An image guided small animal radiation therapy platform (SmART) to monitor glioblastoma progression and therapy response.

BACKGROUND AND PURPOSE: Glioblastoma multiforme is the most common malignant brain tumor. Standard treatment including surgery, radiotherapy and chemotherapy with temozolomide is not curative. There is a great need for in vitro and in vivo models closely mimicking clinical practice to ensure better translation of novel preclinical findings. METHODS AND MATERIALS: A 3D spheroid model was established using the U87MG cell line. The efficacy of temozolomide, RT and combinations was assessed using growth delay assays. Orthotopic glioblastoma tumors were established, different radiation doses delivered based on micro-CT based treatment planning (SmART-plan) and dose volume histograms (DVH) were determined. Tumor growth was monitored using bioluminescent imaging. RESULTS: 3D spheroid cultures showed a dose-dependent growth delay upon single and combination treatments. Precise uniform radiation was achieved in all in vivo treatment groups at all doses tested, and DVHs showed accurate dose coverage in the planning target volume which resulted in tumor growth delay. CONCLUSION: We demonstrate that 3D spheroid technology can be reliably used for treatment efficacy evaluation and that mimicking a clinical setting is also possible in small animals. Both these in vitro and in vivo techniques can be combined for clinically relevant testing of novel drugs combined with radiation.

Spores of Clostridium engineered for clinical efficacy and safety cause regression and cure of tumors in vivo.

Spores of some species of the strictly anaerobic bacteria Clostridium naturally target and partially lyse the hypoxic cores of tumors, which tend to be refractory to conventional therapies. The anti-tumor effect can be augmented by engineering strains to convert a non-toxic prodrug into a cytotoxic drug specifically at the tumor site by expressing a prodrug-converting enzyme (PCE). Safe doses of the favored prodrug CB1954 lead to peak concentrations of 6.3 µM in patient sera, but at these concentration(s) known nitroreductase (NTR) PCEs for this prodrug show low activity. Furthermore, efficacious and safe Clostridium strains that stably express a PCE have not been reported. Here we identify a novel nitroreductase from Neisseria meningitidis, NmeNTR, which is able to activate CB1954 at clinically-achievable serum concentrations. An NmeNTR expression cassette, which does not contain an antibiotic resistance marker, was stably localized to the chromosome of Clostridium sporogenes using a new integration method, and the strain was disabled for safety and containment by making it a uracil auxotroph. The efficacy of Clostridium-Directed Enzyme Prodrug Therapy (CDEPT) using this system was demonstrated in a mouse xenograft model of human colon carcinoma. Substantial tumor suppression was achieved, and several animals were cured. These encouraging data suggest that the novel enzyme and strain engineering approach represent a promising platform for the clinical development of CDEPT.

High NOTCH activity induces radiation resistance in non small cell lung cancer.

BACKGROUND AND PURPOSE: Patients with advanced NSCLC have survival rates <15%. The NOTCH pathway plays an important role during lung development and physiology but is often deregulated in lung cancer, making it a potential therapeutic target. We investigated NOTCH signaling in NSCLC and hypothesized that high NOTCH activity contributes to radiation resistance. MATERIALS AND METHODS: NOTCH signaling in NSCLC patient samples was investigated using quantitative RT-PCR. H460 NSCLC cells with either high or blocked NOTCH activity were generated and their radiation sensitivity monitored using clonogenic assays. In vivo, xenograft tumors were irradiated and response assessed using growth delay. Microenvironmental parameters were analyzed by immunohistochemistry. RESULTS: Patients with high NOTCH activity in tumors showed significantly worse disease-free survival. In vitro, NOTCH activity did not affect the proliferation or intrinsic radiosensitivity of NSCLC cells. In contrast, xenografts with blocked NOTCH activity grew slower than wild type tumors. Tumors with high NOTCH activity grew significantly faster, were more hypoxic and showed a radioresistant phenotype. CONCLUSIONS: We demonstrate an important role for NOTCH in tumor growth and correlate high NOTCH activity with poor prognosis and radioresistance. Blocking NOTCH activity in NSCLC might be a promising intervention to improve outcome after radiotherapy.

E-Cadherin loss associated with EMT promotes radioresistance in human tumor cells.

BACKGROUND AND PURPOSE: Hypoxia is a hallmark of solid cancers and associated with metastases and treatment failure. During tumor progression epithelial cells often acquire mesenchymal features, a phenomenon known as epithelial-to-mesenchymal transition (EMT). Intratumoral hypoxia has been linked to EMT induction. We hypothesized that signals from the tumor microenvironment such as growth factors and tumor oxygenation collaborate to promote EMT and thereby contribute to radioresistance. MATERIALS AND METHODS: Gene expression changes under hypoxia were analyzed using microarray and validated by qRT-PCR. Conversion of epithelial phenotype upon hypoxic exposure, TGFß addition or oncogene activation was investigated by Western blot and immunofluorescence. Cell survival following ionizing radiation was assayed using clonogenic survival. RESULTS: Upon hypoxia, TGFß addition or EGFRvIII expression, MCF7, A549 and NMuMG epithelial cells acquired a spindle shape and lost cell-cell contacts. Expression of epithelial markers such as E-cadherin decreased, whereas mesenchymal markers such as vimentin and N-cadherin increased. Combining hypoxia with TGFß or EGFRvIII expression, lead to more rapid and pronounced EMT-like phenotype. Interestingly, E-cadherin expression and the mesenchymal appearance were reversible upon reoxygenation. Mesenchymal conversion and E-cadherin loss were associated with radioresistance. CONCLUSIONS: Our findings describe a mechanism by which the tumor microenvironment may contribute to tumor radioresistance via E-cadherin loss and EMT.

The deletion mutant EGFRvIII significantly contributes to stress resistance typical for the tumour microenvironment.

BACKGROUND AND PURPOSE: The epidermal growth factor receptor (EGFR) is overexpressed or mutated in many tumour types. The truncated, constitutively active EGFRvIII variant has not been detected in normal tissues but is found in many malignancies. In the current study, we have investigated the hypothesis that EGFRvIII contributes to a growth and survival advantage under tumour microenvironment-related stress conditions. MATERIALS AND METHODS: U373MG doxycycline-regulated isogenic cells expressing EGFRwt or EGFRvIII were created and validated using Western blot, FACS and qRT-PCR. In vitro proliferation was evaluated with standard growth assays. Cell survival was assayed using clonogenic survival. Animal experiments were performed using NMRI-nu-xenografted mice. RESULTS: Inducible isogenic cell lines were created and showed high induction of EGFRwt and EGFRvIII upon doxycycline addition. Overexpression of EGFRvIII but not of EGFRwt in this model resulted in a growth and survival advantage upon different tumour microenvironment-related stress conditions in vitro. Induction of EGFRvIII increased tumour growth in vivo, which was reversible upon loss of expression. CONCLUSIONS: Under conditions where nutrients are limited and stress is apparent, as in the tumour microenvironment, expression of EGFRvIII leads to a growth and survival advantage. These data indicate a potential selection of EGFRvIII-expressing tumour cells under such stress conditions.

Potential and limitations of bacterial-mediated cancer therapy.

Bacterial-based tumor-targeted therapy is an area of growing interest and holds promise for the treatment of solid tumors. Upon systemic administration, various types of non-pathogenic obligate anaerobes and facultative anaerobes have been shown to infiltrate and selectively replicate within solid tumors. The tumor specificity is based upon the unique physiology of solid tumors, which is often characterized by regions of hypoxia and necrosis. Prokaryotic vectors can be safely administered and their potential to deliver therapeutic proteins has been demonstrated in a variety of preclinical models. Although the amount of clinical experience with bacterial vectors is limited to date, the available data clearly demonstrated the feasibility of bacterial-mediated therapy in humans. There are several issues however that are still unknown and remain major challenges. In this review, using Clostridium and modified Salmonella as prototypical agents, we will discuss the major advantages, challenges and shortcomings of bacterial systems for tumor-specific therapy. In addition, we will highlight the requirements needed to advance the approach into clinical trials.