Reovirus Type 3 Dearing Variants Do Not Induce Necroptosis in RIPK3-Expressing Human Tumor Cell Lines
Diana J M van den Wollenberg1, Vera Kemp1, Martijn J W E Rabelink1
1Department of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
Abstract:
Reoviruses are used as oncolytic viruses to destroy tumor cells. The concomitant induction of anti-tumor immune responses enhances the efficacy of therapy in tumors with low amounts of immune infiltrates before treatment. The reoviruses should provoke immunogenic cell death (ICD) to stimulate a tumor cell-directed immune response. Necroptosis is considered a major form of ICD, and involves receptor-interacting protein kinase 1 (RIPK1), RIPK3 and phosphorylation of mixed-lineage kinase domain-like protein (MLKL). This leads to cell membrane disintegration and the release of damage-associated molecular patterns that can activate immune responses. Reovirus Type 3 Dearing (T3D) can induce necroptosis in mouse L929 fibroblast cells and mouse embryonic fibroblasts. Most human tumor cell lines have a defect in RIPK3 expression and consequently fail to induce necroptosis as measured by MLKL phosphorylation. We used the human colorectal adenocarcinoma HT29 cell line as a model to study necroptosis in human cells since this cell line has frequently been described in necroptosis-related studies. To stimulate MLKL phosphorylation and induce necroptosis, HT29 cells were treated with a cocktail consisting of TNFα, the SMAC mimetic BV6, and the caspase inhibitor Z-VAD-FMK. While this treatment induced necroptosis, three different reovirus T3D variants, i.e., the plasmid-based reverse genetics generated virus (T3DK), the wild-type reovirus T3D isolate R124, and the junction adhesion molecule-A-independent reovirus mutant (jin-1) failed to induce necroptosis in HT29 cells. In contrast, these viruses induced MLKL phosphorylation in murine L929 cells, albeit with varying efficiencies. Our study shows that while reoviruses efficiently induce necroptosis in L929 cells, this is not a common phenotype in human cell lines. This study emphasizes the difficulties of translating the results of ICD studies from murine cells to human cells.
Insights
Reoviruses can trigger immunogenic cell death (ICD) to enhance cancer therapy. However, they fail to induce necroptosis, a key ICD form, in most human tumor cells, unlike in mouse cells.
Area of Science:
- Oncolytic virotherapy
- Immunogenic cell death (ICD)
- Necroptosis signaling pathways
Background:
- Reoviruses are explored as oncolytic viruses to eliminate tumor cells.
- Therapeutic efficacy is enhanced by inducing anti-tumor immune responses, particularly in immune-infiltrated tumors.
- Immunogenic cell death (ICD) induction by reoviruses is crucial for stimulating tumor-specific immune responses.
Purpose of the Study:
- To investigate the capacity of Reovirus Type 3 Dearing (T3D) variants to induce necroptosis in human tumor cells.
- To compare reovirus-induced necroptosis in human HT29 cells versus murine L929 cells.
- To assess the translational relevance of murine cell-based ICD studies to human cancer therapy.
Main Methods:
- Human colorectal adenocarcinoma HT29 cells and murine L929 fibroblast cells were utilized.
- Necroptosis was induced in HT29 cells using TNFα, BV6 (SMAC mimetic), and Z-VAD-FMK (caspase inhibitor).
- Three T3D reovirus variants were tested for their ability to induce necroptosis, assessed by MLKL phosphorylation.
Main Results:
- Reovirus T3D variants efficiently induced necroptosis in murine L929 cells.
- These reovirus variants failed to induce necroptosis in human HT29 cells, despite successful induction via chemical inducers.
- Human tumor cell lines commonly exhibit defects in RIPK3 expression, hindering necroptosis induction.
Conclusions:
- Reovirus-induced necroptosis is not a common phenotype in human tumor cell lines.
- Significant challenges exist in translating findings from murine models to human systems for ICD-based cancer therapies.
- Further research is needed to understand and overcome species-specific barriers in reovirus oncolytic virotherapy.
