Figure 2

PBMC and MΦ display divergent patterns of transcript induction and ligand secretion upon priming by tumor cells. (A). Primed PBMC induce EREG, TGFA and OSM transcripts. qPCR of control-treated (Ctrl) or MDA-MB-231-primed (Primed) PBMC. Means ± 95% confidence intervals; *p < 0.05 by Mann-Whitney’s U-Test; n = 4. (B). Primed PBMC secrete EREG and OSM. PBMC supernatants were preincubated with control sera or blocking antibodies, then used to stimulate reporter cells in which pEGFR, pSTAT3 and tubulin levels were visualised. Western blots with densitometric analysis. Means ± SEM; n = 4; C: control Ig; T: TGFα blocking antibody; E: EREG blocking antibody; O: OSM blocking antibody; *p < 0.02; **p < 0.006 by Student’s T-Test. (C). Primed PBMC secrete elevated OSM levels. ELISA of control-treated or MDA-MB-231-primed PBMC. N: culture medium; P: priming medium; MC: control PBMC supernatant ; P-MC: primed PBMC supernatant; means ± SEM; n = 7; *p < 0.002 by Mann-Whitney’s U-Test. (D). Primed MΦ upregulate AREG, EREG, HBEGF and OSM transcription. qPCR of control-treated or MDA-MB-231-primed MΦ. Means ± 95% confidence intervals; *p < 0.02 by Mann-Whitney’s U-Test; n = 4. (E). Primed MΦ secrete HB-EGF and OSM. MΦ were control-treated or MDA-MB-231-primed for 24 hrs. MΦ supernatants were preincubated with control sera or blocking antibodies, then used to stimulate reporter cells in which pEGFR, pSTAT3 and tubulin levels were visualised. Western blots with densitometric analysis. Means ± SEM; pEGFR: n = 6; pSTAT3: n = 12; H: HB-EGF blocking antibody; A: AREG blocking antibody; O: OSM blocking antibody. **p < 0.00006 by Student’s T-Test. (F). Primed MΦ secrete elevated HB-EGF and OSM levels. ELISA of control-treated or MDA-MB-231-primed MΦ. N: culture medium; P: priming medium; MΦ: control macrophage supernatant; P-MΦ: primed macrophage supernatant; means ± SEM; n = 6; *p < 0.004 by Mann-Whitney’s U-Test.