Hemoglobin-based oxygen carrier mitigates transfusion-mediated pancreas cancer progression.

BACKGROUND: Perioperative blood transfusion in pancreatic cancer patients is linked to decreased survival; however, a causal mechanism has not been determined. Previously we have shown that the plasma fraction of stored packed red blood cells (pRBCs) promotes pancreas cancer progression and associated morbidity. We hypothesize these untoward effects will be mitigated by use of a hemoglobin-based oxygen carrier (HBOC). METHODS: Cytokines and growth factors were measured in the plasma fraction from stored pRBCs and in an HBOC via cytokine array followed by formal enzyme-linked immunosorbent assay (ELISA). In an immunocompetent murine model, pancreas cancer progression was determined in vivo by bioluminescence, tumor weight, and number of metastases. RESULTS: Elevated levels of epidermal growth factor (EGF), platelet-derived growth factor BB (PDGF-BB), and regulated upon activation, normal T cell expressed and secreted (RANTES) were present in the plasma fraction of stored pRBCs, but were not found in the HBOC. Intravenous delivery of plasma fraction to mice with pancreatic cancer resulted in increased bioluminescence activity compared with mice that received HBOC. Metastatic events and pancreatic primary tumor weights were significantly higher in animals receiving plasma fraction from stored pRBCs compared with animals receiving HBOC. CONCLUSIONS: Intravenous receipt of the acellular plasma fraction of stored pRBCs promotes pancreatic cancer progression in an immunocompetent mouse model. These untoward events are mitigated by use of an HBOC.

Activation state of stromal inflammatory cells in murine metastatic pancreatic adenocarcinoma.

The histologic presence of macrophages (tumor-associated macrophages, TAMs) and neutrophils (tumor-associated neutrophils, TANs) has been linked to poor clinical outcomes for solid tumors. The exact mechanism for this association with worsened prognosis is unclear. It has been theorized that TAMs are immunomodulated to an alternatively activated state and promote tumor progression. Similarly, TANs have been shown to promote angiogenesis and tumor detachment. TAMs and TANs were characterized for activation state and production of prometastatic mediators in an immunocompetent murine model of pancreatic adenocarcinoma. Specimens from liver metastases were evaluated by immunofluorescence and immunoblotting. TAMS have upregulated expression of CD206 and CD163 markers of alternative activation, (4.14 ± 0.55-fold and 7.36 ± 1.13-fold over control, respectively, P < 0.001) but do not have increased expression of classically activated macrophage markers CCR2 and CCR5. TAMs also express oncostatin M (OSM). We found that TANs, not TAMs, predominantly produce matrix metalloproteinase-9 (MMP-9) in this metastatic tumor microenvironment, while MMP-2 production is pan-tumoral. Moreover, increased expression of VEGF colocalized with TAMs as opposed to TANs. TAMs and TANs may act as distinct effector cells, with TAMs phenotypically exhibiting alternative activation and releasing OSM and VEGF. TANs are localized at the invasive front of the metastasis, where they colocalize with MMP-9. Improved understanding of these interactions may lead to targeted therapies for pancreas adenocarcinoma.

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.

Gender-specific transfusion affects tumor-associated neutrophil: macrophage ratios in murine pancreatic adenocarcinoma.

INTRODUCTION: Perioperative blood transfusion has been linked to decreased survival for pancreas cancer. Noting clinical data associating female blood products with increased morbidity, our lab has demonstrated that transfusion of female blood augments metastatic events compared to male blood in an immunocompetent murine pancreatic cancer model. It has been suggested that tumor-associated macrophages correlate with tumor progression by promoting angiogenesis. More recently, tumor-associated neutrophils have been implicated in aggressive tumor behavior. We hypothesize that differences in gender-specific transfusion-mediated pancreatic cancer progression are due to microenvironmental changes within the tumor. To test this hypothesis, we examined tumor-associated neutrophils and macrophage ratios in male and female mice with pancreatic cancer receiving blood transfusion from male or female donors. METHODS: C57/BL6 mice, age 7-9 weeks, underwent splenic inoculation with 2.5 × 10(5) PanO2 murine pancreatic adenocarcinoma cells. Mice were transfused on post-op day 7 with 1 ml/kg supernatant from day 42 male or female packed red cells. Necropsy was performed at 5 weeks or earlier for clinical deterioration, and tumors harvested. Frozen sections (5 µm) were stained for neutrophils and macrophages by immunofluorescence. Data were analyzed using ANOVA; p ≤ 0.05 was used to determine significance; N ≥ 3 per group. RESULTS: Clinically, male mice had greater morbidity and mortality than female mice when receiving female blood products, with roughened hair coat, development of ascites and death due to bowel obstruction. In evaluating the tumor microenvironment from mice receiving female blood products, male mice were noted to have a greater neutrophil to macrophage ratio than female mice, 0.176 ± 0.028 vs. 0.073 ± 0.012, p = 0.03. When examining neutrophil to macrophage ratio in mice receiving male blood products, no difference was noted (p = 0.48). CONCLUSIONS: Male mice with pancreas cancer have greater morbidity than female mice when receiving female blood products. Furthermore, the difference in neutrophil to macrophage ratio suggests that gender-specific blood transfusion promotes aggressive tumor behavior in male mice via microenvironmental changes. These data warrant further study to delineate sex-related differences in pancreatic cancer progression.