Figure 6.

SNALPs containing mOX40L and siPDL1 significantly delay tumor growth and alter leukocyte populations in both tumor an TDLN. C57/Bl6 (n = 8 per group) were subcutaneously implanted with B16F10 cells (1 × 10⁶ cells/mouse). Once tumors were palpable (day 5 post implantation), SNALPs containing either mOX40L and siPDL1 or mLuc and siNeg were injected intratumorally (13 μg of total RNA per dose) or left untreated (NT). The SNALPs were administered two further times (days 7 and 10). (A) Tumor growth was monitored over the time course, injection time point is indicated by dotted lines. The group mean ± SD is shown in each case. Once the control group reached its humane end point mice were culled, and tumors and TDLNs were isolated. Single-cell suspensions were obtained using physical dissociation of tissues. Cells extracted from the tumors were stained with monoclonal antibodies targeting CD4 (B) and CD8 (C). Cells obtained from the TDLN were stained with anti-CD4, -CD44, and -CD69 (D, E) or anti-CD8, -CD44, and -CD69 (F, G). Absolute cell counts were obtained by including precision counting beads prior to acquisition on flow cytometer. For tumors, the cell count is normalized to tumor weight (B, C); for TDLN, it is presented as the whole cell fraction obtained from the TDLN (D, F). Lymphocyte activation in the TDLN was assessed by first gating on either CD4 or CD8 before the CD44+, CD69+ dual-positive population was identified. Data are presented as CD44+ CD69+ as percentage of the parent population. Each point represents an individual mouse; error bars correspond to the SD. Statistical analysis was carried out using a Student’s t-test. *, p < 0.05; **, p < 0.005; ***, p < 0.001; ns, nonsignificant.