J. A. Yi1, K. El Kasmi1, E. E. Moore1,2, C. C. Barnett1,2 1University Of Colorado Denver,Aurora, CO, USA 2Denver Health Medical Center,Aurora, CO, USA
Introduction:
Tumor-associated macrophages (TAMs) mediate tumor progression. Macrophage polarization is categorized as M1 (classical) or M2 (alternative) activation pathways. M1 cells are pro-inflammatory and involved in innate immunity, while M2 cells function in wound healing and cell proliferation. The M2 phenotype is most commonly seen among TAMs, correlates with poor patient prognosis, and is considered pro-tumorigenic. Hypoxia inducible factor-1α (HIF1α) is a transcription factor activated in hypoxia and inflammation that modulates numerous critical functions for tumor growth and metastasis. Increased HIF1α expression in pancreatic adenocarcinoma is a marker of advanced malignancy. We hypothesize that pancreatic cancer modulates local macrophages towards a pro-tumorigenic M2 phenotype via a HIF1α-dependent mechanism.
Methods:
A murine pancreas adenocarcinoma cell line (Pan02) was modified using short-hairpin RNA targeting HIF1α via lentiviral transduction to create Pan02 SH+ cells lacking HIF1α activity. Pan02 and Pan02 SH+ cells were then incubated until 80% confluent prior to collection of supernatant. RAW 264.7 cells, a murine macrophage line, were exposed to Pan02 or Pan02 SH+ supernatant in culture for 24 hours. At 24 hours, the RAW 264.7 cells were harvested for mRNA extraction. Real-time polymerase chain reaction was used to determine macrophage phenotype based on suppressor of cytokine signaling (SOCS) 1 or 3 expression, accepted markers of M1 and M2 phenotypes respectively. Statistical analysis was performed using one-way analysis of variance with significance determined by α<0.05.
Results:
There was a modest increase in SOCS1 expression by RAW 264.7 cells exposed to either Pan02 or Pan02 SH+. The relative expression of SOCS1 was not significantly different between the two treatment groups (mean fold change in expression of Pan02-exposed = 1.5399 ± 0.2151, Pan02 SH+-exposed = 10.3159 ± 5.1322, p=0.1384). SOCS3 expression was significantly greater than SOCS1 in both Pan02 and Pan02 SH+ treatment groups (p=0.0015). Among RAW 264.7 cells exposed to Pan02 SH+ supernatant, SOCS3 expression was significantly reduced as compared to the Pan02-exposed cells (mean fold change in expression of Pan02-exposed = 152.206 ± 25.795, Pan02 SH+-exposed = 66.088 ± 10.617, p=0.0368).
Conclusions:
There was an increase in M2 versus M1 differentiation upon exposure to both Pan02 and Pan02 SH+ cell supernatant. Importantly, exposure to Pan02 supernatant significantly increased M2 differentiation compared to Pan02 SH+ supernatant, implicating HIF1α in the modulation of a pro-tumorigenic M2 macrophage activation state. This may represent a therapeutic target for treatment of pancreatic cancer.