American Association for Cancer Research
cir-22-0595_supplementary_data_suppsf1-sf7_st1.pdf (640.26 kB)

Supplementary Data from Tumor Cell–Derived Microparticles Induced by Methotrexate Augment T-cell Antitumor Responses by Downregulating Expression of PD-1 in Neutrophils

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journal contribution
posted on 2023-04-03, 08:40 authored by Pingwei Xu, Xiaojie Zhang, Kai Chen, Meng Zhu, Ru Jia, Qingwei Zhou, Jintao Yang, Juqin Dai, Yuepeng Jin, Keqing Shi

supplementary figures 1-7 and table S1


National Natural Science Foundation of China (NSFC)

Medical Health Science and Technology Project of Zhejiang Provincial Health Commission

the Provinces and Ministries Co-Contribution of Zhejiang

Key Project of Wenzhou Science and Technolog Bureau



Neutrophils act as a “double-edged sword” in the tumor microenvironment by either supporting or suppressing tumor progression. Thus, eliciting a neutrophil antitumor response remains challenging. Here, we showed that tumor cell–derived microparticles induced by methotrexate (MTX-MP) acts as an immunotherapeutic agent to activate neutrophils, increasing the tumor-killing effect of the cells and augmenting T-cell antitumor responses. We found that lactate induced tumor-associated neutrophils to elevate expression of programmed cell death protein 1 (PD-1) and that PD-1+ neutrophils had the properties of N2 neutrophils and suppressed T-cell activation through PD-1/programmed death-ligand 1 (PD-L1) signaling. By performing ex vivo experiments, we found that MTX-MPs–activated neutrophils had reduced surface expression of PD-1 as a result of PD-1 internalization and degradation in the lysosomes, leading to the cells showing a decreased capacity to suppress T-cell responses. In addition, we also found that MTX-MP–activated neutrophils released neutrophil elastase which could kill tumor cells and disrupt tumor stroma, leading to increased T-cell infiltration. Furthermore, using a combination of anti–PD-L1 and MTX-MPs, we observed that long-term survival increased in a mouse model of lung cancer. Collectively, these findings highlight the potential use of a combination of anti–PD-L1 and MTX-MPs to enhance the therapeutic effect of anti–PD-L1 alone.

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