In vivo RNAi screen to identify new inhibitory targets in tumour-specific T lymphocytes during liver cancer development

我们最近发现，诱导th1极化的适应性免疫对于抑制表达nrasg12v的肝脏肿瘤至关重要（Kang & Yevsa et al, Nature, 2011）。我们的数据以及从肝脏和其他恶性肿瘤患者获得的数据表明，T细胞在调节肿瘤进展中起着核心作用。然而，肝脏肿瘤微环境持续抑制T细胞免疫应答。我们假设，T细胞中的大多数抑制因子（基因）仍然是未知的，为了鉴定主要的T细胞抑制基因，我们建议使用主题集中的shRNA文库进行体内RNAi筛选，针对肝癌发展过程中记忆T细胞中上调的基因列表。我们将使用最近在我们的研究中建立的基于mir30的RNAi-和转座子介导的癌基因传递平台（Kang & Yevsa等人，Nature, 2011; w<e:1> stefeld等人，Cell, 2013）。重要的是，在我们将要使用的肝癌模型中，肝脏肿瘤的发展与人类肿瘤相似，并且不是基于细胞移植。据我们所知，到目前为止，还没有人在患有肝脏恶性肿瘤的小鼠的免疫系统中进行过类似的RNAi筛选。提出的项目对癌症患者的T细胞免疫治疗的临床前和临床策略有很大的希望。单独或联合其他（免疫-）治疗阻断T细胞中的抑制分子将显著抑制肿瘤生长并增加肝恶性肿瘤患者的总生存期。

We have recently found that induction of Th1-polarized adaptive immunity was crucial for suppression of NrasG12V-expressing liver tumours (Kang & Yevsa et al, Nature, 2011). Our data and data obtained from patients with liver- and other malignancies imply a central role of T cells in modulating tumour progression. However, liver tumor microenvironment continuously inhibits T cell immune responses. We hypothesize, that the majority of inhibitory factors (genes) in T cells remains unknown and to identify the main T cell inhibitory genes, we propose to perform an in vivo RNAi screen using a thematically focused shRNA library targeting a list of genes up-regulated in memory T cells during liver cancer development. We will use a combination of mir30-based RNAi- and transposon mediated oncogene delivery platforms, recently established in our studies (Kang & Yevsa, et al, Nature, 2011; Wüstefeld et al, Cell, 2013). Importantly, in the liver cancer model that we are going to use, liver tumours develop similarly to human tumours and are not based on cell transplantation. To our knowledge until now nobody has performed a similar RNAi screen in immune system in mice bearing liver malignancies. The proposed project has a great promise for later preclinical and clinical strategies for T cell-immunotherapy in cancer patients. Blocking of inhibitory molecules in T cells either alone or in combination with additional (immuno-) therapy will significantly inhibit tumour growth and increase the overall survival of patients with hepatic malignancies.