On TRAIL for glioma therapy?

Despite conventional treatment protocols (surgery, radiotherapy and chemotherapy), is the prognosis for patients with a glioblastoma, extremely poor. There is an urgency to develop new treatment strategies to improve outcome. Escapism of apoptosis, the intrinsic suicide program of the cell, is a hallmark of human cancer. Defects in apoptosis may confer resistance to therapy because most current treatment approaches initiate cell death through activation of an apoptosis pathway. Current attempts to improve the survival of glioblastoma patients will have to include strategies that specifically target tumor cell resistance to apoptosis. Of interest for GBM therapy is the selective induction of apoptosis using the pro-apoptotic tumor necrosis factor-related apoptosis inducing ligand (TRAIL).
TRAIL acts primarily through induction of the extrinsic apoptosis pathway in contrast to chemo and radiotherapy who act through the intrinsic mitochondrial pathway. This thesis elucidates TRAIL as a potential anti-neoplastic drug as TRAIL can bypass intrinsic (mitochondrial) resistance of tumor cells by acting its death inducing effect through the extrinsic pathway, evidently without causing systemic toxicity. Therefore TRAIL might be a new adjuvant therapy in the battle against GBM.
Chapter 1 outlines the aim of this thesis. Chapter 2 presents a review which addresses several aspects of TRAIL-TRAIL receptor interaction and the potential of TRAIL as an anti-neoplastic agent in the treatment of malignant gliomas.
TRAIL receptor expression is found in many normal tissues and malignant cells of various origin. Preclinical studies show a variable sensitivity of tumor cells for TRAIL, including GBM cells, without major systemic-or neuro toxicity. Animal in vivo studies showed no major systemic toxicity after treatment with TRAIL. Phase 1 studies have investigated the applicability of rhTRAIL and TRAIL receptor antibodies in solid and hematological tumors (no GBM tumors) have been executed or are still in progress. In concordance with the preclinical studies, these trials showed (preliminary data) again that rhTRAIL and TRAIL receptor treatment is feasible without major systemic toxicity. Although systemic toxicity seems not to be a major obstacle for the introduction of TRAIL treatment for patients with a GBM, other restrictions can be. TRAIL induction of the apoptosis pathway can be restrained by intracellular resistance mechanisms. Several in vitro studies point out that combination therapy with various (non)conventional drugs and TRAIL overrides resistance of glioma cells to TRAIL. Yet, it is not known if these combination therapies do lead to induction of systemic- or neurotoxicity. As highlighted in this chapter, TRAIL can be combined with a variety of different conventional and novel therapeutic strategies to yield synergistic apoptotic activity. Of particular interest is the use of dual purpose TRAIL-based molecules, such as the EGFR-targeted TRAIL fusion protein scFv425:sTRAIL. This fusion protein simultaneously blocks EGFR signaling, thereby sensitizing tumor cells to apoptosis, and induces apoptosis via TRAIL receptor signaling. It has been shown in the literature that this fusion protein efficiently activates apoptosis and shows promising in vivo activity. Chapter 2 also elaborates on other TRAIL- combination strategies which may help to optimize anti-GBM activity.
In Chapter 3 we describe the amount of TRAIL-R1 and TRAIL-R2 expression on primary GBM specimens. A requirement for success of TRAIL therapy, in patients with a GBM, is the presence of TRAIL receptors on GBM cells. Studies investigating TRAIL receptor expression were mainly focused on homogeneic cell lines which differ from primary GBM tumor tissue. Therefore, quantification of TRAIL receptor expression in primary GBM cells was instigated. Furthermore a possible association between expression of TRAIL-R1/TRAIL-R2 and survival was evaluated.
It was concluded that TRAIL-R1 and TRAIL-R2 expression patterns on tumor cells are independent prognostic factors for survival in patients with a GBM. Both receptors could be targets for TRAIL therapy. However since TRAIL-R2 is more expressed, on GBM tumor cells than TRAIL-R1, TRAIL-R2 seems to be the most important target for future TRAIL therapy.
In Chapter 4 the development, production and properties of a single chain –TRAIL fusion protein (scFvC54:sTRAIL) were described. The scFvC54:sTRAIL fusion protein was designed to induce apoptosis by cross-linking of agonistic TRAIL-R2 receptors only after specific binding of scFvC54:sTRAIL to the abundantly expressed carcinoma-associated cell surface antigen EGP2 (epithelial glycoprotein 2). Analysis of the solution of the scFvC54:sTRAIL showed soluble stable homogeneous trimers of scFvC54:sTRAIL with no or only minimal aggregate formation. This is important as certain aggregated TRAIL forms have shown organ-specific toxicity. When EGP2-positive tumor cells were subjected to treatment with scFvC54:sTRAIL, an efficient induction of apoptosis was observed thereby concluding that, after production, the biological functionality of the fusion protein was maintained. Interesting was the fact that the target antigen restricted apoptosis inducing capacity of scFvC54:sTRAIL was directly proportional to the degree of TRAIL-R2 receptor crosslinking. The conclusion of this chapter was that the scFvC54:sTRAIL fusion protein efficiently induced bi- or multi-cellular reciprocal apoptosis in a target antigen restricted fashion.
As discussed in the review article presented in Chapter 2, the method of drug delivery is an important factor for success in the drug treatment of GBM tumors. In Chapter 5 we evaluated the alginate microencapsulation method. Chinese Hamster Ovary cells (CHO-K1) were engineered to produce the scFv425:sTRAIL protein. The CHO-K1 producer cells were encapsulated in an alginate capsule with a semi-permeable membrane through which the scFv425:sTRAIL protein could be released. Utilizing this method a constant production of fusion protein was enabled, thereby creating a biological scFv425:sTRAIL producing factory. The biological functionality of the apoptosis inducing scFv425:sTRAIL protein, which was released through the microencapsulation method, was studied in vitro and analysis of the intracerebral biocompatibility of alginate capsules was performed by implantation of empty alginate capsules in the brain of mice. The study concluded that the biological functionality of the produced scFv425:sTRAIL protein was maintained and intracerebral biocompatibility of the capsules was warranted. The next step is to design an in vivo study with a rat or mouse brain tumor model with intracerebral application of alginate encapsulated scFv425:sTRAIL - producer cells and to evaluate the antitumor effects of this delivery method.
Previous publications showed that the scFv425:sTRAIL fusion protein resulted in enhanced apoptosis in cancer cells. These promising in vitro results justified in vivo experiments and in Chapter 6 the in vivo efficacy of the scFv425:sTRAIL fusion protein in a mouse brain tumor model was investigated. A brain tumor model was developed by xenografting a SW948 cell line in the cerebrum of an SCID mouse. Since the scFv425:sTRAIL fusion protein induced a strong apoptotic signal in the SW948 cell line and xenografting of this cell line into the cerebrum of SCID mice resulted in a more than 90 % acceptance of the graft, this seemed a good animal model for testing the in vivo death inducing potential of the scFv425:sTRAIL fusion protein. Through the convection enhanced delivery method (CED), with an Alzet® osmotic pump, the scFv425:sTRAIL fusion protein or a placebo was intracerebrally, intratumorally infused. Pre and post treatment MRI was used to detect in vivo efficacy of the scFv425:sTRAIL fusion protein. Eventually, the in vivo efficacy of the scFv425:sTRAIL fusion protein, in the animal brain tumor model used in this study, could not be shown. After careful analysis it was concluded that the doses of the infused scFv425:sTRAIL was to low to exert a proper anti-tumor effect. New animal (CED) brain tumor experiments with gradual dose increment should be initiated to evaluate the minimal effective dose.
It has been brought up that radiation can enhance the apoptosis-inducing efficacy of the TNF Related Apoptosis Inducing Ligand (TRAIL). In Chapter 7 we evaluated the effect of combined γ-radiation-TRAIL therapy in a glioblastoma (A172) cell line, measuring both early apoptotic cell death and clonogenic ability as endpoints. Although the combination of radiation and TRAIL lead to a small synergy in apoptosis induction measured by short term assays, this did not translate to synergy in ultimate loss of clonogenicity, where radiation and TRAIL merely showed additive effects. Besides the conclusion that glioma cells may not show (much) synergy between radiation and TRAIL, our data also argue for re-evaluation of the observed synergy between these modalities seen in rapid death endpoint assays in other studies. In fact, our finding that the extent of TRAIL-induced clonogenic death exceeded the extent of rapid apoptotic cell death underscores the need for such re-evaluation. This study shows an additive effect on cell death after combination treatment of radiation and TRAIL and therefore TRAIL might have a place within the treatment of patients with a GBM.

It can be concluded that TRAIL and TRAIL deratives have unique properties and can play a role in the treatment of GBM. Multimodality treatment is the key factor for successful treatment of GBM. TRAIL in combination with (non) conventional treatment modalities must be explored. TRAIL-combination therapy might overcome resistance of GBM cells to chemo-radiotherapeutic approaches and hopefully prolonging overall survival for this patient group.

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