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

Author:

Li Wenliang12,Jin Xun13,Xu Xing14,Yang Yongchang14,Liu Yancheng14,Ge Xianglian13,Yi Tailong13,Xie Yang14,Ning Chunlan14,Shen Shengfu14,Sun Zengfeng12,Zhang Zhen12,Zhai Qiongli15,Wang Xiaoguang12,Meng Xianghui6,Dong Jun7,Huang Qiang89,Yang Xuejun810

Affiliation:

1. Key Laboratory of Cancer Prevention and Therapy of Tianjin, Tianjin's Clinical Research Center for Cancer, Tianjin, China

2. Department of Neuro-Oncology and Neurosurgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China

3. Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China

4. Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China

5. Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China

6. Department of Neurosurgery, General Hospital of People's Liberation Army, Beijing, China

7. Department of Neurosurgery, The Second Affiliated Hospital of Soochow University, Suzhou, China

8. Department of Neurosurgery, Tianjin Medical University General Hospital, Tianjin, China

9. Laboratory of Neurooncology, Tianjin Neurological Institute, Tianjin, China

10. Laboratory of Neurooncology, Tianjin Neurological Institute, Tianjin, China

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). Method: 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. Result: 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.

Funder

National Natural Science Foundation of China

Science and Technology Support Plan Key Projects of Tianjin

Scientific Research Foundation of Tianjin Medical University Cancer Institute and Hospital

Natural Science Foundation of Tianjin

Publisher

Bentham Science Publishers Ltd.

Subject

Cancer Research,Drug Discovery,Pharmacology,Oncology

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