Turchan William T, Shapiro Ronald H, Sevigny Garrett V, Chin-Sinex Helen, Pruden Benjamin, Mendonca Marc S
a Department of Radiation Oncology, Radiation and Cancer Biology Laboratory , Indianapolis , IN 46202 , USA ;
b Department of Medical and Molecular Genetics , Indiana University School of Medicine , Indianapolis , IN 46202 , USA.
Int J Radiat Biol. 2016 Aug;92(8):427-33. doi: 10.1080/09553002.2016.1186299. Epub 2016 Jun 3.
Purpose To investigate whether irradiated human endothelial progenitor cells (hEPC) could induce bystander killing in the A549 non-small cell lung cancer (NSCLC) cells and help explain the improved radiation-induced tumor cures observed in A549 tumor xenografts co-injected with hEPC. Materials and methods We investigated whether co-injection of CBM3 hEPC with A549 NSCLC cells would alter tumor xenograft growth rate or tumor cure after a single dose of 0 or 5 Gy of X-rays. We then utilized dual chamber Transwell dishes, to test whether medium from irradiated CBM3 and CBM4 hEPC would induce bystander cell killing in A549 cells, and as an additional control, in human pancreatic cancer MIA PaCa-2 cells. The CBM3 and CBM4 hEPC were plated into the upper Transwell chamber and the A549 or MIA PaCa-2 cells were plated in the lower Transwell chamber. The top inserts with the CBM3 or CBM4 hEPC cells were subsequently removed, irradiated, and then placed back into the Transwell dish for 3 h to allow for diffusion of any potential bystander factors from the irradiated hEPC in the upper chamber through the permeable membrane to the unirradiated cancer cells in the lower chamber. After the 3 h incubation, the cancer cells were re-plated for clonogenic survival. Results We found that co-injection of CBM3 hEPC with A549 NSCLC cells significantly increased the tumor growth rate compared to A549 cells alone, but paradoxically also increased A549 tumor cure after a single dose of 5 Gy of X-rays (p < 0.05). We hypothesized that irradiated hEPC may be inducing bystander killing in the A549 NSCLC cells in tumor xenografts, thus improving tumor cure. Bystander studies clearly showed that exposure to the medium from irradiated CBM3 and CBM4 hEPC induced significant bystander killing and decreased the surviving fraction of A549 and MIA PaCa-2 cells to 0.46 (46%) ± 0.22 and 0.74 ± 0.07 (74%) respectively (p < 0.005, p < 0.0001). In addition, antibody depletion studies demonstrated that the bystander killing induced in both A549 and MIA PaCa-2 cells was mediated by the cytokines TNF-α and TGF-β (p < 0.05). Conclusions These data provide evidence that irradiated hEPC can induce strong bystander killing in A549 and MIA PaCa-2 human cancer cells and that this bystander killing is mediated by the cytokines TNF-α and TGF-β.
目的 研究经辐照的人内皮祖细胞（hEPC）是否能诱导A549非小细胞肺癌（NSCLC）细胞发生旁观者杀伤效应，并有助于解释在与hEPC共注射的A549肿瘤异种移植模型中观察到的辐射诱导肿瘤治愈情况改善的现象。材料与方法 我们研究了将CBM3 hEPC与A549 NSCLC细胞共注射是否会改变单剂量0或5 Gy X射线照射后肿瘤异种移植的生长速率或肿瘤治愈率。然后我们使用双室Transwell培养板，测试来自经辐照的CBM3和CBM4 hEPC的培养基是否会诱导A549细胞发生旁观者细胞杀伤效应，作为额外对照，还测试了对人胰腺癌MIA PaCa - 2细胞的效应。将CBM3和CBM4 hEPC接种到上室Transwell培养板中，将A549或MIA PaCa - 2细胞接种到下室Transwell培养板中。随后取出含有CBM3或CBM4 hEPC细胞的上室插入物，进行辐照，然后放回Transwell培养板中3小时，以使上室中经辐照的hEPC释放的任何潜在旁观者因子通过可渗透膜扩散到下室未辐照的癌细胞中。3小时孵育后，将癌细胞重新接种以进行克隆存活分析。结果 我们发现，与单独的A549细胞相比，将CBM3 hEPC与A549 NSCLC细胞共注射显著提高了肿瘤生长速率，但矛盾的是，单剂量5 Gy X射线照射后A549肿瘤治愈率也提高了（p < 0.05）。我们推测经辐照的hEPC可能在肿瘤异种移植中诱导A549 NSCLC细胞发生旁观者杀伤效应，从而提高肿瘤治愈率。旁观者效应研究清楚地表明，暴露于经辐照的CBM3和CBM4 hEPC的培养基会诱导显著的旁观者杀伤效应，并使A549和MIA PaCa - 2细胞的存活分数分别降至0.46（46%）±0.22和0.74±0.07（74%）（p < 0.005，p < 0.
