Noninvasive Theranostic Imaging of HSV1-sr39TK-NTR/GCV-CB1954 Dual-Prodrug Therapy in Metastatic Lung Lesions of MDA-MB-231 Triple Negative Breast Cancer in Mice

Abstract

Metastatic breast cancer is an obdurate cancer type that is not amenable to chemotherapy regimens currently used in clinic. There is a desperate need for alternative therapies to treat this resistant cancer type. Gene-Directed Enzyme Prodrug Therapy (GDEPT) is a superior gene therapy method when compared to chemotherapy and radiotherapy procedures, proven to be effective against many types of cancer in pre-clinical evaluations and clinical trials. Gene therapy that utilizes a single enzyme/prodrug combination targeting a single cellular mechanism needs significant overexpression of delivered therapeutic gene in order to achieve therapy response. Hence, to overcome this obstacle we recently developed a dual therapeutic reporter gene fusion that uses two different prodrugs, targeting two distinct cellular mechanisms in order to achieve effective therapy with a limited expression of delivered transgenes. In addition, imaging therapeutic reporter genes offers additional information that indirectly correlates gene delivery, expression, and functional effectiveness as a theranostic approach. In the present study, we evaluate the therapeutic potential of HSV1-sr39TK-NTR fusion dual suicide gene therapy system that we recently developed, in MDA-MB-231 triple negative breast cancer lung-metastatic lesions in a mouse model. We compared the therapeutic potential of HSV1-sr39TK-NTR fusion with respective dual prodrugs GCV-CB1954 with HSV1-sr39TK/GCV and NTR/CB1954 single enzyme prodrug system in this highly resistant metastatic lesion of the lungs. In vitro optimization of dose and duration of exposure to GCV and CB1954 was performed in MDA-MB-231 cells. Drug combinations of 1 μg/ml GCV and 10 μM CB1954 for 3 days was found to be optimal regimen for induction of significant cell death, as assessed by FACS analysis. In vivo therapeutic evaluation in animal models showed a complete ablation of lung metastatic nodules of MDA-MB-231 triple negative breast cancer cells following two consecutive doses of a combination of GCV (40 mg/kg) and CB1954 (40 mg/kg) administered at 5 day intervals. In contrast, the respective treatment condition in animals expressing HSV1-sr39TK or NTR separately, showed minimal or no effect on tumor reduction as measured by bioluminescence (tumor mass) and [18F]-FHBG microPET (TK expression) imaging. These highlight the strong therapeutic effect of the dual fusion prodrug therapy and its use in theranostic imaging of tumor monitoring in living animals by multimodality molecular imaging.

Development of a Bioluminescent Nitroreductase Probe for Preclinical Imaging

“…As the next step we applied NCL for imaging of NTR activity in breast cancer cells, stably transfected with NTR and luciferase (MDA-MB-231-NTR+luc+) [ 25 ]. The expression of active NTR was confirmed by using the previously described NTR-specific fluorescent CytoCy5S probe ( S9 Fig ).…”

“…Cell line MDA-MB-231-NTR-Fluc-EGFP (NTR+luc+) used for in vitro and in vivo experiments in this study was kindly provided by Dr. Ramasamy Paulmurugan (Stanford University School of Medicine, Stanford, USA) [ 25 ]. The cells were generated as described below with a reference to Sekar et al [ 25 ]. The cloning vectors, expressing bacterial nitroreductase gene (NTR2) and Fluc-EGFP fusion constructs, were from the plasmid bank (Cellular Pathway Imaging Laboratory, Stanford).…”

“…Several activatable fluorescent probes for NTR imaging in vitro have been previously described [ 18 – 22 ]. However, the only reported probe for imaging of NTR in vivo relies on fluorescence (CytoCy5S) [ 23 – 25 ] and therefore possesses the limitations mentioned above.…”

An electrochemical biosensor for double-stranded Wnt7B gene detection based on enzymatic isothermal amplification

“…S10). The real sample was extracted from MDA-MB-231 cell, which was the cancer cell about triple negative breast cancer (Sekar et al, 2014;Yeo et al, 2014). Besides, we chose 293T cell as negative control, which was non-carcinous cell coming from human renal epithelial (Wang et al, 2013).…”

Drug Discovery by Molecular Imaging and Monitoring Therapy Response in Lymphoma

“…Over the past decade, various enzyme/prodrug systems, such as yeast cytosine deaminase/5-fluorocytosine (yCD/5-FC), thymidine kinase/ganciclovir (TK/GCV), and nitroreductase/CB1954 (NTR/CB1954), have been used for suicide gene therapy in cancer treatment. CD [ 49 , 50 , 51 ], herpes simplex virus type 1 thymidine kinase (HSV1-TK) [ 51 , 52 ], NTR [ 53 ], and NTR mutant deoxycytidine kinase (dCK) [ 54 ] can convert a prodrug into a cytotoxic drug; thus, a therapeutic radionuclide becomes obsolete in therapy. Receptor-based monitoring of somatostatin receptor type 2 [ 55 ], bombesin receptor [ 56 ] and transported based monitoring of sodium iodide symporter [ 57 ], norepinephrine [ 58 ], can be used for molecular imaging.…”