NETosis是一种特殊的中性粒细胞死亡方式，它在促进肿瘤发展方面起着重要作用，同时也抑制了癌症免疫治疗。因此，非侵入式实时成像对于癌症免疫治疗的预测至关重要，但仍具有挑战性。在此，我们报道了一种串联锁定的NETosis报告因子1(TNR1)，只有在中性粒细胞弹性蛋白酶(NE)和猫hep蛋白酶G(CTSG)同时存在时才会激活荧光信号，实现对NETosis的特异性成像。在分子设计方面，生物标记物特异性串联肽段序列可以大大影响对NETosis的检测特异性。在活细胞成像方面，串联锁定设计使得TNR1可以区分NETosis和中性粒细胞活化，而单锁定报告因子则无法实现。来自活体小鼠肿瘤的活性TNR1近红外信号与组织学结果中的肿瘤内NETosis水平一致。此外，活性TNR1近红外信号与免疫治疗后的肿瘤抑制效果呈负相关，从而为癌症免疫治疗提供了预测。因此，我们的研究不仅证明了第一个灵敏的光学报告因子，用于非侵入式监测NETosis水平和评估肿瘤内免疫治疗功效，同时还提出了一种通用的串联锁定探针设计方法。© 2023 Wiley-VCH GmbH。

NETosis, the peculiar type of neutrophil death, plays important roles in pro-tumorigenic functions and inhibits cancer im-munotherapy. Non-invasive real-time imaging is thus imperative for prognosis of cancer immunotherapy yet remains chal-lenging. Herein, we report a tandem-locked NETosis Reporter 1 (TNR1) that activates fluorescence signals only in the pres-ence of both neutrophil elastase (NE) and cathepsin G (CTSG) for specific imaging of NETosis. In the aspect of molecular design, the sequence of biomarker-specific tandem peptide blocks can largely affect the detection specificity towards NETosis. In live cell imaging, the tandem-locked design allows TNR1 to differentiate NETosis from neutrophil activation, while single-locked reporters fail to do so. The near-infrared signals from activated TNR1 in tumor from living mice were consistent with the intra-tumoral NETosis levels from histological results. Moreover, the near-infrared signals from activated TNR1 negatively correlated with tumor inhibition effect after immunotherapy, thereby providing prognosis for cancer immu-notherapy. Thus, our study not only demonstrates the first sensitive optical reporter for noninvasive monitoring of NETosis levels and evaluation of cancer immunotherapeutic efficacy in tumor-bearing living mice, but also proposes a generic approach for tandem-locked probe design.© 2023 Wiley-VCH GmbH.