Mark A. Schroeder

Stretch induces cytokine release by alveolar epithelial cells in vitro

Mechanical ventilation can injure the lung, causing edema and alveolar inflammation. Interleukin-8 (IL-8) plays an important role in this inflammatory response. We postulated that cyclic cell stretch upregulates the production and release of IL-8 by human alveolar epithelium in the absence of structural cell damage or paracrine stimulation. To test this hypothesis, alveolar epithelial cells (A549 cells) were cultured on a deformable silicoelastic membrane. When stretched by 30% for up to 48 h, the cells released 49 ± 34% more IL-8 ( P < 0.001) than static controls. Smaller deformations (20% stretch) produced no consistent increase in IL-8. Stretch of 4 h duration increased IL-8 gene transcription fourfold above baseline. Stretch had no effect on cell proliferation, cell viability as assessed by51Cr release assay, or the release of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-α. We conclude that deformation per se can trigger inflammatory signaling and that alveolar epithelial cells may be active participants in the alveolitis associated with ventilator-induced lung injury.

The histone H3.3K27M mutation in pediatric glioma reprograms H3K27 methylation and gene expression

Recent studies have identified a Lys 27-to-methionine (K27M) mutation at one allele of H3F3A, one of the two genes encoding histone H3 variant H3.3, in 60% of high-grade pediatric glioma cases. The median survival of this group of patients after diagnosis is ∼1 yr. Here we show that the levels of H3K27 di- and trimethylation (H3K27me2 and H3K27me3) are reduced globally in H3.3K27M patient samples due to the expression of the H3.3K27M mutant allele. Remarkably, we also observed that H3K27me3 and Ezh2 (the catalytic subunit of H3K27 methyltransferase) at chromatin are dramatically increased locally at hundreds of gene loci in H3.3K27M patient cells. Moreover, the gain of H3K27me3 and Ezh2 at gene promoters alters the expression of genes that are associated with various cancer pathways. These results indicate that H3.3K27M mutation reprograms epigenetic landscape and gene expression, which may drive tumorigenesis.

Patient tumor EGFR and PDGFRA gene amplifications retained in an invasive intracranial xenograft model of glioblastoma multiforme

We have previously described a panel of serially transplantable glioblastoma multiforme xenograft lines established by direct subcutaneous injection of patient tumor tissue in the flanks of nude mice. Here we report the characterization of four of these lines with respect to their histopathologic, genetic, and growth properties following heterotopic-to-orthotopic (flank-to-intracranial) transfer. Cells from short-term cultures, established from excised flank xenografts, were harvested and injected into the brains of nude mice (10(6) cells per injection). The intracranial tumors generated from these injections were all highly mitotic as well as highly invasive, but they lacked necrotic features in most instances and failed to show endothelial cell proliferation in all instances. For mice receiving injections from a common explant culture, tumor intracranial growth rate was consistent, as indicated by relatively narrow ranges in survival time. In contrast to the loss of epidermal growth factor receptor gene (EGFR) amplification in cell culture, high-level amplification and overexpression of EGFR were retained in intracranial tumors established from two EGFR-amplified flank tumors. A third intracranial tumor retained patient tumor amplification and high-level expression of platelet-derived growth factor receptor alpha gene. Because the heterotopic-to-orthotopic transfer and propagation of glioblastoma multiforme preserves the receptor tyrosine kinase (RTK) gene amplification of patient tumors, this approach should facilitate investigations for determining the extent to which RTK amplification status influences tumor response to RTK-directed therapies. The fact that such studies were carried out by using an invasive tumor model in an anatomically appropriate context should ensure a rigorous preclinical assessment of agent efficacy.

Identification of molecular characteristics correlated with glioblastoma sensitivity to EGFR kinase inhibition through use of an intracranial xenograft test panel

In the current study, we examined a panel of serially passaged glioblastoma xenografts, in the context of an intracranial tumor therapy response model, to identify associations between glioblastoma molecular characteristics and tumor sensitivity to the epidermal growth factor receptor (EGFR) kinase inhibitor erlotinib. From an initial evaluation of 11 distinct glioblastoma xenografts, two erlotinib-sensitive tumors were identified, each having amplified EGFR and expressing wild-type PTEN. One of these tumors expressed truncated EGFRvIII, whereas the other expressed full-length EGFR. Subsequent cDNA sequence analysis revealed the latter tumor as expressing an EGFR sequence variant with arginine, rather than leucine, at amino acid position 62; this was the only EGFR sequence variant identified among the 11 xenografts, other than the aforementioned vIII sequence variant. EGFR cDNAs were then examined from 12 more xenografts to determine whether additional missense sequence alterations were evident, and this analysis revealed one such case, expressing threonine, rather than alanine, at amino acid position 289 of the extracellular domain. This glioblastoma was also amplified for EGFR, but did not display significant erlotinib sensitivity, presumably due to its lacking PTEN expression. In total, our study identified two erlotinib-sensitive glioblastoma xenografts, with the common molecular characteristics shared by each being the expression of wild-type PTEN in combination with the expression of amplified and aberrant EGFR. [Mol Cancer Ther 2007;6(3):1167–74]

Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts

Temozolomide (TMZ)-based therapy is the standard of care for patients with glioblastoma multiforme (GBM), and resistance to this drug in GBM is modulated by the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Expression of MGMT is silenced by promoter methylation in approximately half of GBM tumors, and clinical studies have shown that elevated MGMT protein levels or lack of MGMT promoter methylation is associated with TMZ resistance in some, but not all, GBM tumors. In this study, the relationship between MGMT protein expression and tumor response to TMZ was evaluated in four GBM xenograft lines that had been established from patient specimens and maintained by serial subcutaneous passaging in nude mice. Three MGMT unmethylated tumors displayed elevated basal MGMT protein expression, but only two of these were resistant to TMZ therapy (tumors GBM43 and GBM44), while the other (GBM14) displayed a level of TMZ sensitivity that was similar in extent to that seen in a single MGMT hypermethylated line (GBM12). In tissue culture and animal studies, TMZ treatment resulted in robust and prolonged induction of MGMT expression in the resistant GBM43 and GBM44 xenograft lines, while MGMT induction was blunted and abbreviated in GBM14. Consistent with a functional significance of MGMT induction, treatment of GBM43 with a protracted low-dose TMZ regimen was significantly less effective than a shorter high-dose regimen, while survival for GBM14 was improved with the protracted dosing regimen. In conclusion, MGMT expression is dynamically regulated in some MGMT nonmethylated tumors, and in these tumors, protracted dosing regimens may not be effective.

Retargeted Oncolytic Measles Strains Entering via the EGFRvIII Receptor Maintain Significant Antitumor Activity against Gliomas with Increased Tumor Specificity

Among the best-characterized genetic alterations in gliomas is the amplification of the epidermal growth factor receptor (EGFR) gene, present in approximately 40% of glioblastoma multiforme, and frequently associated with the EGFRvIII gene rearrangement. We have previously shown that attenuated vaccine strains of measles virus have potent antitumor activity against gliomas, and identified H protein mutations, which ablate recognition of the natural measles virus receptors CD46 and SLAM. Retargeted recombinant viruses were generated from the measles Edmonston-NSe vaccine strain displaying a single-chain antibody against EGFRvIII at the COOH terminus of H and containing the marker green fluorescent protein (GFP) gene in position 1. Two different H mutants were employed: H(SNS) (V451S, Y481N, and A527S)-CD46 blind, and H(AA) (Y481A and R533A)-CD46 and SLAM blind. MV-GFP virus was used as a positive control. Both EGFRvIII-retargeted viruses had significant antitumor activity against EGFRvIII-expressing glioblastoma multiforme but no cytopathic effect against normal cells. In an orthotopic model of EGFRvIII-expressing GBM39 xenografts, there was comparable therapeutic efficacy between retargeted strains and unmodified MV-GFP and statistically significant prolongation of survival in treated animals compared with the control group (P = 0.001). Formation of syncytia was observed in tumors treated with retargeted viruses, with a surrounding infiltrate consisting of macrophages and natural killer cells. In summary, EGFRvIII-retargeted oncolytic measles virus strains have comparable therapeutic efficacy with the unmodified MV-GFP strain against EGFRvIII-expressing glioma lines and xenografts with improved therapeutic index, a finding with potential translational implications in glioma virotherapy.

Gemcitabine and ISIS-2503 for Patients With Locally Advanced or Metastatic Pancreatic Adenocarcinoma: A North Central Cancer Treatment Group Phase II Trial

PURPOSEnGemcitabine remains the standard therapy for metastatic pancreatic adenocarcinoma (ACA), but has limited activity. ISIS-2503 is an antisense compound directed against H-ras with preclinical activity against pancreatic ACA in tumor models. The combination of ISIS-2503 and gemcitabine has been evaluated in a prior phase I study.nnnMETHODSnPatients with metastatic or locally advanced pancreatic ACA not amenable to surgery or local radiation received gemcitabine 1,000 mg/m(2) intravenously over 30 minutes on days 1 and 8 and ISIS-2503 6 mg/kg/d as a continuous intravenous infusion over 14 days of an every-3-weeks cycle. Responses were monitored by radiologic imaging every 6 weeks.nnnRESULTSnForty-eight eligible patients were enrolled, 43 with metastatic disease. Median follow-up was 12.6 months (range, 2.2 to 16.8 months) for living patients. A median of four cycles of treatment was given (range, 1 to 18 cycles). All patients were assessable for response and toxicity. The 6-month survival percentage was 57.5% (95% CI, 44.9% to 73.5%) and the median survival was 6.6 months. The response rate was 10.4% (one complete response, four partial responses). Clinically significant toxicity was limited except for one fatal pulmonary embolism.nnnCONCLUSIONnThis study shows a promising response rate to the combination of gemcitabine and ISIS-2503 in patients with pancreatic ACA. The observed 6-month survival rate in these patients met our protocol-defined criteria for success. This regimen is tolerable, but is of unclear benefit. Additional studies evaluating the role of gemcitabine and ISIS-2503 in the treatment of pancreatic ACA should be considered.

Establishment, Maintenance, and In Vitro and In Vivo Applications of Primary Human Glioblastoma Multiforme (GBM) Xenograft Models for Translational Biology Studies and Drug Discovery

Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro‐oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short‐term explant cultures or intracranial xenografts. This unit describes detailed procedures for establishment, maintenance, and utilization of a primary GBM xenograft panel for the purpose of using them as tumor models for basic or translational studies. Curr. Protoc. Pharmacol. 52:14.16.1‐14.16.23.

p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells

Deregulation of the p16(INK4a)-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16(INK4a)-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16(INK4a)-Cdk4/6-Rb pathway. In vitro evaluation using short-term explant cultures from selected GBM xenograft lines showed that PD0332991 effectively arrested cell cycle in G1-phase and inhibited cell proliferation dose-dependently in lines deleted for CDKN2A/B-p16(INK4a) and either single-copy deletion of CDK4 (GBM22), high-level CDK6 amplification (GBM34), or deletion of CDKN2C/p18(INK4c) (GBM43). In contrast, 2 GBM lines with p16(INK4a) expression and either CDK4 amplification (GBM5) or RB mutation (GBM28) were completely resistant to PD0332991. Additional xenograft lines were screened, and GBM63 was identified to have p16(INK4a) expression and CDK4 amplification. Similar to the results with GBM5, GBM63 was resistant to PD0332991 treatment. In an orthotopic survival model, treatment of GBM6 xenografts (CDKN2A/B-deleted and CDK4 wild-type) with PD0332991 significantly suppressed tumor cell proliferation and prolonged survival. Collectively, these data support the concept that GBM tumors lacking p16(INK4a) expression and with nonamplified CDK4 and wild-type RB status may be more susceptible to Cdk4/6 inhibition using PD0332991.

Bioluminescence monitoring of intracranial glioblastoma xenograft: response to primary and salvage temozolomide therapy

OBJECTnBioluminescence imaging (BLI) offers a rapid and accurate means for longitudinal study of tumor cell growth and response to therapy in rodent models. Because this technology has only recently come into use in the field of small animal imaging, applications in this area have been limited. In the current study we have applied BLI to the analysis of clinically relevant issues involving use of the DNA methylating agent temozolomide (TMZ) in a mouse model.nnnMETHODSnAn invasive glioblastoma multiforme xenograft was modified for BLI via transduction with a luciferase-encoding lentivirus. Supratentorial tumors were established in athymic nude mice that were subsequently assigned randomly to control and TMZ treatment groups, and the extent of intracranial tumor was monitored using BLI.nnnRESULTSnIn an experiment designed to compare the extent of antitumor effect between a single high-dose TMZ treatment and a protracted low-dose TMZ regimen, BLI revealed the protracted regimen as having superior antitumor effect, and this interpretation was consistent with results from a survival comparison between the two TMZ treatment groups. In a second experiment designed to assess the utility of BLI for testing therapies against recurrent glioblastoma multiforme, mice with intracranial tumors were retreated with TMZ at a time when BLI monitoring revealed tumor regrowth following initial TMZ treatment, and retreatment was successful in providing additional survival benefit.nnnCONCLUSIONSnThe results of these experiments indicate that BLI monitoring can be used as a surrogate for predicting survival benefit from TMZ treatment, permits early determination of relative survival benefit associated with distinct TMZ therapeutic regimens, and offers a means of investigating secondary/salvage therapy efficacy following tumor regrowth from initial therapy.