Accumulation of Pro-Cancer Cytokines in the Plasma Fraction of Stored Packed Red Cells

Introduction: Perioperative blood transfusion has been linked to decreased survival in pancreatic cancer; however, the exact causal mechanism has not been elucidated. Allogeneic transfusions are known to expose patients to foreign cells and lipid mediators. We hypothesize that stored packed red cells (pRBCs) contain pro-cancer cytokines that augment tumor progression. We analyzed the plasma fraction of stored pRBCs for pro-cancer cytokines and evaluated the affect of both storage time and leukocyte reduction on these mediators. Methods: Chemiarray™ analysis for pro-cancer cytokines was performed on the acellular plasma fraction of stored leukocyte-reduced (LR) and non-leukocyte-reduced (NLR) pRBCs at day 1 (D. 1-fresh) and day 42 (D. 42-outdate) of storage. Elevated expression of monocyte chemotactic protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), angiogenin, tumor necrosis factor-alpha (TNF-α), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) was found. Specific enzyme-linked immunosorbent assay was performed for each of these factors in LR and NLR blood at D. 1, day 28, and D. 42. Data were analyzed by ANOVA. A p value ≤0. 05 was considered significant; N ≥ 4 per group. Migration assays were performed using inhibitors of EGF (gefitinib) and PDGF (imatinib) on murine pancreatic adenocarcinoma cells (Pan02) exposed to D. 1 and D. 42 LR and NLR plasma. Proliferation assays were performed on Pan02 cells to test the inhibition of PDGF. Results: MCP-1 levels increased with storage time in LR blood, 86. 3 ± 6. 3 pg/ml at D. 1 vs. 121. 2 ± 6. 1 pg/ml at D. 42 (p = 0. 007), and NLR blood, 78. 2 ± 7. 3 pg/ml at D. 1 vs. 647. 8 ± 220. 7 pg/ml at D. 42 (p = 0. 02). RANTES levels are lower in LR compared to NLR stored blood, 3. 0 ± 1. 9 vs. 15. 8 ± 0. 7 pg/ml at D. 42 (p < 0. 001), but similar in D. 1 blood, 13. 8 ± 1. 8 pg/ml in LR vs. 12. 0 ± 1. 6 pg/ml in NLR. Angiogenin levels were different between LR and NLR blood, 0 pg/ml (undetectable) vs. 44. 2 ± 3. 7 pg/ml (p < 0. 001). Storage time did not affect concentration. TNF-α levels were not different between LR and NLR blood, and there was no storage time effect on concentration. EGF and PDGF levels increased with storage time in NLR blood only, 216. 4 ± 3. 8 pg/ml at D. 1 vs. 1,436. 4 ± 238. 6 pg/ml at D. 42 for EGF (p = 0. 001), and 61. 6 ± 6. 0 pg/ml at D. 1 vs. 76. 5 ± 1. 7 pg/ml at D. 42 (p = 0. 003) for PDGF. Inhibition of EGF reduced migration in Pan02 cells treated with D. 42 NLR blood, 245. 9 ± 11. 2 vs. 164. 6 ± 10. 6 cells/hpf (p < 0. 001). Inhibition of PDGF had no effect on Pan02 migration and reduced cell proliferation in cells treated with D. 42 NLR, 181. 1 ± 1. 5% over control vs. 157. 5 ± 2. 1% (p < 0. 001). Conclusion: Pro-cancer cytokines that can augment tumor progression were identified in pRBCs. Some of these factors are present in fresh blood. The soluble factors identified herein may represent possible therapeutic targets to offset negative effects of transfusion. These data stress the need for efforts in cancer patients to reduce transfusion requirements if needed.