STEM-02. IN VIVO PHAGE DISPLAY IDENTIFIES PEPTIDE TARGETING N-CADHERIN ON GLIOMA STEM CELLS

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with high mortality rates and resistance to conventional therapy. Glioma stem cells (GSCs) comprise a sub-population of glioma tumor cells with the ability of self-renewal and tumor recapitulation, and may be responsible for GBM’s treatment resistant properties. Identification of surface receptors that are novel and specific to GSCs may be the key to the development of effective therapeutic strategies. We have selected a GSC specific targeting peptide isolated through in vitro and in vivo phage display biopanning. This screening technique allowed us to determine a peptide (GBM-IC2) which binds specifically to GSCs in vitro, and to GBM tissue in vivo. Although this screening process allows for isolation of cell specific targeting peptides, it does so without identification of the cellular binding partner. Given the specificity of the peptide, identification of the cellular receptor may allow for discovery of novel markers to identify GSCs. To identify the peptide binding partner of GBM-IC2, the biotinylated peptide was incubated with GSC protein lysate. The peptide, along with its binding partner, was isolated using streptavidin agarose resin. The binding partner protein was then identified using mass spectroscopy. This revealed N-cadherin (CDH2) as a potential binding partner for the GBM-IC2 peptide. GBM-IC2 demonstrated specificity for targeting CDH2 compared to control peptide using ELISA. Lentiviral induced overexpression of CDH2 in HEK293 cells allowed for GBM-IC2 peptide binding. Competition assay was performed by applying anti-CDH2 antibody to GBM-IC2 peptide and GSCs in culture. Application of anti-CDH2 antibody decreased peptide binding to GSCs, confirming CDH2 as the binding partner for GBM-IC2. These results demonstrate that cell specific targeting peptides isolated through phage display may lead to the isolation of novel cell specific proteins through immunoprecipitation isolation and mass spectroscopy analysis.

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Prrx1 promotes stemness and angiogenesis via activating TGF-β/smad pathway and upregulating proangiogenic factors in glioma

Cell Death and Disease ◽

10.1038/s41419-021-03882-7 ◽

2021 ◽

Vol 12 (6) ◽

Author(s):

Zetao Chen ◽

Yihong Chen ◽

Yan Li ◽

Weidong Lian ◽

Kehong Zheng ◽

...

Keyword(s):

Stem Cells ◽

Therapeutic Strategy ◽

Critical Role ◽

Glioma Stem Cells ◽

Promoter Regions ◽

Aberrant Expression ◽

Smad Pathway ◽

Tgf Β1

AbstractGlioma is one of the most lethal cancers with highly vascularized networks and growing evidences have identified glioma stem cells (GSCs) to account for excessive angiogenesis in glioma. Aberrant expression of paired-related homeobox1 (Prrx1) has been functionally associated with cancer stem cells including GSCs. In this study, Prrx1 was found to be markedly upregulated in glioma specimens and elevated Prrx1 expression was inversely correlated with prognosis of glioma patients. Prrx1 potentiated stemness acquisition in non-stem tumor cells (NSTCs) and stemness maintenance in GSCs, accompanied with increased expression of stemness markers such as SOX2. Prrx1 also promoted glioma angiogenesis by upregulating proangiogenic factors such as VEGF. Consistently, silencing Prrx1 markedly inhibited glioma proliferation, stemness, and angiogenesis in vivo. Using a combination of subcellular proteomics and in vitro analyses, we revealed that Prrx1 directly bound to the promoter regions of TGF-β1 gene, upregulated TGF-β1 expression, and ultimately activated the TGF-β/smad pathway. Silencing TGF-β1 mitigated the malignant behaviors induced by Prrx1. Activation of this pathway cooperates with Prrx1 to upregulate the expression of stemness-related genes and proangiogenic factors. In summary, our findings revealed that Prrx1/TGF-β/smad signal axis exerted a critical role in glioma stemness and angiogeneis. Disrupting the function of this signal axis might represent a new therapeutic strategy in glioma patients.

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Enhanced invasion in vitro and the distribution patterns in vivo of CD133+ glioma stem cells

Yearbook of Pathology and Laboratory Medicine ◽

10.1016/j.ypat.2011.11.133 ◽

2012 ◽

Vol 2012 ◽

pp. 224-226

Author(s):

D.M. Grzybicki

Keyword(s):

Stem Cells ◽

Distribution Patterns ◽

Glioma Stem Cells

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Erratum to “Targeting metabolic plasticity in glioma stem cells in vitro and in vivo through specific inhibition of c-Src by TAT-Cx43266-283” [EBioMedicine 62, December 2020] DOI: https://doi.org/10.1016/j.ebiom.2020.103134

EBioMedicine ◽

10.1016/j.ebiom.2021.103752 ◽

2021 ◽

Vol 74 ◽

pp. 103752

Author(s):

Sara G. Pelaz ◽

Myriam Jaraíz-Rodríguez ◽

Andrea Álvarez-Vázquez ◽

Rocío Talaverón ◽

Laura García-Vicente ◽

...

Keyword(s):

Stem Cells ◽

Glioma Stem Cells ◽

Specific Inhibition ◽

Metabolic Plasticity

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Adenovirus infection promotes the formation of glioma stem cells by glioblastoma cells through the TLR9/NEAT1/STAT3 pathway

10.21203/rs.3.rs-15785/v1 ◽

2020 ◽

Author(s):

Jian Zang ◽

Min-hua Zheng ◽

Xiu-li Cao ◽

Yi-zhe Zhang ◽

Yu-fei Zhang ◽

...

Keyword(s):

Stem Cells ◽

Glioma Cells ◽

Tumor Model ◽

Glioma Stem Cells ◽

Stem Cell Markers ◽

Qrt Pcr ◽

Lineage Differentiation ◽

Stat3 Signaling

Abstract BackgroundGlioma stem cells (GSCs) are glioma cells with stemness and are responsible for a variety of malignant behaviors of glioma. Evidence has shown that signals from tumor microenvironment (TME) enhance stemness of glioma cells, but the identity of the signaling molecules and underlying mechanisms have been incompletely elucidated.MethodsHuman samples and glioma cell lines were cultured in vitro to determine the effects of viral infection by sphere formation, qRT-PCR, Western blot, FACS and immunofluorescence; for in vivo analysis, mice subcutaneous tumor model was carried; while bioinformatics analysis and qRT-PCR were applied for further mechanistic studies.ResultsIn this study, we show that infection of patient-derived glioma cells with adenovirus (ADV) increases the formation of tumor spheres. ADV infection upregulated stem cell markers, and the resultant tumor spheres held the capacities of self-renewal and multi-lineage differentiation, and had stronger potential to form xenograft tumors in immune-compromised mice. ADV promoted GSC formation likely via TLR9, because TLR9 was upregulated after ADV infection, and knockdown of TLR9 reduced ADV-induced GSCs. Consistently, MYD88, as well as total STAT3 and phosphorylated (p-)STAT3, were also upregulated in ADV-induced GSCs. Knockdown of MYD88 or pharmaceutical inhibition of STAT3 attenuated stemness of ADV-induced GSCs. Moreover, we found that ADV infection upregulated lncRNA NEAT1, which is downstream to TLRs and play important roles in cancer stem cells via multiple mechanisms including strengthening STAT3 signaling. Indeed, knockdown of NEAT1 impaired stemness of ADV-induced GSCs. Lastly, we show that HMGB1, a damage associated molecular pattern (DAMP) that also triggers TLR signaling, upregulated stemness markers in glioma cells.ConclusionsIn summary, our data indicate that ADV, which has been developed as vectors for gene therapy and oncolytic virus, promotes the formation of GSCs via TLR9/NEAT1/STAT3 signaling.

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The Development and Applications of a Dual Optical Imaging System for Studying Glioma Stem Cells

Molecular Imaging ◽

10.1177/1536012119870899 ◽

2019 ◽

Vol 18 ◽

pp. 153601211987089 ◽

Cited By ~ 1

Author(s):

Po-An Tai ◽

Yen-Lin Liu ◽

Ya-Ting Wen ◽

Chien-Min Lin ◽

Thanh-Tuan Huynh ◽

...

Keyword(s):

Stem Cells ◽

Brain Tumor ◽

Optical Imaging ◽

Fluorescent Protein ◽

Imaging System ◽

High Rate ◽

Glioma Stem Cells ◽

Optical Imaging System

Glioblastoma multiforme represents one of the deadliest brain tumor types, manifested by a high rate of recurrence and poor prognosis. The presence of glioma stem cells (GSCs) can repopulate the tumor posttreatment and resist therapeutics. A better understanding of GSC biology is essential for developing more effective interventions. We established a CD133 promoter-driven dual reporter, expressing green fluorescent protein (GFP) and firefly luciferase (CD133-LG), capable for in vitro and in vivo imaging of CD133+ GSCs. We first demonstrated the reporter enabled in vitro analyses of GSCs. DBTRG-05MG (Denver Brain Tumor Research Group 05) carrying CD133-LG (DBTRG-05MG-CD133-LG) system reported increased GFP/luciferase activities in neurospheres. Additionally, we identified and isolated CD133+/GFP+ cells with increased tumorigenic properties, stemness markers, Notch1, β-catenin, and Bruton’s tyrosine kinase (Btk). Furthermore, prolonged temozolomide (TMZ) treatment enriched GSCs (reflected by increased percentage of CD133+ cells). Subsequently, Btk inhibitor, ibrutinib, suppressed GSC generation and stemness markers. Finally, we demonstrated real-time evaluation of anti-GSC function of ibrutinib in vivo with TMZ-enriched GSCs. Tumorigenesis was noninvasively monitored by bioluminescence imaging and mice that received ibrutinib showed a significantly lower tumor burden, indicating ibrutinib as a potential GSC inhibitor. In conclusion, we established a dual optical imaging system which enables the identification of CD133+ GSCs and screening for anti-GSC drugs.

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FRAT1 Enhances the Proliferation and Tumorigenesis of CD133+Nestin+ Glioma Stem Cells In Vitro and In Vivo

Journal of Cancer ◽

10.7150/jca.37622 ◽

2020 ◽

Vol 11 (9) ◽

pp. 2421-2430

Author(s):

Geng Guo ◽

Jing Liu ◽

Yeqing Ren ◽

Xinggang Mao ◽

Yining Hao ◽

...

Keyword(s):

Stem Cells ◽

Glioma Stem Cells

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Targeting Folate Metabolism Is Selectively Cytotoxic to Glioma Stem Cells and Effectively Cooperates with Differentiation Therapy to Eliminate Tumor-Initiating Cells in Glioma Xenografts

International Journal of Molecular Sciences ◽

10.3390/ijms222111633 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11633

Author(s):

Masashi Okada ◽

Shuhei Suzuki ◽

Keita Togashi ◽

Asuka Sugai ◽

Masahiro Yamamoto ◽

...

Keyword(s):

Stem Cells ◽

Cancer Stem Cells ◽

High Sensitivity ◽

Therapy Resistance ◽

Folate Metabolism ◽

Glioma Stem Cells ◽

Tumor Initiating Cells ◽

Dismal Prognosis

Glioblastoma (GBM) is one of the deadliest of all human cancers. Developing therapies targeting GBM cancer stem cells or glioma stem cells (GSCs), which are deemed responsible for the malignancy of GBM due to their therapy resistance and tumor-initiating capacity, is considered key to improving the dismal prognosis of GBM patients. In this study, we found that folate antagonists, such as methotrexate (MTX) and pemetrexed, are selectively cytotoxic to GSCs, but not to their differentiated counterparts, normal fibroblasts, or neural stem cells in vitro, and that the high sensitivity of GCSs to anti-folates may be due to the increased expression of RFC-1/SLC19A1, the reduced folate carrier that transports MTX into cells, in GSCs. Of note, in an in vivo serial transplantation model, MTX alone failed to exhibit anti-GSC effects but promoted the anti-GSC effects of CEP1347, an inducer of GSC differentiation. This suggests that folate metabolism, which plays an essential role specifically in GSCs, is a promising target of anti-GSC therapy, and that the combination of cytotoxic and differentiation therapies may be a novel and promising approach to effectively eliminate cancer stem cells.

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ET-33 * PLACENTA-DERIVED MESENCHYMAL STEM CELLS AND THEIR SECRETED EXOSOMES INHIBIT THE SELF-RENEWAL AND STEMNESS OF GLIOMA STEM CELLS IN VITRO AND IN VIVO

Neuro-Oncology ◽

10.1093/neuonc/nou255.33 ◽

2014 ◽

Vol 16 (suppl 5) ◽

pp. v86-v87

Author(s):

H. K. Lee ◽

E. Buchris ◽

S. Finniss ◽

S. Cazacu ◽

C. Xiang ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

The Self ◽

Glioma Stem Cells ◽

Self Renewal

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BSCI-15. METASTATIC BRAIN TUMOR TARGETING PEPTIDES ISOLATED THROUGH PHAGE DISPLAY BIOPANNING AGAINST BRAIN METASTASIS-INITIATING CELLS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdz014.013 ◽

2019 ◽

Vol 1 (Supplement_1) ◽

pp. i4-i4

Author(s):

JongMyung Kim ◽

James Liu

Keyword(s):

Lung Cancer ◽

Brain Metastasis ◽

Phage Display ◽

Metastatic Brain Tumors ◽

Serum Free ◽

Phage Display Biopanning ◽

Free Media ◽

In Vivo Phage Display

Abstract To effectively target metastatic brain tumors (MBTs), the paradigm of treating MBTs after visualization on clinical imaging needs to be shifted to an understanding of the mechanisms that drive the formation and maintenance of brain metastasis-initiating cells (BMICs). Targeting this tumor sub-population, which may form as a result from activation of epithelial-mesenchymal transition, may allow for more effective means of isolating and targeting brain metastasis. In order to isolate BMICs, we have harvested cells from patient derived MBTs originating from lung cancer and cultured the cells using serum-free media conditions. In vivo phage display biopanning was used to isolate 12-amino acid length peptides that specifically target BMICs. Of the peptides recovered, one peptide, LBM4, was tested for specificity of binding to MBTs through in vitro and in vivo binding assays. When comparing patient derived metastatic brain tumors cells against brain metastasis cell lines, we found that both types of cells demonstrated similar morphology when grown in serum media conditions, but when grown in serum-free media, both demonstrated a tumor sphere morphology, similar to a stem cell-like state. LBM4 demonstrated specific binding to MBT cells over primary lung cancer cells in vitro through flow cytometry analysis and immunocytochemistry. Fluorescent tagged LBM4 intravenously injected into mice harboring intracranial BM demonstrated peptide localization to the tumor within the intracranial cavity visualized with live animal imaging. In vivo phage display biopanning is an effective tool that is able to isolate cell specific targeting peptides. MBT targeting peptides can potentially result in a shifting of the clinical treatment paradigm of brain metastases, through the development of more effective targeted therapeutics aimed at BMICs, as well as improving detection of MBT cells which may result in earlier tumor visualization as well as delineation of tumor recurrence versus radiation effects.

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