Figure 2

IK14004 inhibits growth of Lewis lung cancer (LCC) and enhances IL-2/IL-12 receptor expression in splenocyte-derived T and NK cells. Peptide biodistribution and tumour studies were undertaken with ethics approval and experiments to determine the effects of IK14004 in vitro/in vivo were performed as described in the “Materials and methods”. Five technical replicates per experiment were included in each of duplicate biological replicates to assess in vitro cytotoxicity of IK14004 and Doxorubicin. Biodistribution experiments were performed on 3 mice administered with ⁶⁴NOTA-IK14004 and flow cytometry/ELISA experiments were performed with fresh splenocytes/supernatants from overnight splenocyte cultures, respectively. Two LLC models (subcutaneous allograft and lung metastasis model are identified below as AM, i.e., allograft model and LMM, i.e., lung metastasis model) were used to test effects of IK14004 administered I.P. twice per week for 2 weeks to groups of 8 mice (IK14004: 8 mice and vehicle control: 8 mice). Overnight splenocyte cultures were either left unstimulated or stimulated with anti-CD3 mAb alone or in combination with anti-CD28 mAb. All error bars represent standard error of the mean (SEM). Flow cytometry data are shown as percentage viability and mean fluorescence intensity (MFI). Dot plots and gating strategies are shown in Supplementary Figs. S2–S4. (a) Proliferation of LLC in vitro in the presence of IK14004 and Doxorubicin after 72 h. (b) Uptake of ⁶⁴Cu-NOTA-conjugated IK14004 assessed in vivo within the lungs, spleen and heart (surrogate for blood) during the first 4 h. (c) Percentage retention of ⁶⁴Cu-NOTA-conjugated IK14004 within the lungs, spleen and blood assessed ex vivo after 24 h. (d) LLC allograft growth after 2 weeks of treatment with IK14004. (e) Viability of freshly harvested splenocytes from the allografted mice (AM). (f) CD25 expression in CD4+ T cells within harvested splenocytes from the AM. (g) IL-12Rβ1 expression in CD4+ T cells within harvested splenocytes from the AM. (h) IL-12Rβ2 expression in CD4+ T cells within harvested splenocytes from the AM. (i) CD25 expression in NK cells within harvested splenocytes from the AM. (j) IL-12Rβ1 expression in NK cells within harvested splenocytes from the AM. (k) IL-12Rβ2 expression in NK cells within harvested splenocytes from the AM. (l) Percentage of lung area occupied by tumour in the LLC lung metastasis model (LMM). (m) CD28 expression in CD4+ T cells within harvested splenocytes in the LMM. (n) Viability of unstimulated and TCR-stimulated splenocytes following overnight culture in the LMM. (o) IL-2 in supernatants from unstimulated and TCR-stimulated splenocytes following overnight culture in the LMM. (p) IFN-γ in supernatants from unstimulated and TCR-stimulated splenocytes following overnight culture in the LMM. (q) IL-12Rβ1 expression in CD4+ T cells within unstimulated and TCR-stimulated splenocytes after overnight culture in the LMM. (r) IL-12Rβ2 expression in CD4+ T cells within unstimulated and TCR-stimulated splenocytes after overnight culture in the LMM. For tumour growth comparisons, data were analysed using an unpaired two-tailed t-test to assess differences between the two groups in both the allograft and the lung metastasis study. All other data were analysed using two-way ANOVA with Sidak’s multiple comparison. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.