SDEPT-Sonoporation-directed enzyme prodrug therapy in pancreatic cancer
Study of Nitroreductase based translational imaging has been concluded and sent in for submission awaiting reviewing process and response. In vitro work regarding uptake of drugs/dye under sonoporation process is still under attention. Acoustic Cluster Therapy has been summed up and submitted in the Journal of Control Release as of January 2017.Research on Nitroreductase based gene therapy against cancer has been hampered in part due to lak of available imaging modalities assessing therapeutic response. In this project this has been adressed. Breast cancer cells have been modified to express nitroreductase and put in mice for disease development. Subsequently when tumours developed the mice were images using translational PET imaging with FMISO as substrate. The results came out positive for nitroreductase expressing tumours and showed significant contrast to tumours not expressing nitroreductase. The results show potential for future investigation into gene therapy using nitroreductase as both reporter gene but also as activator for prodrugs that can selectively induce cytotoxic effect locally in tumours that have been targeted. Furthermore, the study show that using PET imaging can bridge the gap from preclinical to clinical research pushing this kind of treatment forward. The study has during spring been written in an original article format and sent in for submission in the Journal of Nuclear Medicine. The paper is currently under review and awaiting response. Alongside work on nitroreductase a study on the use of Acoustic Cluster Therapy (ACT) has been accepted and published in Jan-17 in the Journal Control Release. The preclinical study looked at using a combination of oil droplets loaded with standard cytostatica Paclitaxel together with comercially available microbubbles Sonazoid activated by ultrasound treatment achieveing the effect called sonoporation. Sonoporation causes transient pores in the cell membrane and the rational behind this form of therapy is that the effect caused by sonoporation will increase the uptake of the drugs in the treated area. The results of the study indicated that ACT bubbles alone had no toxic effect, while ACT bubbles in combination with paclitaxel could transiently reduce tumour volume. In vitro investigation of optimal ultrasound settings and bubble type -and concentration has been investigated to find the full effect of sonoporation induced uptake of dye/drugs in pancreatic cancer cells. The work is still ongoing but protocols have been established to run the experiments needed. The set-up include a bespoke ultrasound device that allows cells growing in a specific cell chamber to be sonicated submerged in water. Evaluation of sonoporation efficacy is done by measuring uptake of fluorescent dye by the cells in a flow cytometer. The established protocol and preliminary results have been presented in poster format at European Molecular Imaging Meeting in 2016.
Evaluation of codon optimized nitroreductase: in progress. Results at this time is presented in EMIM-2015, Tubingen. Multimodal Preclinical Imaging of Nitroreductase: Concluded. Manuscript in progress. Work presented in WMIC-2015, Honolulu. Sonoporation-directed enzyme prodrug therapy in pancreatic adenocarcinoma: work in progress, in vitro.Evaluation of codon optimized nitroreductase: The nfsB gene from E. coli has been used to synthesize a codon-optimized version, NTRo, where the codons were subjected to silent mutations in order to change the codon usage to mammalian preference. A study was performed where two plasmids were made each expressing etiher NTR (nfsB) or NTRo, and transduced in to different cell lines. Comparison of activity was made between NTR (nfsB) and NTRo expressing cells based on cell fluorescence by nitroreductase activation of CytoCy5S. Fluorescence intensity showed a 20% increase in cells expressing NTRo. The results have been presented (poster)(1). Multimodal Preclinical Imaging of Nitroreductase: Positron emission tomography (PET) has not yet been available for nitroreductase based Gene-directed enzyme prodrug therapy (GDEPT). Studies have been done to investigate the use of radiolabelled substrates for nitroreduction. Cells expressing NTR were implanted in mice to induce subcutaneous tumour growth and orthotopic metastatic development. Mice have been evaluated using bioluminescence and PET imaging. The study investigated the use of hypoxia PET tracer 18F-FMISO as substrate for NTR and compared the signals reported by NTR with signals obtained under hypoxic conditions. The study has been concluded and the results have been presented (oral presentation) (2). Manuscript is in progress. Sonoporation-directed enzyme prodrug therapy in pancreatic adenocarcinoma: Based on poor gene transfer efficacy by viral vectors in GDEPT applications investigations are in progress and will be done assessing ultrasound-mediated gene transfer. Ultrasound-mediated gene delivery has shown promise as alternative to viral delivery. By using microbubbles both drugs and DNA can be targeted to selective tissue in a localized acoustic field due to sonoporation of microbubbles and target cells. A new protocol using flow cytometry was developed to examine the effect of ultrasonic treatment conditions on “sonoporation” efficacy in vitro using a pancreatic adenocarcinoma cell line in hypoxic bioreactors mimicking in situ conditions. This protocol will subsequently be used to delineate the optimal sonoporation conditions in several pancreatic adenocarcinoma cell lines allowing improved therapeutic efficacy. A preclinical pancreatic in vivo study testing the efficacy of a novel microbubble formulation (Acoustic Cluster Therapy – ACT™) for treating pancreatic adenocarcinoma was performed. Results show that this combined therapy can transiently reduce tumour volume following treatment. 1. Stigen E, Lund KB, Garcia de Jalon E, Popa M, Grohmann M, Micklem D, et al. Preclinical evaluation of mammalianized nitroreductase. In Tubingen, Germany; 2015. 2. Lund KB, Stigen E, Popa M, Rygh CB, Adamsen TCH, Kvernenes OH, et al. Preclinical PET/CT imaging of a nitroreductase reporter gene with 18F-FMISO. In Honolulu, HI, USA; 2015.