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Current Cancer Drug Targets

Author(s): Xing Xu, Yongchang Yang, Yancheng Liu, Xianglian Ge, Tailong Yi, Yang Xie, Chunlan Ning, Shengfu Shen, Zengfeng Sun, Zhen Zhang, Qiongli Zhai, Xiaoguang Wang, Xianghui Meng, Jun Dong, Qiang Huang, Xuejun Yang, Wenliang Li* and Xun Jin*

DOI: 10.2174/1568009623666221027101637

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Combinatorial Application of Papain and CD66B for Isolating Glioma- Associated Neutrophils

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Abstract

Background: Stromal cells in the tumor microenvironment play crucial roles in glioma development. Current methods for isolating tumor-associated stromal cells (such as neutrophils) are inefficient due to the conflict between tissue dissociation and cell surface protein protection, which hampers the research on patient-derived stromal cells. Our study aims to establish a novel method for isolating glioma-associated neutrophils (GANs).

Methods: To observe neutrophil-like polymorphonuclear cells, we performed Hematoxylin-Eosin staining on glioma tissues. For isolating single cells from glioma tissues, we evaluated the efficiency of tissue dissociation with FastPrep Grinder-mediated homogenization or proteases (trypsin or papain) digestion. To definite specific markers of GANs, fluorescence-activated cell sorting (FACS) and immunofluorescence staining were performed. FACS and Ficoll were performed for the separation of neutrophils from glioma tissue-derived single-cell or whole blood pool. To identify the isolated neutrophils, FACS and RT-PCR were carried out.

Results: Neutrophil-like cells were abundant in high-grade glioma tissues. Among the three tissue dissociation methods, papain digestion produced a 5.1-fold and 1.7-fold more living cells from glioma mass than physical trituration and trypsin digestion, respectively, and it preserved over 97% of neutrophil surface protein markers. CD66B could be adopted as a unique neutrophil surface protein marker for FACS sorting in glioma. Glioma-derived CD66B+ cells specifically expressed neutrophil marker genes.

Conclusion: A combination of papain-mediated tissue dissociation and CD66B-mediated FACS sorting is an effective novel method for the isolation of GANs from glioma tissues.

Graphical Abstract

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