J. Yi1, E. E. Moore1,2, R. D. Schulick1, B. Edil1, K. C. El Kasmi1, C. C. Barnett1,2 1University Of Colorado Denver,Aurora, CO, USA 2Denver Health Medical Center,Aurora, CO, USA
Introduction: Alternative activation of tumor-associated macrophages is pro-tumorigenic and associated with poor prognosis. Similarly, upregulation of tumor-expressed hypoxia inducible factor-1α (HIF1α) augments tumor progression. We hypothesize that tumor derived mediators activate macrophages towards an alternative activation phenotype. Further, inhibition of macrophage HIF1α signaling will mitigate cancer cell-induced alternative macrophage activation.
Methods: Pan02 pancreatic and MC38 colon adenocarcinoma murine cell lines were grown under uniform conditions to create cancer-conditioned media (CCM) rich in tumor-derived factors. YC1, a HIF1α inhibitor, and dimethyloxalylglycine (DMOG), a HIF1α inducer, were used to modulate HIF1α activity of RAW 264.7 murine macrophages. Macrophages were pretreated with YC1 or DMOG for 6 hours prior to 18 hours of CCM exposure and compared to vehicle controls. Polymerase chain reaction was used to phenotype macrophages based on expression of canonical activation genes (Socs1=classical activation, Socs3=alternative activation). Expression of HIF1α target gene vascular endothelial growth factor (Vegfa) was used to determine HIF1α modulation.
Results: CCM exposure induced mild Socs1 expression in macrophages, representing minimal classical/pro-inflammatory activation (Pan02 1.55±0.02 fold-increase, p<0.0001; MC38 2.09±0.19 fold-increase, p<0.0001). CCM exposure greatly induced Socs3 expression (Pan02 11.34 ±0.29 fold-increase, p<0.0001; MC38 962.90±53.88 fold-increase, p<0.0001). YC-1 pretreatment reduced Socs3 expression (Pan02 0.19±0.03 fold-decrease, p<0.0001; MC38 0.13±0.01 fold-decrease, p<0.0001), while DMOG pretreatment increased Socs3 expression (Pan02 6.09±2.80 fold-increase, p=0.0118; MC38 8.73±0.71 fold-increase, p=0.0006). Correspondingly, Vegfa expression increased with DMOG (Pan02 1.75±0.11 fold-increase, p<0.0001) and decreased with YC1 pre-treatment among CCM-exposed macrophages (Pan02 0.44±0.02 fold-decrease, p<0.0001) as compared to CCM alone. (See Figure)
Conclusion: Conditioned media from pancreatic and colon adenocarcinoma induces alternative activation as represented by increased Socs3 expression. Pretreatment with the HIF1α inhibitor YC1 mitigates CCM-induced alternative activation, while pretreatment with the HIF1α inducer DMOG augments alternative activation. This was dependent upon HIF1α activity, as shown by the modulation in expression of HIF1α target gene Vegfa among treated macrophages. These data demonstrate that targeting HIF1α effectively modulates pro-tumorigenic alternative macrophage activation.
