Cytoplasm protein GFAP magnetic beads construction and application as cell separation target for brain tumors

Yang Zhao,Kai Wang,Jiajia Wang,Junping Ao,Yipeng Han,Qinhua Wang,Xiaofei Liang,Jie Ma,Jian Yang,Xunxiang Guo,Feng Jiang,Baocheng Wang

doi:10.1186/s12951-020-00729-9

Abstract

BackgroundIt is very important to develop a highly efficient cerebrospinal fluid (CSF) detection system with diagnosis and prediction function, for which the detection of circulating tumor cells (CTCs) in CSF is a good choice. In contrast to the past use of epithelial EpCAM as CTCs separation target, a cytoplasm protein of GFAP antibody was first selected to construct highly-sensitive immunomagnetic liposome beads (IMLs). The validation and efficiency of this system in capturing CTCs for brain tumors were measured both in vitro and in vivo. The associations between the numbers of CTCs in patients with their clinical characteristics were further analyzed.ResultsOur data show that CTCs can be successfully isolated from CSF and blood samples from 32 children with brain tumors. The numbers of CTCs in CSF were significantly higher than those in blood. The level of CTCs in CSF was related to the type and location of the tumor rather than its stage. The higher the CTCs number is, the more possibly the patient will suffer from poor prognosis. Genetic testing in GFAP CTC-DNA by sanger sequencing, q-PCR and NGS methods indicated that the isolated CTCs (GFAP+/EGFR+) are the related tumor cell. For example, the high expression of NPR3 gene in CSF CTCs was consistent with that of tumor tissue.ConclusionsThe results indicated that GFAP-IML CTCs isolation system, combined with an EGFR immunofluorescence assay of antitumor marker, can serve as a brand-new method for the identification of CTCs for brain tumors. Via lumbar puncture, a minimally invasive procedure, this technique may play a significant role in the clinical diagnosis and drug evaluation of brain tumors.

Highlights

It is very important to develop a highly efficient cerebrospinal fluid (CSF) detection system with diagnosis and prediction function, for which the detection of circulating tumor cells (CTCs) in CSF is a good choice
Can cytoplasmic proteins be selected as an effective target for CTCs separation, especially in tumors with unreliable surface markers? In this study, for the first time, we introduced the endocytic GFAP antibody of neuroepithelial tumors (NT) [20, 21] as a positive CTCs sorting marker to verify its feasibility in the diagnosis of brain tumors in children
GFAP-immunomagnetic liposome beads (IMLs) were prepared by reverse emulsification method by using GFAP-Glycidyl hexadecyl dimethylammonium chloride (GHDC), DOPC and cholesterol. Fe3O4 nanoparticles were wrapped in liposomes, and the long chain alkyl part of amphiphilic GFAP-GHDC was inserted into the lipid bilayer membrane, and most of the GFAP antibodies were distributed on the surface of the liposomes

Summary

Introduction

It is very important to develop a highly efficient cerebrospinal fluid (CSF) detection system with diagnosis and prediction function, for which the detection of circulating tumor cells (CTCs) in CSF is a good choice. The validation and efficiency of this system in capturing CTCs for brain tumors were measured both in vitro and in vivo. The associations between the numbers of CTCs in patients with their clinical characteristics were further analyzed. The clinical assessment of brain tumors mainly relies. Zhao et al J Nanobiotechnol (2020) 18:169 on radiological examinations, such as CT and MRI [5,6,7] Factors such as radiation necrosis, hemorrhage, and inflammation may vastly affect radiologists, preventing a correct judgment [8]. The lack of circulating serum markers limits valuable methods for early clinical assessment and reduces the available options for monitoring disease progression

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