Broad spectrum assessment of resistance phenomena to virotherapeutics

Introduction: Measles vaccine virus (MeV) has proven its oncolytic potential in several tumor models. Nevertheless primary and secondary resistance phenomena often emerge. In order to increase efficiency and to overcome resistance phenomena, a recombinant armed MeV vaccine strain (MeV ld-SCD) was generated. The inserted suicide gene encodes a fusion protein composed both of yeast cytosine deaminase (YCD) and yeast uracil phosphoribosyltransferase (YUPRT), which converts the non-toxic prodrug 5-fluorocytosine (5-FC) into the cytotoxic 5-fluorouracil (5-FU). Because of its ability to diffuse freely, 5-FU can also kill neighbouring cells which are not infected primarily („bystander effect“).

Aim: We set out to systematically unravel defect mechanisms of the oncolytic cell death and to develop means to overcome such resistances to virotherapeutics.

Methods: All experiments were done based on the well-characterized NCI-60 cell panel which consists of 60 tumor cell lines, comprising tumors of both gastrointestinal and other origin. In order to detect possible resistancies to MeV-based virotherapy, tumor cells were infected with MeV ld-SCD using several multiplicities of infection (MOI) in the presence and in the absence of the prodrug 5-FC. 96 hrs after infection the remaining cell mass was determined using a viability assay (Sulforhodamine B (SRB) assay). Expression of the MeV vaccine strain CD46 receptor was proven via FACS analysis. Percentages of infected tumor cells were quantified by infection with marker gene encoding vector MeV ld-GFP and subsequent FACS analysis. To investigate replication of MeV vectors, viral growth curves were generated. Viral protein expression was confirmed by Western Blot.

Results: Six cell lines proved to be completely resistant, i.e. loss of cell mass was less than 25% under standardized conditions (96 hrs after infection with MeV ld-SCD, MOI 1, no prodrug added). Nevertheless, resistance phenomena were overcome by increased MOI and addition of 5-FC.

Conclusion: Resistance phenomena of many different tumor entities can be overcome by usage of the suicide gene function of MeV ld-SCD. This probably will enable a much more efficient treatment of patients exhibiting tumors of both gastrointestinal and non-gastrointestinal origin.