Patient-derived glioblastoma cells show significant heterogeneity in treatment responses to the inhibitor-of-apoptosis-protein antagonist birinapant.

BACKGROUND: Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Here we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist birinapant to enhance treatment responses to TMZ in both commercially available and patient-derived GBM cells. METHODS: Responses to TMZ and birinapant were analysed in a panel of commercial and patient-derived GBM cell lines using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of pro- and antiapoptotic proteins. Responses in vivo were analysed in an orthotopic xenograft GBM model. RESULTS: Single-agent treatment experiments categorised GBM cells into TMZ-sensitive cells, birinapant-sensitive cells, and cells that were insensitive to either treatment. Combination treatment allowed sensitisation to therapy in only a subset of resistant GBM cells. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ while addition of birinapant further sensitised the cells to TMZ-induced cell death; Type B cells that readily activated caspase-8 and cell death in response to birinapant but did not show further sensitisation with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm. Furthermore, in vivo, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus birinapant treatments. CONCLUSIONS: Our results demonstrate remarkable differences in responses of patient-derived GBM cells to birinapant single and combination treatments, and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment.

Eph receptors as therapeutic targets in glioblastoma.

The dismal outlook for patients with the most aggressive and common form of adult brain cancer, glioblastoma (GBM), motivates a search for new therapeutic strategies and targets for this aggressive disease. Here we review the findings to date on the role of Eph family receptor tyrosine kinases and their ephrin ligands in brain cancer. Expression of the Eph family of cell surface proteins is generally downregulated to very low levels in normal adult tissues making them particularly attractive for directed therapeutic targeting. Recent Eph targeting studies in pre-clinical models of GBM have been very encouraging and may provide an avenue to treat these highly refractory aggressive tumours.

Mechanism of effector-cell blockade. I. Antigen-induced suppression of Ig synthesis in a hybridoma cell line, and correlation with cell-associated antigen.

A mouse hybridoma cell line, FluIgM-1, which secretes IgM specific for the hapten fluorescein (FLU) was developed to allow detailed analysis of the effector-cell blockade (ECB) phenomenon, in which contact of antibody-forming cells (AFC) with specific antigen results in marked reduction of antibody secretion. Treatment of hybridoma cells with highly substituted FLU conjugates (e.g., Flu20gelatin) resulted in inhibition of plaque formation. The data indicated close parallels with the ECB of normal spleen AFC, both in speed of onset and the dose of antigen required. The inhibition of antibody secretion was confirmed with a biosynthetic-labeling procedure which demonstrated that this was a result of reduced Ig synthesis. The inhibitory effect appeared to be confined to antibody synthesis, in the total protein synthesis, DNA synthesis, and cell-doubling times were unaffected. The association of FLU conjugates with the cells during and following ECB was studied directly using fluorescence microscopy and the fluorescence-activated cell sorter. These experiments showed that FLU conjugates capable of causing blockade aggregated on the cell surface, that the clearance of cell-associated antigen correlated with recovery from ECB, and that at all times when cell associated antigen was detectable, a portion remained bound to the cell surface and was susceptible to enzymatic removal. The latter observations supported previous findings suggesting that ECB was mediated by extracellular antigen. The direct observation of aggregates of antigen on the surface of blockaded cells is consistent with a mechanism involving cross-linking of Ig receptors. Finally, Fc receptors were not present on hybridoma cells, excluding their involvement in induction of ECB.

TLiSA1, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors.

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.

Knob-independent cytoadherence of Plasmodium falciparum to the leukocyte differentiation antigen CD36.

The survival of Plasmodium falciparum-infected erythrocytes is enhanced by the sequestration of mature trophozoites and schizonts from the peripheral circulation. Cytoadherence of infected erythrocytes in vivo is associated with the presence of knobs on the erythrocyte surface, but we and others have shown recently that cytoadherence to C32 melanoma cells may occur in vitro in the absence of knobs. We show here that a knobless clone of P. falciparum adheres to the leukocyte differentiation antigen, CD36, suggesting that binding to CD36 is independent of the presence of knobs on the surface of the infected erythrocyte. This clone showed little cytoadherence to immobilized thrombospondin or to endothelial cells expressing the intercellular adhesion molecule 1. Furthermore, an Mr approximately 300-kD trypsin-sensitive protein doublet was immunoprecipitated from knobless trophozoite-infected erythrocytes. Finding a P. falciparum erythrocyte membrane protein 1 (PfEMP1)-like molecule on these infected erythrocytes is consistent with a role for PfEMP1 in cytoadherence to CD36 and C32 melanoma cells.

Epigenetic silencing of EphA1 expression in colorectal cancer is correlated with poor survival.

Aberrant expression of Eph and ephrin proteins has well-established functions in oncogenesis and tumour progression. We describe EphA1 expression in 6 colorectal cancer (CRC) cell lines, 18 controls and 125 CRC specimens. In addition, a well-characterised cohort of 53 paired normal colon and CRCs was also assessed. Expression of EphA1 mRNA was assessed by quantitative real-time PCR and correlated with protein expression by flow cytometry, immunoprecipitation, western blotting and immunohistochemistry. Significant upregulation (2- to 10-fold) of EphA1 was seen in over 50% of cases (P=0.005) whereas many of the remainder showed downregulation of EphA1. Intriguingly, EphA1 over-expression was more prevalent in stage II compared to stage III CRCs (P=0.02). Low EphA1 expression significantly correlated with poor survival (P=0.02). Epigenetic silencing appeared to explain the loss of EphA1 expression as methylation of the EphA1 CpG island strongly correlated with low EphA1 expression (P<0.01). Furthermore, EphA1 re-expression could be induced by treatment with demethylating agents. Our findings identify EphA1 as a potential prognostic marker in CRC. Although therapies targeting high EphA1 expression seem plausible in CRC, the loss of expression in advanced disease suggests a potential risk that targeted therapy, by selecting for loss of expression, might contribute to disease progression.

Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways.

The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES+/CD133+) and their differentiated (diff) progeny cells (NES-/CD133-). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFß1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53ß in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.

Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo.

The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal lobes. To study EphA4 kinase signaling in these processes, we generated mice expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. We demonstrate that EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. Our findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts.

Decreased signalling of EphA4 improves functional performance and motor neuron survival in the SOD1G93A ALS mouse model.

Amyotrophic lateral sclerosis (ALS) is an untreatable, progressive, neurodegenerative disease specifically affecting motor neurons. Recently, the tyrosine kinase receptor EphA4 was directly implicated in ALS disease progression. We report that a long-lived mutated form of the EphA4 antagonist EphA4-Fc (mutEphA4-Fc), which blocks EphA4 binding to its ligands and inhibits its function, significantly improved functional performance in SOD1G93A ALS model mice, as assessed by rotarod and hind-limb grip strength tests. Further, heterozygous motor neuron-specific EphA4 gene deletion in SOD1G93A mice promoted significant improvement in functional performance during the disease course and a delay in disease onset relative to control mice. Importantly, mice in the heterozygous deletion group showed significantly improved survival of motor neurons and architecture of endplates of neuromuscular junctions compared with control and homozygous EphA4-deletion groups. Our novel results show that EphA4 signalling directly regulates motor neuron survival and that mutEphA4-Fc is a promising therapeutic candidate to slow disease progression in ALS.

B cell origin of non-T cell acute lymphoblastic leukemia. A model for discrete stages of neoplastic and normal pre-B cell differentiation.

The expression of B cell associated and restricted antigens on tumor cells isolated from 138 patients with non-T cell acute lymphoblastic leukemia (non-T cell ALL) was investigated by flow cytometric analysis by means of a panel of monoclonal antibodies. Tumor cells from these patients could be assigned to one of four subgroups: human leukocyte antigen-DR-related Ia-like antigens (Ia) alone (4%, stage I); IaB4 (14%, stage II); IaB4CALLA (33%, stage III); and IaB4CALLAB1 (49%, stage IV). The expression of B cell-restricted antigens (B4 and B1) and rearrangements of Ig heavy chain genes provided strong evidence for the B cell lineage of stages II, III, and IV tumors. The lineage of the Ia alone group is still unknown. The B4 antigen was expressed on approximately 95% of all non-T cell ALLs tested, and given its absence on T cell and myeloid tumors, it appears to be an exceptional marker to define cells of B lineage. The demonstration that Ia alone, IaB4, IaB4CALLA, and IaB4CALLAB1 positive cells can be readily identified by dual fluorescence analysis in normal fetal and adult bone marrow provided critical support for the view that these leukemic pre-B cell phenotypes were representative of the stages of normal pre-B cell differentiation. It was interesting that the IaB4+ cell was more frequently identified in fetal bone marrow than in adult marrow, whereas the predominant cell found in adult marrow expressed the IaB4CALLAB1 phenotype. These data suggest that the leukemogenic event may be random, since the predominant pre-B cell leukemic phenotype appears to correspond to the normal pre-B cell phenotype present in these hematopoietic organs. Our observations provide an additional distinction between adult and childhood ALL, since these studies show that most non-T cell ALLs seen in children less than 2 yr old are of stage II phenotype, whereas the majority of non-T ALLs in adults are of stage IV phenotype. Finally, it should be noted that the present study suggests that the analysis of leukemic B cell phenotypes and their normal counterparts can provide a mechanism for the investigation and orderly definition of stages of pre-B cell differentiation in man.

EphA3 as a target for antibody immunotherapy in acute lymphoblastic leukemia.

The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting 213Bismuth payload.

Expression of the Wilms' tumor suppressor gene, WT1, reduces the tumorigenicity of the leukemic cell line M1 in C.B-17 scid/scid mice.

The Wilms' tumor suppressor gene, WT1, encodes a transcription of the Cys2-His2 zinc finger type. Loss of WT1 gene function has been implicated in the development of malignancies including Wilms' tumor and acute leukemias. We have shown previously that ectopic expression of WT1 +KTS isoforms in murine M1 leukemic cells spontaneously induces monocytic differentiation without the requirement for external differentiation-inducing stimuli. To determine whether these observed effects in vitro corresponded to a reduction in tumorigenicity in vivo, parental M1, control M1.Neo, and M1.WT1 +KTS cells were transplanted into C.B-17 scid/scid mice, and the growth and metastatic behavior of the cell lines were monitored for a period of 20 weeks. Mice inoculated either s.c. on the flank or directly into the peritoneal cavity, with M1 cells stably expressing WT1 +KTS isoforms exhibited a marked decrease in tumor formation compared with control groups. Moreover, tumors arising in mice after the injection of M1.WT1 +KTS cells exhibited a loss in ectopic WT1 protein expression. Confirmation that the tumors arose from M1.WT1 +KTS cells was achieved by the amplification of the introduced transgene from tumor samples and indicates that the tumorigenicity of leukemic M1 cells in these animals correlates with a loss in WT1 expression. This investigation is the first to demonstrate the tumor-suppressive effects of WT1 expression in a leukemic cell line, further advancing the notion that WT1 acts as a differentiation-promoting gene during hematopoiesis and that loss of functional WT1 expression may contribute to leukemogenesis in vivo.

An early developmental role for eph-ephrin interaction during vertebrate gastrulation.

Eph receptor tyrosine kinases (RTK) and their ephrin ligands are involved in the transmission of signals which regulate cytoskeletal organisation and cell migration, and are expressed in spatially restricted patterns at discrete phases during embryogenesis. Loss of function mutants of Eph RTK or ephrin genes result in defects in neuronal pathfinding or cell migration. In this report we show that soluble forms of human EphA3 and ephrin-A5, acting as dominant negative inhibitors, interfere with early events in zebrafish embryogenesis. Exogenous expression of both proteins results in dose-dependent defects in somite development and organisation of the midbrain-hindbrain boundary and hindbrain. The nature of the defects as well as the distribution and timing of expression of endogenous ligands/receptors for both proteins suggest that Eph-ephrin interaction is required for the organisation of embryonic structures by coordinating the cellular movements of convergence during gastrulation.

Signals from Eph and ephrin proteins: a developmental tool kit.

Interactions between Eph receptors and their ligands the ephrin proteins are critically important in many key developmental processes. Emerging evidence also supports a role for these molecules in postembryonic tissues, particularly in pathological processes, including tissue injury and tumor metastasis. We review the signaling mechanisms that allow the 14 Eph and nine ephrin proteins to deliver intracellular signals that regulate cell shape and movement. What emerges is that the initiation of these signals is critically dependent on which Eph and ephrin proteins are expressed, the level of their expression, and, in some cases, which splice variants are expressed. Diversity at the level of initial interaction and in the downstream signaling processes regulated by Eph-ephrin signaling provides a subtle, versatile system of regulation of intercellular adhesion, cell shape, and cell motility.

Self-renewal of a transplantable murine leukemia induced by co-culture with human stromal cell lines.

The PGM-1 murine leukemic cell line can be serially transplanted in syngeneic C3H/HeJ mice but cannot be maintained in in vitro culture. In response to a wide range of known growth factors, the PGM-1 cells either die or differentiate into mature granulocytes and macrophages with loss of all clonogenic (i.e. agar culture colony-forming) cells within 7 days. In this report we show that coculture with human, but not mouse, bone marrow stromal cell lines allows maintenance of clonogenic cells. One line in particular (197/17) allowed continuous expansion of clonogenic cells with no evidence of differentiation. The maintenance of clonogenic cells correlated with maintenance of tumor stem cells. Even after 9 months continuous passaging on stromal cells, the cultured cells produced tumors on injection into syngeneic mice with the same latency as cells from explanted tumors. We demonstrated that this activity was due to a soluble factor in 197/17 conditioned medium. An extensive survey of known factors, either alone or in combination, failed to reproduce this effect, implying that the effect was due to a novel factor acting on self-renewal of early stem cells.

Expression of B cell activation antigens on normal and malignant B cells.

In an attempt to relate the functional events of B cell activation with changes in cell surface molecules, we have used a panel of monoclonal antibodies directed against cell surface antigens expressed on activated but not resting B cells, to determine a sequence of activation antigen expression following anti-immunoglobulin stimulation. Within the first 24 hr of culture with anti-Ig, resting splenic B cells were induced to express B5 and interleukin-2 receptor (IL-2R) and subsequently express T9 and BB1 by 48 hr. Maximum antigen expression was seen by day 3 with the majority of cells expressing B5, IL-2R, T9, and BB1, and fewer numbers of cells expressing Blast-1 and Blast-2. By day 6, the expression of these antigens significantly decreased. Dual fluorochrome staining of anti-Ig activated B cells demonstrated heterogeneity of activation antigen expression, suggesting the existence of subpopulations of activated B cells. In an attempt to relate the non-Hodgkin's lymphomas (NHLs) to this sequence of activation, 69 tumor samples from patients with B cell NHLs were then examined for expression of these activation antigens. Histologically defined subgroups of B cell NHLs demonstrated differential expression of activation antigens with B5, BB1, and T9 exhibiting the widest distribution, whereas IL-2R, Blast-1, and Blast-2 demonstrated more limited expression. The finding that no B cell malignancy phenotypically resembles the small resting B lymphocyte coupled with the observation that virtually all B cell NHLs examined expressed activation antigens suggests that these tumors may be the neoplastic counterparts of subpopulations of activated B lymphocytes.

Mechanism of effector cell blockade--IV. Induction by monoclonal anti-mu or anti-idiotypic antibody, role of secreted IgM and mechanism of decreased secretion.

Effector cell blockade (ECB), the inhibition of antibody synthesis by antigen or antigen-antibody complexes, was shown to be induced by monoclonal anti-mu or anti-idiotypic antibodies. An initial event in the induction of blockade was demonstrated to be the build up on the cell surface of a complex of membrane Ig, and either antigen or anti-mu or anti-Id antibody, together with secreted IgM. Trapping of secreted IgM in this complex was not, however, sufficient to account for the overall reduction in antibody secretion. This reduction was not due to reduced translation of mu mRNA, but rather to blockade of the normal process of assembly of the IgM molecule which was followed by increased intracellular degradation of intermediate precursor molecules. These findings have shown ECB to be a rapidly induced negative feedback control of IgM secretion which, at least initially, could be reversed. ECB may act in vivo as a flexible first-line mechanism for prevention of overproduction of IgM antibodies.

Detection of nonrandom clustering of colonies in agar culture.

We report a quantitative method for the detection of clustering in agar cultures. A statistical model was constructed on the assumption that colony formation by a cell in agar culture was independent of its spatial relationship to other cells in the culture. Deviation from this model was then tested by a chi-squared test of the actual observations compared with values obtained from the model. This method provides a technique that will identify interactions between cells of like or unlike type. Using a cell line with an extremely high cloning frequency, we show that the assumptions of the method are valid. Two situations are examined in which there was a suggestion of colony clustering on observational grounds. In both cases the test confirmed this impression and allowed us to analyze the possible causes of this behavior. We believe that this method will be of value in the identification of as yet unknown growth factors or cell-cell interactions.

The effect of cytokines on CD34+ Rh-123high and low progenitor cells from human umbilical cord blood.

In this study, we show how Rhodamine-123 (Rh-123), as in other hematopoietic populations, can be used to define functionally distinct progenitor cells from human umbilical cord blood (HUCB). CD34+ cells were subdivided into Rh-123high (78.2 +/- 4.5%) and Rh-123low (21.8 +/- 3.6%). While 9.3 +/- 1.6% of the CD34+Rh-123high cells formed colonies in agar, only 0.4 +/- 0.2% of the CD34+Rh-123low population did so. However, the CD34+Rh-123low cells resulted in the greatest expansion of colony-forming cells (CFC) when cultured in liquid medium with different cytokine combinations. When the CD34+Rh-123low cells were cultured for 7 days with stem cell factor (SCF) and erythropoietin (Epo), the CD34+Rh-123low cells resulted in a 94-fold increase in CFC compared with a 2.5-fold increase from the CD34+Rh-123high cells. The combination of SCF and Epo or granulocyte-macrophage colony-stimulating factor (GM-CSF) supported the production and maintenance of CFC from CD34+Rh-123low cells > 28 days compared with only 21 days for the CD34+Rh-123high cells. Coculture of CD34+Rh-123low cells with stromal cell line 11 (SCL11) demonstrated that long-term culture initiating cells (LTCIC) were present within this population, as CFC could be recovered for > 10 weeks compared with < 6 weeks in cocultures with CD34+Rh-123high cells. The duration of maintenance of CFC in liquid culture could be further enhanced by the addition of an antibody (Ab) directed against the binding site of the GM-CSF receptor. The addition of anti-GM-CSF receptor Ab to cultures of CD34+Rh-123high and low cells supplemented with SCF, interleukin-3 (IL-3), and IL-6 resulted in an initial 10-fold decrease in CFC in cultures of both the CD34+Rh-123high and low cells. Although very few CFCs were present by 42 days in liquid cultures of CD34+Rh-123high cells, the number of CFCs in these cultures was significantly increased when anti-GM-CSF receptor Ab was added. Although this effect was also observed in cultures of CD34+Rh-123low cells, it was less dramatic as more CFC persisted even in the absence of Ab. The possible mechanism of this effect is discussed.