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

抗血管生成疗法的使用，如贝伐单抗(阿瓦斯丁)，扰乱血管内皮生长因子(VEGF)信号，显著扩大了转移性结直肠癌(CRC)患者的治疗选择。然而，相当多的患者对这种昂贵的治疗没有反应；而另一些患者则遭受着严重的副作用，如伤口愈合受损和中风风险增加。从肿瘤患者的血液中检测到从肿瘤脱落的循环肿瘤细胞(CTCs)和从骨髓(BM)动员的循环内皮祖细胞(CEP)，这一事实使这些问题变得更加复杂，因为目前还没有明确的标记来选择可能受益于抗血管内皮生长因子治疗的患者。这两类细胞都被证明与肿瘤进展和对包括抗血管生成治疗在内的一系列治疗耐药有关。然而，首次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研究揭示了有争议的结果。目前定义的CTCs/CEPs描述了具有遗传和表型异质性的复杂细胞群体。目前，尚不清楚哪些特性能使单个CTCs生长到固体转移。此外，在治疗耐药中起关键作用的CEP亚群尚未确定。因此，本项目的目标是(I)使用miRNA表达谱来研究CTCs/CEPs的异质性；(Ii)开发一组miRNAs，它将与CTCs/CEPs的单细胞分析相结合，以确定患者对贝伐单抗治疗的反应。利用原位杂交(ISH)和免疫细胞化学(ICC)相结合的方法，在单个CEPS/CTCs上分析与恶性肿瘤/肿瘤血管生成有关的miRNAs的表达。此外，接受Bevacizumab治疗的结直肠癌患者将在六个不同的治疗时间点进行CTCS/CEPs和基于细胞的miRNA表达的分析。与临床数据的相关性将导致识别在CTCs/EPC中表达的预测疾病进展的miRNAs。这些miRNAs及其功能将通过细胞转染实验进一步研究，我相信这些实验的结果将有助于开发新的策略来克服治疗耐药性。

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.