Carbon irradiation overcomes glioma radioresistance by eradicating stem cells and forming an antiangiogenic and immunopermissive niche.

Tumor radioresistance leading to local therapy failure remains a major obstacle for successful treatment of high-grade glioma. We hypothesized that distinct radiobiological features of particle therapy with carbon ions may circumvent glioma radioresistance. We demonstrate that carbon irradiation (CIR) efficiently eradicates radioresistant patient-derived glioma stem cells (GSCs), leading to growth inhibition and prolonged survival. The impact of CIR at the tumor-stroma interface was further investigated in 2 syngeneic mouse and 2 orthotopic GSC xenograft models. Intriguingly, tumor regressions and long-term local controls were observed at doses greater than or equal to 15-Gy CIR. Fractionated CIR further prolonged survival. The enhanced relative biological effectiveness of CIR in vivo was attributed to its potent antiangiogenic effects and eradication of radioresistant hypoxic tumor cells. Blockade of the HIF1-α/stromal cell-derived factor 1/CXCR4 axis by CIR reduced the recruitment of microglia and myeloid-derived suppressor cells (CD11b+Gr1+). Consequently, CIR abrogated M2-like immune polarization and enhanced the influx of CD8+ cells, generating an immunopermissive niche. We report that radiotherapy with carbon ions could surmount several central glioma resistance mechanisms by eradicating hypoxic and stem cell-like tumor cells, as well as modulating the glioma niche toward an antiangiogenic and less immunosuppressive state. Conclusively, potentially novel rationales for CIR in conquering glioma radioresistance are provided.

K-Ras and cyclooxygenase-2 coactivation augments intraductal papillary mucinous neoplasm and Notch1 mimicking human pancreas lesions.

Mutational activation of K-Ras is an initiating event of pancreatic ductal adenocarcinomas (PDAC) that may develop either from pancreatic intraepithelial neoplasia (PanIN) or intraductal papillary mucinous neoplasms (IPMN). Cyclooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) is causally related to pancreatic carcinogenesis. Here, we deciphered the impact of COX-2, a key modulator of inflammation, in concert with active mutant K-Ras(G12D) on tumor burden and gene expression signature using compound mutant mouse lines. Concomitant activation of COX-2 and K-Ras(G12D) accelerated the progression of pancreatic intraepithelial lesions predominantly with a cystic papillary phenotype resembling human IPMN. Transcriptomes derived from laser capture microdissected preneoplastic lesions of single and compound mutants revealed a signature that was significantly enriched in Notch1 signaling components. In vitro, Notch1 signaling was COX-2-dependent. In line with these findings, human IPMN stratified into intestinal, gastric and pancreatobillary types displayed Notch1 immunosignals with high prevalence, especially in the gastric lesions. In conclusion, a yet unknown link between activated Ras, protumorigenic COX-2 and Notch1 in IPMN onset was unraveled.

Correlated magnetic resonance imaging and ultramicroscopy (MR-UM) is a tool kit to assess the dynamics of glioma angiogenesis.

Neoangiogenesis is a pivotal therapeutic target in glioblastoma. Tumor monitoring requires imaging methods to assess treatment effects and disease progression. Until now mapping of the tumor vasculature has been difficult. We have developed a combined magnetic resonance and optical toolkit to study neoangiogenesis in glioma models. We use in vivo magnetic resonance imaging (MRI) and correlative ultramicroscopy (UM) of ex vivo cleared whole brains to track neovascularization. T2* imaging allows the identification of single vessels in glioma development and the quantification of neovessels over time. Pharmacological VEGF inhibition leads to partial vascular normalization with decreased vessel caliber, density, and permeability. To further resolve the tumor microvasculature, we performed correlated UM of fluorescently labeled microvessels in cleared brains. UM resolved typical features of neoangiogenesis and tumor cell invasion with a spatial resolution of ~5 µm. MR-UM can be used as a platform for three-dimensional mapping and high-resolution quantification of tumor angiogenesis.

Inhibition of Tumor Growth and Metastasis in Pancreatic Cancer Models by Interference With CD44v6 Signaling.

BACKGROUND & AIMS: Cancer cells with high metastatic potential and stem cell-like characteristics express the cell surface marker CD44. CD44 isoforms that include the v6 exon are co-receptors for the receptor tyrosine kinases MET and Vascular Endothelial Growth factor Receptor-2 (VEGFR-2). We studied CD44v6 signaling in several pancreatic cancer cell lines, and its role in tumor growth and metastasis in several models of pancreatic cancer. METHODS: We analyzed the effects of v6 peptides that interfere with the co-receptor functions of CD44v6 for MET and VEGFR-2 in tumors and metastases grown from cells that express different CD44 isoforms, including CD44v6. The peptides were injected into rats with syngeneic tumors and mice with orthotopic or xenograft tumors. We also tested the effects of the peptides in mice with xenograft tumors grown from patient tumor samples and mice that express an oncogenic form of RAS and develop spontaneous pancreatic cancer (KPC mice). We measured levels of CD44v6 messenger RNA (mRNA) in pancreatic cancer tissues from 136 patients. RESULTS: Xenograft tumors grown from human cancer cells injected with v6 peptides were smaller and formed fewer metastases in mice. The v6 peptide was more efficient than the MET inhibitor crizotinib and/or the VEGFR-2 inhibitor pazopanib in reducing xenograft tumor growth and metastasis. Injection of KPC mice with the v6 peptide increased their survival time. Injection of mice and rats bearing metastases with the v6 peptide induced regression of metastases. Higher levels of CD44v6 mRNA in human pancreatic tumor tissues were associated with increased expression of MET, tumor metastasis, and shorter patient survival times. CONCLUSIONS: Peptide inhibitors of CD44v6 isoforms block tumor growth and metastasis in several independent models of pancreatic cancer. The v6 peptides induced regression of metastases. Levels of CD44v6 mRNA are increased, along with those of MET mRNA, in patients with metastatic pancreatic tumors, compared with nonmetastatic tumors; the increased levels correlated with shorter patient survival time.

Radiosensitivity of Patient-Derived Glioma Stem Cell 3-Dimensional Cultures to Photon, Proton, and Carbon Irradiation.

PURPOSE: To investigate the radiosensitivity of primary glioma stem cell (GSC) cultures with different CD133 status in a 3-dimensional (3D) model after photon versus proton versus carbon irradiation. METHODS AND MATERIALS: Human primary GSC spheroid cultures were established from tumor specimens of six consented glioblastoma patients. Human U87MG was used as a classical glioblastoma radioresistant cell line. Cell suspensions were generated by mechanical dissociation of GSC spheroids and embedded in a semi-solid 3D matrix before irradiation. Spheroid-like colonies were manually counted by microscopy. Cells were also recovered and quantified by fluorescence. CD133 expression and DNA damage were evaluated by flow cytometry. RESULTS: The fraction of CD133(+) cells varied between 0.014% and 96% in the six GSC cultures and showed a nonsignificant correlation with plating efficiency and survival fractions. The 4 most photon-radioresistant GSC cultures were NCH644, NCH421k, NCH441, and NCH636. Clonogenic survival for proton irradiation revealed relative biologic effectiveness (RBE) in the range of 0.7-1.20. However, carbon irradiation rendered the photon-resistant GSC cultures sensitive, with average RBE of 1.87-3.44. This effect was partly attributed to impaired capability of GSC to repair carbon ion-induced DNA double-strand breaks as determined by residual DNA repair foci. Interestingly, radiosensitivity of U87 cells was comparable to GSC cultures using clonogenic survival as the standard readout. CONCLUSIONS: Carbon irradiation is effective in GSC eradication with similar RBE ranges approximately 2-3 as compared with non-stem GSC cultures (U87). Our data strongly suggest further exploration of GSC using classic radiobiology endpoints such as the here-used 3D clonogenic survival assay and integration of additional GSC-specific markers.

Lessons we learned from high-throughput and top-down systems biology analyses about glioma stem cells.

A growing body of evidence suggests that glioma stem cells (GSCs) account for tumor initiation, therapy resistance, and the subsequent regrowth of gliomas. Thus, continuous efforts have been undertaken to further characterize this subpopulation of less differentiated tumor cells. Although we are able to enrich GSCs, we still lack a comprehensive understanding of GSC phenotypes and behavior. The advent of high-throughput technologies raised hope that incorporation of these newly developed platforms would help to tackle such questions. Since then a couple of comparative genome-, transcriptome- and proteome-wide studies on GSCs have been conducted giving new insights in GSC biology. However, lessons had to be learned in designing high-throughput experiments and some of the resulting conclusions fell short of expectations because they were performed on only a few GSC lines or at one molecular level instead of an integrative poly-omics approach. Despite these shortcomings, our knowledge of GSC biology has markedly expanded due to a number of survival-associated biomarkers as well as glioma-relevant signaling pathways and therapeutic targets being identified. In this article we review recent findings obtained by comparative high-throughput analyses of GSCs. We further summarize fundamental concepts of systems biology as well as its applications for glioma stem cell research.

Identification of stable endogenous control genes for transcriptional profiling of photon, proton and carbon-ion irradiated cells.

BACKGROUND: Quantitative analysis of transcriptional regulation of genes is a prerequisite for a better understanding of the molecular mechanisms of action of different radiation qualities such as photon, proton or carbon ion irradiation. Microarrays and real-time quantitative RT-PCR (qRT-PCR) are considered the two cornerstones of gene expression analysis. In interpreting these results it is critical to normalize the expression levels of the target genes by that of appropriately selected endogenous control genes (ECGs) or housekeeping genes. We sought to systematically investigate common ECG candidates for their stability after different radiation modalities in different human cell lines by qRT-PCR. We aimed to identify the most robust set of ECGs or housekeeping genes for transcriptional analysis in irradiation studies. METHODS: We tested the expression stability of 32 ECGs in three human cancer cell lines. The epidermoid carcinoma cells (A431), the non small cell lung carcinoma cells (A549) and the pancreatic adenocarincoma cells (BxPC3) were irradiated with photon, proton and carbon ions. Expression Heat maps, clustering and statistic algorithms were employed using SUMO software package. The expression stability was evaluated by computing: mean, standard deviation, ANOVA, coefficient of variation and the stability measure (M) given by the geNorm algorithm. RESULTS: Expression analysis revealed significant cell type specific regulation of 18 out of 32 ECGs (p < 0.05). A549 and A431 cells shared a similar pattern of ECG expression as the function of different radiation qualities as compared to BxPC3. Of note, the ribosomal protein 18S, one of the most frequently used ECG, was differentially regulated as the function of different radiation qualities (p ≤ 0.01). A comprehensive search for the most stable ECGs using the geNorm algorithm identified 3 ECGs for A431 and BxPC3 to be sufficient for normalization. In contrast, 6 ECGs were required to properly normalize expression data in the more variable A549 cells. Considering both variables tested, i.e. cell type and radiation qualities, 5 genes-- RPLP0, UBC, PPIA, TBP and PSMC4-- were identified as the consensus set of stable ECGs. CONCLUSIONS: Caution is warranted when selecting the internal control gene for the qRT-PCR gene expression studies. Here, we provide a template of stable ECGs for investigation of radiation induced gene expression.

Origin of pancreatic ductal adenocarcinoma from atypical flat lesions: a comparative study in transgenic mice and human tissues.

Pancreatic ductal adenocarcinoma (PDAC) and its precursor lesions, pancreatic intraepithelial neoplasia (PanIN), display a ductal phenotype. However, there is evidence in genetically defined mouse models for PDAC harbouring a mutated kras under the control of a pancreas-specific promoter that ductal cancer might arise in the centroacinar-acinar region, possibly through a process of acinar-ductal metaplasia (ADM). In order to further elucidate this model of PDAC development, an extensive expression analysis and molecular characterization of the putative and already established (PanIN) precursor lesions were performed in the Kras(G12D/+) ; Ptf1a-Cre(ex1/+) mouse model and in human tissues, focusing on lineage markers, developmental pathways, cell cycle regulators, apomucins, and stromal activation markers. The results of this study show that areas of ADM are very frequent in the murine and human pancreas and represent regions of increased proliferation of cells with precursor potential. Moreover, atypical flat lesions originating in areas of ADM are the most probable precursors of PDAC in the Kras(G12D/+); Ptf1a-Cre(ex1/+) mice and similar lesions were also found in the pancreas of three patients with a strong family history of PDAC. In conclusion, PDAC development in Kras(G12D/+); Ptf1a-Cre(ex1/+) mice starts from ADM and a similar process might also take place in patients with a strong family history of PDAC.

Preinvasive duct-derived neoplasms in pancreas of keratin 5-promoter cyclooxygenase-2 transgenic mice.

BACKGROUND & AIMS: Basic research aimed at a better understanding of pancreatic carcinogenesis and improving the treatment of this disease is crucial because the majority of pancreatic cancers are highly aggressive and therapeutically nonaccessible. Cyclooxygenase (COX)-2, which is a key enzyme of prostaglandin (PG) biosynthesis, is overexpressed in around 75% of human carcinomas including those of the pancreas. METHODS: The pathologic changes of transgenic mouse pancreas with keratin 5-promoter-driven expression and activity of COX-2 were characterized. RESULTS: Aberrant expression of COX-2 in a few ductal cells and COX-2-mediated PG synthesis in the transgenic mice resulted in keratin 19- and mucin-positive intraductal papillary mucinous neoplasm- and pancreatic intraepithelial neoplasia-like structures, characterized by an increased proliferation index and serous cystadenomas. Moreover, Ras activation was enhanced and the HER-2/Neu receptor was overexpressed. Loss of acini, fibrosis, and inflammation were pronounced. Feeding a COX-2-selective inhibitor to the transgenic mice suppressed the accumulation of PG and the phenotype. The changes resemble the human disease in which COX-2 was overexpressed consistently. CONCLUSIONS: We present strong evidence for a causal relationship between aberrant COX-2 overexpression and COX-2-mediated PG synthesis and the development of serous cystadenoma, intraductal papillary mucinous, and pancreatic intraepithelial neoplasms. This model offers the unique possibility of identifying molecular pathways leading to the formation and malignant progression of the various types of preinvasive lesions of pancreatic adenocarcinomas that show different dismal outcomes.