Ziegler Verena G, Knaup Julia, Stahl Dorothea, Krammer Barbara, Plaetzer Kristjan
Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
Lasers Surg Med. 2011 Sep;43(7):548-56. doi: 10.1002/lsm.21089.
A major obstacle for permanent cancer eradication is the persistence of circulating tumor cells (CTCs) in blood, which often escape radio- or chemotherapy. Currently no efficient strategy to remove CTCs from peripheral blood in order to lower the risk of metastases or tumor recurrence exists. Photodynamic treatment (PDT) using aminolevulinic acid (ALA) induced protoporphyrin IX (PPIX) as photosensitizer offers an innovative approach to overcome this problem. This study aims at providing basic evidence towards fluorescence detection and photodynamic depletion of scattered cancer cells from blood preparations.
The breast cancer cell line T47D, endothelial GP8 cells, red blood cells (RBCs) and peripheral blood mononuclear cells (MNCs) have been tested for ALA-induced formation kinetics of PPIX by flow cytometry and microplate fluorescence analysis. The influence of the presence of RBCs on the PPIX-accumulation in cancer cells was evaluated by flow cytometry; the efficacy of PDT on cancer cells and MNCs has been tested by resazurin assay. Mixtures of T47D and GP8 cells and MNCs spiked with cancer cells were tested to determine the limit of fluorescence detection by flow cytometry and antibody co-staining.
T47D cells accumulated significantly higher PPIX-amounts after ALA-incubation than any other cell type tested. The presence of RBCs had no impact on PPIX-formation in T47D cells. Experiments towards the fluorescence detection of cancer cells in blood revealed that the sensitivity of this method is yet limited. Viability testing after PDT showed that cancer cells where almost completely eradicated after illumination whereas MNCs were almost spared.
We clearly demonstrate in vitro tumor cell selectivity of PPIX-accumulation over endothelial cells, MNCs and RBCs. Breast cancer cells are efficiently killed by PDT with minor depletion of MNCs. Our findings provide a basis for the PDT of blood samples for a future depletion of CTCs.
彻底根除癌症的一个主要障碍是血液中循环肿瘤细胞（CTC）的持续存在，这些细胞常常能逃避放疗或化疗。目前，尚无有效的策略从外周血中清除CTC以降低转移或肿瘤复发的风险。使用氨基乙酰丙酸（ALA）诱导的原卟啉IX（PPIX）作为光敏剂的光动力治疗（PDT）提供了一种创新方法来克服这一问题。本研究旨在为血液制品中散在癌细胞的荧光检测和光动力清除提供基础证据。
通过流式细胞术和微孔板荧光分析，检测了乳腺癌细胞系T47D、内皮GP8细胞、红细胞（RBC）和外周血单个核细胞（MNC）中ALA诱导的PPIX形成动力学。通过流式细胞术评估RBC的存在对T47D细胞中PPIX积累的影响；通过刃天青检测法测试了PDT对癌细胞和MNC的疗效。测试了T47D和GP8细胞以及掺入癌细胞的MNC的混合物，以通过流式细胞术和抗体共染色确定荧光检测限。
ALA孵育后，T47D细胞积累的PPIX量显著高于其他任何测试细胞类型。RBC的存在对T47D细胞中PPIX的形成没有影响。对血液中癌细胞进行荧光检测的实验表明，该方法的灵敏度仍然有限。PDT后的活力测试表明，光照后癌细胞几乎被完全根除，而MNC几乎未受影响。
我们清楚地证明了在体外，PPIX在肿瘤细胞中的积累相对于内皮细胞、MNC和RBC具有选择性。PDT能有效杀死乳腺癌细胞，对MNC的损耗较小。我们的研究结果为未来用于清除CTC的血液样本PDT提供了基础。
