A three-dimensional assay for measurement of viral-induced oncolysis

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-D24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-D24 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.

Development of Multicellular Tumor Spheroid (MCTS) Culture from Breast Cancer Cell and a High Throughput Screening Method Using the MTT Assay

“…The method was then compared to the LDH release assay, one of the most common approaches for cytotoxicity testing of cells grown as spheroids [14], [15], [16]. To validate the results from both assays, a flow cytometric analysis of phosphatidyl externalization was also carried out.…”

The chicken chorioallantoic membrane tumor assay as model for qualitative testing of oncolytic adenoviruses

“…24 They have also been used to test oncolysis and infectivity of adenoviral vectors. 28 However, their structure is much simpler than a real tumor environment, as only one type of cell is grown in spheroids. Recently, advanced models of mixed spheroids have been developed, using co-culture of tumor cells with HUVEC and/or fibroblasts.…”

A Targeted Multifunctional Platform for Imaging and Treatment of Breast Cancer and Its Metastases Based on Adenoviral Vectors and Magnetic Nanoparticles

“…Thus, novel assay systems are being developed to aid in pre-clinical analysis of the oncolytic potency of the viruses. For example, Lam et al have developed a tumor-spheroid three-dimensional system as compared to two-dimensional cell culture mono-layers to measure the viral penetration and oncolytic potency (Lam et al 2007).…”

“…For example, cyclooxygenase-2 (Cox-2) has been shown to be highly expressed in a number of epithelial tumors (Lam et al 2007). Based on this consideration, an infectivity enhanced CRAd with the E1 region under transcriptional control of the Cox-2 promoter was constructed.…”