Cell based miRNA Expression as a Marker of Response to Bevacizumab (Avastin) Therapy of Patients with Colorectal Cancer

使用抗血管生成疗法，如贝伐单抗（Avastin），破坏血管内皮生长因子（VEGF）信号传导，显着扩大了转移性结直肠癌（CRC）患者的治疗选择。然而，大量患者对这种高成本疗法没有反应;而其他人则遭受严重的副作用，如伤口愈合受损和中风风险增加。在癌症患者的血液中检测到从肿瘤脱落的循环肿瘤细胞（CTC）和从骨髓（BM）动员的循环内皮祖细胞（CEP）。这两种细胞群已被证明与肿瘤进展和对一系列治疗（包括抗血管生成治疗）的耐药性相关。然而，研究这些细胞在接受贝伐单抗治疗的患者中的预测价值的第一项研究揭示了有争议的结果。目前定义的CTC/CEP描述了具有遗传和表型异质性的复杂细胞群。目前，尚不清楚是哪些特性使个体CTC能够生长成实体转移。此外，在治疗耐药的发展中起关键作用的CEP亚群尚未被确定，因此，我的项目的目的是（i）使用miRNA表达谱研究CTC/CEP的异质性，（ii）开发一组miRNA，将与CTC/CEP的单细胞分析结合使用，以确定患者对贝伐单抗治疗的反应。将使用原位杂交（ISH）和免疫细胞化学（ICC）的组合在单个CEP/CTC上分析与恶性肿瘤/肿瘤血管生成病理相关的miRNA的表达。此外，将在6个不同的治疗时间点分析接受贝伐珠单抗的CRC患者的CTC/CEP和基于细胞的miRNA表达。与临床数据的相关性将导致鉴定CTC/EPCs中表达的可预测疾病进展的miRNA。这些miRNAs及其功能将通过细胞转染实验进一步研究，我相信其结果将有助于开发克服治疗耐药性的新策略。

The use of anti-angiogenic therapies, such as Bevacizumab (Avastin), which disrupt vascular endothelial growth factor (VEGF) signalling, has significantly extended the treatment options for patients with metastatic colorectal cancer (CRC). Nevertheless, a significant number of patients does not respond to this high-cost therapy; while others suffer from severe side effects such as impaired wound healing and an increased risk of stroke. These problems are compounded by the fact that there is as yet no clear marker for selecting patients that may benefit from anti-VEGF-therapy.Circulating tumor cells (CTCs), shed from the tumor, and circulating endothelial progenitor cells (CEPs), mobilized from the bone marrow (BM), are detected in the blood from cancer patients. Both populations of cells have been shown to be associated with tumor progression and resistance to a range of treatments, including anti-angiogenesis therapy. However, first studies investigating the predictive value of these cells in patients receiving Bevacizumab treatment have revealed controversial results. CTCs/CEPs as they are currently defined describe a complex cell population of cells with genetic and phenotypic heterogeneity. Currently, it is still unknown which properties enable individual CTCs to grow out to solid metastasis. Additionally, a subpopulation of CEPs that plays a crucial role in the development of therapy resistance has not yet been identified.Thus, the objective of my project is (i) to investigate the heterogeneity of CTCs/CEPs using miRNA expression profiling and (ii) to develop a panel of miRNAs that will be used in combination with single cell analysis of CTCs/CEPs to determine patients´ response to Bevacizumab treatment. The expression of miRNAs that are pathologically relevant in malignancy/tumor angiogenesis will be analyzed on single CEPs/CTCs using a combination of In Situ hybridization (ISH) and immunocytochemistry (ICC). Furthermore, CRC patients receiving Bevacizumab will be analyzed for CTCs/CEPs and cell based miRNA-expression at six different time points of treatment. The correlation to clinical data will lead to the identification of miRNAs expressed in CTCs/EPCs that are predictive of disease progression. These miRNAs and their function will be further studied using cell transfection experiments the results of which I believe will contribute to the development of novel strategies to overcome therapy resistance.