We investigated whether treatment with the T helper cell 1 (TH1) cytokines TNF-α and IFN-γ drives neuroblastoma (NB) cell lines into permanent growth arrest. Introduction: Most cancer immunotherapies focus on cytotoxic treatment strategies mediated by CD8-positive cytotoxic T lymphocyte or NK cell responses. However, besides killing, induction of permanent tumor growth arrest is another important mechanism of cancer control. TNF-α and IFN-γ are known to induce senescence in a large number of human cancers. Thus, we investigated their potential to drive NB cell lines into senescence as well. Methods: We evaluated growth arrest in 7 different NB cell lines (LS, LAN-1, SH-SY5Y, SHEP, SK-N-AS, SK-N-BE(2) and Kelly), and in the TH1 cytokine-sensitive melanoma cell line WM115, which was used as a positive control. Cells were cultivated in medium alone (control group) or in medium supplemented with TNF-α (10 ng/ml) and IFN-γ (100 ng/ml) for 96 hrs (treated group). After treatment, cell cycle was evaluated using a 5-ethynyl-2´-deoxyuridine (EdU) assay. Additionally, we investigated whether or not growth arrest persists upon withdrawal of TNF-α and IFN-γ. For growth arrest assays, cells were first treated as described above (2 passages) and subsequently reseeded without addition of TNF-α and IFN-γ. Results: Cell cycle analysis: 5 out of 7 NB cell lines showed reduced percentage of S-phase cells (S) while the G1/G0 fraction (G) increased. This change was significant (p<0.05). Shown are the mean percentages of S- and G-phase fractions and the G/S ratio (R) (n=3; control vs. treated). A significant change was observed for LS (S: 57% vs. 27% / G: 27% vs. 55%; R: 0.48 vs. 2.11), SH-SY5Y (S: 60% vs. 11% / G: 22% vs. 51%; R: 0.40 vs. 5.97), SK-N-AS (S: 34% vs. 18% / G: 47% vs. 56%; R: 1.40 vs. 3.04), SHEP (S: 58% vs. 32% / G: 24% vs. 45%; R: 0.43 vs. 1.26), Kelly (S: 57% vs. 45% / G 29% vs. 42%; R: 0.51 vs. 0.94), and the WM115 positive control (S: 26% vs. 4% / G: 46% vs. 50% (ns); R: 1.78 vs. 15.97). Growth assays: Cells were defined as senescent if the proliferation factor (PF) calculated for the reseeding phase of the previously treated group was between 0.5 and 1.5 and ≤ 50% of the PF calculated for the control group. Partial senescence was defined as PF > 1.5 but still ≤ 50% of the PF calculated for the control group. Shown is control vs. treated group. According to this definition cell lines are defined as (A) senescent: LS (2.3 vs. 1), SHEP (13 vs. 0.7), Kelly (6.4 vs. 1.5), WM115 (3.8 vs. 0.7) (B) partial senescent: SH-SY5Y (5.6 vs. 2.4), LAN-1 (7.0 vs. 1.6) (C) not senescent: SK-N-AS (6.1 vs. 3.3), SK-N-BE(2) (9.6 vs. 9.4). Conclusion: TNF-α und IFN-γ mediate inhibition of the cell cycle in the majority of tested NB cell lines as determined by the EdU assay. For 3 cell lines, inhibition of the cell cycle was confirmed in growth assays and shown to be permanent (LS, Kelly and SHEP). This was not true for the other two cell lines SH-SY5Y and SK-N-AS that both restarted proliferation upon withdrawal of TNF-α and IFN-γ. On the other hand, LAN-1 was apparently not affected in the cell cycle assay but drastically slowed down its proliferation rate after repeated TNF-α and IFN-γ treatment in the cell growth assays. SK-N-BE(2) was not affected in any of those experiments. In summary, these results suggest that some of the NB cell lines are able to completely arresT-cell growth upon treatment with TNF-α and IFN-γ while other cell lines are only temporarily or not at all affected. Future experiments will aim to confirm cytokine-induced senescence in NB cell lines by detection of several senescence markers, e.g., senesecence-associated beta-galactosidase or induction of p16Ink4a.
Citation Format: Theresa Harmuth, Florian Heubach, Thomas Wieder, Rupert Handgretinger, Peter Lang. Cytokine-induced senescence in neuroblastoma cell lines: Therapeutic option or idle wish? [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A080.