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Keywords:

  • transfusion;
  • pancreatic cancer;
  • metastasis;
  • proliferation;
  • erythrocytes;
  • immunomodulation

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

BACKGROUND:

Perioperative blood transfusion in pancreatic cancer patients has been linked to decreased survival; however, a causal mechanism has not been determined. During the processing and storage of packed erythrocytes, biologically active molecules accumulated in the acellular plasma fraction; therefore, the authors hypothesized that the plasma fraction of stored packed erythrocytes promoted tumor progression.

METHODS:

Proliferation and migration of murine pancreatic cancer and control cells were determined in vitro in response to the plasma fraction from leukocyte and nonleukocyte-reduced fresh versus stored packed erythrocytes. Last, an immunocompetent murine model was used to assess the effect of the plasma fraction of stored and processed packed erythrocytes on pancreatic cancer progression.

RESULTS:

Incubation of pancreatic cancer cells with the plasma fraction of packed erythrocytes increased proliferation and migration. Intravenous delivery of the acellular plasma fraction to mice with pancreatic cancer significantly increased the tumor weight in both leukocyte-reduced and nonleukocyte-reduced packed-erythrocyte groups (P < .01), although tumor growth and morbidity were greatest in the nonleukocyte-reduced group.

CONCLUSIONS:

The plasma fraction of stored packed erythrocytes promoted murine pancreatic cancer proliferation and migration in vitro and when administered intravenously, significantly augmented pancreatic cancer growth in immunocompetent mice. Cancer 2010. © 2010 American Cancer Society.

Transfusion of stored packed erythrocytes often represents a lifesaving clinical intervention designed to rapidly correct deficits of volume-carrying and oxygen-carrying capacity. Unfortunately, allogeneic blood transfusions expose patients to foreign cells, antigens, and soluble factors that may affect host immune function, a clinical phenomenon described as transfusion-related immunomodulation. Transfusion-related immunomodulation was first described in 1973 when renal transplant allografts were found to survive longer in patients who had previously received blood transfusions.1 Based upon these observations, Gantt2 questioned whether this transfusion-related immunomodulation effect might also be associated with an increased risk of cancer recurrence in patients undergoing resection for malignancy. The clinical corollary to this hypothesis proposes that allogeneic blood transfusion modulates the host immune system or directly affects cellular activities, resulting in increased tumor growth and metastasis, thus hastening cancer-related death.

Since these initial studies, more than 150 clinical studies have examined the association of perioperative allogeneic blood transfusion with cancer recurrence.3 Most of these are observational cohort studies, comparing patients who did or did not receive a transfusion.4 Notably, several studies showed worsened outcomes in patients who were carefully matched for stage and other pertinent clinicopathologic characteristics, such as stage and operative difficulty, that could otherwise be confounding in terms of separating tumor biology from transfusion effect.5-7 Interestingly, it has been shown in 1 study that even the recipients of autologous blood transfusions have increased cancer-related morbidity and mortality when compared with patients who do not require transfusions.8 These data suggest that something other than the immunogenicity of an allogeneic transfusion is responsible for poorer outcomes wherein processing or blood storage may play a role. In a seminal preclinical study by Blajchman et al, it was found that prestorage leukocyte depletion reduced pulmonary metastatic events, as did syngeneic versus allogeneic transfusions.9 Furthermore, at least 8 randomized control trials have compared the risk of cancer recurrence between patients receiving packed erythrocytes with and without processing to remove elements believed to be responsible for transfusion-related immunomodulation.10, 11 Likely due to limitations inherent to clinical trials, especially sample size, these studies failed to demonstrate a causal relation between perioperative allogeneic blood transfusion and cancer recurrence or death due to malignant progression.12, 13 Nevertheless, clinical14-16 and laboratory observations support Gantt's original hypothesis and have lead to continued investigations. More recently, 2 relatively large clinical studies have shown that blood transfusion negatively impacts survival in patients with periampullary (pancreas) cancer.17, 18

During routine storage, cytokines and other bioactive substances accumulate in the plasma fraction within packed erythrocytes.19-21 Plasma from stored, but not fresh, packed erythrocytes contain lipids, which prime neutrophils, causing neutrophil-mediated cytotoxicity, induce acute lung injury as the second event in a 2-event model, and have been implicated in transfusion-related acute lung injury.21-26 As the ability of the plasma from stored packed erythrocytes has never been tested on tumor growth and metastasis, we hypothesized that the plasma from stored packed erythrocytes may be pro-oncogenic and augment the growth of solid tumors, or affect host immunity, thereby allowing more rapid tumor progression.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

Plasma Preparation From Stored Packed Erythrocytes

After obtaining informed consent under a protocol approved by the Colorado Multi-institutional Review Board, 10 healthy donors donated 450 mL of whole blood per American Association of Blood Banks (AABB) criteria.27 The whole blood was separated into components: 50% (by weight) of the packed erythrocytes were prestorage leukoreduced by filtration by using a Pall BPF4 leukoreduction filter (Pall Corporation, Port Washington, NY), and the other 50% of the unit was left as an unmodified control. The division of these units was completed without air contamination in a closed system and stored in accordance with AABB Guidelines.28 Samples from these packed erythrocytes units, both prestorage leukoreduced units and nonleukoreduced units on Day 1, Day 28, and Day 42 were obtained by means of sterile couplers, and the plasma was separated by centrifugation.27 After plasma isolation, a second spin (12,500 g for 5 minutes) was performed to remove acellular debris.21 Plasma was aliquotted and stored at −80°C.

Cell Proliferation

Cells were maintained at 37°C in a mixture of 5% CO2 and 95% air in Dulbecco modified Eagle medium ([DMEM] Gibco; Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gemini Bio-Products, Woodland, Calif). bEnd.3 (mouse vascular endothelial; American Type Culture Collection [ATCC], Manassas, Va), RAW 274.1 (mouse macrophage; ATCC, Manassas, Va), and Pan02 (mouse pancreatic carcinoma; National Cancer Institute, Frederick, Md) cells were cultured and harvested by using 1% Trypsin-EDTA (Invitrogen, Carlsbad, Calif). The cells were placed in 96-well dishes (Falcon; BD Biosciences, San Jose, Calif) in DMEM + 10% fetal bovine serum (FBS) for 6 hours to allow the cells to adhere. Media was removed, and experimental media was added with various conditions as follows: DMEM alone (control), DMEM + 10% FBS, DMEM + 10% Day 1, Day 28, and Day 42 from both leukoreduced and nonleukoreduced plasma from packed erythrocytes. Cell proliferation was determined in triplicate at 24 hours by using a commercially available MTS assay (Cell Titer 96 Aqueous 1 Solution Proliferation Assay; Promega Corporation, Madison, Wis, Cat. no. G3580) according to the manufacturer's guidelines, and absorbance was quantified by using a FLUOstar OPTIMA plate reader (BMG LabTech, Offenburg, Germany). Data are presented as a percentage of control (DMEM alone).

Cell Migration

Pan02 cells were harvested into DMEM alone in a modified Boyden chamber with experimental media in the lower chamber, which contained the following reagents: DMEM alone (control), DMEM + 10% FBS, DMEM & plus; Day 1 leukoreduced and nonleukoreduced plasma or Day 42 leukoreduced and nonleukoreduced plasma. Cells were kept at 37°C for 24 hours. Membranes were then stained, excised, mounted on slides, and examined by using a Nikon inverted microscope (Eclipse E600; Nikon, Tokyo, Japan) at ×200 total magnification. Cell migration across the membrane was quantified in 6 fields of view for each membrane, with 2 membranes for each treatment condition. Data were presented as the mean number of cells that migrated across the membrane for each field (total magnification, ×200).

Orthotopic Immunocompetent Murine Model of Pancreatic Cancer

Two sets of animal experiments were performed by using plasma from different donors. All murine studies were carried out in accordance with the guidelines of the American Association for Accreditation of Laboratory Care and The University of Texas Southwestern Animal Care and Use Committee. Mice underwent orthotopic injection of 3 × 105 GFP-expressing Pan02 cells as previously described.29 Mice were randomized 1 week post-tumor injection to receive a lateral tail-vein injection of either saline or Day 1 leukoreduced or nonleukoreduced or either saline or Day 42 leukoreduced or nonleukoreduced plasma extract, while undergoing a second sham surgery. Two nontumor-bearing groups (n = 3 per group) were given either Day 1 leukoreduced or nonleukoreduced plasma or Day 42 leukoreduced or nonleukoreduced plasma as a control. Units of packed erythrocytes contain 60-80 mL of acellular plasma. Thus, if one assumes that the average weight of a healthy human adult is 70 kg, then the estimated acellular plasma dose for a patient receiving a single unit of blood would be approximately 1 mL/kg. Therefore, all animals were given a dose of 1 mL/kg of this acellular plasma diluted into 100 μL of saline. Mice were clinically followed,30 weighed 3 times each week, and sacrificed 3 weeks after the orthotopic injection of tumor cells. Nontumor-bearing mice were maintained for 4 weeks after lateral tail-vein injection of plasma extract to observe them for any adverse effects from injection of human plasma.

Necropsy was performed, and the extent of disease was quantified, noting tumor size, visible metastatic disease, and tumor sequelae. Necropsy was performed by 2 individuals blinded to randomization (authors S.E.H. and C.C.B). Lesions were examined under blue light (485 nm) to confirm presence of green-fluorescent protein (GFP) tumor cells. Selected lymph nodes were embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to confirm the presence of metastasis. Jaundice was determined subjectively by direct visual inspection of the abdominal viscera, and visceral obstruction was assessed by inspection of dilated viscera proximal to GFP-positive implants.

Tumor Immunohistochemistry

Tumors from Control-Saline, Day 42 leukoreduced plasma from stored erythrocytes, and Day 42 nonleukoreduced plasma from stored erythrocytes were examined for microvessel density, mature macrophage (F4/80 antigen) infiltration, and activated (Mac-3 antibody) macrophage infiltration by using fluorescent immunohistochemistry (IHC). Sectioning was performed in 10 μm serial sections of frozen tumors. Sections were air-dried for 30 minutes, fixed to slides with acetone for 30 seconds, rehydrated with tris-buffered saline (TBS) and 0.2% Tween (TBST), and blocked with 20% Aquablock (East Coast Biologics, North Berwick, Me, Cat. no, PP82-A3021), and diluted in TBST for 2 hours. Primary antibody (Rat anti-CD 31; [MEC13.3; BD Biosciences, San Jose, Calif; Cat. no. 550274]), a vascular endothelial cell surface antibody; rat anti-F4/80 (AbD Serotec, Raleigh, NC; Cat no. MCA497R), a surface marker for mature macrophages; rat anti-MAC-3 (M3/84; BD Biosciences; Cat. no. 553322), a surface marker for activated macrophages), diluted in DAKO antibody diluent (DAKO, Carpentaria, Calif; Code no. S3022) at 10 μg/mL and was then placed onto separate tissue sections. These were incubated overnight at 4°C, washed 3 times with TBST, and fluorescently labeled secondary antibody was then placed on the tissue for 1 hour (goat antirat IgG). After washing the slides 3 times in TBST, the tissue was mounted with ProLong Gold Antifade reagent (Invitrogen, Carlsbad, Calif; Cat. no. P36930), and the fluorescent signal was quantified. Pixel quantification was performed by using previously described techniques.31

Statistical Analysis

Data are expressed as mean ± the standard error of the mean. One-way analysis of variance (ANOVA) testing was performed to determine the significance of observed differences. Fisher protected least significant difference (PLSD) was performed for post hoc comparisons. Statistical significance was determined at P < .05.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

Cellular Proliferation

Three cell lines were examined, Pan02 (cancer), bEnd.3 (control), and RAW 274.1 (control), and the cells demonstrated similar proliferation effects in response to the acellular plasma from fresh (Day 1) and stored (Day 42) packed erythrocytes. All data are represented as percentages over control (DMEM alone). The presence of 10% FBS caused significant proliferation in all cell lines tested and was used as a positive control. When specifically examining response to the plasma fraction of packed erythrocytes, RAW cells showed a significant increase in proliferation when comparing plasma from leukoreduced and nonleukoreduced packed erythrocytes, whether the units were fresh, Day 1 (P < .05), or stored, Day 42 (P < .01). Moreover, increasing storage time of the packed erythrocytes from identical donors also increased RAW cell proliferation, whereas plasma from stored packed erythrocytes showed a significant increase over fresh from both leukoreduced packed erythrocytes and nonleukoreduced packed erythrocytes: Day 1 versus Day 42 leukoreduced (P < .01) and Day 1 nonleukoreduced versus Day 42 nonleukoreduced (P < .01). Concerning bEnd.3 proliferation, there was no difference in proliferation between plasma from Day 1 leukoreduced packed erythrocytes versus Day 1 nonleukoreduced packed erythrocytes (P = .11). However, there was a significant difference when comparing stored Day 42 leukoreduced versus Day 42 nonleukoreduced (P < .01) plasma from stored erythrocytes. Storage time did not significantly affect the leukoreduced group when comparing plasma from fresh or stored units, although there was a trend that approached statistical significance (P = .07). However, storage time did have a significant effect on bEnd.3 proliferation when examining nonleukoreduced plasma comparing fresh plasma from packed erythrocytes to plasma from nonleukoreduced stored erythrocytes (P < .01). Finally, upon examining Pan02 proliferation, there was a significant difference between the plasma from leukoreduced and nonleukoreduced packed erythrocytes in both the fresh and the stored groups (P = .04 and P < .01, respectively). Packed erythrocyte storage time did not significantly affect Pan02 proliferation in the leukoreduced group (although the trend was in this direction); conversely, it did significantly affect the nonleukoreduced group such that the plasma from stored nonleukoreduced packed erythrocytes evinced greater proliferation when compared with Pan02 cells treated with plasma from fresh nonleukoreduced packed erythrocytes (Fig. 1).

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Figure 1. Cell proliferation was significantly increased in Pan02, bEnd.3, and RAW cells when exposed to nonleukoreduced plasma as storage time increased.

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Cellular Migration

Cellular migration was measured in Pan02 cells. As in the proliferation experiments, differences in leukocyte reduction and storage time were compared, such that storage time was compared within a single group (either leukoreduced or nonleukoreduced). Interestingly, exposure to the acellular plasma fraction from Day 1 leukoreduced packed erythrocytes provoked more Pan02 migration than the plasma from Day 1 nonleukoreduced packed erythrocytes (90 ± 6.3 vs 66 ± 7.8 cells per high-power field, not statistically significant). However, with storage, the plasma from Day 42 nonleukoreduced packed erythrocytes significantly enhanced the Pan02 migration when compared with plasma from Day 42 leukoreduced packed erythrocytes (Day 42 nonleukoreduced packed erythrocytes, 206 ± 22.9 vs Day 42 leukoreduced packed erythrocytes, 161 ± 15.1 cells per high-power field; P < .01). Within groups, storage time had a significant effect on migration, in that the plasma from Day 42 leukoreduced packed erythrocytes increased Pan02 migration compared with the plasma from Day 1 leukoreduced packed erythrocytes (161 ± 15.1 vs 90 ± 6.3 cells per high-power field; P < .01), and plasma from Day 42 nonleukoreduced packed erythrocytes caused a similar increased migration versus the plasma from these identical units on Day 1 (206 ± 22.9 vs 66 ± 7.8 cells per high-power field; P < .01; Fig. 2).

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Figure 2. Pan02 cell migration was significantly increased when cells were exposed to nonleukoreduced plasma from stored packed erythrocytes at “outdate,” Day 42 of storage.

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Orthotopic Pancreatic Cancer Progression

Animals with established tumors received intravenous plasma from fresh (Day 1) or stored (Day 42) leukoreduced or nonleukoreduced packed erythrocytes, whereas control animals received intravenous saline. The mean tumor weight in the control and treated animals was as follows: controls, 56.0 ± 11.7 mg; Day 1 leukoreduced packed erythrocytes, 146 ± 7.5 mg; Day 1 nonleukoreduced packed erythrocytes, 200.6 ± 16.8 mg; Day 42 leukoreduced packed erythrocytes, 150.4 ± 13.3 mg; Day 42 nonleukoreduced packed erythrocytes, 213 ± 14.1 mg. All mice transfused with packed-erythrocyte plasma had statistically larger tumors than the control group (P < .01). Animals infused with plasma from fresh or stored nonleukoreduced packed erythrocytes had tumors that were statistically larger than mice infused with plasma from fresh or stored leukoreduced packed erythrocytes (P = .04 and P = .01, respectively). Storage time did not affect tumor growth in animals transfused with plasma from either the leukoreduced or nonleukoreduced packed erythrocytes (P = .88 and P = .50, respectively) (Fig. 3).

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Figure 3. All mice transfused with packed-erythrocyte plasma had statistically larger tumors than the control group (P < .01). Animals infused with plasma from fresh or stored nonleukoreduced packed erythrocytes had tumors that were statistically larger than mice infused with plasma from fresh or stored leukoreduced packed erythrocytes (P = .04 and P = .01, respectively). Storage time did not affect tumor growth in animals transfused with plasma from either the leukoreduced or nonleukoreduced packed erythrocytes (P = .88 and P = .50, respectively). PRBC indicates processing and storage of packed erythrocytes.

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Determination of Metastatic Disease

Animals developed metastatic lesions with the same pattern and site-dependent frequency (hepatic, peritoneal, and lymph node metastases) as has been observed in human patients with adenocarcinoma of the pancreas.32

Hepatic metastases

The difference in the number of visible liver metastases did not reach statistical significance among any of the groups. However, 3 of the mice receiving plasma from nonleukoreduced packed erythrocytes, 1 mouse on Day 1 and 2 mice on Day 42, developed a miliary pattern of liver metastases that made visual inspection and counting difficult. This miliary pattern was not observed in the saline-treated controls or mice infused with plasma from Day 1 or Day 42 leukoreduced packed erythrocytes (Fig. 4).

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Figure 4. There was no difference in hepatic metastasis when comparing fresh versus stored plasma from nonleukoreduced stored packed erythrocytes. However, mice receiving stored plasma had significantly more peritoneal metastases.

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Peritoneal metastases

In comparing groups, leukocyte reduction did not reduce peritoneal metastases in mice treated with plasma from fresh packed erythrocytes, leukoreduced packed erythrocytes versus nonleukoreduced packed erythrocytes (P = .3), whereas in mice infused with plasma from Day 42 nonleukoreduced packed erythrocytes, there were significantly more metastases as compared with controls (P = .04). Storage time did not increase the metastatic potential for animals infused with plasma from leukoreduced packed erythrocytes; however, for Day 42 nonleukoreduced packed erythrocytes, the difference between stored, Day 42, and fresh, Day 1, approached significance (P = .08) (Fig. 4).

Lymph lymph node metastases

There was a significant difference in the number of lymph node metastases in mice when comparing the infusion of plasma from nonleukoreduced packed erythrocytes to leukoreduced packed erythrocytes from both fresh (Day 1 nonleukoreduced packed erythrocytes vs Day 1 leukoreduced packed erythrocytes, P = .02) and stored groups (Day 42 nonleukoreduced packed erythrocytes vs Day 42 leukoreduced packed erythrocytes, P = .03). However, storage time did not increase the number of lymph node metastases in the homologous groups, plasma from Day 1 versus Day 42 leukoreduced packed erythrocytes or nonleukoreduced packed erythrocytes (Fig. 5).

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Figure 5. Lymph node metastases were significantly higher in mice receiving plasma from nonleukoreduced stored packed erythrocytes versus fresh (data from mice receiving plasma from stored nonleukoreduced plasma are shown). Inset: GFP expression in lymph node metastases and histology of metastatic adenocarcinoma within a lymph node are shown.

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Clinical Status of Mice

Within 2 weeks of intravenous injection of plasma from both fresh and stored nonleukoreduced packed erythrocytes, the tumor-bearing mice demonstrated roughened coat, loss of body condition, and inactivity,33 whereas tumor-bearing control animals (saline) appeared normal. Tumor-bearing animals transfused with plasma from leukoreduced packed erythrocytes had variable rates of distress, with most animals appearing well (data not shown). The change in animal weight for each group closely paralleled these qualitative observations (data not shown). Mice without cancer, which received plasma from Day 42 nonleukoreduced packed erythrocytes, gained weight appropriately for C57BL/6J mice,30 and control tumor-bearing animals gained in parallel until approximately14 days after tumor-cell injection when weight gain tapered off (presumably because of increasing tumor burden). Mice receiving intravenous, nonleukoreduced, packed erythrocytes plasma, whether fresh or stored, demonstrated poor weight gain compared with controls initially; however, mice receiving stored packed-erythrocytes plasma gained weight rapidly after 1 week (Fig. 6). Findings at necropsy offer some explanation for the poor clinical course of these mice. Ascites was present in 7 of 12 (58%) animals receiving plasma from stored nonleukoreduced packed erythrocytes and only 1 of 11 of the mice infused with plasma from Day 1 nonleukoreduced packed erythrocytes, and none of the control mice had ascites. Interestingly, no animals in the infused with plasma from fresh leukoreduced packed erythrocytes group and only 1 animal (20%) infused with plasma from stored leukoreduced packed erythrocytes developed ascites. Clinical jaundice, along with a distended gallbladder and biliary tree, were present in 6 (50%) mice infused with plasma from stored nonleukoreduced packed erythrocytes versus 2 (16%) animals treated with plasma from fresh nonleukoreduced packed erythrocytes, and none of the control mice or those receiving plasma from Day 1 leukoreduced packed erythrocytes (Fig. 7). These differences represent a trend toward more severe disease in the mice receiving plasma from stored packed erythrocytes, with the difference in ascites formation reaching statistical significance (P < .05) and biliary obstruction and jaundice trending toward a difference (P = .11). Taken together, these data demonstrate a more virulent pattern of tumor growth in plasma-treated mice over control mice and mitigation of the severe clinical effects from prestorage leukoreduction.

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Figure 6. Mice receiving plasma from fresh and stored nonleukoreduced stored packed erythrocytes demonstrated failure to gain compared with controls initially. Interestingly, mice receiving stored plasma developed rapid weight gain after 7-8 days, representing ascites formation in >50%.

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Figure 7. Mice receiving either fresh or stored nonleukoreduced plasma developed clinical morbidity. Mice receiving plasma from nonleukoreduced stored packed erythrocytes developed significant ascites (58% vs 9%) compared with fresh. Inset left: A normal mouse gallbladder compared with an enlarged gallbladder due to biliary obstruction is shown. Inset right: A bowel obstruction is developing because of lymph node metastasis.

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Tumor Immunohistochemistry

Tumors from animals treated with plasma from both leukoreduced and nonleukoreduced packed erythrocytes had significantly higher microvessel density than tumors in saline-treated animals (P < .001). Mature macrophage infiltration as determined by F4/80 staining did not differ between groups, but activated macrophage infiltration was higher in mice infused with stored nonleukoreduced packed erythrocytes or leukoreduced packed erythrocytes compared with saline-treated controls. In addition, tumors from animals receiving plasma from stored nonleukoreduced packed erythrocytes had significantly more activated macrophage infiltration than tumors from animals receiving stored or fresh plasma from leukoreduced packed erythrocytes (P < .001) (Fig. 8).

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Figure 8. Tumors from mice receiving plasma from stored nonleukoreduced packed erythrocytes had significantly more activated macrophage infiltration than tumors in animals receiving plasma from stored or fresh leukoreduced packed erythrocytes. PRBC indicates processing and storage of packed erythrocytes.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

The role of perioperative blood transfusion in augmenting pancreatic cancer progression remains unclear because of clinical variability among patients and individual tumor biology, although large clinical series of patients with pancreatic cancer suggest a negative impact of transfusion on survival.17, 18 Similarly, the specific constituent(s) of allogeneic blood that mediate transfusion-related immunomodulation as it relates to cancer progression are unclear. Plasma, platelets, and leukocytes, which inherently accompany nonleukoreduced packed erythrocytes, and proteins (cytokines or chemokines) that accumulate during packed-erythrocyte storage have been implicated in the pathogenesis of transfusion-related immunomodulation.34-37 Clinical studies in noncancer patients support the link between transfusion-related immunomodulation and the length of storage of banked blood.24, 38-40 Furthermore, in vitro data have demonstrated that tumor cell growth is enhanced by blood that has been stored for increasing time periods.41 The release of bioactive substances by leukocytes and platelets and the accumulation of these substances during storage is generally accepted,21, 42, 43 and animal studies suggest that early removal of leukocytes from banked blood ameliorates the tumor growth-promoting effects of blood transfusion.36, 37, 44 However, 5 randomized controlled trials designed to evaluate the effect of allogeneic white blood cell transfusion on cancer recurrence failed to yield convincing results with regard to an enhanced transfusion-related immunomodulation effect from stored versus fresh packed erythrocytes.11

This study has demonstrated that the acellular plasma fraction from stored packed erythrocytes promotes cellular proliferation, and Pan02 migration (surrogates for cancer growth and metastasis) promotes in vivo pancreatic cancer growth. Although previous clinical studies have shown similar conclusions,10, 17, 18, 45 these studies are difficult to interpret given the large number of variables inherent in any cancer patient population, including tumor biology, tumor stage, and untoward events during surgery, as well as innumerable host factors. Use of this well-established immunocompetent model of pancreatic cancer29 has allowed us to control for many factors that cause clinical trials to be dismissed. First, a clonal pancreatic cancer-cell tumor was surgically introduced into the pancreas itself, which allowed for consistent tumor biology within all test animals. Second, the use of an identical tumor inoculum (cell number) in all mice as well as ultrasound examination to confirm tumor presence allowed control for tumor stage before administration of plasma from packed erythrocytes. Third was the use of syngeneic animals controls to stand in for patient variability, and fourth, the same surgical stress was applied to all animals.

Interestingly, when examining the effect of the plasma from packed erythrocytes on cellular proliferation in vitro, it appears that the presence of leukocytes is the predominant factor that promotes proliferation, although increasing storage time trended toward increasing proliferation in the Pan02 group as well. Moreover, as opposed to proliferation, storage time appeared to be more important in Pan02 migration, although the presence of leukocytes was clearly important in the stored Day 42 blood.

The significant difference in tumor growth and metastasis observed in the animals that received plasma from packed erythrocytes suggests that infusion of bioactive substances within the plasma of packed erythrocytes promotes tumor progression, regardless of storage time. In addition, acellular plasma harvested from stored Day 42 packed erythrocytes did not differentially affect tumor size and metastatic burden when infused into tumor-bearing animals. However, there was a trend toward more severe disease in the Day 42 animals, with these animals demonstrating a higher rate of jaundice and ascites formation. Current transfusion literature supports these findings somewhat, suggesting that transfusion of blood with increased storage time augments the detrimental effects of transfusion-related immunomodulation.40, 41, 43, 46, 47 Although levels of biologically active cytokines increase during storage of packed erythrocytes, prestorage leukoreduction decreases the cancer-promoting effects of such transfusions21, 48, 49; moreover, contrary to the presented data, these studies showed that reduction of white blood cells before storage reduced the metastasis formation to control levels. However, until recently, little was known about the effects of prestorage leukoreduction on platelet-derived growth factors, eg, vascular endothelial growth factor, platelet-derived growth factor, or endothelial growth factor, all of which may have a role in tumor growth and may be eliminated from packed erythrocytes by the current generation of filters. Interestingly, the infusion of the plasma fraction from Day 1 and Day 42 of storage from nonleukoreduced packed erythrocytes was not different. These findings suggest that substances with sufficient bioactivity to promote tumor growth are present in the plasma after only 1 day of storage.

An unexpected finding was the observed rapid decline in the health of the mice harboring pancreatic cancer and receiving plasma versus the tumor-bearing control animals. After sham surgery and plasma transfusion, mice with tumors appeared ill within days and failed to gain appropriate weight, whereas tumor-bearing control animals gained weight in parallel with nontumor-bearing controls until late in the study. The causes for this “clinical” picture appear to have been identified at necropsy, where animals predominantly receiving plasma from Day 1 and Day 42 nonleukoreduced packed erythrocytes suffered biliary, gastrointestinal, and urinary tract obstruction, and a large portion of the mice treated with the plasma from Day 42 nonleukoreduced packed erythrocytes plasma developed ascites within the study period. This difference may be more profound than is initially apparent, as human clinical studies have demonstrated that the presence of malignant cells in the peritoneum is a strong negative prognostic marker in pancreatic cancer, independent of the size of the primary tumor.50 Thus, there may be a differential effect of stored packed erythrocytes versus fresher erythrocytes. The rapid decline of health in these animals closely parallels cancer-related morbidity witnessed in human patients and suggests that substances within plasma are promoting a virulent cancer progression that is not because of surgical trauma or the presence of tumor alone but rather a combination of plasma transfusion and surgery in a tumor-bearing host. Although the observed increased morbidity may be related to storage age of the blood, it will require further study for elucidation. Furthermore, these findings suggest that transfusion of packed erythrocytes may “tip the balance” of the tumor-host interaction in favor of the tumor.

There are several limitations of this study that warrant discussion. 1) Plasma contains many substances that could affect the host response, and the effect of individual agents is difficult to determine. 2) Human plasma was used in this model, and the mouse immune response to these foreign proteins is difficult to predict. Using blood from a syngeneic source would be ideal, but it would be logistically difficult considering the need for similar harvesting, processing, and storage techniques to make the model clinically relevant. Multiple studies by Silliman et al23, 51 have demonstrated acute lung injury after transfusion of plasma from stored, but not fresh, packed erythrocytes that is reproducible by using multiple blood donors with no idiosyncratic effects noted in those studies.23, 51, 52 3) Many of the parameters used to determine disease severity, such as identification of cancerous lesions (lymph node, liver, and peritoneal metastases) as well as jaundice and visceral obstruction, are subjective. Two blinded observers were used for confirmation of findings at necropsy to avert bias. Finally, statistical comparisons were necessarily made at high multiplicity comparing tumor growth, which should be viewed with some caution. Despite these issues, this model has clinical relevance and warrants further confirmation with more sophisticated studies in the future.

Blood products are given intravenously to cancer patients in the perioperative period every day, and the effects on cancer-related morbidity and death have yet to be proven. This study has attempted to control for many of the variables present in the clinical setting. In summary, these data suggest that bioactive substances capable of affecting the tumor biology within an organism are present within 24 hours of storage of erythrocytes. The substances and mechanism of action remain to be elucidated; however, these studies offer an avenue for investigation and identification of strategies for intervention.

CONFLICT OF INTEREST DISCLOSURES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES

This work supported in part by ACS Grant no. MRSG-09-034-01-CCE (to Carlton C. Barnett Jr) and NHLBI Grant no. R01 HL59355, NIGMS GM49222 (to Christopher C. Silliman).

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. CONFLICT OF INTEREST DISCLOSURES
  7. REFERENCES