Functional precision medicine in AML often relies on short-term in vitro drug sensitivity screening (DSS) of primary patient cells in standard culture conditions. We designed a niche-like DSS assay combining physiologic hypoxia (O2 3%) and mesenchymal stromal cell (MSC) co-culture with multiparameter flow cytometry to enumerate lymphocytes and differentiating (CD11/CD14/CD15+) or leukemic stem cell (LSC)-enriched (GPR56+) cells within the leukemic bulk. After functional validation of GPR56 expression as a surrogate for LSC enrichment, the assay identified three patterns of response, including cytotoxicity on blasts sparing LSCs, induction of differentiation, and selective impairment of LSCs. We refined our niche-like culture by including plasma-like amino-acid and cytokine concentrations identified by targeted metabolomics and proteomics of primary AML bone marrow plasma samples. Systematic interrogation revealed distinct contributions of each niche-like component to leukemic outgrowth and drug response. Short-term niche-like culture preserved clonal architecture and transcriptional states of primary leukemic cells. In a cohort of 45 AML samples enriched for NPM1c AML, the niche-like multiparametric assay could predict morphologically (p = 0.02) and molecular (NPM1c MRD, p = 0.04) response to anthracycline-cytarabine induction chemotherapy. In this cohort, a 23-drug screen nominated ruxolitinib as a sensitizer to anthracycline-cytarabine. This finding was validated in an NPM1c PDX model.
在急性髓系白血病中，功能性精准医学通常依赖于在标准培养条件下对原代患者细胞进行短期体外药物敏感性筛选。我们设计了一种模拟微环境的药物敏感性筛选方法，结合生理性低氧（3%氧浓度）、间充质基质细胞共培养以及多参数流式细胞术，用于定量分析白血病细胞群中的淋巴细胞、分化细胞（CD11/CD14/CD15+阳性）及富含白血病干细胞（GPR56+阳性）的细胞群。在验证GPR56表达可作为白血病干细胞富集替代标志物后，该方法识别出三种药物反应模式：包括对原始细胞具有细胞毒性而保留白血病干细胞、诱导分化作用以及选择性抑制白血病干细胞。我们通过对原代急性髓系白血病骨髓血浆样本进行靶向代谢组学和蛋白质组学分析，确定了血浆样氨基酸和细胞因子浓度，进一步优化了模拟微环境培养体系。系统研究发现，每种微环境组分对白血病细胞扩增和药物反应均具有独特影响。短期模拟微环境培养能够保持原代白血病细胞的克隆结构和转录状态。在45例富含NPM1c突变急性髓系白血病样本的队列研究中，该模拟微环境多参数检测方法能够预测患者对蒽环-阿糖胞苷诱导化疗的形态学应答（p=0.02）和分子应答（NPM1c突变微小残留病，p=0.04）。在该队列中，针对23种药物的筛选实验确定鲁索替尼可作为蒽环-阿糖胞苷的增敏剂，这一发现在NPM1c突变人源肿瘤异种移植模型中得到验证。
A multiparametric niche-like drug screening platform in acute myeloid leukemia
肿瘤(癌症)患者之家