Figure 3.

Conditioned media from primary PDA (PDA-CM) or metastatic epithelial cells (Met-CM) promotes the survival of Gr-MDSC. (A) Sorted Gr-MDSC cultured in basic media or PDA-CM for 48h. (B) PDA-CM protects a cultured Gr-MDSC from apoptosis (48h). (C) Relative gene expression of select cytokines and chemokines as determined by quantitative PCR (data represent mean ± SEM of 3 independently derived primary invasive tumor (PDA) and paired metastatic cell preparations and are normalized to expression in preinvasive cells). (D) Impact of conditioned media and cytokines on apoptosis of splenic-derived Gr-MDSC. Bar graphs represent mean ± SEM from 4 independent experiments performed. Treated samples were compared to the basic media (Basic) control and significance was determined by using a one-way ANOVA followed by a Dunnett’s multiple comparison test. (E) Sorted splenic Gr-MDSC from KPC mice with PDA were incubated with the indicated media or cytokines and the percentage of Annexin-V⁺ Gr-MDSC at 48h determined by FACS (representative results of 4 independent experiments). (F) Sorted splenic Gr-MDSC were incubated with PDA-CM ± the indicated blocking mAb for 48h. The percentage of live cells was determined by FACS analyses of Annexin-V^neg Gr-MDSC. (G) Clusters of PDA cells contain GM-CSF at their stromal surface. CK, cytokeratin. Scale bar, 25 µm. *, p<0.05; **, p<0.005; ***, p<0.0005.