Spatial-Temporal Analysis Revealed the Dynamic Changes of Leukemia Neighborhood After Receiving Pembrolizumab and Decitabine

Acute myeloid leukemia (AML) is a genetically heterogeneous disease that is characterized by abnormal clonal proliferation of myeloid progenitor cells found predominately within the bone marrow (BM) and blood. Recent studies suggest that genetic and phenotypic alterations in the BM microenvironment support leukemogenesis and allow leukemic cells to survive and evade chemotherapy-induced death. However, despite substantial evidence indicating the role of tumor-host interactions in AML pathogenesis, little is known about the complex microenvironment of the BM. To address this, we performed novel proteomic profiling of the non-cellular compartment of the BM microenvironment in AML patients (n=10) and age- and sex-matched healthy controls (n=10) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). We demonstrate that proteomic assessment of blood or RNA-sequencing of BM are suboptimal alternate screening strategies to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. Proteomic analysis showed 168 proteins significantly differed in abundance, with 91 proteins up-regulated and 77 proteins down-regulated in leukemic BM. A highly connected signaling network of cytokines and chemokines, including IL-8, was found to be the most prominent proteomic signature associated with AML in the BM microenvironment. We report the first description of significantly elevated levels of the myelosuppressive chemokine CCL23 (MPIF–1) in both AML and MDS patients and perform functional experiments supportive of a role in the suppression of normal hematopoiesis. This unique paired RNA-sequencing and proteomics dataset provides innovative mechanistic insights into AML and healthy aging and should serve as a useful public resource. For bulk RNAseq: Total RNA sequencing of rRNA-depleted RNA was performed using the Illumina TruSeq Stranded Total RNA kit. Sequencing was performed on RNA isolated from bone marrow PAXgene tubes of 10 acute myeloid leukemia patients at multiple follow-up time points. For single-cell: Bone marrow aspirate was collected from enrolled patients at different time points. The 10x Genomics 3’v3 Single Cell Immune profiling platform was used for scRNA-seq of BMMCs. cDNA amplification was performed according to manufacturer’s instructions with the addition of 1 ul 0.2 uM ADT additive primer per sample to amply ADT tags.

急性髓系白血病（Acute myeloid leukemia, AML）是一种遗传异质性疾病，以主要分布于骨髓（bone marrow, BM）与血液中的髓系祖细胞发生异常克隆性增殖为核心特征。
近期研究表明，骨髓微环境中的遗传与表型改变可促进白血病发生，并使白血病细胞得以存活且逃避化疗诱导的细胞死亡。
然而，尽管已有大量证据证实肿瘤-宿主互作在AML发病机制中的关键作用，但学界对骨髓复杂的微环境仍知之甚少。
为解决这一研究空白，本研究采用基于适配体的高通量亲和蛋白质组学平台（SOMAscan），对10名AML患者与10名年龄、性别匹配的健康对照者的骨髓微环境非细胞组分开展了新型蛋白质组学分析。
本研究证实，通过血液蛋白质组检测或骨髓RNA测序来评估AML骨髓微环境细胞外组分的真实蛋白质组组成，均为次优的替代筛查策略。
蛋白质组分析结果显示，白血病骨髓中有168种蛋白质的丰度存在显著差异，其中91种蛋白表达上调，77种蛋白表达下调。
包含IL-8在内的细胞因子与趋化因子构成的高度连接信号网络，被确定为骨髓微环境中与AML相关的最显著蛋白质组特征。
本研究首次报道了髓系抑制性趋化因子CCL23（MPIF–1）在AML与骨髓增生异常综合征（myelodysplastic syndrome, MDS）患者体内均显著升高，并通过功能实验证实其可抑制正常造血功能。
这套独特的配对RNA测序与蛋白质组学数据集为AML及健康衰老的机制研究提供了创新性见解，可作为一项极具价值的公共研究资源。
关于批量RNA测序（bulk RNA-seq）：本研究采用Illumina TruSeq Stranded Total RNA试剂盒，对经核糖体RNA去除的总RNA进行全转录组测序。测序样本取自10名急性髓系白血病患者多个随访时间点的骨髓PAXgene管分离得到的RNA。
关于单细胞测序：研究人员于不同时间点从入组患者中采集骨髓穿刺液。采用10x Genomics 3’v3 单细胞免疫分析平台对骨髓单个核细胞（bone marrow mononuclear cells, BMMCs）进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。依照制造商说明书进行cDNA扩增，并向每份样本中添加1 μl 0.2 μM ADT添加引物，以扩增ADT标签。