Department of Radiation Oncology, Ludwig-Maximilians-University, Munich, Germany.
Radiat Oncol. 2014 Mar 26;9(1):85. doi: 10.1186/1748-717X-9-85.
Radiotherapy, administered in fractionated as well as ablative settings, is an essential treatment component for breast cancer. Besides the direct tumor cell death inducing effects, there is growing evidence that immune mechanisms contribute - at least in part - to its therapeutic success. The present study was designed to characterize the type and the extent of cell death induced by fractionated and ablative radiotherapy as well as its impact on the release of monocyte migration stimulating factors by dying breast cancer cells.
Cell death and senescence assays were employed to characterize the response of a panel of breast cancer cell lines with different receptor and p53 status towards γ-irradiation applied in a fractionated (daily doses of 2 Gy) or ablative setting (single dose of 20 Gy). Cell-free culture supernatants were examined for their monocyte migration stimulating potential in transwell migration and 2D chemotaxis/chemokinesis assays. Irradiation-induced transcriptional responses were analyzed by qRT-PCR, and CD39 surface expression was measured by flow cytometry.
Fast proliferating, hormone receptor negative breast cancer cell lines with defective p53 predominantly underwent primary necrosis in response to γ-irradiation when applied at a single, ablative dose of 20 Gy, whereas hormone receptor positive, p53 wildtype cells revealed a combination of apoptosis, primary, and secondary (post-apoptotic) necrosis. During necrosis the dying tumor cells released apyrase-sensitive nucleotides, which effectively stimulated monocyte migration and chemokinesis. In hormone receptor positive cells with functional p53 this was hampered by irradiation-induced surface expression of the ectonucleotidase CD39.
Our study shows that ablative radiotherapy potently induces necrosis in fast proliferating, hormone receptor negative breast cancer cell lines with mutant p53, which in turn release monocyte migration and chemokinesis stimulating nucleotides. Future studies have to elucidate, whether these mechanisms might be utilized in order to stimulate intra-tumoral monocyte recruitment and subsequent priming of adaptive anti-tumor immune responses, and which breast cancer subtypes might be best suited for such approaches.
放疗，包括分次放疗和根治性放疗，是乳腺癌治疗的重要组成部分。除了直接诱导肿瘤细胞死亡的作用外，越来越多的证据表明免疫机制至少在一定程度上有助于其治疗效果。本研究旨在描述分次放疗和根治性放疗诱导的细胞死亡类型和程度，以及其对死亡乳腺癌细胞释放单核细胞迁移刺激因子的影响。
采用细胞死亡和衰老测定法，研究一组不同受体和 p53 状态的乳腺癌细胞系对γ射线的反应，γ射线应用于分次（每天 2 Gy）或根治性（单次 20 Gy）照射。在 Transwell 迁移和 2D 趋化/趋化测定中检测无细胞培养上清液的单核细胞迁移刺激潜能。通过 qRT-PCR 分析照射诱导的转录反应，并用流式细胞术测量 CD39 表面表达。
快速增殖、激素受体阴性、p53 缺陷的乳腺癌细胞系在单次根治性 20 Gy 照射时主要发生原发性坏死，而激素受体阳性、p53 野生型细胞则显示凋亡、原发性和继发性（凋亡后）坏死的组合。在垂死的肿瘤细胞释放无酶敏感核苷酸时，坏死发生，这些核苷酸有效地刺激单核细胞迁移和趋化作用。在具有功能性 p53 的激素受体阳性细胞中，这种情况受到照射诱导的表面表达ectonucleotidase CD39 的阻碍。
我们的研究表明，根治性放疗强烈诱导快速增殖、激素受体阴性、p53 突变的乳腺癌细胞系发生坏死，进而释放单核细胞迁移和趋化刺激核苷酸。未来的研究必须阐明这些机制是否可用于刺激肿瘤内单核细胞募集和随后的适应性抗肿瘤免疫反应的启动，以及哪些乳腺癌亚型最适合这些方法。
