR and Axl RTKs are unknown. The purpose of this study was to investigate this novel and unconventional interaction among RTKs of different families in human GBM cells. With the use of western blotting, in vitro kinase activity, co-immunoprecipitation and bimolecular fluorescence complementation assays, we show that EGF stimulates activation of Axl kinase and that there is a hetero-interaction between the two RTKs. Through small interfering RNA knockdown and quantitative PCR screening, we identified distinct gene expression patterns in GBM cells that were specifically regulated by signalling from EGFR-EGFR, Axl-Axl and EGFR-Axl RTK parings. These included genes that promote invasion, which were activated only via the EGFR-Axl axis (MMP9), while EGFR-EGFR distinctly regulated the cell cycle and Axl-Axl regulated invasion. Our findings provide critical insights into the role of EGFR-Axl hetero-dimerisation in cancer cells and reveal regulation of cell invasion via Axl as a novel function of EGFR signalling.

Cancer stem cells in the mammalian central nervous system.

Malignant tumours intrinsic to the central nervous system (CNS) are among the most difficult of neoplasms to treat effectively. The major biological features of these tumours that preclude successful therapy include their cellular heterogeneity, which renders them highly resistant to both chemotherapy and radiotherapy, and the propensity of the component tumour cells to invade, diffusely, the contiguous nervous tissues. The tumours are classified according to perceived cell of origin, gliomas being the most common generic group. In the 1970s transplacental administration of the potent neurocarcinogen, N-ethyl-N-nitrosourea (ENU), enabled investigation of the sequential development of brain and spinal neoplasms by electron microscopy and immunohistochemistry. The significance of the primitive cells of the subependymal plate in cellular origin and evolution of a variety of glial tumours was thereby established. Since then, the development of new cell culture methods, including the in vitro growth of neurospheres and multicellular tumour spheroids, and new antigenic markers of stem cells and glial/neuronal cell precursor cells, including nestin, Mushashi-1 and CD133, have led to a reappraisal of the histological classification and origins of CNS tumours. Moreover, neural stem cells may also provide new vectors in exciting novel therapeutic strategies for these tumours. In addition to the gliomas, stem cells may have been identified in paediatric tumours including cerebellar medulloblastoma, thought to be of external granule cell neuronal derivation. Interestingly, while the stem cell marker CD133 is expressed in these primitive neuroectodermal tumours (PNETs), the chondroitin sulphate proteoglycan neuronal/glial 2 (NG2), which appears to denote increased proliferative, but reduced migratory activity in adult gliomas, is rarely expressed. This is in contrast to the situation in the histologically similar supratentorial PNETs. A possible functional 'switch' between proliferation and migration in developing neural tumour cells may exist between NG2 and ganglioside GD3. The divergent pathways of differentiation of CNS tumours and the possibility of stem cell origin, for some, if not all, such neoplasms remain a matter for debate and continued research, but the presence of self-renewing neural stem cells in the CNS of both children and adults strongly suggests a role for these cells in tumour initiation and resistance to current therapeutic strategies.

Morphological, immunocytochemical and flow cytometric in vitro characterisation of a surface-adherent medulloblastoma.

Well-characterised cell lines derived from paediatric intrinsic brain tumours are rare. The different repertoire of cell adhesion molecules expressed by primitive neuro-ectodermal tumours, when compared with gliomas, results in a general lack of propensity for surface adherence. In this study, a highly cellular, medulloblastoma biopsy with a Ki-67 index of 20%, obtained by posterior fossa craniotomy of a two-year-old boy, was maintained in surface- adherent culture for twelve sequential in vitro passages. The culture (VC312R) was characterised by immunocytochemistry and flow cytometry using antibodies against cluster of differentiation 44 (CD44), glialfibrillary acidic protein (GFAP), intermediate filament proteins (Nestin and Vimentin), neural cell adhesion molecules (NCAMs) (ERIC and UJ13A), ganglioside (GD3) and neuron-glial 2 (NG2). GD3, GFAP, ERIC-1, UJ13A and NG2 were detected by neither immunocytochemistry nor flow cytometry. It is of particular interest that we have previously reported that the progenitor cell-associated NG2 heparan sulphate proteoglycan was not expressed in a series of medulloblastoma biopsy sections in our laboratories, while NG2 positivity was seen in supratentorial primitive neuro-ectodermal tumours (PNETs). Strong CD44 positivity was detected on most cells (mean = 93.5% of cells on flow cytometry). In one previous case of medulloblastoma, maintained in our laboratories (IPNN-8) as a substrate-adherent culture, no CD44 staining was detected. Twenty-five percent of cells were strongly Vimentin-positive while 54.5% of cells showed Nestin positivity. The expression of Nestin, Vimentin and CD44 is consistent with primitive neural cell evolution. Non-expression of NCAMs may be consistent with the lack of cell-cell adhesion in this culture, which results in surface adherence. The high expression of CD44 may also indicate a distinct phenotype within primitive neuroectodermal tumours, which determines cell-cell and cell-extra cellular matrix adhesive properties.

Tumour cell migration in the central nervous system.

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 millimeters 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, ganglioside, paracrine and autocrine growth and motility factors and matrix metalloproteinases (MMPs) and their inhibitors probably all play important and inter-dependent roles in the migration of neoplastic neural cells.

Extracellular matrix proteins inhibit proliferation, upregulate migration and induce morphological changes in human glioma cell lines.

The influence of an artificial basement membrane (BM), Matrigel, and four individual extracellular matrix proteins, fibronectin, laminin, collagen I and vitronectin, on cell proliferation, morphology and migration was assessed in four glioma cell lines. Matrigel and individual BM proteins differentially inhibited cell proliferation of all cell lines studied. In addition, Matrigel was found to induce extensive morphological changes in glioma cells. Polycarbonate filters, of 8-microns porosity in modified Boyden chambers, were used to assess the chemoattraction activity of Matrigel and the individual proteins on glioma cells. All these components were found to stimulate cell migration, albeit to different extents but laminin proved to be the most effective chemoattractant for glioma cells in vitro. These data suggest that basement membrane proteins may inhibit proliferation and stimulate migration in order to facilitate invasion.

The effect of exogenous gangliosides on matrix metalloproteinase secretion by human glioma cells in vitro.

Matrix metalloproteinases (MMPs) are zinc-dependent peptidases and are amongst those enzymes responsible for extracellular matrix (ECM) degradation during tumour-cell migration. Gangliosides are a family of acidic membrane glycolipids thought to play a role during cell development, differentiation and oncogenic transformation. In this descriptive study, we investigated the effects of six exogenous gangliosides (GM1, GM3, GD1a, GD1b, GD3 and GT1b) on the secretion of MMP-2 (72 kDa gelatinase or gelatinase-A) and MMP-9 (92 kDa gelatinase or gelatinase-B). Cell-conditioned media from eight human glioma-derived cell-lines served as the source of MMPs and were investigated using SDS-PAGE zymography. Six of the cell lines showed upregulation of secretion of both enzymes by all six gangliosides. Of the remaining two cell lines, one showed inhibition of MMP secretion by all gangliosides and the other had a small but differential response to the range of gangliosides investigated. These results suggest that gangliosides may stimulate glioma cell invasiveness by promoting MMP expression.

Vascular and perivascular GD3 expression in human glioma.

A major hallmark of gliomas is their intense neovascularisation. Ganglioside GD3, is one of the major gangliosides which has been implicated in tumour angiogenesis. Recently we reported that GD3 was a potent stimulator of vascular endothelial growth factor release in human glioma cell lines. In the present study we were able to detect GD3-immunoreactivity in 10 out of 10 cases of glioblastoma multiforme and 7 out of 10 cases of anaplastic astrocytoma while low grade tumours were negative. Interestingly, GD3 was intensively expressed in hypervascularised areas of high grade gliomas. These data support the involvement of this ganglioside in brain tumour angiogenesis.

Vascular endothelial growth factor production is stimulated by gangliosides and TGF-beta isoforms in human glioma cells in vitro.

Vascular endothelial growth factor (VEGF) is an angiogenic factor which is known to be expressed in several malignancies including glioma. The effect of transforming growth factor-beta (TGF-beta) isoforms as well as gangliosides on VEGF production was investigated in human glioma cell lines. TGF-beta isoforms and gangliosides were found to differentially stimulate VEGF production by these cells. The ganglioside GD3 enhanced this release to the greatest extent and the stimulation was more marked in a glioblastoma cell line than in the two other anaplastic astrocytoma cell lines. These results suggest that both TGF-betas and gangliosides may act as indirect angiogenic factors by stimulating VEGF secretion.

An inverse correlation between expression of NCAM-A and the matrix-metalloproteinases gelatinase-A and gelatinase-B in human glioma cells in vitro.

Matrix metalloproteinases (MMPs) are an homologous family of proteolytic enzymes capable of degrading components of the extracellular matrix (ECM) and thereby facilitating the invasion of tumour cells into normal tissues. The neural cell adhesion molecules (NCAMs) of neuronal and glial cells provide a Ca2+-independent mechanism for cell-cell and cell-ECM adhesion. NCAMs are downregulated to promote cell disaggregation during cell migration in the developing nervous system whereas MMPs facilitate migration. Recent studies have shown downregulation of MMP secretion in rat glioma cells transfected with an NCAM cDNA, implying an inverse correlation between NCAM and MMP expression. The purpose of this study was to establish whether such a correlation could be demonstrated in a panel of nine human glioma cell-lines, one metastatic carcinoma and one foetal astrocyte derived cell line. The secretion of two MMPs, 72 kDa gelatinase (MMP-2 or gelatinase-A) and 92 kDa gelatinase (MMP-9 or gelatinase-B), was investigated using SDS-PAGE zymography; NCAM-A was assayed by an immunochemiluminescent assay following SDS-PAGE of whole-cell extracts. An inverse correlation was found between the expression of NCAM-A and that of both MMPs studied although the patterns of expression showed no obvious correlation with histological type or grade of the parent tumours. Our results suggest that downregulation of NCAM-A may contribute to tumour invasiveness by promoting both cell disaggregation and protease secretion.

CD44 expression in human meningiomas: An immunocytochemical, immunohistochemical and flow cytometric analysis.

CD44 is a polymorphic family of cell adhesion molecules that has been implicated in tumour invasion and metastasis. In this comparative analysis study, we investigated the expression of the standard form of CD44 (CD44s or CD44H) in 25 early passage cultures of meningiomas and histological sections, using immunohistochemical, immunocytochemical and flow cytometry techniques. There were 20 grade I, 3 grade II and 2 grade III meningiomas in the study which also included 2 recurrent meningiomas and 1 meningioma arising some time after previous radiotherapy. Immunocytochemistry and flow cytometry results on early passage culture cells show that although the majority of the meningiomas were strongly positive for CD44H, some were only weakly positive. Immunohistochemical studies revealed a great variability in staining patterns both within individual tumours and between different tumours. Generally, the intensity varied between strong and negative, and in most tumours that were immunopositive, there was a multifocal pattern of staining. Five meningiomas did not stain at all for CD44H. Taken together, these findings suggest that generally the flow cytometry results correspond well with those of both immunocytochemical and immunohistochemical techniques, with a few exceptions. It is concluded that microenvironmental factors may be responsible for the differential expression seen with different techniques.

Comparative analysis of matrix metalloproteinases by immunocytochemistry, immunohistochemistry and zymography in human primary brain tumours.

Matrix metalloproteinases (MMPs) are a growing family of zinc-dependent endopeptidases which are characterised by their ability to degrade various extracellular matrix (ECM) components. The family includes collagenases, gelatinases, stromelysins, metalloelastase and membrane type metalloproteinases. Consistent with their proteolytic activities, MMPs have been implicated in a variety of physiological and pathological conditions, such as normal embryogenesis, tissue morphogenesis and are thought to play a role in facilitating tumour cell invasion of the normal brain. In this comparative study, we have used zymography, immunohistochemical and immunocytochemical techniques to demonstrate the expression of gelatinase-A and B (MMP-2 and 9, respectively) and membrane type metalloproteinase (MMP-14) in 8 intrinsic human primary brain tumours of various histological type and grade. Zymography results showed that MMP-2 was the most prominent proteolytic enzyme in all the cell lines studied (with one exception), while MMP-9 was only faintly expressed. However, the corresponding paraffin sections showed no expression of either MMP-2, 9 or 14 within the tumour cells, positivity being confined to haematogenous cells and the vascular endothelium. Fluorescence immunocytochemical studies, using monoclonal antibodies to MMP-2, 9 and 14, showed granular cytoplasmic reactivity in vitro. In addition, there was strong focal positivity at the cell membrane with MMP-14 in some high grade tumours suggesting that MMPs are produced at the leading edge of the cell by individual subpopulations of invading glia, in small quantities and on demand in vivo. It can be concluded that local microenvironmental conditions in vitro appear to stimulate such MMP activity.

Expression of serotonin receptors in human fetal astrocytes and glioma cell lines: a possible role in glioma cell proliferation and migration.

Expression of seven serotonin or 5-hydroxytryptamine (5-HT) receptors (5-HT1D alpha, 5-HT1E, 5-HT2, 5-HT1A, 5-HT1C, 5-HT1D beta, and 5-HT6) was investigated in human normal fetal astrocytes and eight glioma cell lines by reverse transcription and polymerase chain reaction (RT-PCR). No expression of 5-HT1D beta and 5-HT6 was observed in any of the cell lines studied. The 5-HT1D alpha receptor was found to be expressed in two human glioma cell lines but not in normal astrocytes. In addition, only three glioma cell lines expressed the 5-HT1E receptor. The 5-HT1C receptor was expressed in six glioma cell lines but not in normal astrocytes while the 5-HT1A was found to be expressed in normal astrocytes from the left hemisphere and in six glioma cell lines but not in normal astrocytes from the cerebellum. Interestingly, the 5-HT2 receptor was expressed in all cells studied but very weakly in normal astrocytes. The effect of 5-HT on glioma cell proliferation, migration, and invasion was also investigated. Serotonin was found to positively modulate these three processes in vitro. These results suggest that 5-HT may play an important role in the control of the biological properties of human glioma cells.

Fibronectin staining detects micro-organisms in aged and Alzheimer's disease brain.

Filamentous, fibronectin-immunopositive structures, previously described in Alzheimer's disease and control brains were negative for neuronal, glial, and macrophage markers. The present study sought to determine the nature of these entities and to further characterize their morphology, immunoreactivity and distribution between neuropathologies. Ultrastructural analysis shows these formations to be filamentous micro-organisms, which may belong to the actinomycetes. Immunohistochemistry for the cell-stress protein ubiquitin is consistently positive in these organisms. They are also present in Down's syndrome, dementia pugilistica, amyotrophic lateral sclerosis with dementia, and Parkinson's disease. The pattern of tissue distribution implies a pre-mortem invasion of the brain, and, as the micro-organism is present at a four to five-fold higher frequency in Alzheimer's disease, it may act pathogenically in this dementing illness.

Differential binding of anti-CD44 on human gliomas in vitro.

Recent studies have shown that the membrane glycoprotein, CD44, is expressed on both normal and neoplastic astrocytes. Moreover, CD44 has been shown to recognise the receptor for hyaluronic acid (HA), an extracellular matrix protein thought to be involved in tumour invasion. The present study has established, by immunocytochemical staining, that CD44 is present on the majority of cells in long and short term cultures derived from astrocytic gliomas. In glioblastoma multiforme, medulloblastoma, ependymoma and dysembryoplastic neuroepithelial tumour, however, staining is either absent or restricted to isolated cells. This differential binding suggests that HA receptor expression may be related to (a) the cell cycle and (b) local invasiveness of brain tumours.

Recombinant nef HIV-IIIB protein is toxic to human neurons in culture.

The expression of HIV-1 negative factor (nef) has been positively correlated with HIV disease progression [Z. Hanna, D.G. Kay, N. Rebai, A. Guimond, S. Jothy, P. Jocicoeur, Nef harbors a makor determinant of pathogenicity for an AIDS-like disease induced by HIV-1 in transgenic mice. Cell 95 (1998) 163-175]. Nef expression has been detected in HIV infected human brains with neuronal damage [A. Ranki, M. Nyberg, V. Ovod, M. Haltia, I. Elovaara, R. Raininko, H. Haapsalo, K. Krohn, Abundant expression of HIV Nef and Rev proteins in brain astrocytes in associated with dementia, AIDS 9(9) (1995) 1001-1008; Y. Saito, L.R. Sharer, M.G. Epstein, J. Michaels, M. Mintz, M. Londer, K. Golding, B.M. Blumberg, Overexpression of nef as a marker for restricted HIV-1 infection of astrocytes in postmorten paediatric central tissues, Neurology 14 (1994) 474-480]. It is postulated that nef may contribute to the neuronal damage observed in the brain of those with late HIV disease. To test this, the potential toxicity of recombinant nef (from HIV-1 IIIB) was compared to the neurotoxin human tumour necrosis alpha (TNFalpha) on human brain cells in culture. SK-N-SH neuroblastoma, primary human neurons and glial cells were exposed to recombinant nef or TNFalpha protein for 3 days or twice over 6 days. Cell viability was assessed by Trypan Blue, lactate dehydrogenase (LDH) release and MTT assays. Nuclear fragmentation was detected using the Hoechst Blue nuclear dye assay. Both nef and TNFalpha (100 ng/ml) caused a significant 30% reduction of SK-N-SH cell numbers after 3 days exposure (P=0. 001). At this time, exposure to nef caused evident fragmented nuclei in these cultures. Human neuronal cultures had a 32 and 33% decrease in cell number after 6 days exposure to either nef or TNFalpha, respectively (P<0.001). Furthermore, as previously shown [J. He, C.M. DeCastro, G.R. Vandenbark, J. Busciglio, D. Gabuzda, Astrocyte apoptosis induced by HIV-1 transactivation of the c-kit protoonocogene, Proc. Natl. Acad. Sci. 94 (1997) 3954-3959], a 3-day exposure to nef significantly reduced human glial cell number by 25% (P=0.001). Recombinant nef and TNFalpha compromise human neurons in culture. Thus, like other virotoxins, it is shown for the first time that nef may also contribute to neuronal damage that has been reported in dementia in late HIV disease.

The NG2 chondroitin sulfate proteoglycan: role in malignant progression of human brain tumours.

The expression and function of NG2, a transmembrane chondroitin sulfate proteoglycan was studied in human gliomas of various histological types in culture using immunocytochemistry and flow cytometry. NG2 was differentially expressed in the neoplasms, with higher expression in high compared to low-grade gliomas. In acutely isolated cells from human biopsies, NG2 +ve and NG2 -ve populations were morphologically distinct from each other, and NG2 +ve cells were more proliferative than NG2 -ve cells. The mitogens platelet derived growth factor (PDGF-AA) and basic fibroblast growth factor (bFGF) added in combination to serum-free medium (SFM) upregulated NG2 expression on glioblastoma multiforme cells in culture but had little effect on NG2 expression on the anaplastic astrocytoma cells. Furthermore, NG2 was colocalised with the platelet derived growth factor alpha receptor (PDGFalphaR) and antibody blockade of the PDGF-alphaR ablated NG2 expression on the glioblastoma multiforme cells, suggesting that increased NG2 expression in the presence of PDGF-AA is mediated via the PDGF-alphaR. Assays of migration and invasion indicate that NG2 +ve glioma cells migrated more efficiently on collagen IV and that NG2 -ve cells were more invasive than their NG2 +ve counterparts. The results indicate that NG2 may be, respectively, positively and negatively related to the proliferative and invasive capacity of glioma cells. Thus, expression of the NG2 proteoglycan may have major implications for malignant progression in glial neoplasms and may prove a useful target for future therapeutic regimens.