Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jul;86(13):7280-91.
doi: 10.1128/JVI.00227-12. Epub 2012 May 2.

LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells

Affiliations

LuIII parvovirus selectively and efficiently targets, replicates in, and kills human glioma cells

Justin C Paglino et al. J Virol. 2012 Jul.

Abstract

Because productive infection by parvoviruses requires cell division and is enhanced by oncogenic transformation, some parvoviruses may have potential utility in killing cancer cells. To identify the parvovirus(es) with the optimal oncolytic effect against human glioblastomas, we screened 12 parvoviruses at a high multiplicity of infection (MOI). MVMi, MVMc, MVM-G17, tumor virus X (TVX), canine parvovirus (CPV), porcine parvovirus (PPV), rat parvovirus 1A (RPV1A), and H-3 were relatively ineffective. The four viruses with the greatest oncolytic activity, LuIII, H-1, MVMp, and MVM-G52, were tested for the ability, at a low MOI, to progressively infect the culture over time, causing cell death at a rate higher than that of cell proliferation. LuIII alone was effective in all five human glioblastomas tested. H-1 progressively infected only two of five; MVMp and MVM-G52 were ineffective in all five. To investigate the underlying mechanism of LuIII's phenotype, we used recombinant parvoviruses with the LuIII capsid replacing the MVMp capsid or with molecular alteration of the P4 promoter. The LuIII capsid enhanced efficient replication and oncolysis in MO59J gliomas cells; other gliomas tested required the entire LuIII genome to exhibit enhanced infection. LuIII selectively infected glioma cells over normal glial cells in vitro. In mouse models, human glioblastoma xenografts were selectively infected by LuIII when administered intratumorally; LuIII reduced tumor growth by 75%. LuIII also had the capacity to selectively infect subcutaneous or intracranial gliomas after intravenous inoculation. Intravenous or intracranial LuIII caused no adverse effects. Intracranial LuIII caused no infection of mature mouse neurons or glia in vivo but showed a modest infection of developing neurons.

PubMed Disclaimer

Figures

Fig 1
Fig 1
Screening 12 parvoviruses against human glioblastoma U87. (A) U87 cells were infected with a panel of 12 parvoviruses at a high multiplicity of infection (MOI) (30,000 genomes per cell) or mock infected. Cell density was assessed 0, 1, 3, 5, and 7 dpi by microscopy. G52, MVM-G52; G17, MVM-G17. Infection phenotypes were grouped as described in the text by the magnitude of the oncosuppressive effect at this high MOI, demonstrating negligible (dotted lines), moderate (dashed lines), or strong (solid lines) oncosuppression. Bars, standard errors of the means. (B) Representative micrographs from 7 dpi are shown.
Fig 2
Fig 2
Screening 12 parvoviruses against glioblastoma U373. (A) U373 cells were infected with a panel of 12 parvoviruses at a high MOI (30,000 genomes per cell) or mock infected. Cell density was assessed 0, 1, 3, 5, and 7 dpi by microscopy. G52, MVM-G52; G17, MVM-G17. Infection phenotypes were grouped as described in the text by the magnitude of the oncosuppressive effect at this high MOI, demonstrating negligible (dotted lines), moderate (dashed lines), or strong (solid lines) oncosuppression. Bars, standard errors of the means. (B) Representative micrographs from 7 dpi are shown.
Fig 3
Fig 3
Low multiplicity of infection of five human glioblastomas: virus expansion and cell growth. (A) Expansion assay. Five human glioblastoma lines were infected at a low MOI (1,000 genomes per cell) with LuIII, H-1, MVMp, or MVM-G52 (G52). The percentage of cells expressing viral NS1 was assessed at 24 and 72 hpi. Here, infection is characterized as progressive when the percentage of cells infected 72 hpi is greater than the percentage infected 24 hpi. Progressively infecting viruses are shown by solid lines (others are shown by dotted lines). (B) Cell growth with low-MOI parvovirus. The growth of five human glioblastomas was assessed at a low MOI (1,000 genomes per cell) with LuIII, H-1, MVMp, or MVM-G52 (G52) or after mock infection. Cell density at 0, 1, 3, 5, and 7 dpi was assessed by microscopy. Viruses able to suppress or reverse cell growth are shown by solid lines (others are shown by dotted lines).
Fig 4
Fig 4
Influence of parvoviral genotype on GBM tropism and killing. (A) Genetic map indicating the design of chimeric MVM-LuCap and MVM-LuCap/mP4. P4 and P38 promoters are shown; the “4-between” P4 mutation (mP4) is indicated by an asterisk. (B) Oncolysis assay. Serially diluted inocula of five different parvoviruses were tested for killing against broadly susceptible control cells 324K and against three human glioblastoma lines—M0595, U373, and A172. Subconfluent cells were infected at the indicated number of genomes per cell and were stained for viable cells 7 dpi with Leishman's solution. A dark well is indicative of a healthy GBM monolayer; wells that appear white or clear are ones in which the GBM cells have been killed by the respective parvovirus. LC, MVM-LuCap; LC4′, MVM-LuCap/mP4.
Fig 5
Fig 5
Oncolytic effect of LuIII on U87 glioma. Cultures were uninfected (A and B) or infected with LuIII at 10,000 genomes per cell (C and D). Cell density and ethidium homodimer (EtHD) staining for dead cells (red fluorescence) were assessed in parallel wells at 72 and 96 hpi (shown). Incidence of cell death as measured by EtHD staining was at least 5 times greater in LuIII-infected cultures (E).
Fig 6
Fig 6
LuIII is highly selective for glioblastoma versus normal glia in vitro irrespective of cell proliferation rate. Human glial cultures (hGlia) and human glioblastoma (U87) were infected with LuIII at 8,000 genomes per cell. In parallel cultures, U87 were grown in 1% FBS, to increase the cell doubling time. Cell doubling times (B) were determined in parallel wells. At 24 hpi, cells were fixed and immunostained for viral NS protein to determine infectivity rates (A). Representative micrographs are shown in panel C.
Fig 7
Fig 7
Intratumoral LuIII inhibits growth of subcutaneous human GBM tumors. (A) Tumor volume. LuIII parvovirus (1 × 1010 genomes) was injected in 7 tumors, and 5 tumors were left uninfected. Tumor dimensions were measured daily by caliper; the average of tumor volumes relative to day 0 is shown. Bars, standard errors of the means. (B) Histology, 12 dpi. Phase-contrast, red filter (red fluorescent rU87 tumor cells), and green filter (immunostaining for viral NS and VP proteins) micrographs of a treated tumor at 12 dpi are shown.
Fig 8
Fig 8
rU87 tumors are selectively infected in vivo by LuIII. Intracranial tumors were stereotactically injected directly with 1 × 1010 genomes of LuIII. Panel A (phase contrast) shows the tumor and normal surrounding tissue; panels B (red fluorescent tumor) and C (green immunostaining for NS1) are from a representative mouse sacrificed 6 dpi. The virus infects only the tumor; no infection is seen in normal brain tissue. Enlargements in panels D and E show the specificity of LuIII infection. Liver (F) and spleen (H) from the mouse shown in panel A show no immunostaining for virus (G and I, respectively). Four days after intravenous administration of LuIII in SCID mice, subcutaneous tumors (phase contrast [J] and red fluorescent tumor [K]) show substantial infection by immunofluorescence (L); intracranial tumors (M and N) were also partly infected 3 days after intravenous LuIII (O).
Fig 9
Fig 9
LuIII infection of murine brain and glioma. (A and B) LuIII was injected directly into the brain (striatum) of normal adult Swiss Webster mice. Immunostaining showed no evidence of virus infection in a set of serial sections through the injection sites. (A and B) Phase-contrast (A) and green fluorescence (B) images are shown; bar, 150 μm. (C) Eight-day-old Swiss Webster pups were injected in the cerebellum with LuIII. Infected cells were found in the internal granule cell layer (IGL), as detected with LuIII NS immunostaining. The micrograph shown is 6 days postinoculation. Bar, 14 μm. EGL, external granule cell layer; PCL, Purkinje cell layer. (D and E) Murine glioma was infected with LuIII (8,000 genomes/cell) and immunostained for viral NS1 protein 24 hpi. Phase-contrast (D) and immunofluorescence (E) images are shown.

Similar articles

Cited by

References

    1. Abschuetz A, et al. 2006. Oncolytic murine autonomous parvovirus, a candidate vector for glioma gene therapy, is innocuous to normal and immunocompetent mouse glial cells. Cell Tissue Res. 325:423–436 - PubMed
    1. Arruda VR, Xiao W. 2007. It's all about the clothing: capsid domination in the adeno-associated viral vector world. J. Thromb. Haemost. 5:12–15 - PubMed
    1. Arstila P. 1976. Quantitative studies on adsorption, elution, and haemagglutination of vesicular stomatitis virus. Arch. Virol. 51:51–58 - PubMed
    1. Ball-Goodrich LJ, Tattersall P. 1992. Two amino acid substitutions within the capsid are coordinately required for acquisition of fibrotropism by the lymphotropic strain of minute virus of mice. J. Virol. 66:3415–3423 - PMC - PubMed
    1. Besselsen DG, Romero MJ, Wagner AM, Henderson KS, Livingston RS. 2006. Identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice. J. Gen. Virol. 87:1543–1556 - PubMed

Publication types

MeSH terms

LinkOut - more resources