Replication of an integrin targeted conditionally replicating adenovirus on primary ovarian cancer spheroids

Abstract

Replication competent viruses hold promise for treatment of advanced cancers resistant to available therapeutic modalities. Although preliminary clinical results have substantiated their efficacy, preclinical development of these novel approaches is limited by assay substrates. The evaluation of candidate agents could be confounded by differences between primary tumor cells and tumor cell lines, as discordance in the levels of surface receptors relevant for viral entry has been reported. Since primary tumor cells are difficult to analyze ex vivo for longitudinal observation of virus replication, we developed three-dimensional aggregates or spheroids of unpassaged and purified ovarian cancer cells as a means for prolonging primary tumor cell viability and as a threedimensional in vitro model for replicative viral infection. Ovarian cancer cells purified from ascites samples were sustained for 30 days while retaining the infection profile with tropism modified and unmodified adenoviruses (Ads). Cell line and primary cell spheroids were used to quantitate the replication and oncolytic potency of replicative Ads in preclinical testing for human ovarian cancer trials. Therefore, spheroids provide a method to sustain purified unpassaged primary ovarian cancer cells for extended periods and to allow evaluation of replicative viruses in a three-dimensional model.

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

“…5 Therefore, we have developed three-dimensional aggregates or spheroids of unpassaged and purified ovarian cancer cells as a means for prolonging primary tumor cell viability. 15 Spheroids also provide a threedimensional in vitro model for replicative viral infection. 15 As viral penetration of solid tumor proceeds in three dimensions, conventional two-dimensional cell culture assays might produce data that poorly represent these in vivo events.…”

“…15 Spheroids also provide a threedimensional in vitro model for replicative viral infection. 15 As viral penetration of solid tumor proceeds in three dimensions, conventional two-dimensional cell culture assays might produce data that poorly represent these in vivo events. Spheroids, however, are often greater than 1 mm in diameter 16 and therefore allow viral spread through a solid rather than planar substrate.…”

“…In a previous study, we measured firefly luciferase released from human cancer cell spheroids to quantify tumor cell killing by a replicative Ad with a luciferase transgene expression cassette. 15 As Ads kill infected cells by effecting membrane disruption, detection of intracellular luciferase protein 17 indicates cellular membrane disruption and consequent cell death. This method, however, can only test CRAds that incorporate the luciferase gene, but clinical therapeutic agents rarely accommodate such reporter genes.…”

“…Photomicrographs of ovarian cancer spheroids, including those undergoing Ad replication-mediated oncolysis, have been recently reported. 15,16 Materials and methods…”

A heparan sulfate-targeted conditionally replicative adenovirus, Ad5.pk7-Δ24, for the treatment of advanced breast cancer

“…21,22,[35][36][37] Also, relatively good correlation has been seen between tumor cell lines grown two-dimensionally on plates or as three-dimensional spheroids. [38][39][40] With regard to breast cancer, this has not been studied previously. However, a recent report suggested that the level and location of CAR were different when the same breast cancer cells were grown on plates versus in a Figure 4 Noninvasive imaging of viable tumor cells following treatment with Ad5.pK7-D24.…”