Overcoming of primary & secondary chemotherapy resistance of advanced staged pancreatic cancer by additive virotherapy

Abstract

Oncolytic virotherapy utilizes naturally occurring or genetically modified viruses for targeted cancer treatment. Such oncolytic viruses (OVs) are designed to specifically infect and replicate in tumor cells ultimately leading to their lysis (so-called oncolysis). Additionally, the release of both tumor antigens and immuno-genic viral particles is believed to strongly induce antitumor immunity. However, in line with the consideration that multimodal cancer therapy is likely to be more effective than monotherapeutic treatment protocols, chemovirotherapy focuses on adding the oncolytic and immunotherapeutic potential of OVs to already es-tablished chemotherapeutic treatment protocols. Especially for pancreatic ductal adenocarcinoma (PDA) chemotherapy still fails to considerably improve patient survival. Additionally to PDA’s substantial intrin-sic resistance to chemothera¬peutic agents the dense and highly immunosup-pressive tumor microenvironment impedes the efficacy of current chemotherapy protocols. Then again, such conditions have been shown to favor oncolytic viro-therapy. This thesis therefore focused on the design of a novel chemovirotherapeutic pro-tocol to improve the currently poor treatment outcome of pancreatic cancer. For this purpose, 4 established and well-characterized tumor cell lines of pancreatic adenocarcinoma (AsPc-1, BxPc-3, MIA PaCa-2, Panc-1) were treated in vitro with the oncolytic vaccinia virus GLV-1h68 in combination with selected chemo-therapeutic agents. Moreover, the influence of different administration sequenc-es on the therapeutic outcome of chemovirotherapy was analyzed. Cytotoxicity after treatment was measured by sulforhodamine B (SRB) assay and confirmed by CellTiter Blue (CTB) and MTT assays, respectively. Due to its insertion of green fluorescent protein (GFP) GLV-1h68 additionally enabled the non-invasive visualization of viral gene expression and replication. In a first step, for each agent doses having only subtherapeutic tumoricidal ef-fects had to be determined. Accordingly, all 4 tumor cell lines were treated with ascending doses of the respective chemo- or virotherapeutic agents in mono-therapy. As a result, drug- and dose-dependent antitumor responses were seen indicating resistance against some cytotoxic agents. In a next step, the actual chemovirotherapy was performed. Combinations of GLV-1h68 with either 5-fluorouracil (5-FU), gemcitabine or oxaliplatin were found to result only in a moderate increase of cytotoxicity regardless of their admin-istration sequence. Only the combination with the mitotic inhibitor nab-paclitaxel showed promising signs of potent tumor cell killing. However, given the fact that for metastatic pancreatic cancer nab-paclitaxel is only approved in combination with gemcitabine a triple-therapy protocol combining GLV-1h68 with the dual chemotherapy nab-paclitaxel + gemcitabine was devised. Interestingly, this tri-ple-therapy resulted in 2 out of 4 tumor cell lines (BxPc-3, MIA PaCa-2) in a strongly improved treatment outcome. Notably, in the other tumor cell lines in which no enhanced response was seen after the triple-therapy (AsPc-1, Panc-1) viral titers were found to be considerably reduced under the influence of the dual chemotherapy. Thus, therapeutic success of this therapeutic regimen was linked to an unaltered viral replication of GLV-1h68 and - vice versa - failure to a miss-ing suppression of fabricating the progeny GLV-1h68 particles. Further investigations indicated that the interference with viral replication specifi-cally was the result of overdosing gemcitabine. When delaying gemcitabine ad-ministration viral GFP expression could be somewhat conserved. Similarly, pre-vious reports had demonstrated the benefit of unraveling a chemovirotherapeutic treatment protocol by administering the chemo- and virotherapeutic agents sepa-rately. Thus, the next step of promoting the combination of GLV-1h68 with nab-paclitaxel and gemcitabine would be to investigate the influence of such measures on therapeutic success, at best in an immunocompetent animal model while non-invasively monitoring viral replication and therapeutic efficacy.