Natural products constitute and significantly impact many current anti-cancer medical interventions. A subset of natural products induces injury processes in malignant cells that recruit and activate host immune cells to produce an adaptive anti-cancer immune response, a process known as immunogenic cell death. However, a challenge in the field is to delineate forms of cell death and injury that best promote durable antitumor immunity. Addressing this with a single-cell chemical biology natural product discovery platform, like multiplex activity metabolomics, would be especially valuable in human leukemia, where cancer cells are heterogeneous and may react differently to the same compounds. Herein, a new ten-color, fluorescent cell barcoding-compatible module measuring six immunogenic cell injury signaling readouts are as follows: DNA damage response (γH2AX), apoptosis (cCAS3), necroptosis (p-MLKL), mitosis (p-Histone H3), autophagy (LC3), and the unfolded protein response (p-EIF2α). A proof-of-concept screen was performed to validate functional changes in single cells induced by secondary metabolites with known mechanisms within bacterial extracts. This assay was then applied in multiplexed activity metabolomics to reveal an unexpected mammalian cell injury profile induced by the natural product narbomycin. Finally, the functional consequences of injury pathways on immunogenicity were compared with three canonical assays for immunogenic hallmarks, ATP, HMGB1, and calreticulin, to correlate secondary metabolite-induced cell injury profiles with canonical markers of immunogenic cell death. In total, this work demonstrated a new phenotypic screen for discovery of natural products that modulate injury response pathways that can contribute to cancer immunogenicity.

天然产物构成并显着影响许多当前的抗癌医学干预措施。一部分天然产物在恶性细胞中诱导损伤过程，这些细胞募集和激活宿主免疫细胞以产生适应性抗癌免疫反应，这一过程称为免疫原性细胞死亡。然而，该领域的一个挑战是描述最能促进持久抗肿瘤免疫的细胞死亡和损伤形式。用单细胞化学生物学天然产物发现平台解决这个问题，如多重活性代谢组学，在人类白血病中特别有价值，因为癌细胞是异质的，可能对相同的化合物有不同的反应。在此，一种新的十色荧光细胞条形码兼容模块测量六个免疫原性细胞损伤信号读数如下：DNA 损伤反应 (γH2AX)、细胞凋亡 (cCAS3)、坏死性凋亡 (p-MLKL)、有丝分裂 (p-组蛋白 H3)、自噬 (LC3) 和未折叠蛋白反应 (p-EIF2α)。进行了概念验证筛选，以验证细菌提取物中具有已知机制的次级代谢物诱导的单个细胞的功能变化。然后将该测定应用于多重活性代谢组学，以揭示由天然产物纳博霉素诱导的意想不到的哺乳动物细胞损伤谱。最后，将损伤途径对免疫原性的功能后果与免疫原性标志的三种典型测定法 ATP、HMGB1 和钙网蛋白进行比较，以将次级代谢物诱导的细胞损伤谱与免疫原性细胞死亡的典型标志物相关联。总共，