Hast J, Schiffer I B, Neugebauer B, Teichman E, Schreiber W, Brieger J, Kim D W, Gebhard S, Born C J, Strugala M, Sagemüller J, Brenner W, Mann W J, Oesch F, Thelen M, Hengstler J G
Department of Radiology, Johannes Gutenberg-University Mainz, Germany.
Anticancer Res. 2002 Mar-Apr;22(2A):677-88.
Recently, the combination of ionizing radiation with inhibitors of angiogenesis has been reported to improve tumor eradication compared to treatment with irradiation alone. However, the mechanisms of this effect have not been defined. For this purpose [corrected] we established a non-small cell lung cancer model in nude mice. Tumor vascularization was visualized in vivo by MRI using gadolinium-DTPA as contrast agent. Further, cryosections were produced as close as possible to the MRI slice positions. Since we were interested in examining the formation of a recurrent tumor, irradiation was performed with a single fraction of 4 Gy. This dose caused a partial remission followed by recurrent tumor growth 25 to 35 days after therapy. The process of partial remission as well as formation of the recurrent tumor was examined in 28 nude mice analysing the following parameters: (i) contrast agent enhancement using high-resolution MRI, (ii) proliferation of tumor cells and fibroblasts using Ki-67 immunohistochemistry and (iii) formation of microvessels using CD31 immunohistochemistry. The latter analyses led to differentiation of three stages. Stage 1 (day 1 to day 15 after irradiation) was characterized by increasing areas of dead cell mass in hematoxylin-eosin-stained slides that corresponded to a decrease in tumor cell proliferation as well as contrast agent enhancement in MRI. The percentage of Ki-67-positive tumor cells decreased from initially 45.1% +/- 6.0% (mean +/- standard deviation) to 1.4% +/- 1.2% (mean +/- standard deviation) on day 15. Stage 2 (day 6 to day 20 after irradiation; overlapping with stage 1) was characterized by proliferation of fibroblasts leading to formation of fibrotic septae with abundant microvessels. Already during late stage 2, MRI identified new contrast agent enhancing areas. Stage 3 (day 20 to day 40 after irradiation) was characterized by new tumor cell proliferation. Interestingly, tumor cells almost exclusively proliferated in the direct neighbourhood of the fibrotic septae that had been formed in stage 2. Obviously, proliferation of fibroblasts and blood vessels was a condition prior to formation of recurrent tumor tissue. Thus, our results are in contrast with the view that tumors or recurrent tumors begin as avascular masses that later induce neovascularization. With respect to clinical practice, our results suggest that: (i) adjuvant anti-angiogenic therapy should not be limited to the day of irradiation but should cover a critical period until day 5 to day 20 after radiotherapy, (ii) adjuvant therapy should also include inhibition of fibroblast proliferation and (iii) MRI can identify a recurrent tumor 10 to 15 days before occurrence of new tumor growth.
最近，有报道称，与单独使用放疗相比，电离辐射与血管生成抑制剂联合使用可提高肿瘤根除率。然而，这种效应的机制尚未明确。为此，我们在裸鼠中建立了非小细胞肺癌模型。使用钆-DTPA作为造影剂，通过磁共振成像（MRI）在体内观察肿瘤血管生成。此外，尽可能在与MRI切片位置相近处制作冷冻切片。由于我们感兴趣的是研究复发性肿瘤的形成，因此采用单次4 Gy的剂量进行放疗。该剂量导致部分缓解，随后在治疗后25至35天出现复发性肿瘤生长。在28只裸鼠中分析了部分缓解过程以及复发性肿瘤的形成，检测了以下参数：（i）使用高分辨率MRI检测造影剂增强情况；（ii）使用Ki-67免疫组织化学检测肿瘤细胞和成纤维细胞的增殖情况；（iii）使用CD31免疫组织化学检测微血管的形成情况。后一项分析得出了三个阶段。第1阶段（放疗后第1天至第15天）的特征是苏木精-伊红染色切片中死细胞团区域增加，这与肿瘤细胞增殖减少以及MRI造影剂增强减少相对应。Ki-67阳性肿瘤细胞的百分比从最初的45.1%±6.0%（平均值±标准差）降至第15天的1.4%±1.2%（平均值±标准差）。第2阶段（放疗后第6天至第20天；与第1阶段重叠）的特征是成纤维细胞增殖，导致形成含有丰富微血管且有纤维化间隔的结构。在第2阶段后期，MRI就已识别出新的造影剂增强区域。第3阶段（放疗后第20天至第40天）的特征是新的肿瘤细胞增殖。有趣的是，肿瘤细胞几乎只在第2阶段形成的纤维化间隔的直接邻近区域增殖。显然，成纤维细胞和血管的增殖是复发性肿瘤组织形成的前提条件。因此，我们的结果与肿瘤或复发性肿瘤始于无血管肿块、随后诱导新血管生成的观点相反。就临床实践而言，我们的结果表明：（i）辅助抗血管生成治疗不应仅限于放疗当天，而应涵盖放疗后第5天至第20天这一关键时期；（ii）辅助治疗还应包括抑制成纤维细胞增殖；（iii）MRI可在新肿瘤生长出现前10至15天识别出复发性肿瘤。
