Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Oct;10(5):772-779.
doi: 10.1016/j.tranon.2017.07.002. Epub 2017 Aug 4.

Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas

Minna Niittykoski  1 , Mikael von Und Zu Fraunberg  2 , Miika Martikainen  3 , Tuomas Rauramaa  4 , Arto Immonen  5 , Susanna Koponen  6 , Ville Leinonen  7 , Markus Vähä-Koskela  8 , Qiwei Zhang  9 , Florian Kühnel  10 , Ya-Fang Mei  11 , Seppo Ylä-Herttuala  12 , Juha E Jääskeläinen  13 , Ari Hinkkanen  14
Affiliations

Immunohistochemical Characterization and Sensitivity to Human Adenovirus Serotypes 3, 5, and 11p of New Cell Lines Derived from Human Diffuse Grade II to IV Gliomas

Minna Niittykoski et al. Transl Oncol. 2017 Oct.

Abstract

Background: Oncolytic adenoviruses show promise in targeting gliomas because they do not replicate in normal brain cells. However, clinical responses occur only in a subset of patients. One explanation could be the heterogenic expression level of virus receptors. Another contributing factor could be variable activity of tumor antiviral defenses in different glioma subtypes.

Methods: We established a collection of primary low-passage cell lines from different glioma subtypes (3 glioblastomas, 3 oligoastrocytomas, and 2 oligodendrogliomas) and assessed them for receptor expression and sensitivity to human adenovirus (HAd) serotypes 3, 5, and 11p. To gauge the impact of antiviral defenses, we also compared the infectivity of the oncolytic adenoviruses in interferon (IFN)-pretreated cells with IFN-sensitive Semliki Forest virus (SFV).

Results: Immunostaining revealed generally low expression of HAd5 receptor CAR in both primary tumors and derived cell lines. HAd11p receptor CD46 levels were maintained at moderate levels in both primary tumor samples and derived cell lines. HAd3 receptor DSG-2 was reduced in the cell lines compared to the tumors. Yet, at equal multiplicities of infection, the oncolytic potency of HAd5 in vitro in tumor-derived cells was comparable to HAd11p, whereas HAd3 lysed fewer cells than either of the other two HAd serotypes in 72 hours. IFN blocked replication of SFV, while HAds were rather unaffected.

Conclusions: Adenovirus receptor levels on glioma-derived cell lines did not correlate with infection efficacy and may not be a relevant indicator of clinical oncolytic potency. Adenovirus receptor analysis should be preferentially performed on biopsies obtained perioperatively.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression of adenovirus receptors (CAR, CD46, and DSG-2) in glioma tissues and cultured glioma cells. (A) Adenovirus receptor expression (brown color) was variable in paraffin sections of patient gliomas for which hematoxylin (blue color) was used as counterstain. Anaplastic oligodendroglioma 014-AOD was used as positive control in CAR and CD46 staining and rat kidney in DSG-2 staining. Among gliomas especially, 014-AOD had abundant expression of adenovirus receptors. Scale bar 100 μm. (B) Fluorescence images of immunocytochemically (red color) stained cultured cells. Nuclei were stained with DAPI (blue color). Especially CD46, receptor for serotype 11p, was well preserved among patient glioma-derived cells. A549 and 293 cells were used as positive controls. Scale bar 20 μm.
Figure 2
Figure 2
Relationship between viability and receptor expression level in glioma cell lines. Viability is determined 3 days after infection with adenovirus (HAd) or SFV and is given as percentage of control (100%; average ± S.E.M., n = 3 parallel wells/group) at the same time point. Cells were derived from glioblastoma (003-GB, 027-GB, and 038-GB), oligodendroglioma (004-OD and 020-OD), and oligoastrocytoma (015-OA, 025-OA, and 026-OA) patients. MOI was 10 for adenoviruses (HAd5, HAd3, and HAd11p) and 1 for SFV.
Figure 3
Figure 3
Effect of exogenous interferon on infection with adenovirus (HAd) and SFV. Viability of human cells is determined 3 days after infection and is given as percentage of control (ctrl, 100%; average ± S.E.M., n = at least 3 parallel wells/group) at the same time point. Cells were derived from glioblastoma (003-GB, 027-GB, and 038-GB), oligodendroglioma (004-OD and 020-OD), and oligoastrocytoma (015-OA, 025-OA, and 026-OA) patients. A549 human lung carcinoma cells were used as controls. MOI was 10 for adenoviruses (HAd5, HAd3, and HAd11p) and 1 for SFV. IFN-β (2000 U/ml) was applied 4 hours before cells were challenged with viruses. *P < .05, **P < .01, ***P < .001 (unpaired t test, comparisons: control versus virus infected or virus infected versus virus infected + IFN).
Supplementary Figure S1
Supplementary Figure S1
Magnetic resonance imaging characteristics of gliomas. Postcontrast T1W and T2W/FLAIR images were obtained preoperatively. Contrast enhancement was present for 003-GB, 027-GB, 038-GB, 014-AOD, 015-OA, and 026-OA but not for 004-OD, 020-OD, and 025-OA. Postcontrast T1W images are shown for enhancing and T2W/FLAIR images for nonenhancing gliomas.
Supplementary Figure S2
Supplementary Figure S2
Western blot analysis of IDH1, IDH1 (R132H), EGFR, p53, MGMT, and S100 in the established glioma cell lines. “+” indicates positive control cells. IDH1 represents wild type, and IDH1 (R132H) is the most common mutant of IDH1.
Supplementary Figure S3
Supplementary Figure S3
Glial (GFAP and CNPase), neuronal (NeuN), and proliferating (Ki-67) cells in glioma tissues and cultured glioma cells. (A) Diversity of tumors was evident from paraffin sections of patient gliomas in which hematoxylin (blue color) was used as counterstain. Some tumors, e.g., oligodendroglioma 004-OD, were morphologically compact. H&E staining shows gemistocytes with large eosinophilic cytoplasm and peripheral nuclei in 015-OA parental tumor. Notable are, for example, the highly proliferating, Ki-67–positive (brown color) cells in glioblastomas and anaplastic oligodendroglioma. 014-OAD was used as control in all cases except in NeuN staining in which neurons from rat brain are shown. Scale bar 100 μm. (B) Fluorescence images of immunocytochemically (red color) stained cultured cells. GFAP images do not contain nuclei stain. In the other images, nuclei are stained with DAPI (blue color). Cultured cells derived from patient gliomas possessed astrocytic marker GFAP and, to a lesser extent, oligodendrocytic marker CNPase. A172 cells and primary cultures from rat cortex were used as positive controls. Scale bar 50 μm and 20 μm in GFAP and other images, respectively.
Supplementary Figure S4
Supplementary Figure S4
Western blot analysis of adenovirus receptor expression in established and commercially available glioma cell lines (A172, U251, and U87-MG). “+” indicates positive control cells; A431 for DSG-2, 025 (original tumor lysate) for CAR, and U251 for CD46.
Supplementary Figure S5
Supplementary Figure S5
Human cells infected with adenoviruses (HAd5, HAd3, and HAd11p) and SFV VA7. Phase contrast (upper) and fluorescence (lower) microscopy images were captured before infection and up to 3 days after infection. 038-GB cells were derived from glioblastoma patient, while A549 human lung carcinoma cells served as controls. Scale bar 500 μm.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, Ohgaki H, Wiestler OD, Kleihues P, Ellison DW. The 2016 World Health Organization classification of tumors of the central nervous system: a summary. Acta Neuropathol. 2016;131:803–820. - PubMed
    1. Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, Ludwin SK, Allgeier A, Fisher B, Belanger K. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol. 2009;10:459–466. - PubMed
    1. Walker C, Baborie A, Crooks D, Wilkins S, Jenkinson MD. Biology, genetics and imaging of glial cell tumours. Br J Radiol. 2011;84:S90–S106. - PMC - PubMed
    1. Weller M, Pfister SM, Wick W, Hegi ME, Reifenberger G, Stupp R. Molecular neuro-oncology in clinical practice: a new horizon. Lancet Oncol. 2013;14:e370–379. - PubMed
    1. Chiocca EA, Abbed KM, Tatter S, Louis DN, Hochberg FH, Barker F, Kracher J, Grossman SA, Fisher JD, Carson K. A phase I open-label, dose-escalation, multi-institutional trial of injection with an E1B-attenuated adenovirus, ONYX-015, into the peritumoral region of recurrent malignant gliomas, in the adjuvant setting. Mol Ther. 2004;10:958–966. - PubMed