The Barretos Cancer Hospital Animal Facility: Implementation and Results of a Dedicated Platform for Preclinical Oncology Models.

The Barretos Cancer Hospital Animal Facility (BCHAF) is a unique facility in Brazil exclusively dedicated to working with animal models for cancer research. In this article, we briefly present our modern facility and the main experiments performed, focusing on mutant strains of mice (PTCH-knockout and ApcMin mice), xenograft models, and patient-derived xenografts (PDXs). Our results show the progress and challenges in establishing these models and the need for having an appropriate representation of our cancer population to better understand tumor biology and to identify cancer biomarkers, which could be putatively targeted, allowing for personalized therapy.

Alpha2beta1 Integrin Polymorphism in Diffuse Astrocytoma Patients.

Integrins are heterodimeric transmembrane glycoproteins resulting from the non-covalent association of an α and ß chain. The major integrin receptor for collagen/laminin, α2ß1 is expressed on a wide variety of cell types and plays an essential role in the adhesion of normal and tumor cells to the extracellular matrix. Integrin-triggered signaling pathways promote the invasion and survival of glioma cells by modifying the brain microenvironment. In this study, we investigated the association of a specific genetic polymorphism of integrin α2ß1 with the incidence of diffusely infiltrating astrocytoma and the progression of these tumors. Single-nucleotide polymorphism in intron 7 of the integrin ITGA2 gene was examined in 158 patients and 162 controls using polymerase chain reaction and restriction enzyme analysis. The ITGA2 genotype +/+ (with a BglII restriction site in both alleles) exhibited higher frequency in grade II astrocytoma compared to control (P = 0.02) whereas the genotype -/- (lacking the BglII site) correlated with the poorest survival rate (P = 0.04). In addition, in silico analyses of ITGA2 expression from low-grade gliomas (LGG, n = 515) and glioblastomas (GBM, n = 159) indicated that the higher expression of ITGA2 in LGG was associated with poor overall survival (P < 0.0001). However, the distribution of integrin ITGA2 BglII genotypes (+/+, +/-, -/-) was not significantly different between astrocytoma subgroups III and IV (P = 0.65, 0.24 and 0.33; 0.29, 0.48, 0.25, respectively) compared to control. These results suggest a narrow association between the presence of this SNP and indicate that further studies with larger samples are warranted to analyze the relation between tumor grade and overall survival, highlighting the importance of determining these polymorphisms for prognosis of astrocytomas.

Indisulam Reduces Viability and Regulates Apoptotic Gene Expression in Pediatric High-Grade Glioma Cells.

Pediatric high-grade glioma (pHGG) is one of the most aggressive brain tumors. Treatment includes surgery, radiotherapy, chemotherapy, or combination therapy in children older than 3−5 years of age. These devastating tumors are influenced by the hypoxic microenvironment that coordinatively increases the expression of carbonic anhydrases (CA9 and CA12) that are involved in pH regulation, metabolism, cell invasion, and resistance to therapy. The synthetic sulphonamide Indisulam is a potent inhibitor of CAs. The aim of this study was to evaluate the effects of Indisulam on CA9 and CA12 enzymes in pHGG cell lines. Our results indicated that, under hypoxia, the gene and protein expression of CA9 and CA12 are increased in pHGG cells. The functional effects of Indisulam on cell proliferation, clonogenic capacity, and apoptosis were measured in vitro. CA9 and CA12 gene and protein expression were analyzed by RT-PCR and western blot. The treatment with Indisulam significantly reduced cell proliferation (dose-time-dependent) and clonogenic capacity (p < 0.05) and potentiated the effect of apoptosis (p < 0.01). Indisulam promoted an imbalance in the anti-apoptotic BCL2 and pro-apoptotic BAX protein expression. Our results demonstrate that Indisulam contributes to apoptosis via imbalance of apoptotic proteins (BAX/BCL2) and suggests a potential to overcome chemotherapy resistance caused by the regulation these proteins.

The Carbonic Anhydrase Inhibitor E7070 Sensitizes Glioblastoma Cells to Radio- and Chemotherapy and Reduces Tumor Growth.

Glioblastomas (GBMs), the most common and lethal primary brain tumor, show inherent infiltrative nature and high molecular heterogeneity that make complete surgical resection unfeasible and unresponsive to conventional adjuvant therapy. Due to their fast growth rate even under hypoxic and acidic conditions, GBM cells can conserve the intracellular pH at physiological range by overexpressing membrane-bound carbonic anhydrases (CAs). The synthetic sulfonamide E7070 is a potent inhibitor of CAs that harbors putative anticancer properties; however, this drug has still not been tested in GBMs. The present study aimed to evaluate the effects of E7070 on CA9 and CA12 enzymes in GBM cells as well as in the tumor cell growth, migration, invasion, and resistance to radiotherapy and chemotherapy. We found that E7070 treatment significantly reduced tumor cell growth and increased radio- and chemotherapy efficacy against GBM cells under hypoxia. Our data suggests that E7070 has therapeutic potential as a radio-chemo-sensitizing in drug-resistant GBMs, representing an attractive strategy to improve the adjuvant therapy. We showed that CA9 and CA12 represent potentially valuable therapeutic targets that should be further investigated as useful diagnostic and prognostic biomarkers for GBM tailored therapy.

Cancer cell aggregate hypoxia visualized in vitro via biocompatible fiber sensors.

To fully understand biological behavior in vitro often dictates that oxygen be reported at either a local or a cellular level. Oxygen sensors based on the luminescent quenching of a specific form of electrospun fiber were developed for measurement of both gaseous and dissolved oxygen concentrations. Electrospinning was used to fabricate "core-shell" fiber configurations in which oxygen-sensitive transition-metal porphyrin complexes are embedded in an optically clear, gas permeable polycarbonate polymer 'core' while polycaprolactone provided a protective yet biocompatible 'shell'. By taking advantage of the resulting high sensitivity and fast response of electrospun core-shell fiber sensors, we were able to locate and image hypoxic regions in contact with aggregates of glioblastoma cells. Nanoscale, biomimetic sensors containing oxygen-sensitive porphyrins are particularly well suited to biological applications. These 'smart' nanofiber based sensors do not consume oxygen, their mechanical and chemical characteristics can be finely tuned allowing tailoring of biocompatibility and microstructure. Core-shell nanofiber oxygen sensing fibers could provide real-time assessments of tumor cell response to pharmacological innovations designed to target hypoxic regions driving new knowledge and technological advancement.

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR.

BACKGROUND: Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue. RESULTS: Here we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold. CONCLUSION: Altogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

The novel lectin KM+ detects a specific subset of mannosyl-glycoconjugates in the rat cerebellum.

KM+ is a D(+)mannose-specific lectin with a carbohydrate structure-affinity relationship different from those of most mannose-binding lectins. KM+ elicits carbohydrate-dependent biological effects in several mammalian cell types, but it has not yet been employed as a probe for the detection of its specific ligands. We show here for the first time the screening and partial identification of cerebellar mannosyl-glycoconjugates recognized by KM+, by means of lectin-histochemistry and lectin-blotting. Biotinylated KM+ stained most cellular structures in the adult rat cerebellum, particularly Purkinje cells bodies and the surface of granule cells, but not cellular processes. Capillaries in the choroid plexus were also strongly decorated, while blood vessels in the cerebellar parenchyma remained unstained. D(+)mannose, but not D(+)galactose, abolished the staining of all cerebellar structures. Higher inhibitory potencies were found for mannosyl-glycans such as mannotriose (man-alpha1,3-[man-alpha1,6]-man) and the biantennary heptasaccharide carried by the enzyme horseradish peroxidase. After separation of cerebellar proteins by SDS-PAGE, KM+ recognized three major unidentified mannosyl-glycoproteins of 132, 83 and 49 kDa. KM+ also detected high-Mw bands corresponding to the light and heavy chains of Type-I laminin, but not a 160-kDa cleavage product of laminin. We conclude that KM+ binds preferentially to a specific subset of mannose-containing glycoproteins in cerebellar tissue, thus being much more restricted than other mannose-specific lectins. KM+ can be used as a novel probe to screen the central nervous system for this specific subset of complex mannosyl-glycoconjugates.