Irradiation Can Selectively Kill Tumor Cells while Preserving Erythrocyte Viability in a Co-Culture System

Abstract

An understanding of how to safely apply intraoperative blood salvage (IBS) in cancer surgery has not yet been obtained. Here, we investigated the optimal dose of 137Cs gamma-ray irradiation for killing human hepatocarcinoma (HepG2), gastrocarcinoma (SGC7901), and colonic carcinoma (SW620) tumor cells while preserving co-cultured erythrocytes obtained from 14 healthy adult volunteers. HepG2, SGC7901, or SW620 cells were mixed into the aliquots of erythrocytes. After the mixed cells were treated with 137Cs gamma-ray irradiation (30, 50, and 100 Gy), tumor cells and erythrocytes were separated by density gradient centrifugation in Percoll with a density of 1.063 g/ml. The viability, clonogenicity, DNA synthesis, tumorigenicity, and apoptosis of the tumor cells were determined by MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, subcutaneous xenograft implantation into immunocompromised mice, and annexin V/7-AAD staining, respectively. The ATP concentration, 2,3-DPG level, free Hb concentration, osmotic fragility, membrane phosphatidylserine externalization, blood gas variables, reactive oxygen species levels, and superoxide dismutase levels in erythrocytes were analyzed. We found that 137Cs gamma-ray irradiation at 50 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SGC7901, and SW620 cells without markedly damaging the oxygen-carrying ability or membrane integrity or increasing the oxidative stress of erythrocytes in vitro. These results demonstrated that 50 Gy irradiation in a standard 137Cs blood irradiator might be a safe and effective method of inactivating HepG2, SGC7901, and SW620 cells mixed with erythrocytes, which might help to safely allow IBS in cancer surgery.

Effect of X-ray irradiation on hepatocarcinoma cells and erythrocytes in salvaged blood

“…After irradiation, necrosis played a key role in cell death. These findings were consistent with those of our previous reports that tumor cells showed necrosis after gamma-ray irradiation 5 . Necrosis in these three tumor cell lines caused by irradiation may be attributed to mitotic catastrophe due to DNA destruction, chromosomal aberrations and dysfunction of cell cycle checkpoints 24 .…”

“…The effects on the erythrocytes by X-ray irradiation were similar to gamma irradiation in our previous study 5 . However, the X-ray irradiation was more efficient than gamma irradiation in inhibiting the colony formation of HepG2 cells in vitro .…”

“…We irradiated all mixed cells with 6 MV LINAC (Varian Trilogy, USA). When the irradiation process was completed, we used single-step density gradient centrifugation in Percoll (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), as described in our previous study 5 . Next, the viability of the separated tumor cells was determined by the MTT assay, plate colony formation, EdU incorporation assay, and subcutaneous xenografting into immunocompromised mice.…”

“…When the irradiation process was completed, we separated tumor cells and erythrocytes. Then, ATP, 2,3-DPG, free Hb concentration, osmotic fragility and blood gas variables in separated erythrocytes were assayed immediately as previously described 5 . The morphology, ATP, 2,3-DPG, free Hb concentration of separated erythrocytes were assayed after culturing for 12 h, 24 h, 48 h, and 72 as described in the previous study 25 .…”

“…Currently, there are two methods that can be used to remove contaminating tumor cells from salvaged blood: leukoreduction filtration (LDF) 3 and gamma irradiation 4 , 5 . However, LDF is limited to the re-transfusion of salvaged blood containing less than 10 7 cells 6 ,7] .…”