Spatial Epigenomic Profiling of Immune Cell Signatures at Subcellular Resolution in Health and Disease

免疫细胞亚细胞信号的空间表型分析 健康与疾病的解决方案 超过10%的儿童癌症仍然无法治愈，需要新的治疗方法。表观遗传治疗 由于其特异性和降低的毒性，应得到特别关注。在这里，我计划探索表观遗传概况， 在Garry的指导下，正常发育和血癌患者的免疫细胞和癌细胞 Nolan负责单细胞蛋白质组学技术开发，与霍华德·张合作负责 实施表观基因组方法，如染色体可及性测定，并与卡拉戴维斯为 急性淋巴细胞白血病（ALL）中治疗抗性B细胞亚型的表观遗传学研究。后生 测量已限于批量水平测序和连接测定或有限数量的成像 标记。为了解决这些局限性，我将使用新兴的三维（3D）蛋白质组成像 在单个细胞中的3D多路复用离子束成像（MIBI）技术被称为3D MIBI。Epigenetics 3D MIBI的研究受益于高度多路复用（多达100个标记）和超分辨率成像 能力（20 nm x-y; 5 nm z分辨率），为研究基因组位点、甲基化 DNA、蛋白质因子和染色体可及性，都在单次免疫和 异常（白血病）细胞。为了系统地确定表观遗传状态，我计划利用克隆B细胞系， 通过一种新的方法来破译表观遗传组分的变异性，包括染色质状态，蛋白质因子和修饰剂， 50标记3D MIBI面板（Aim 1）。这些实验将显示表观遗传因子的分布（线性或非线性）。 对数标度）和染色质状态的空间变化（全局或局部）。然后我将 用分离自正常人的六种不同骨髓抽吸物的原代B细胞进行实验 目标2（Aim 2）。我将把每个B细胞亚型的表观遗传特征与相应的发育相关联 状态（前体、前体、后体或成熟）。然后，我将在OP 9上进行原代B细胞的离体共培养 培养1-6周的基质细胞，随后通过3D MIBI进行固定和分析。这些 干扰实验将显示来自邻近细胞的信号事件如何驱动必要的表观遗传 达到B细胞亚群所需的条件。最后，我将转向分离的原代B细胞， 来自20名新诊断的ALL患者（目标3）。我将剖析分化和空间表观基因组 来自骨髓抽吸物的应答B细胞亚群和治疗抗性B细胞亚型的重塑 使用OP 9共培养。这些将显示治疗抗性如何来自单一的表观遗传状态或 多种不同的表观遗传特征然后，我将筛选组蛋白去乙酰化酶抑制剂（HDACi）上相同的 来自ALL的B细胞亚型和基质细胞的共培养。通过改变抑制的浓度和持续时间 条件下，我将剖析表观遗传药物在空间染色质重塑对发展的作用， 表观遗传疗法在ALL中的应用总之，这些实验将揭示表观遗传编程的作用 用于从正常受试者和血癌患者中的免疫细胞签名的癌症治疗应用。

SPATIAL EPIGENOMIC PROFILING OF IMMUNE CELL SIGNATURES AT SUBCELLULAR RESOLUTION IN HEALTH AND DISEASE More than ten percent of childhood cancers are still incurable and need novel therapies. Epigenetic treatments deserve special attention with their specificity and reduced toxicity. Here I plan to explore epigenetic profiles of immune and cancer cells in normal development and blood cancer patients under the mentorship of Garry Nolan for single cell proteomics technology development, in collaboration with Howard Chang for implementation of epigenomic methods such as chromosome accessibility assays, and with Kara Davis for epigenetics studies of treatment resistant B cell subtypes in acute lymphoblastic leukemia (ALL). Epigenetic measurements have been limited to bulk level sequencing and ligation assays or limited number of imaging markers. To address these limitations, I will use an emerging three dimensional (3D) proteomic imaging technology in individual cells, termed as 3D Multiplexed ion beam imaging (MIBI) or 3D MIBI. Epigenetics research by 3D MIBI benefits from high degree multiplexing (up to 100 markers) and super resolution imaging capability (20 nm x-y; 5 nm z resolution), providing exciting opportunities to study genomic sites, methylated DNA, protein factors, and chromosome accessibility, all within the same experiments in single immune and aberrant (leukemic) cells. To systematically determine epigenetic states, I plan to utilize clonal B cell lines to decipher variability of epigenetic components including chromatin states, protein factors and modifiers by a fifty-marker 3D MIBI panel (Aim 1). These experiments will show distribution of epigenetic factors (linear or log-scale) in their expression levels and spatial variations (global or local) in the chromatin states. I will then perform experiments with primary B cells isolated from six different bone marrow aspirates of normal human subjects (Aim 2). I will correlate epigenetic signatures of each B cell subtype to corresponding development state (progenitor, pre, post, or mature). I will then perform an ex vivo co-culture of primary B cells on OP9 stromal cells over 1-6 weeks of culturing, which will be followed by fixation and profiling by 3D MIBI. These perturbation experiments will show how signaling events from neighboring cells drive necessary epigenetic conditions that are required for reaching a B cell subset. Finally, I will turn to primary B cells that are isolated from twenty newly diagnosed ALL patients (Aim 3). I will dissect differentiation and spatial epigenomic remodeling of responder B cell subsets and treatment resistant B cell subtypes from bone marrow aspirates using the OP9 co-culture. These will show how treatment resistance arises from a single epigenetic state or multiple distinct epigenetic signatures. I will then screen Histone deacetylase inhibitors (HDACi) on the same co-culture of B cell subtypes from ALL and stromal cells. By varying concentration and duration of inhibition conditions, I will dissect the role of epigenetic drugs in spatial chromatin remodeling toward development of epigenetic therapies in ALL. Together, these experiments will shed light on the role of epigenetic programming for cancer treatment applications from immune cell signatures in normal subjects and blood cancer patients.