Related Experiment Videos
Quantitation of bystander effects in nitroreductase suicide gene therapy using three-dimensional cell cultures
William R Wilson1, Susan M Pullen, Alison Hogg
1Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland, New Zealand. wr.wilson@auckland.ac.nz
Abstract:
The efficacy of cancer gene therapy depends critically on "bystander effects" by which genetic modification of tumor cells results in killing of unmodified cells in the local microenvironment. In gene-dependent enzyme-prodrug therapy, expression of a prodrug-activating suicide gene is used to generate a cytotoxic metabolite that diffuses to nontransduced cells. The objective of this study was to develop a physiologically relevant tissue culture model for quantifying bystander effects and to validate the model using as an example the activation of dinitrobenzamide prodrugs (e.g., CB 1954) by Escherichia coli aerobic nitroreductase (NTR). Bystander effects were measured in three-dimensional multilayer cocultures of NTR+ and NTR- cells by determining clonogenic survival curves for both cell types using V79, Skov3, or WiDr as parental cell lines. Bystander killing by CB 1954 was much more efficient in multilayers than monolayers at equivalent cell:medium ratios, whereas the chloromustard analogue of CB 1954 showed even greater efficiency. For a series of dinitrobenzamides, bystander killing in multilayers showed a positive correlation with prodrug lipophilicity and also correlated with the bystander effect in mixed tumor xenografts grown from the same NTR+ and NTR- WiDr cell lines (r(2) = 0.84; P < 0.001). The multilayer model identified a bromomustard prodrug (SN 24927) with superior therapeutic activity to CB 1954 that provided curative activity against WiDr tumors comprising 1:1 mixtures of NTR+ and NTR- cells. This study demonstrates the utility of the multilayer tissue culture model for quantifying and optimizing bystander effects in tumors and identifies a new lead prodrug for NTR gene-dependent enzyme-prodrug therapy.
Insights
Cancer gene therapy relies on bystander effects, where modified tumor cells kill nearby unmodified cells. A new 3D model accurately quantifies these effects, identifying a potent prodrug for enhanced cancer treatment.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Cancer gene therapy efficacy is enhanced by bystander effects, where modified tumor cells induce death in adjacent unmodified cells.
- Enzyme-prodrug therapy utilizes suicide genes to activate prodrugs, generating cytotoxic metabolites that diffuse to kill neighboring cancer cells.
Purpose of the Study:
- To develop a physiologically relevant 3D tissue culture model for quantifying cancer gene therapy bystander effects.
- To validate the model using Escherichia coli aerobic nitroreductase (NTR) mediated activation of dinitrobenzamide prodrugs.
- To identify novel prodrugs with improved therapeutic potential.
Main Methods:
- Utilized three-dimensional multilayer cocultures of NTR-expressing (NTR+) and non-expressing (NTR-) cells (V79, Skov3, WiDr).
- Quantified bystander effects by determining clonogenic survival curves for both cell types.
- Correlated in vitro bystander killing with in vivo xenograft data.
Main Results:
- Bystander killing was significantly more efficient in 3D multilayers compared to 2D monolayers.
- Prodrug lipophilicity positively correlated with bystander killing efficiency in the multilayer model.
- The model identified a bromomustard prodrug (SN 24927) with superior efficacy to CB 1954, achieving curative activity in mixed cell tumors.
Conclusions:
- The developed 3D multilayer model is effective for quantifying and optimizing bystander effects in cancer gene therapy.
- This model facilitated the identification of SN 24927 as a promising lead prodrug for NTR gene-dependent enzyme-prodrug therapy.
- The findings support the advancement of enzyme-prodrug strategies for improved cancer treatment outcomes.