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This article has been retracted at the request of the Editor-in-Chief due to concerns regarding the legitimacy of images and data presented in the paper.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.

Improved antigen binding by a CD20-specific single-chain antibody fragment with a mutation in CDRH1.

We have prepared single-chain immunoglobulin Fv fragments from the CD20-specific hybridoma HB13d. One scFv clone demonstrated strong binding to a CD20-derived peptide by ELISA and to CD20-positive cells by flow cytometry, a second had reduced binding, and a third clone did not bind the target antigen. Sequence analysis showed that all three constructs contained shared and unique amino acid changes when compared to the nearest germline match. Molecular modelling of the scFv variants revealed that several of the mutations are located in regions predicted to contact antigen, including a mutation in the heavy chain CDR1 of the strongest binding scFv construct. No similar mutation is present in the highly conserved protein sequences of a number of CD20-specific monoclonal antibodies. BIACORE analysis demonstrated that the mutated scFv had approximately three-fold greater antigen-binding activity than another clone. Competition studies showed that the scFv is able to compete with intact CD20 monoclonal antibody for binding to the target antigen. The improved antigen binding of this scFv will permit the construction of novel CD20-specific reagents for the therapy of lymphomas.

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.

Molecular profile of an antibody response to HIV-1 as probed by combinatorial libraries.

A large number (33) of human Fab fragments reacting with HIV-1 surface glycoprotein gp120 have been generated by selection from a combinatorial IgG1 kappa library displayed on the surface of phage. The library was prepared from a long term asymptomatic HIV-seropositive donor. Analysis of the sequences from these Fabs shows the heavy chains can be placed in groups, many of which contain intraclonal variants, almost certainly corresponding to chains used in vivo. Further variants can be accessed via chain shuffling experiments in which a given light chain is recombined with a library of heavy chains. Heavy chain promiscuity, i.e. the ability of heavy chains to pair with different light chains with retention of antigen binding, is dependent on the particular heavy chain considered and probably excludes the identification of in vivo light chain partners. The antibodies examined here are primarily to the CD4 binding site on gp120 and broadly reflect the serum profile of the donor. The antibodies show evidence of extensive somatic modification indicative of an antigen-driven response. The heavy chain CDR3 regions of the antibodies show a remarkably conserved extended length. A number also show strong sequence conservation in CDR3 against a background of considerable diversity in the rest of the VH gene supporting a central role for this region in antigen recognition.

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.

Fc gamma RII-mediated superoxide production by phagocytes is augmented by GM-CSF without a change in Fc gamma RII expression.

Freshly purified neutrophils and monocytes respond to multiple cross-linking of Fc gamma RII with the IgG1 monoclonal antibody, CIKM5, with a rapid rise in Ca(2+)i, but not with a respiratory burst, although superoxide is generated by these cells when stimulated with the chemotactic peptide, FMLP, or phorbol ester (TPA). Incubation in vitro for 30-60 min at 37 degrees C in medium + 0.1% FCS had no effect on the neutrophil superoxide response to CIKM5 but induced a weak monocyte response in 11/13 experiments. However, incubation with rhGM-CSF (10 ng/ml) under similar conditions induced a neutrophil respiratory burst in response to cross-linking Fc gamma RII in 12/14 experiments and enhanced the monocyte response by 181%. GM-CSF also enhanced the response of neutrophils and monocytes to FMLP by 308% and 165%, respectively. The response to TPA was not significantly enhanced by GM-CSF. rhIFN-gamma (100 mu/ml) was ineffective as a priming agent for all agonists tested in short-term incubations but augmented the monocyte response to CIKM5 after 5 d exposure in vitro. Whilst GM-CSF induced neutrophil superoxide production in response to cross-linking Fc gamma RII, there was no concomitant change in Fc gamma RII expression either in in vitro studies of neutrophils from healthy individuals or in in vivo studies of patients receiving GM-CSF. Stimulation of unprimed neutrophils with CIKM5 induced a rapid transient increase in intracellular calcium levels to 181% of resting levels. However, incubation with GM-CSF did not further augment the calcium transients above the stimulated level. The mechanism by which GM-CSF induces an enhanced respiratory burst in response to cross-linking of Fc gamma RII remains to be elucidated, but is not related to receptor expression or increases in receptor mediated calcium mobilization.

Recombinant human Fab antibody fragments to HIV-1 Rev and Tat regulatory proteins: direct selection from a combinatorial phage display library.

A human Fab phage display library has been produced from peripheral blood lymphocytes of an individual who was asymptomatic after 10 years of infection with human immunodeficiency virus type-1 (HIV-1). The library was panned against the HIV-1 Rev and Tat regulatory proteins and several clones, producing Fab binding to these proteins, were isolated (3 to Rev and 4 to Tat) with binding constants varying from 10(-6)M to 10(-8)M. DNA sequencing demonstrated two unique anti-Rev Fab clones, but the four anti-Tat Fab comprised only two unique IgG1 heavy chain Fd fragments, illustrating redundancy of light chains. Peptide mapping of the epitopes recognized by these Fab indicated that three of the anti-Tat Fab were directed to the functional domain between amino acid residues 22-33 of the Tat molecule, and that binding was inhibited by reduction of this cysteine-rich region with dithiothreitol. The anti-Rev Fab were directed to sites adjacent to the Rev basic nucleolar localization sequence (residues 52-64) and to the Rev activation domain (residues 75-88). Binding constants were of a similar order to that of an anti-Rev single-chain Fv fragment (SFv) used successfully for intracellular immunization, and as such intracellular effects with the human anti-Tat and anti-Rev Fab are not precluded. These newly described human antibody fragments to HIV-1 regulatory proteins may be critical moieties for gene therapeutic protocols, to control HIV-1 replication in human cells.

Fc gamma RII, but not erythropoietin or GM-CSF, mediates calcium mobilization in fetal hemopoietic blast cells.

A proportion of fetal liver hemopoietic blast cells express Fc gamma RII, and addition of the anti-Fc gamma RII monoclonal antibody CIKM5 induces a rise in calcium in these cells in suspension. Although these cells are thus capable of mobilizing intracellular calcium in response to surface receptor mediated events, neither granulocyte-macrophage colony-stimulating factor (GM-CSF) nor erythropoietin produced detectable changes in intracellular calcium ion concentration in these cells.

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.

Evidence against potassium as an endothelium-derived hyperpolarizing factor in rat mesenteric small arteries.

1. Endothelium-derived hyperpolarizing factor (EDHF) has recently been identified as potassium released from endothelial cells into the myo-endothelial space. The present study was designed to test this hypothesis. 2. In rat small mesenteric arteries, mounted in a wire myograph, relaxation to acetylcholine or potassium was not significantly changed following incubation with oxadiazolo-quinoxalin-1-one (ODQ, 4 microM) and indomethacin (10 microM, n = 9). 3. Maximal relaxations to acetylcholine occurred in all arteries, were maintained and were significantly greater (P < 0.01, n = 9) than the transient relaxations to potassium, which only occurred in 30-40% of vessels. 4. Removal of the vascular endothelium abolished relaxant responses both to potassium and acetylcholine (P < 0.005, n = 9). 5. Compared with responses in 5.5 mM potassium PSS, relaxation responses to added potassium in arteries maintained in 1.5 mM potassium PSS were more marked and were not dependent on the presence of an intact endothelium (n = 8). 6. Incubation with BaCl2 (50 microM) significantly inhibited the maximal relaxant response to potassium in the presence of an intact endothelium in 5.5 mM potassium PSS (P < 0.05, n = 4), but had no effect on relaxation of de-endothelialized preparations in 1.5 mM potassium PSS (n = 5). 7. Treatment with ouabain (0.1 mM) abolished the relaxant response to potassium in 1.5 mM potassium PSS (P < 0.001, n = 9), but only partly inhibited the maximal relaxant response to acetylcholine in 5.5 mM potassium PSS (P < 0.01, n = 5). 8. These data show that at physiological concentrations of potassium an intact endothelium is necessary for potassium-induced relaxation in rat mesenteric arteries. Furthermore, the response to potassium is clearly different to that from acetylcholine, indicating that potassium does not mimic EDHF released by acetylcholine in these arteries.

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.

Antibody against CD20 in patients with B cell malignancy.

Cancer patients may make antibodies against antigens on the surface of their malignant cells due either to the expression of unique antigens or to dysregulated responses to self antigens. Patients with B cell malignancy frequently produce autoantibodies and may therefore be a source of immunoglobulin genes for the production of phage display antibody libraries directed against tumour-associated antigens. Patients with autoimmune disease have circulating antibodies against lymphocyte surface antigens, and may also provide a good starting point for the production of a library of lymphocyte-reactive antibody structures. In this study, plasma and serum samples from patients with B cell malignancy or Sjogren's syndrome and from healthy controls were screened for antibodies against the B cell membrane antigens CD20. While the majority of samples showed very low reactivity, some individuals did show significant and reproducible binding to CD20. To identify a good donor for library construction, it would be advisable to screen donors for antibody against the antigens of interest.