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A three-dimensional assay for measurement of viral-induced oncolysis
J T Lam1, A Hemminki, A Kanerva
1Department of Pathology, University of Mississippi Medical Center, Jackson, MS, USA.
Abstract:
Oncolytic viruses represent a novel cancer treatment strategy. Despite their promising preclinical data, however, corresponding clinical trials have disappointed. To aid preclinical analyses, we hypothesized that three-dimensional tumor cell clusters or spheroids might provide an assay system superior to conventional monolayer cell cultures. Spheroids show viral infection, replication and oncolytic patterns distinct from conventional monolayer assays. Therefore, viral tumor penetration and oncolysis measurements may be improved with such three-dimensional models. Also, preclinical analyses of oncolytic viruses frequently measure mitochondrial activity, but more accurate measures of oncolysis might involve quantitation of intracellular protein release. Therefore, we measured luciferase released from luciferase-expressing spheroids and found unique patterns that maintained consistency with various viruses and doses. The relative variations between viruses and doses may represent temporal differences in oncolysis dynamics. Analysis of five recombinant replicative adenoviruses with promise for clinical application showed that Ad5/3-Delta24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-Delta24 also effected the earliest subtotal spheroid cell death. These findings closely parallel monolayer oncolysis assays with these agents. Therefore, the luciferase-expressing tumor spheroid assay represents a promising three-dimensional model for preclinical analysis of replicative oncolytic agents.
Insights
Three-dimensional tumor spheroids offer a superior model for studying oncolytic viruses compared to traditional cell cultures. This new assay system accurately measures viral oncolysis by quantifying protein release, aiding cancer therapy development.
Area of Science:
- Oncology
- Virology
- Biotechnology
Background:
- Oncolytic viruses show promise for cancer treatment but have faced challenges in clinical trials.
- Preclinical studies often use monolayer cell cultures, which may not fully represent tumor complexity.
Purpose of the Study:
- To evaluate three-dimensional (3D) tumor spheroids as an improved preclinical model for oncolytic virus assessment.
- To compare oncolysis measurements in spheroids versus traditional monolayer assays.
- To quantify viral oncolysis by measuring intracellular protein release.
Main Methods:
- Developed and utilized luciferase-expressing tumor spheroids.
- Infected spheroids with various oncolytic adenoviruses at different doses.
- Measured released luciferase as an indicator of oncolysis.
- Compared spheroid results with conventional monolayer assays.
Main Results:
- Tumor spheroids exhibited distinct viral infection, replication, and oncolysis patterns compared to monolayers.
- Luciferase release from spheroids provided consistent and unique patterns across different viruses and doses.
- Ad5/3-Delta24 demonstrated the earliest and highest peak luciferase release and spheroid cell death.
- Spheroid assay results correlated well with monolayer assay findings.
Conclusions:
- Luciferase-expressing tumor spheroids represent a promising 3D model for preclinical evaluation of oncolytic viruses.
- This spheroid assay system offers a more accurate method for assessing viral tumor penetration and oncolysis.
- The model aids in understanding oncolysis dynamics and selecting effective oncolytic agents for clinical application.
