Christoph P. Beier

Chemoresistance of glioblastoma cancer stem cells - much more complex than expected

Glioblastomas (GBM) are a paradigm for the investigation of cancer stem cells (CSC) in solid malignancies. The susceptibility of GBM CSC to standard chemotherapeutic drugs is controversial as the existing literature presents conflicting experimental data. Here, we summarize the experimental evidence on the resistance of GBM CSC to alkylating chemotherapeutic agents, with a special focus on temozolomide (TMZ). The data suggests that CSC are neither resistant nor susceptible to chemotherapy per se. Detoxifying proteins such as O6-methylguanine-DNA-methyltransferase (MGMT) confer a strong intrinsic resistance to CSC in all studies. Extrinsic factors may also contribute to the resistance of CSC to TMZ. These may include TMZ concentrations in the brain parenchyma, TMZ dosing schemes, hypoxic microenvironments, niche factors, and the re-acquisition of stem cell properties by non-stem cells. Thus, the interaction of CSC and chemotherapy is more complex than may be expected and it is necessary to consider these factors in order to overcome chemoresistance in the patient.

CD133 Expression and Cancer Stem Cells Predict Prognosis in High-grade Oligodendroglial Tumors

High‐grade oligodendroglial tumors, that is, anaplastic oligodendroglial tumors and glioblastomas with oligodendroglial component, differ significantly in terms of prognosis and response to chemotherapy. Differentiation might be difficult because the histological differences are vague and reliable markers are not established. We correlated the presence of putative cancer stem cells (CSC) in high‐grade oligodendroglial tumors (WHO grades III and IV) with clinical outcome. Tumors with favorable prognosis neither contained CSC nor did they show CD133 expression. Tumor cells resembled lineage‐restricted progenitor cells with limited proliferative capacity and differentiation profile. In contrast, CD133 expression and stem cell‐like tumor cells characterized tumors with poor prognosis. They showed neurosphere‐like growth, differentiated into cells of all neural lineages, and were tumorigenic in nude mice. In our series, CSC and expression of CD133 predicted the clinical course of disease better than the histological grading. To confirm these results, we retrospectively analyzed 36 high‐grade oligodendroglial tumors. Again, CD133 expression indicated shorter survival and predicted clinical outcome more reliable than the histological assessment.

CAMTA1 is a novel tumour suppressor regulated by miR-9/9* in glioblastoma stem cells.

Cancer stem cells or cancer initiating cells are believed to contribute to cancer recurrence after therapy. MicroRNAs (miRNAs) are short RNA molecules with fundamental roles in gene regulation. The role of miRNAs in cancer stem cells is only poorly understood. Here, we report miRNA expression profiles of glioblastoma stem cell‐containing CD133+ cell populations. We find that miR‐9, miR‐9* (referred to as miR‐9/9*), miR‐17 and miR‐106b are highly abundant in CD133+ cells. Furthermore, inhibition of miR‐9/9* or miR‐17 leads to reduced neurosphere formation and stimulates cell differentiation. Calmodulin‐binding transcription activator 1 (CAMTA1) is a putative transcription factor, which induces the expression of the anti‐proliferative cardiac hormone natriuretic peptide A (NPPA). We identify CAMTA1 as an miR‐9/9* and miR‐17 target. CAMTA1 expression leads to reduced neurosphere formation and tumour growth in nude mice, suggesting that CAMTA1 can function as tumour suppressor. Consistently, CAMTA1 and NPPA expression correlate with patient survival. Our findings could provide a basis for novel strategies of glioblastoma therapy.

Aldehyde dehydrogenase 1A1—a new mediator of resistance to temozolomide in glioblastoma

Implementation of chemotherapy with the drug temozolomide increased the overall survival of patients with glioblastoma multiforme (GBM; WHO grade IV), in particular when the O(6)-methylguanine DNA methyltransferase (MGMT) promoter is epigenetically silenced. Nevertheless, the prognosis remains poor, and relapse in GBM occurs regularly. This clinical behavior seems to be due to the existence of a therapy-resistant subpopulation of cells that induce tumor regrowth. The objective of this work was to analyze the role of aldehyde dehydrogenase (ALDH) 1A1 in mediating temozolomide resistance and its value as a predictor of clinical outcome in GBM patients. Nine GBM cell lines were treated with temozolomide alone or in combination with 4-diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH1A1, or with ALDH1A1 short hairpin (sh)RNA. ALDH1A1 expression and MGMT status of 70 primary GBM patients were correlated with median survival. ALDH1A1 overexpression predicted temozolomide resistance in vitro. Sensitivity of ALDH1A1 positive/MGMT-positive cells to temozolomide could be restored by inhibition of ALDH1A1 by DEAB or by knockdown with shRNA, as indicated by increased cytotoxicity, reduced clonogenicity, and accumulation in the G2/M cell-cycle phase. The prognosis of patients with a high level of ALDH1A1 expression was poor compared with that of patients with low levels (P < .0001). ALDH1A1 is a new mediator for resistance of GBM to temozolomide and a reliable predictor of clinical outcome and may serve as a potential target to improve treatment of human GBM.

Glioblastoma cancer stem cells – From concept to clinical application

Ten years after the first description of cancer stem cells (CSCs) in glioblastoma (GBM), the initial concept of CSC has been challenged and our understanding of cellular heterogeneity within malignant brain tumors became more complex. The increasing knowledge on CSC also influences preclinical research and clinical practice. This review therefore describes current concepts and controversies on CSC in GBM and summarizes the recent progress how the CSC hypothesis is about to translate into preclinical and clinical application.

RNOP-09: Pegylated liposomal doxorubicine and prolonged temozolomide in addition to radiotherapy in newly diagnosed glioblastoma - a phase II study

BackgroundAlthough Temozolomide is effective against glioblastoma, the prognosis remains dismal and new regimens with synergistic activity are sought for.MethodsIn this phase-I/II trial, pegylated liposomal doxorubicin (Caelyx™, PEG-Dox) and prolonged administration of Temozolomide in addition to radiotherapy was investigated in 63 patients with newly diagnosed glioblastoma. In phase-I, PEG-Dox was administered in a 3-by-3 dose-escalation regimen. In phase-II, 20 mg/m2 PEG-Dox was given once prior to radiotherapy and on days 1 and 15 of each 28-day cycle starting 4 weeks after radiotherapy. Temozolomide was given in a dose of 75 mg/m2 daily during radiotherapy (60 Gy) and 150-200 mg/m2 on days 1-5 of each 28-day cycle for 12 cycles or until disease progression.ResultsThe toxicity of the combination of PEG-Dox, prolonged administration of Temozolomide, and radiotherapy was tolerable. The progression free survival after 12 months (PFS-12) was 30.2%, the median overall survival was 17.6 months in all patients including the ones from Phase-I. None of the endpoints differed significantly from the EORTC26981/NCIC-CE.3 data in a post-hoc statistical comparison.ConclusionTogether, the investigated combination is tolerable and feasible. Neither the addition of PEG-Dox nor the prolonged administration of Temozolomide resulted in a meaningful improvement of the patients outcome as compared to the EORTC26981/NCIC-CE.3 dataTrial registrationclinicaltrials.gov NCT00944801.

Efficacy of clinically relevant temozolomide dosing schemes in glioblastoma cancer stem cell lines

The effectiveness of temozolomide (TMZ) dosing schemes and the “rechallenge” of recurrent glioblastoma (GBM) with TMZ are controversial. We therefore compared the efficacy of different TMZ dosing schemes against GBM cancer stem cell (CSC) lines inxa0vitro. In O6-methyl-guanidine-methyl-transferase (MGMT)-negative CSC lines, all schedules (1xa0day on/27xa0days off, 5xa0days on/23xa0days off, 7xa0days on/7xa0days off, 21xa0days on/7xa0days off, continuous low-dose TMZ) depleted clonogenic cells. In TMZ-resistant CSC lines, the 7xa0days on/7xa0days off scheme showed higher toxicity as compared with the other schemes. However, clinically feasible concentrations remained ineffective in highly resistant CSC lines. In addition, none of the schedules induced long-term depletion of clonogenic cells even at the highest concentrations (up to 250xa0μM). After sublethal TMZ treatment for 5xa0days, TMZ rechallenge of recovering CSC lines remained effective. Our data advocate CSC lines as inxa0vitro model to address clinical questions. Using this model, our data suggest the effectiveness of TMZ in MGMT-negative CSC lines and support the concept of TMZ rechallenge. The 7xa0days on/7xa0days off scheme consistently showed the best activity of all schedules in TMZ-resistant CSC lines.

Identification of CD133−/Telomeraselow Progenitor Cells in Glioblastoma-Derived Cancer Stem Cell Lines

Glioblastoma multiforme (GBM) is paradigmatic for the investigation of cancer stem cells (CSC) in solid tumors. The CSC hypothesis implies that tumors are maintained by a rare subpopulation of CSC that gives rise to rapidly proliferating progenitor cells. Although the presence of progenitor cells is crucial for the CSC hypothesis, progenitor cells derived from GBM CSC are yet uncharacterized. We analyzed human CD133+ CSC lines that were directly derived from CD133+ primary astrocytic GBM. In these CSC lines, CD133+/telomerasehigh CSC give rise to non-tumorigenic, CD133−/telomeraselow progenitor cells. The proliferation of the progenitor cell population results in significant telomere shortening as compared to the CD133+ compartment comprising CSC. The average difference in telomere length as determined by a modified multi-color flow fluorescent in situ hybridization was 320xa0bp corresponding to 4–8 cell divisions. Taken together, we demonstrate that CD133+ primary astrocytic GBM comprise proliferating, CD133−/telomeraselow progenitor cell population characterized by low telomerase activity and shortened telomeres as compared to CSC.

Comprehensive Small Animal Imaging Strategies on a Clinical 3 T Dedicated Head MR-Scanner; Adapted Methods and Sequence Protocols in CNS Pathologies

Background Small animal models of human diseases are an indispensable aspect of pre-clinical research. Being dynamic, most pathologies demand extensive longitudinal monitoring to understand disease mechanisms, drug efficacy and side effects. These considerations often demand the concomitant development of monitoring systems with sufficient temporal and spatial resolution. Methodology and Results This study attempts to configure and optimize a clinical 3 Tesla magnetic resonance scanner to facilitate imaging of small animal central nervous system pathologies. The hardware of the scanner was complemented by a custom-built, 4-channel phased array coil system. Extensive modification of standard sequence protocols was carried out based on tissue relaxometric calculations. Proton density differences between the gray and white matter of the rodent spinal cord along with transverse relaxation due to magnetic susceptibility differences at the cortex and striatum of both rats and mice demonstrated statistically significant differences. The employed parallel imaging reconstruction algorithms had distinct properties dependent on the sequence type and in the presence of the contrast agent. The attempt to morphologically phenotype a normal healthy rat brain in multiple planes delineated a number of anatomical regions, and all the clinically relevant sequels following acute cerebral ischemia could be adequately characterized. Changes in blood-brain-barrier permeability following ischemia-reperfusion were also apparent at a later time. Typical characteristics of intra-cerebral haemorrhage at acute and chronic stages were also visualized up to one month. Two models of rodent spinal cord injury were adequately characterized and closely mimicked the results of histological studies. In the employed rodent animal handling system a mouse model of glioblastoma was also studied with unequivocal results. Conclusions The implemented customizations including extensive sequence protocol modifications resulted in images of high diagnostic quality. These results prove that lack of dedicated animal scanners shouldnt discourage conventional small animal imaging studies.

Chemotherapy increases long-term survival in patients with adult medulloblastoma—a literature-based meta-analysis

BACKGROUNDnAdult medulloblastoma is a potentially curable malignant entity with an incidence of 0.5-1 per million. Valid data on prognosis, treatment, and demographics are lacking, as most current knowledge stems from retrospective studies. Surgical resection followed by radiotherapy are accepted parts of treatment regimes; however, established prognostic factors and data clarifying the role of chemotherapy are missing.nnnMETHODSnWe investigated 227 publications from 1969-2013, with 907 identifiable, individual patients being available for meta-analysis. Demographic data, risk stratification, and treatment of these patients were similar to previous cohorts.nnnRESULTSnThe median overall survival (mOS) was 65 months (95% CI: 54.6-75.3) , the 5-year overall survival was 50.9% with 16% of the patients dying more than 5 years after diagnosis. Incomplete resection, clinical and radiological signs for brainstem infiltration, and abstinence from radiotherapy were predictive of worse outcome. Metastatic disease at tumor recurrence was identified as a new prognostic factor, while neither metastasis at initial diagnosis nor desmoplastic/classic histology was correlated with survival. Patients receiving chemotherapy first-line survived significantly longer (mOS: 108 mo, 95% CI: 68.6-148.4) than patients treated with radiation alone (mOS: 57 mo, 95% CI: 39.6-74.4) or patients who received chemotherapy at tumor recurrence. This effect was not biased by tumor stage or decade of treatment. Importantly, (neo)adjuvant chemotherapy also significantly increased the chance for long-term survival (>5 y) compared with radiotherapy alone or chemotherapy at tumor recurrence.nnnCONCLUSIONSnThis meta-analysis clarifies relevant prognostic factors and suggests that chemotherapy as part of first-line therapy improves overall survival and increases the proportion of patients with long-term survival.