Expression of ABCB1, ABCC1 and 3 and ABCG2 in glioblastoma and their relevance in relation to clinical survival surrogates.

PURPOSE: Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumors in adults. Patients invariably relapse during or after first-line therapy and the median overall survival is 14.6 months. Such poor clinical response is partly ascribed to the activity of ATP-binding cassette (ABC) transporters. The activity of these proteins, severely reduces the amount of therapeutics that penetrates the tumor cells. We hypothesized that ABC transporter expression could correlate with survival surrogates. In this study, we assessed the expression of four commonly expressed ABC transporters in GBM samples and investigated if mRNA levels could serve as a prognostic biomarker. METHODS: Human specimens were analyzed by qPCR to assess ABCB1, ABCC1/3 and ABCG2 expression. Kaplan-Meier and multivariate analyses were then used to evaluate the correlation with overall survival (OS) and progression-free survival (PFS). RESULTS: Our cohort included 22 non-tumoral samples as well as 159 GBM tumor specimens. ABC transporters were significantly more expressed in GBM samples compared to non-tumoral tissue. Moreover ABCC1 and 3 mRNA expression were significantly increased at recurrence. Statistical analyses revealed that increased expression of either ABCC1 or ABCC3 did not confer a poorer prognosis. However, increased ABCC1 mRNA levels did correlate with a significantly shorter PFS. CONCLUSION: In this manuscript, the analyses we conducted suggest that the expression of the four ABC transporters evaluated would not be suitable prognostic biomarkers. We believe that, when estimating prognosis, the plethora of mechanisms implicated in chemoresistance should be analyzed as a multi-facetted entity rather than isolated units.

Liposomal formulations of carboplatin injected by convection-enhanced delivery increases the median survival time of F98 glioma bearing rats.

BACKGROUND: Effectiveness of chemotherapy for treating glioblastoma (GBM) brain tumors is hampered by the blood-brain barrier which limits the entry into the brain of most drugs from the blood. To bypass this barrier, convection-enhanced delivery (CED) was proposed to directly inject drugs in tumor. However, the benefit of CED may be hampered when drugs diffuse outside the tumor to then induce neurotoxicity. Encapsulation of drugs into liposome aims at increasing tumor cells specificity and reduces neurotoxicity. However, the most appropriate liposomal formulation to inject drugs into brain tumor by CED still remains to be determined. In this study, four liposomal carboplatin formulations were prepared and tested in vitro on F98 glioma cells and in Fischer rats carrying F98 tumor implanted in the brain. Impact of pegylation on liposomal surface and relevance of positive or negative charge were assessed. RESULTS: The cationic non-pegylated (L1) and pegylated (L2) liposomes greatly improved the toxicity of carboplatin in vitro compared to free carboplatin, whereas only a modest improvement and even a reduction of efficiency were measured with the anionic non-pegylated (L3) and the pegylated (L4) liposomes. Conversely, only the L4 liposome significantly increased the median survival time of Fisher rats implanted with the F98 tumor, compared to free carboplatin. Neurotoxicity assays performed with the empty L4' liposome showed that the lipid components of L4 were not toxic. These results suggest that the positive charge on liposomes L1 and L2, which is known to promote binding to cell membrane, facilitates carboplatin accumulation in cancer cells explaining their higher efficacy in vitro. Conversely, negatively charged and pegylated liposome (L4) seems to diffuse over a larger distance in the tumor, and consequently significantly increased the median survival time of the animals. CONCLUSIONS: Selection of the best liposomal formulation based on in vitro studies or animal model can result in contradictory conclusions. The negatively charged and pegylated liposome (L4) which was the less efficient formulation in vitro showed the best therapeutic effect in animal model of GBM. These results support that relevant animal model of GBM must be considered to determine the optimal physicochemical properties of liposomal formulations.

Differential Expression and Clinical Significance of Transforming Growth Factor-Beta Isoforms in GBM Tumors.

Glioblastoma (GBM) represents the most common and aggressive malignant primary brain tumors in adults. Response to standard treatment is transitory and the survival of clinical trial cohorts are little more than 14 months. GBM are characterized by excessive proliferation, invasiveness, and radio-/chemoresistance features; which are strongly upregulated by transforming growth factor-beta (TGF-ß). We hypothesized that TGF-ß gene expression could correlate with overall survival (OS) and serve as a prognostic biomarker. TGF-ß1 and -ß2 expression were analyzed by qPCR in 159 GBM tumor specimens. Kaplan-Meier and multivariate analyses were used to correlate expression with OS and progression-free survival (PFS). In GBM, TGF-ß1 and -ß2 levels were 33- and 11-fold higher respectively than in non-tumoral samples. Kaplan-Meier and multivariate analyses revealed that high to moderate expressions of TGF-ß1 significantly conferred a strikingly poorer OS and PFS in newly diagnosed patients. Interestingly, at relapse, neither isoforms had meaningful impact on clinical evolution. We demonstrate that TGF-ß1 is the dominant isoform in newly diagnosed GBM rather than the previously acknowledged TGF-ß2. We believe our study is the first to unveil a significant relationship between TGF-ß1 expression and OS or PFS in newly diagnosed GBM. TGF-ß1 could serve as a prognostic biomarker or target affecting treatment planning and patient follow-up.

Chloroquine inhibits the malignant phenotype of glioblastoma partially by suppressing TGF-beta.

BACKGROUND: Glioblastoma (GBM), the most common and aggressive primary brain tumor, is characterized by excessive brain infiltration which prevents the complete surgical resection. These tumors also display treatment non-compliance and responses to standard therapy are invariably transient; consequently, the prognosis barely exceeds 14 months and recurrence is inevitable. Accordingly, several new treatment strategies have been studied. One such option is the use of chloroquine (CQ), a lysosomotropic weak base and renowned antimalarial drug, that has shown promising results in several pre-clinical studies. In this paper, we investigate the efficiency of CQ to hinder the malignant phenotype of GBM, namely extensive proliferation, invasion and radio-resistance. RESULTS: In cell cycle analysis, proliferation assays and immunofluorescence, CQ treatments halved proliferation of primary cultures from GBM specimens and GBM cell lines (U-373 MG et U-87 MG). Gelatin zymography and Matrigel(TM)-coated transwell invasion assays also revealed a 50 % CQ induced inhibition of MMP-2 activity and GBM invasion. Concomitant treatment with CQ and radiation also radiosensitized GBM cells as shown by an accumulation in the G2/M phase, increased cell death and reduced clonogenic formation. Moreover, radiation-induced invasion was considerably restrained by CQ. We also observe that these effects are owed to CQ-induced inhibition of TGF-ß secretion and signaling pathway, a predominant growth factor in GBM progression. CONCLUSION: These results suggest that CQ, alone or as an adjuvant therapeutic, could be used to inhibit the GBM malignant phenotype and could benefit GBM afflicted patients.

The development of a rapid patient-derived xenograft model to predict chemotherapeutic drug sensitivity/resistance in malignant glial tumors.

BACKGROUND: High-grade gliomas (HGG) are aggressive brain tumors associated with short median patient survival and limited response to therapies, driving the need to develop tools to improve patient outcomes. Patient-derived xenograft (PDX) models, such as mouse PDX, have emerged as potential Avatar platforms for personalized oncology approaches, but the difficulty for some human grafts to grow successfully and the long time required for mice to develop tumors preclude their use for HGG. METHODS: We used a rapid and efficient ex-ovo chicken embryo chorioallantoic membrane (CAM) culture system to evaluate the efficacy of oncologic drug options for HGG patients. RESULTS: Implantation of fresh glioma tissue fragments from 59 of 60 patients, that include difficult-to-grow IDH-mutated samples, successfully established CAM tumor xenografts within 7 days, with a tumor take rate of 98.3%. These xenografts faithfully recapitulate the histological and molecular characteristics of the primary tumor, and the ability of individual fragments to form tumors was predictive of poor patient prognosis. Treatment of drug-sensitive or drug-resistant xenografts indicates that the CAM-glioma assay enables testing tumor sensitivity to temozolomide and carboplatin at doses consistent with those administered to patients. In a proof-of-concept study involving 14 HGG patients, we observed a correlation of 100% between the CAM xenograft response to temozolomide or carboplatin and the clinical response of patients. CONCLUSION: The CAM-glioma model is a fast and reliable assay that has the potential to serve as a complementary model to drug discovery and a real-time Avatar platform to predict the best treatment for HGG patients.

Transforming growth factor-beta and its implication in the malignancy of gliomas.

Malignant gliomas are the most common type of primary malignant brain tumors. They are characterized by enhanced growing capabilities, neoangiogenic proliferation, and extensive infiltration of the brain parenchyma, which make their complete surgical resection impossible. Together with transient and refractory responses to standard therapy, these aggressive neoplasms are incurable and present a median survival of 12 to 14 months. Transforming growth factor-beta (TGF-ß) is a pleiotropic cytokine of which two of the three isoforms expressed in humans have been shown to be overexpressed proportionally to the histologic grade of glioma malignancy. The increase of chromosomal aberrations and genetic mutations observed in glioma cells turns TGF-ß into an oncogene. For that reason, it plays critical roles in glioma progression through induction of several genes implicated in many carcinogenic processes such as proliferation, angiogenesis, and invasion. Consequently, investigators have begun developing innovative therapeutics targeting this growth factor or its signaling pathway in an attempt to hinder TGF-ß's appalling effects in order to refine the treatment of malignant gliomas and improve their prognosis. In this paper, we extensively review the TGF-ß-induced oncogenic pathways and discuss the diverse new molecules targeting this growth factor.