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Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.

Binder ZA, Wilson KM, Salmasi V, Orr BA, Eberhart CG, Siu IM, Lim M, Weingart JD, Quinones-Hinojosa A, Bettegowda C, Kassam AB, Olivi A, Brem H, Riggins GJ, Gallia GL - PLoS ONE (2016)

Bottom Line: When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity.Multipotency was confirmed using in vitro differentiation.These oncosphere cell lines will expand the resources available for preclinical study.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.

ABSTRACT

Objective: Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants.

Methods: Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling.

Results: Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors.

Conclusions: We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.

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Related in: MedlinePlus

Cell line growth and multipotency.(A-B) Phase contrast images of oncospheres from JHU-0879 (A) and JHU-0937 (B). (C-F) Immunofluorescence images demonstrating the multipotency of the oncospheres. (C-D) Astrocytic-like cells stained for glial fibrillary acidic protein in lines JHU-0879 (C) and JHU-0937 (D). (E-F) Neuronal-like cells stained for class III β-tubulin in lines JHU-0879 (E) and JHU-0937 (F). All images taken at 400x.
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pone.0150271.g003: Cell line growth and multipotency.(A-B) Phase contrast images of oncospheres from JHU-0879 (A) and JHU-0937 (B). (C-F) Immunofluorescence images demonstrating the multipotency of the oncospheres. (C-D) Astrocytic-like cells stained for glial fibrillary acidic protein in lines JHU-0879 (C) and JHU-0937 (D). (E-F) Neuronal-like cells stained for class III β-tubulin in lines JHU-0879 (E) and JHU-0937 (F). All images taken at 400x.

Mentions: All 13 cell lines demonstrated growth of spheres beyond passage four (Fig 3A and 3B). We observed the cell lines grew at different rates. They all grew noticeably slower than U87, one of the most widely used adherent GBM cell lines [24]. To quantitate this variability, we performed proliferation assays on several lines. The cell lines were separated into two groups based on their doubling times. The slower cell lines had doubling times of 4.5 days and greater. The faster group had doubling times 2.5 days and shorter. Based on the doubling times of these 4 lines, we subjectively grouped the remaining 9 cell lines as either slow- or fast-growing (Table 2).


Establishment and Biological Characterization of a Panel of Glioblastoma Multiforme (GBM) and GBM Variant Oncosphere Cell Lines.

Binder ZA, Wilson KM, Salmasi V, Orr BA, Eberhart CG, Siu IM, Lim M, Weingart JD, Quinones-Hinojosa A, Bettegowda C, Kassam AB, Olivi A, Brem H, Riggins GJ, Gallia GL - PLoS ONE (2016)

Cell line growth and multipotency.(A-B) Phase contrast images of oncospheres from JHU-0879 (A) and JHU-0937 (B). (C-F) Immunofluorescence images demonstrating the multipotency of the oncospheres. (C-D) Astrocytic-like cells stained for glial fibrillary acidic protein in lines JHU-0879 (C) and JHU-0937 (D). (E-F) Neuronal-like cells stained for class III β-tubulin in lines JHU-0879 (E) and JHU-0937 (F). All images taken at 400x.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814135&req=5

pone.0150271.g003: Cell line growth and multipotency.(A-B) Phase contrast images of oncospheres from JHU-0879 (A) and JHU-0937 (B). (C-F) Immunofluorescence images demonstrating the multipotency of the oncospheres. (C-D) Astrocytic-like cells stained for glial fibrillary acidic protein in lines JHU-0879 (C) and JHU-0937 (D). (E-F) Neuronal-like cells stained for class III β-tubulin in lines JHU-0879 (E) and JHU-0937 (F). All images taken at 400x.
Mentions: All 13 cell lines demonstrated growth of spheres beyond passage four (Fig 3A and 3B). We observed the cell lines grew at different rates. They all grew noticeably slower than U87, one of the most widely used adherent GBM cell lines [24]. To quantitate this variability, we performed proliferation assays on several lines. The cell lines were separated into two groups based on their doubling times. The slower cell lines had doubling times of 4.5 days and greater. The faster group had doubling times 2.5 days and shorter. Based on the doubling times of these 4 lines, we subjectively grouped the remaining 9 cell lines as either slow- or fast-growing (Table 2).

Bottom Line: When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity.Multipotency was confirmed using in vitro differentiation.These oncosphere cell lines will expand the resources available for preclinical study.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Johns Hopkins University School of Medicine, Baltimore, MD, United States of America.

ABSTRACT

Objective: Human tumor cell lines form the basis of the majority of present day laboratory cancer research. These models are vital to studying the molecular biology of tumors and preclinical testing of new therapies. When compared to traditional adherent cell lines, suspension cell lines recapitulate the genetic profiles and histologic features of glioblastoma multiforme (GBM) with higher fidelity. Using a modified neural stem cell culture technique, here we report the characterization of GBM cell lines including GBM variants.

Methods: Tumor tissue samples were obtained intra-operatively and cultured in neural stem cell conditions containing growth factors. Tumor lines were characterized in vitro using differentiation assays followed by immunostaining for lineage-specific markers. In vivo tumor formation was assayed by orthotopic injection in nude mice. Genetic uniqueness was confirmed via short tandem repeat (STR) DNA profiling.

Results: Thirteen oncosphere lines derived from GBM and GBM variants, including a GBM with PNET features and a GBM with oligodendroglioma component, were established. All unique lines showed distinct genetic profiles by STR profiling. The lines assayed demonstrated a range of in vitro growth rates. Multipotency was confirmed using in vitro differentiation. Tumor formation demonstrated histologic features consistent with high grade gliomas, including invasion, necrosis, abnormal vascularization, and high mitotic rate. Xenografts derived from the GBM variants maintained histopathological features of the primary tumors.

Conclusions: We have generated and characterized GBM suspension lines derived from patients with GBMs and GBM variants. These oncosphere cell lines will expand the resources available for preclinical study.

Show MeSH
Related in: MedlinePlus