Construction and Clinical Verification of Cytoplasm Protein GFAP as Circulating Tumor Cell Separation target for Pediatric Neuroepithelial Tumors

Abstract BACKGROUND: Pediatric Neuroepithelial Tumors (NT) are one of the most prevalent diseases among children. Developing a highly efficient cerebrospinal fluid (CSF) detection system with diagnosis and prediction function is very important. Circulating tumor cell (CTC) in CSF is a good choice. In contrast to the past use of epithelial EpCAM as CTC separation target, an cytoplasm protein of GFAP antibody was first selected to construct highly-sensitive immunomagnetic liposomes (IMLs). The validation and efficiency of this system in capturing CTCs for NT were measured both in vitro and in vivo. The associations between the numbers of CTCs in patients with their clinical characteristics were further analyzed. RESULTS: Our data show that CTCs can be successfully isolated from CSF and blood samples from 29 children with NT. 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. 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 CTC was consistant with tumor tissue. CONCLUSIONS: GFAP-IML isolation of CTCs, 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 can be clinically significant in diagnosis and efficacy assessments of pediatric NT.

Download Full-text

Cytoplasm Protein GFAP Magnetic Beads Construction and Application as Cell Separation target for Brain Tumors

10.21203/rs.3.rs-49607/v2 ◽

2020 ◽

Author(s):

Yang Zhao ◽

Feng Jiang ◽

Qinhua Wang ◽

Baocheng Wang ◽

Yipeng Han ◽

...

Keyword(s):

Brain Tumors ◽

Magnetic Beads ◽

Detection System ◽

Drug Evaluation ◽

Invasive Procedure ◽

Good Choice ◽

Isolation System ◽

Prediction Function

Abstract BACKGROUND: 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. 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 liposomes (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. RESULTS: Our 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. CONCLUSIONS: The 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.

Download Full-text

BIOL-04. CYTOPLASM PROTEIN GFAP MAGNETIC BEADS CONSTRUCTION AND APPLICATION AS CELL SEPARATION TARGET FOR BRAIN TUMORS

Neuro-Oncology ◽

10.1093/neuonc/noab090.011 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i3-i3

Author(s):

Yang Zhao ◽

Jie Ma

Keyword(s):

Brain Tumors ◽

Magnetic Beads ◽

Detection System ◽

Drug Evaluation ◽

Invasive Procedure ◽

Good Choice ◽

Isolation System ◽

Prediction Function

Abstract Background 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. 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. Results Our 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. Conclusions The 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.

Download Full-text

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

Journal of Nanobiotechnology ◽

10.1186/s12951-020-00729-9 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Yang Zhao ◽

Feng Jiang ◽

Qinhua Wang ◽

Baocheng Wang ◽

Yipeng Han ◽

...

Keyword(s):

Brain Tumors ◽

Magnetic Beads ◽

Detection System ◽

Drug Evaluation ◽

Invasive Procedure ◽

Good Choice ◽

Isolation System ◽

Prediction Function

Download Full-text

Cytoplasm Protein GFAP Magnetic Beads Construction and Application as Cell Separation Target for Brain Tumors

10.21203/rs.3.rs-49607/v1 ◽

2020 ◽

Author(s):

Yang Zhao ◽

Feng Jiang ◽

Qinhua Wang ◽

Baocheng Wang ◽

Yipeng Han ◽

...

Keyword(s):

Brain Tumors ◽

Magnetic Beads ◽

Detection System ◽

Drug Evaluation ◽

Invasive Procedure ◽

Good Choice ◽

Isolation System ◽

Prediction Function

Abstract BACKGROUND: 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. 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. RESULTS: Our 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.CONCLUSIONS: The 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.

Download Full-text

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

Nuklearmedizin ◽

10.1055/s-0038-1624353 ◽

1987 ◽

Vol 26 (01) ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

S. Selvaraj ◽

M. R. Suresh ◽

G. McLean ◽

D. Willans ◽

C. Turner ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Tumor Cell ◽

T Antigen ◽

Human Carcinomas ◽

Animal Tumor ◽

Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

Download Full-text

The peptide mimicking small extracellular ring domain of CD82 inhibits tumor cell migration in vitro and metastasis in vivo

Journal of Cancer Research and Clinical Oncology ◽

10.1007/s00432-021-03595-6 ◽

2021 ◽

Author(s):

Xin He ◽

Xiaoguang Ma ◽

Congcong Wang ◽

Mingchun Luan ◽

Ying Li ◽

...

Keyword(s):

Cell Migration ◽

Tumor Cell ◽

Ring Domain ◽

Tumor Cell Migration

Download Full-text

TMOD-24. GENERATION AND CHARACTERIZATION OF A NOVEL TRANSGENIC IDO REPORTER MOUSE FOR IDO POSTTRANSLATIONAL MODIFICATION ANALYSIS IN SITU

Neuro-Oncology ◽

10.1093/neuonc/noaa215.974 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii233-ii233

Author(s):

April Bell ◽

Lijie Zhai ◽

Erik Ladomersky ◽

Kristen Lauing ◽

Lakshmi Bollu ◽

...

Keyword(s):

Mouse Model ◽

Tumor Cell ◽

Central Nervous System Tumor ◽

Post Translational Modifications ◽

Reporter Mouse ◽

C Terminus ◽

Human Gbm

Abstract Glioblastoma (GBM) is the most common and aggressive primary central nervous system tumor in adults with a median survival of 14.6 months. GBM is a potently immunosuppressive cancer due in-part to the prolific expression of immunosuppressive indoleamine 2,3 dioxygenase 1 (IDO). Tumor cell IDO facilitates the intratumoral accumulation of regulatory T cells (Tregs; CD4+CD25+FoxP3+). Although immunosuppressive IDO activity is canonically characterized by the conversion of tryptophan into kynurenine, we have utilized transgenic and syngeneic mouse models and mutant glioma lines to demonstrate that tumor cell IDO increases Treg accumulation independent of tryptophan metabolism. Here, we address the gap in our understanding of IDO signaling activity in vivo. Subcutaneously-engrafted human GBM expressing human IDO-GFP cDNA was isolated from immunodeficient humanized NSG-SGM3 mice. The tumor was immunoprecipitated for the GFP tag using GFP-TRAP followed by mass spectrometry which revealed a novel methylation site on a lysine residue at amino acid 373 in the IDO C-terminus region. Western blot analysis of IDO protein also revealed the presence of tyrosine phosphorylation. Additionally, we recently created a new transgenic IDO reporter mouse model whereby endogenous IDO is fused to GFP via a T2A linker (IDO→GFP). This model allows for the isolation of IDO+ cells in real-time and without causing cell death, thereby creating the opportunity for downstream molecular analysis of in situ-isolated GFP+ cells. Collectively, our work suggests that IDO non-enzyme activity may involve the post-translational modifications we recently identified. As IDO activity may differ between in vitro and in vivo modeling systems, we will use the new IDO→GFP reporter mouse model for an improved mechanistic understanding of how immunosuppressive IDO facilitates Treg accumulation in vivo.

Download Full-text

NDUFA4L2 promotes glioblastoma progression, is associated with poor survival, and can be effectively targeted by apatinib

Cell Death and Disease ◽

10.1038/s41419-021-03646-3 ◽

2021 ◽

Vol 12 (4) ◽

Author(s):

Zheng Chen ◽

Xiangyu Wei ◽

Xueyi Wang ◽

Xuan Zheng ◽

Bowen Chang ◽

...

Keyword(s):

Tumor Cell ◽

Hypoxia Inducible Factor ◽

Mitochondrial Respiratory Chain ◽

Metabolic Reprogramming ◽

Tumor Cell Apoptosis ◽

Survival Gene ◽

A Subunit ◽

Tumor Types

AbstractNADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) is a subunit of Complex I of the mitochondrial respiratory chain, which is important in metabolic reprogramming and oxidative stress in multiple cancers. However, the biological role and molecular regulation of NDUFA4L2 in glioblastoma (GBM) are poorly understood. Here, we found that NDUFA4L2 was significantly upregulated in GBM; the elevated levels were correlated with reduced patient survival. Gene knockdown of NDUFA4L2 inhibited tumor cell proliferation and enhanced apoptosis, while tumor cells initiated protective mitophagy in vitro and in vivo. We used lentivirus to reduce expression levels of NDUFA4L2 protein in GBM cells exposed to mitophagy blockers, which led to a significant enhancement of tumor cell apoptosis in vitro and inhibited the development of xenografted tumors in vivo. In contrast to other tumor types, NDUFA4L2 expression in GBM may not be directly regulated by hypoxia-inducible factor (HIF)-1α, because HIF-1α inhibitors failed to inhibit NDUFA4L2 in GBM. Apatinib was able to effectively target NDUFA4L2 in GBM, presenting an alternative to the use of lentiviruses, which currently cannot be used in humans. Taken together, our data suggest the use of NDUFA4L2 as a potential therapeutic target in GBM and demonstrate a practical treatment approach.

Download Full-text

Compensation mechanism in tumor cell migration

The Journal of Cell Biology ◽

10.1083/jcb.200209006 ◽

2003 ◽

Vol 160 (2) ◽

pp. 267-277 ◽

Cited By ~ 997

Author(s):

Katarina Wolf ◽

Irina Mazo ◽

Harry Leung ◽

Katharina Engelke ◽

Ulrich H. von Andrian ◽

...

Keyword(s):

Cell Migration ◽

Tumor Cell ◽

Shape Change ◽

Proteolytic Degradation ◽

Amoeboid Movement ◽

Tumor Cell Migration ◽

Tumor Dissemination ◽

Β1 Integrins

Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor–based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of β1 integrins and MT1–matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered β1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.

Download Full-text