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.

AML 中的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 (DSS)。我们设计了一种结合生理性缺氧（O ₂3%) 和间充质基质细胞 (MSC) 共培养与多参数流式细胞仪计数白血病细胞内的淋巴细胞和分化 (CD11/CD14/CD15+) 或富含白血病干细胞 (LSC) (GPR56+) 的细胞。在对作为 LSC 富集替代物的 GPR56 表达进行功能验证后，该测定确定了三种反应模式，包括对保留 LSC 的原始细胞的细胞毒性、诱导分化和 LSC 的选择性损伤。我们通过包括由原发性 AML 骨髓血浆样本的靶向代谢组学和蛋白质组学鉴定的血浆样氨基酸和细胞因子浓度来改进我们的生态位样培养物。系统性研究揭示了每种生态位样成分对白血病生长和药物反应的不同贡献。短期生态位样培养保留了原发性白血病细胞的克隆结构和转录状态。在一组 45 个 AML 样本中NPM1c AML，利基样多参数测定可以预测形态学（p  = 0.02）和分子（NPM1c MRD，p  = 0.04）对蒽环类-阿糖胞苷诱导化疗的反应。在这个队列中，一项 23 种药物筛选将鲁索替尼指定为蒽环类-阿糖胞苷的敏化剂。这一发现在NPM1c PDX 模型中得到验证。