Different susceptibility of osteosarcoma cell lines and primary cells to treatment with oncolytic adenovirus and doxorubicin or cisplatin

Abstract

Despite improvements in treatment regimens for osteosarcoma (OS) patients, survival rate has not increased over the last two decades. New treatment modalities are therefore warranted. Preclinical results with conditionally replicative adenoviruses (CRAds) to treat OS are promising. One type of CRAd that was effective against OS cells is Ad5-Delta24RGD. In other types of cancer, CRAds have been shown to interact synergistically with chemotherapeutic agents. Chemotherapy for OS often includes doxorubicin and cisplatin. Therefore, we explored combination treatment of OS cell lines and primary OS cell cultures with Ad5-Delta24RGD and doxorubicin or cisplatin. On OS cell lines, combination treatment was additive to synergistic. Surprisingly, however, on seven of eight primary OS samples no such combination effects were observed. In contrast, in many cases chemotherapy even inhibited CRAd-mediated cell killing. The inhibitory effect of doxorubicin on Ad5-Delta24RGD in primary OS cells appeared to correlate with slow cell growth rate; reduced viral replication and absence of chemotherapy-induced G2 cell cycle arrest. Our results point to the possibility that, at least for OS, virotherapy and chemotherapy should best not be performed simultaneously. In general, our work underscores the importance of testing new genetic anticancer agents and treatment regimens on primary cancer specimens.

Figure 1

The oncolytic effect of Ad5-Δ24RGD in combination with doxorubicin or cisplatin on OS cell lines. Three OS cell lines (SaOs-2, MG-63 and U2OS) were incubated at a concentration range of 0.063 to 4-times the average IC₅₀ of each treatment agent. (A) Left panels represent cytotoxicity on OS cell lines of Ad5-Δ24RGD (open diamonds), doxorubicin (open squares) as single agents or the combination of both (closed triangles). Right panels represent cytotoxicity obtained with Ad5-Δ24RGD (open diamonds), cisplatin (open squares) or the combination (closed triangles). Cell survival was expressed relative to nontreated controls. Data represent a typical experiment performed in triplicate. (B) Results from the combination experiments were analysed by the CalcuSyn program. Data shown are mean CI values with s.d. from three independent experiments each performed in triplicate. CI values below 0.9 were defined as synergistic, between 0.9 and 1.1 as additive and above 1.1 as antagonistic.

Figure 2

The cytotoxic effect of Ad5-Δ24RGD with doxorubicin or cisplatin on primary OS cell cultures. Eight primary OS cell cultures were subjected to concentrations of Ad5-Δ24RGD (white bars), doxorubicin (hatched bars; upper panel) or cisplatin (hatched bars; lower panel) resulting in 20–60% cell kill or to a combination of CRAd plus chemotherapeutic drug (black bars). Relative cell survival compared to nontreated cultures was measured by WST-1 conversion assay. Data shown are mean values with s.d. from experiments performed in triplicate.

Figure 3

Effect of delayed doxorubicin or cisplatin treatment after infection with Ad5-Δ24RGD on primary OS cell kill. Primary OS cells were infected with Ad5-Δ24RGD and 24, 48 or 72 h later cells were treated with doxorubicin or cisplatin (black bars). Controls treated only with virus or chemotherapeutic drug are shown by white and hatched bars, respectively. Relative cell survival was measured by WST-1 conversion assay. Data shown are mean values with s.d. from experiments performed in triplicate.

Figure 4

Effect of subtoxic dose of doxorubicin or cisplatin on the oncolytic effect of Ad5-Δ24RGD on OS cell lines and primary OS cells. Osteosarcoma cell lines and primary OS cell cultures were treated with approximate IC₇₀ of Ad5-Δ24RGD (white bars) and the approximate IC₁₀ of doxorubicin or cisplatin (hatched bars) or a combination (black bars). Relative cell survival was measured by WST-1 conversion assay. Data shown are mean values with s.d. from experiments performed in triplicate.

Figure 5

Effect of doxorubicin on cell cycle profile of OS cell lines. SaOs-2, MG-63 and U2OS cells were cultured for 31 h in medium containing IC₁₀ or IC₉₀ doxorubicin as indicated. DNA histograms were made by PI staining and FACS flow cytometry. DNA histograms were analysed by ModFitLT cell cycle analysis software. Percent cells in G2/M phase of the cell cycle are indicated in the panels.

Figure 6

Effect of doxorubicin on cell cycle profile of primary OS cells. Osteosarcoma-11, OS-12A and OS-16 cells were cultured in medium containing IC₁₀ doxorubicin for 31 h or 6 days as indicated. DNA histograms were made and analysed as described in the legend to Figure 5.