Molecular characterisation of RNA-seq identified changes in TCR signalling in prostate cancer disease

背景：越来越多的证据表明，CD4+和CD8+ T细胞在肿瘤免疫监视和抗肿瘤免疫中起着关键作用，操纵这些免疫细胞识别和根除肿瘤细胞是治疗侵袭性和转移性癌症患者的一种很有前景的策略。恶性肿瘤产生的肿瘤抑制微环境是有效抗肿瘤免疫和肿瘤免疫治疗成功的主要障碍（1,2）。肿瘤细胞可以利用不同的策略来扩大和招募各种类型的抑制性肿瘤浸润淋巴细胞（til），包括自然发生和适应性诱导的调节性T细胞（Treg）、耐受性树突状细胞（DCs）、肿瘤源性巨噬细胞和骨髓抑制细胞（MSCs）。此外，肿瘤细胞可以分泌抑制因子（IL-10、TGF-b和IDO），表达免疫抑制分子（FasL和PD-L1），直接抑制肿瘤特异性t细胞的扩增和增殖，或诱导t细胞凋亡(1)。更好地了解肿瘤诱导的免疫抑制微环境的产生和维持的分子机制对于开发新的肿瘤疫苗和治疗人类癌症的策略至关重要。我们最近描述了PDE4D7作为一种新的推定肿瘤抑制基因的鉴定，它与侵袭性疾病和不良患者预后显著相关(3)。基于对来自切除患者材料的bbbb500人前列腺癌肿瘤组织的RNAseq数据进行的相关性研究，我们确定了一系列参与调节t细胞激活的基因，并在具有侵袭性疾病特征的前列腺肿瘤中受到干扰。我们假设我们在RNAseq数据中观察到的基因表达变化源于肿瘤浸润淋巴细胞，更具体地说是肿瘤浸润t细胞。这些t细胞的激活电位可能会因这些基因转录的改变而受损。目的：本项目的目的是了解RNAseq鉴定的感兴趣基因在肿瘤浸润淋巴细胞中是否以及在多大程度上失调，是否可以确定受影响淋巴细胞的类型（如CD4+， CD8+），是否可以测量对下游转录因子激活的任何影响。为此，我们将开发检测方法，利用RNAscope和/或免疫组织化学在病理组织切片上测量已鉴定的感兴趣基因。qPCR检测将用于确定从前列腺癌患者组织和血液样本中分离的肿瘤浸润t细胞如何影响这些基因的表达。患者从疾病的不同阶段（原发性，晚期，转移性，去势抵抗）和经过各种治疗（放射，激素，化疗）选择。功能性增殖试验将用于从人类供体纯化的t细胞与不同的前列腺癌细胞系在体外共培养，以确定纯化的t细胞对肿瘤的抑制水平，这取决于它们对感兴趣的基因的激活状态。这将使我们能够将感兴趣的基因的表达状态与免疫细胞诱导的体外前列腺癌细胞生长抑制水平联系起来。参考文献：1)Croci DO等人（2007）。肿瘤和免疫细胞之间的动态串扰在肿瘤微环境中协调免疫抑制网络。中国生物医学工程学报（英文版）（2008）。肿瘤微环境及其对肿瘤生长的促进作用。中国生物医学工程学报（英文版），2016,(5):591 -591。通过翻译有关cAMP降解磷酸二酯酶4d7 （PDE4D7）的新科学发现，创建前列腺癌风险分层的潜在诊断（InformMDxTM）。中国临床医学杂志，32 (2):389 - 389

Keywords: Prostate cancer, immune cells, TCARTBackground: Increasing evidence suggests that both CD4+ and CD8+ T cells play a critical role in cancer immunosurveillance & anti-tumor immunity, and manipulation of these immune cells to recognize and eradicate tumor cells is a promising strategy for treating patients with invasive & metastatic cancers. It has become clear that the tumor suppressive microenvironments created by malignant tumors are a major obstacle for effective anti-tumor immunity and successful tumor immunotherapy (1,2).Tumor cells can utilize different strategies to expand & recruit various types of suppressive tumor-infiltrating lymphocytes (TILs), including naturally occurring & adaptively induced regulatory T (Treg) cells, tolerogenic dendritic cells (DCs), tumor-derived macrophages and myeloid suppressor cells (MSCs). In addition, tumor cells can secrete suppressive factors (IL-10, TGF-b and IDO), express immune inhibitory molecules (FasL & PD-L1), directly inhibit tumor-specific T-cell expansion and proliferation, or induce T-cell apoptosis (1). A better understanding of the molecular mechanisms involved in creating and sustaining the tumor-induced immune suppressive microenvironment is critical for the development of novel tumor vaccines & therapeutic strategies active against human cancers.We have recently described the identification of PDE4D7 as novel putative tumor suppressor gene which is significantly associated with aggressive disease and poor patient outcome (3). Based on correlation studies performed on RNAseq data of >500 human prostate cancer tumor tissues derived from resected patient material we identified a range of genes that are involved in the regulation of T-cell activation & are disturbed in prostate tumors with aggressive disease characteristics. We hypothesize that the gene expression changes that we observe in the RNAseq data is originating from tumor infiltrating lymphocytes, more specifically tumor infiltrating T-cells. The activation potential of these T-cells might be impaired by the change in the transcription of these genes. Aims: The goal of this project is to understand whether and to what extent the RNAseq identified genes of interest are dis-regulated in tumor infiltrating lymphocytes, whether we can determine the type of affected lymphocyte (e.g., CD4+, CD8+) and whether we can measure any effects on the activation of downstream transcription factors. For this we will develop assays to measure the identified genes of interest on pathology tissue sections with RNAscope, and/or immuno-histochemistry. qPCR assays will be used to determine how the expression of these genes is affected in tumor-infiltrating T-cells isolated from patient tissue and blood samples from patients with prostate cancer. Patients are selected from different stages of the disease (primary, advanced, metastatic, castration-resistant) & after various sorts of therapies (radiation, hormones, chemotherapy). Functional proliferation assays will be used to co-culture purified T-cells from human donors with different prostate cancer cell lines in vitro to determine the level of tumor suppression by purified T-cells depending on their activation status of the genes of interest. This will allow us to correlate the expression status of the genes of interest with the level of immune cell induced suppression of growth prostate cancer cells in vitro.References:1) Croci DO et al (2007).Dynamic cross-talk between tumor and immune cells in orchestrating the immunosuppressive network at the tumor microenvironment. Cancer Immunol Immunother 56: 1687-17002) Whiteside TL(2008).The tumor microenvironment and its role in promoting tumor growth. Oncogene 27: 5904-59123) Henderson et al (2019).Creating a potential diagnostic for prostate cancer risk stratification (InformMDxTM by translating novel scientific discoveries concerning cAMP degrading phosphodiesterase-4D7 (PDE4D7). Clinical Science 133(2): 269-28