Defining the impact of mutational drivers on the immune microenvironment of CLL

项目摘要 肿瘤细胞与其微环境的动态相互作用在刺激癌症方面起着至关重要的作用 进展，并可能有助于克隆进化和治疗耐药的速度。新出现的证据 提示癌细胞的遗传特征可能会影响体内免疫细胞的组成和功能 肿瘤微环境(TME)，但这方面的证据仍然很少。最近，我们创作了几部小说 B细胞限制性驱动的慢性淋巴细胞白血病(CLL)条件性基因敲除小鼠模型 人类CLL损伤的表达，为模拟人类巨大的遗传异质性提供了机会 处于免疫能力强的微环境中的癌症。目前的项目寻求使用单细胞RNA 测序(scRNA-seq)和功能分析以确定这些细胞中CLL-微环境的相互作用 CLL小鼠模型，并在人类CLL中验证这一发现。特别是，我们将：(目标1)发现影响 在CLL免疫版图上的CLL驱动程序事件。通过scRNA-seq分析，我们将表征 CLL微环境中的免疫景观，同时检查脾和骨髓(BM) 年龄匹配的对照组(CD-19 Cre转基因小鼠)和4种不同基因定义的CLL的间隔 小鼠模型(CD19-CRE×MDR，MUT-SF3B和ATM，MUT-IKZF3和DNMT3A-缺失)。在我们引人入胜的基础上 在DNMT3A缺失模型中进行的试点scRNA-seq研究，我们现在建议交叉比较 每种模型中免疫细胞的组成和转录状态，并确定是否存在 CLL和免疫群体之间一致或独特的依赖于模型的交互作用。(目标2)验证 CLL-TME相互作用的候选介体。我们预计，我们将确定微环境的变化 感兴趣的或共同或独特的模型，我们将进行流式细胞术和 对独立的小鼠标本进行免疫荧光(IF)/免疫组织化学(IHC)染色以进行验证。 我们还将进行体外共培养实验和体内移植，以研究功能相互作用。 CLL细胞和特定免疫细胞群之间的关系。事实上，我们来自DNMT3A的试验数据- 删除CLL小鼠的脾已经发现了一个扩大的失调树突状细胞亚群，并且 我们将评估是否在其他模型中也观察到了这种变化，以及这种变化的程度 调节CLL的发育。(目的3)证实CLL驱动事件在人类CLL的TME中的作用。我们 将鼠标scRNA-seq数据与已生成的人类CLL scRNA-seq数据进行交叉比较 在吴的实验室。我们将对CLL患者的LN和BM样本进行IF/IHC染色，以进一步验证 有希望的签名。总之，拟议的分析将扩大我们对癌细胞如何内在地 功能可以重塑肿瘤免疫微环境，并有可能有助于合理 个性化癌症免疫疗法的设计。

Project Summary The dynamic interactions of tumor cells with their microenvironment play an essential role in stimulating cancer progression, and likely contribute to the pace of clonal evolution and therapeutic resistance. Emerging evidence suggests that genetic features of cancer cells may impact composition and function of immune cells within the tumor microenvironment (TME), yet evidence for this remains scant. Recently, we have generated several novel conditional knock-in/out mouse models of chronic lymphocytic leukemia (CLL) driven by B-cell restricted expression of human CLL lesions, providing opportunities to model the vast genetic heterogeneity of human cancers within an immune-competent microenvironment. This current projects seeks to use single cell RNA sequencing (scRNA-seq) and functional assays to determine the CLL-microenvironment interactions in these CLL mouse models, and validate the findings in human CLLs. In particular, we will: (Aim 1) Discover the impact of CLL driver events on the CLL immune landscape. Through scRNA-seq analyses, we will characterize the immune landscape within the CLL microenvironment, examining both the spleen and bone marrow (BM) compartments in age-matched controls (Cd-19 Cre transgenic mice) and 4 different genetically-defined CLL mouse models (Cd19-Cre × MDR, mut-Sf3b&Atm, mut-Ikzf3 and Dnmt3a-deleted). Building on our intriguing pilot scRNA-seq study conducted in the Dnmt3a-deleted model, we now propose to cross compare the composition and transcriptional states of immune cells in each model, and to determine whether there are consistent or unique model-dependent interactions between CLL and immune populations. (Aim 2) Validate candidate mediators of CLL-TME interactions. We anticipate that we will identify microenvironment changes of interest either found in common or unique amongst the models, and we will perform flow cytometry and immunofluorescence (IF)/immunohistochemical (IHC) staining on independent mouse specimens for validation. We will also perform in vitro co-culture experiments and in vivo transplantation to study the functional interaction between CLL cells and particular immune cell populations of interest. Indeed, our pilot data from the Dnmt3a- deleted CLL mouse spleen has already identified an expanded subpopulation of dysregulated dendritic cells, and we will evaluate if this change is also observed across the other models and the extent to which this change regulates CLL development. (Aim 3) Confirm the effects of CLL driver events in the TMEs of human CLL. We will cross compare the mouse scRNA-seq data with that of an existing human CLL scRNA-seq data generated in the Wu lab. We will perform IF/IHC staining of LN and BM samples from CLL patients to further validate promising signatures. Altogether, the proposed analyses will expand our knowledge on how cancer cell-intrinsic features can remodel the tumor immune microenvironment, and has the potential to contribute to the rational design of personalized cancer immunotherapy.