Figure 4: Immune cell recruitment and inflammatory tumour growth in Tlr^−/− mice.

(a) Immunohistochemical staining of frozen tumour sections of 8- to 16-week-old C57BL/6 WT and Tlr3/7/9^−/− was performed to assess the frequency of tumour-infiltrating immune cells at different time points (day 1, day 3, day 6, day 10 and day 14). Quantification of the number of positive cells was performed by observer-assisted counting on four randomly selected × 200 magnified images per sample (n=3–7). Single values and mean (horizontal line) are shown. (b) Representative examples of immunohistochemical staining of tumours dissected 14 days after injection. Scale bar, 100 μm. (c) Hierarchical clustering of gene expression: 2 × 10⁶ MOPC tumour cells were injected s.c. into 8- to 16-week-old C57BL/6 WT and Tlr3/7/9^−/− mice, and tumours were dissected 6 days later. RNA was isolated and a microarray analysis was performed using an Affimetrix Chip (n=2). Signal intensity of different cytokines is shown as hierarchical clustering. Red indicates upregulation of gene expression; white indicates no gene regulation. (d) Gene expression of selected cytokines was analysed in a time course by quantitative PCR: day 0 (skin only), day 4, day 6 and day 10 (n=3 mice each). Mean±s.e.m. of relative expression to day 0 is shown for tumours of Tlr3/7/9^−/− mice. (e) Tumours were explanted at day 10 after s.c. tumour cell injection into 10-week-old C57BL/6 WT and Tlr3/7/9^−/−, and cultured for 2 days in medium (20 mg ml⁻¹). Concentration of IL-6, IFN-γ (n=6) and IL-24 (n=9) in the SNs was analysed. Single values and mean (horizontal line) are shown. Results were considered significant at *P⩽0.05, **P⩽0.005 and ***P⩽0.0005 (t-test).