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. Author manuscript; available in PMC: 2014 May 15.
Published in final edited form as: Cancer Res. 2013 Mar 20;73(10):3007–3018. doi: 10.1158/0008-5472.CAN-12-4601

Figure 6.

Figure 6

PSC supernatants modulate MDSC differentiation via IL-6 and STAT3 signal transduction. A, PBMC were cultured with PSC supernatants or relevant cytokines for 20 minutes and analyzed for levels of total STAT3 or phosphorylated STAT3 (pSTAT3). B, recombinant IL-6 (10 ng/mL) or 10% PSC supernatants were preincubated for 30 minutes with 5μg/mL of anti-IL-6–neutralizing antibody and added to normal donor PBMCs. pSTAT3 was analyzed by immunoblot after 20 minutes. C, in a similar manner, the effect of neutralizing IL-6 on PSC-supernatant—induced MDSC generation was also determined by incubating cells for 7 days (changing and neutralizing medium/cytokines/PSC supernatants on days 3 and 5) and analysis of CD11b+CD33+ MDSC by flow cytometry. D, PBMC were cultured with the FLLL32 STAT3 inhibitor (6 μmol/L) or DMSO (vehicle) for 7 days with 10% PSC supernatants or IL-6/GM-CSF (positive control) and analyzed for CD11b+CD33+ cells by flow cytometry. Representative IHC analysis of pSTAT3 staining in a representative human pancreatic tumor tissue (E) or normal pancreatic tissue (F) specimen. Arrows indicate components of the tumor (T; black) and stroma (S; blue) showing positive pSTAT3 staining. Error bars represent SD from independent experiments with PBMC from 3 separate donors. *, statistically significant compared with DMSO control at P value < 0.05.