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. 2018 Jun 14;20(12):1606–1615. doi: 10.1093/neuonc/noy089

An oncolytic measles virus–sensitive Group 3 medulloblastoma model in immune-competent mice

Sangeet Lal 1, Diego Carrera 1, Joanna J Phillips 1, William A Weiss 2, Corey Raffel 1,✉
PMCID: PMC6231208  PMID: 29912438

Abstract

Background

Oncolytic measles virus (MV) is effective in xenograft models of many tumor types in immune-compromised mice. However, no murine cell line exists that is tumorigenic, grows in immune-competent mice, and is killed by MV. The lack of such a model prevents an examination of the effect of the immune system on MV oncotherapy.

Methods

Cerebellar stem cells from human CD46-transgenic immunocompetent mice were transduced to express Sendai virus C-protein, murine C-Myc, and Gfi1b proteins. The resultant cells were injected into the brain of NSG mice, and a cell line, called CSCG, was prepared from the resulting tumor.

Results

CSCG cells are highly proliferative, and express stem cell markers. These cells are permissive for replication of MV and are killed by the virus in a dose- and time-dependent manner. CSCG cells form aggressive tumors that morphologically resemble medulloblastoma when injected into the brains of immune-competent mice. On the molecular level, CSCG tumors overexpress natriuretic peptide receptor 3 and gamma-aminobutyric acid type A receptor alpha 5, markers of Group 3 medulloblastoma. A single intratumoral injection of MV‒green fluorescent protein resulted in complete tumor regression and prolonged survival of animals compared with treatments with phosphate buffered saline (P = 0.0018) or heat-inactivated MV (P = 0.0027).

Conclusions

This immune-competent model provides the first platform to test therapeutic regimens of oncolytic MV for Group 3 medulloblastoma in the presence of anti-measles immunity. The strategy presented here can be used to make MV-sensitive murine models of any human tumor for which the driving mutations are known.

Keywords: measles virus, medulloblastoma, immune-competent model, oncolytic virus

Importance of the study

Oncolytic viruses (OV) have advantageous properties such as tumor-specific growth, replication, spread of viruses between tumor cells, and potential stimulation of systemic anticancer immune responses. However, the efficacy of OV in the clinical setting has not fulfilled the promise suggested by preclinical studies. One major reason may be that most preclinical studies are performed on xenograft models in immune-deficient hosts with intratumoral injection of viruses. Because anti-viral immunity is prevalent in the human population, results from immunodeficient models may not translate to human disease. In this paper, we describe the development of a murine Group 3 medulloblastoma cell line that is infectable by MV, allows replication of MV, and is tumorigenic when injected into the brain of immune-competent mice. The CSCG tumor is killed by MV in vitro and in vivo. In addition, this is the first MV-sensitive murine tumor model of any type that grows in immune-competent mice.

Brain tumors are the most common form of solid tumor in children. Medulloblastoma, with 350–500 new cases diagnosed annually in the United States, is the most common malignant pediatric brain tumor, accounting for nearly 20% of all childhood brain cancers.1–3 Advanced standard-of-care treatments including surgery, craniospinal irradiation, and chemotherapy have resulted in increased survival of up to 60%–70% of patients for 5 years postdiagnosis.4–6 However, many long-term survivors suffer from severe debilitating neurological and cognitive side effects associated with currently used treatments such as craniospinal radiation.7–9 Dissemination of medulloblastoma tumor cells in the subarachnoid space, diagnosed in about 20% of patients at presentation and in about 75% of patients at recurrence, is a grave negative prognostic factor.10,11 Less than 20% of children with subarachnoid disease survive beyond 5 years.12 Clearly, patients with medulloblastoma urgently need alternate effective therapies with fewer side effects for both primary and disseminated tumor.

The use of replication-competent oncolytic viruses (OV) has been recognized as a novel and unique anticancer therapeutic modality.13 OVs have multiple advantages over conventional approaches, including tumor-selective replication, post-cytolysis viral spread between tumor cells, an excellent safety profile, and the potential to stimulate an antitumor response by the host’s immune system.14 However, the overwhelming preclinical success has failed to translate into the clinic, and one major issue is the lack of appropriate immune-competent preclinical models.

We have reported on the therapeutic efficacy of a recombinant Edmonston vaccine strain of measles virus (MV) in mouse orthotopic models of medulloblastoma.15 In those studies, both irradiated severe combined immunodeficient (SCID) and athymic nude mice implanted with cells from human-derived medulloblastoma cell lines in the caudate/putamen demonstrated significantly increased survival when treated with a recombinant MV compared with those mice treated with an ultraviolet-inactivated form of the virus. Similarly, we have shown that medulloblastoma cells injected into the lateral ventricle of immune deficient mice show growth of tumor that recapitulates CSF disseminated disease in patients.16 Using this model, we have demonstrated that injection of MV into the lateral ventricle 3 or 14 days after injection of tumor cells significantly prolongs survival. We also showed that tumor cells in the lumbar subarachnoid space stained for viral N protein, indicating that the virus was capable of circulating throughout the subarachnoid space.

Unfortunately, MV does not grow well in murine cells. The first reason for the resistance of murine cells is the lack of a receptor for the virus. This issue has been overcome by the development of a transgenic mouse that expresses high levels of human CD46, the preferred receptor for the Edmonston strain of MV.17–19 However, though CD46 facilitates cellular entry of the virus, MV still does not replicate well in cells from this transgenic line. In the normal course of infection, MV makes a protein (V protein) that binds to and inactivates the human Stat1/Stat2 complex.20,21 The V protein does not bind to the murine complex. Because Stat1/Stat2 complex activation by interferon results in transcription of antiviral proteins, replication of MV in murine cells, in which the Stat1/Stat2 complex remains active, is prevented. The Sendai virus homolog of the V protein, the Sendai virus C protein (SVC), does bind to and inactivate the murine Stat1/Stat2 complex.22,23 In this paper, we take advantage of this property of SVC to manufacture a murine model of Group 3 medulloblastoma that is tumorigenic in immune-competent mice, that is infected by MV, that is permissive for the replication of MV, and that is killed by MV.

We chose to model Group 3 medulloblastoma as this subgroup has the worst prognosis and poorest outcome of the 4 molecular subtypes of medulloblastoma.24 Overexpression and/or amplification of MYC has been frequently implicated as a driver of Group 3 tumors. Recently, Northcott et al have shown that simultaneous expression of MYC with GFI1 or GFI1B, growth factor independence 1 proteins, was sufficient to form Group 3 tumors.25

To manufacture this model, cerebellar stem cells from human CD46 transgenic mice were transduced to express SVC and to overexpress stabilized c-Myc and Gfi1b proteins. A cell line was established (CSCG cells) that forms medulloblastoma-like tumors when injected into the brain of syngeneic CD46-transgenic mice. CSCG cells are highly proliferative, express stem cell markers, and express the markers of Group 3 medulloblastoma. This cell line allows replication of and is killed by MV, and a single intratumoral injection of MV significantly increased survival of tumor-bearing animals compared with control groups in vivo.

Materials and Methods

Preparation of CSCG Cell Line and Mouse Model

Animal-related procedures were performed according to guidelines of the Institutional Animal Care and Use Committee at UCSF. Immune-competent transgenic mice expressing human CD46 (hCD46TgIC) were obtained from the Mayo Clinic and bred at UCSF. The cerebella from five 6-days-old pups were minced, digested with papain, and filtered through a 40 μm cell strainer. Cells formed neurospheres on an ultra-low attachment surface (Corning) in Neurobasal-A (NBA) medium containing B-27 and N2 growth supplements (Life Technologies), epidermal growth factor (EGF) (Sigma-Aldrich) and basic fibroblast growth factor (bFGF) 2 (Miltenyi Biotec) (NBA media). Spheres were dissociated with Accumax (Innovative Cell Technologies), and cells expressing prominin-1 (Prom1; Miltenyi Biotec) were sorted by fluorescence activated cell sorting (FACS) as previously described.26 Prom1+ cerebellar stem cells were transduced with a lentivirus to express SVC, and firefly luciferase (System Biosciences) and transformed cells were selected with Geneticin-418 (400 μg/mL) antibiotic (CS cells). Next, the CS cells were co-transduced with retroviruses encoding a stabilized form of murine Myc (MycT58A) and murine Gfi1b in conjugation with tdTomato (tdT) and green fluorescent protein (GFP) markers, respectively.25 Cells double positive for tdT and GFP were flow-sorted and maintained in NBA media with G-418 (200 μg/mL). The stable expression of Myc and Gfi1b was monitored by florescence microscopy. To make a tumorigenic cell line, transformed cells were implanted in the cerebellum of immunodeficient NSG (NOD-scid IL2Rgammanull) mice using stereotaxic guidance, and tumor growth was measured with luciferase bioluminescence intensity (BLI). Tumor cells were harvested and a cell line, called CSCG, was established.

In Vitro Cell Killing and Virus Replication

CSCG cells were seeded at 4 × 105 cells per well in a 12-well format plate. Cells were infected with different multiplicities of infection (MOIs) of the Edmonston vaccine strain of measles virus expressing enhanced GFP (MV-GFP)27 for 3 h at 37°C in Opti-MEM media (Cell Culture Facility, UCSF). Control cells were incubated in Opti-MEM only. After 96 h of infection, the number of live cells was quantified with the Countess (Thermo Fisher Scientific). The survival from each treatment was calculated by dividing the number of viable cells in infected wells by the average of live cells in control wells. For virus replication, 105 cells were seeded per well in a 24-well plate and infected with MV-GFP at MOI of 0.1 for 3 h at 37°C. Virus was collected at 24-h intervals by 2 cycles of freezing and thawing of cell pellet in liquid nitrogen. After centrifugation at 5000× g to remove cell debris, supernatant was immediately used to determine viral titer by 50% tissue culture infective dose (TCID50) titration assay on Vero cells.

In Vivo Model

CSCG cells (2 × 105 per mouse) were injected into the caudate putamen (2 mm lateral of bregma at a depth of 3 mm from the skull) of 7- to 8-week-old hCD46TgIC mice. When the BLI increased by 10- to 20-fold higher than the initial value, animals were randomized into 3 treatment groups. A single intratumoral injection of phosphate buffered saline (PBS), 1.5 × 105 TCID50 of heat-inactivated MV-GFP (HI-MV), or 1.5 × 105 TCID50 of live MV-GFP was given in 6 μL volume. For heat inactivation, virus was incubated at 70°C for 20 min, and the loss of cellular infectivity was verified in vitro (data not shown). Animals were imaged weekly for tumor cell bioluminescence using the Xenogen IVIS Spectrum instrument (Caliper Life Sciences).

Intracellular Flow Cytometry

Cells were dissociated with Accumax, washed with PBS, fixed with 2% paraformaldehyde at room temperature (RT) for 15 min, and permeabilized with −20°C methanol for 15 min on ice. After washing with staining buffer (PBS + 0.1% Tween 20 + 1% bovine serum albumin) and centrifugation at 500 × g, cells were incubated with PBS containing 5% normal goat serum and 0.5% bovine serum albumin for 45 min to block nonspecific staining. Cells were stained with unconjugated primary antibodies for 60 min at 4°C followed by Alexa Fluor 647–conjugated secondary antibody incubation for 45 min at 4°C. Unbound antibody was washed off and cells were analyzed on the BD FACS Calibur flow cytometry system.

Immunostaining

For immunohistochemistry, brains were fixed overnight at 4°C in 10% formalin solution and then stored at 4°C in 70% ethanol. After paraffin-embedding, 5-µm-thick sections were affixed on glass slides and stored at −20°C. For staining, tissues were deparaffinized in xylenes, rehydrated in ethanol gradient (100%–25%) and water, and then subjected to heat-induced epitope retrieval using citrate buffer, pH 6.0 (#C999, Sigma-Aldrich) in a decloaking chamber (Biocare Medical) at 20 psi for 25 minutes. Slides were incubated with 10% goat serum for 1 h at RT to block nonspecific binding, followed by overnight incubation with primary antibodies at 4°C in a moist chamber. Antibodies used were anti–natriuretic peptide receptor 3 (NPR3) (1:200, Abcam, #ab37617), anti–gamma-aminobutyric acid receptor alpha 5 (GABRA5) (1:100, Abcam, #ab10098), and rabbit immunoglobulin G (IgG) isotype control (1:100, Abcam, #ab37415). After washing with Tris-buffer with 0.1% Tween-20, slides were incubated in 3% H2O2 for 10 minutes, washed, and then incubated in horseradish peroxidase (HRP)–conjugated goat anti-rabbit antibody (Signal Stain Boost IHC Detection Reagent, HRP, Rabbit #8114, Cell Signaling Technology) at RT for 45 minutes. Diaminobenzidine solution (SignalStain DAB Detection Kit, #8059, Cell Signaling Technology) was applied for 3–5 min. Tissues were then counterstained in Mayer’s hematoxylin for 1 min. After washing with water, all samples were dehydrated in ethanol (25%–100%) and xylenes for mounting using cytoseal 60 media. For immunoflorescence staining, sterile coverslips in a 24-well plate were coated with 0.1% poly-L-lysine (Sigma-Aldrich) for 1 h at RT. After dissociation of neurospheres with Accumax, single cells were added to coverslips for 4 h at 37°C. Adhered cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.25% Triton X-100 for 10 min, and blocked using 5% normal goat serum for 1 h. Primary antibodies were incubated overnight at 4°C, washed and incubated with a fluorophore-conjugated secondary antibody for 1 h at RT. Coverslips were mounted on glass slides with ProLong Gold antifade media containing 4′,6′-diamidino-2-phenylindole (Molecular Probes).

Results

Preparation and Characterization of Medulloblastoma Cell Line from Immune-Competent Mice

To make an MV-susceptible immune-competent mouse model of medulloblastoma, Prom1+ cerebellar stem cells from P6 hCD46TgIC mice were transfected to express SVC, and murine stabilized Myc and Gfi1b proteins. The stable cells grow as neurospheres in the presence of bFGF and EGF and express Myc and Gfi1b as shown by the florescence of conjugated markers tdT and GFP, respectively (Fig. 1A). Next, we examined if these cells are tumorigenic in mice. No tumor could be detected when cells were implanted directly from culture into the cerebellum of hCD46TgIC mice. However, when injected into the cerebellum of immune-deficient NSG mice, large tumors were formed. A cell line, CSCG, was established from the NSG tumors. CSCG cells were highly tumorigenic when injected into the brains of hCD46TgIC mice (Fig. 1B). The immunoblotting of total cell lysates confirmed overexpression of Myc and Gfi1b proteins in CSCG in comparison to CS cells (Fig. 1C). The expression of Gfi1, MycN, and p53 remained unchanged after modification with Myc and Gfi1b.

Fig. 1 .

Fig. 1

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Overexpression of Myc and Gfi1b transforms cerebellar stem cells. (A) The CS and CSCG cells grow as spheres in the presence of EGF and bFGF growth factors. The red (tdT) and green (GFP) florescence show expression of Myc and Gfi1b proteins, respectively. Scale bar = 50 μm. CS: Cerebellar stem cells expressing Sendai virus c-protein; CSCG: Cerebellar stem cells expressing Sendai virus c-protein, Myc, and Gfi1b. (B) CS cells expressing Myc, and Gfi1b proteins form tumors in the cerebellum of NSG immunodeficient mice. The resulting cell line prepared from NSG brain tumors, called CSCG, is highly tumorigenic in hCD46Tg mice. Tumor growth was monitored by bioluminescence imaging. (C) Western blots showing higher expression of Myc and Gfi1b in CSCG cells compared with the parental CS cells. The expression level of MycN, Gfi1, and p53 proteins were the same in both cell lines.

The rate of proliferation of CSCG cells was compared with that of CS cells. The number of live CSCG cells increased by 19-fold in 72 hours (equivalent to more than 4 population doublings), whereas CS cells, expressing SVC but not Myc and Gfi1b, showed only a 3.4-fold increase (less than 2 doublings) in the same period (Fig. 2A). This suggests that Myc and Gfi1b, and not SVC, drives the high proliferation of CSCG cells.

Fig. 2.

Fig. 2

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Myc and Gfi1b enhance proliferation and self-renewal of CSCG cells. (A) CS and CSCG cells were allowed to grow for 72 h, and the number of live cells was counted by trypan blue exclusion method. The graph shows the mean increase in the number of viable cells. N = 3, error bars = SD. (B) CSCG cells were seeded at a subclonal density of 2 cells/mm2 and neurosphere formation from single cells was monitored at 24-h intervals with a florescence microscope. Images were captured with a Zeiss monochrome camera. Scale bar = 50 μm. (C) Cells were immobilized on poly-L-lysine coated glass coverslips and immunostained for the expression of stem cell markers (Sox2 and nestin), and the proliferation marker (Ki67). No staining was observed with an IgG isotype control antibody that was used to check for nonspecific staining. Images were captured with a Zeiss AxioImager M1 microscope. Scale bar = 50 μm. The percentage of cells expressing Ki67, Sox2, and nestin were quantified by flow cytometry, and histograms are shown below the respective florescence panel (gray = IgG isotype; black = marker).

The ability of self-renewal is a putative characteristic of stem cells. When seeded at a subclonal density (2 cells/mm2) in NBA media, single CSCG cells formed neurospheres in as early as 96 h after seeding (Fig. 2B). The sphere-forming ability of single CSCG cells was monitored for 4 consecutive weeks after repeated dissociation and reseeding suggesting stem cell–like behavior of CSCG cells (data not shown). In addition, these cells show positive immunofluorescence staining for the proliferation marker Ki67, sex determining region Y–box 2 (Sox2), and nestin, proteins widely used as markers for neural stem cells (Fig. 2C). The quantification of positive cell staining by flow cytometry showed that 94.9%, 98.7%, and 93% of cells expressed Ki67, Sox2, and nestin markers, respectively (Fig. 2C).

MV Replicates in and Kills CSCG Cells

We verified by flow cytometry that CS cells express high levels of human MV receptor CD46 on the cell surface (Fig. 3A). To determine that CSCG cells allow replication of measles virus, cells were infected at MOI 0.1 and the titer of infectious virus particles was quantified at 24-h intervals for 4 days. The titer increased with time, peaking at 72 h and then dropping at 96 h, presumably due to extensive cell death caused by the replicating virus (Fig. 3B).

Fig. 3.

Fig. 3

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Measles virus replicates in, and kills, CSCG cells. (A) Flow cytometry showing live CS cells express high levels of CD46, the receptor for MV entry. (B) The titer (TCID50/mL) of infectious measles virus from infected CSCG cells increased with time, indicating replication of the virus in CSCG cells. MV-GFP infects and kills CSCG cells in a dose-dependent (C) and time-dependent manner (D). N = 3. Error bars show SEM, *P ≤ 0.05.

CSCG cells are highly susceptible to measles virus (MV-GFP) in a dose-dependent manner. Infection with an MOI 0.1, 1, and 10 killed 55%, 85%, and 92% of cells, respectively, after 96 h of incubation (Fig. 3C). Similarly, a time-dependent increase in cell killing with 49% at 24 h, and 92% at 96 h post-infection was observed when CSCG cells were infected at MOI 1 with MV-GFP (Fig. 3D). These results show that CSCG cells allow replication of and are killed by MV.

MV Significantly Increases Survival of CSCG Tumor-Bearing Mice

CSCG cells (2 × 105/mouse) were implanted in the brains of hCD46TgIC mice, and BLI was recorded to monitor tumor growth. When the BLI intensity increased by 10- to 20-fold higher than initial value, mice were randomized and treated with a single intratumoral injection of 1.5 × 105 TCID50 of live MV-GFP or HI-MV. Another set of mice were included that received equal volume of PBS as a control. Animals were monitored for tumor regression and survival. The decrease in the intensity of luciferase bioluminescence of CSCG cells demonstrated that mice treated with MV-GFP exhibited a significant decrease in tumor burden compared with mice in control groups (Fig. 4A). Mice treated with MV-GFP had significantly prolonged survival compared with animals that received HI-MV (log-rank test P = 0.0027) or PBS (log-rank test P = 0.0018). The median survival for PBS-treated and HI-MV-injected mice was 32 days ± 4.5d, and 34 days ± 2.9d (log-rank test P = 0.5273), respectively (Fig. 4B). In the MV-treatment group, 3 out of 5 animals had complete tumor regression and remained alive and free of tumor for the maximum period of the study (150 days), hence median survival could not be calculated. One mouse in this group had transient abolition of tumor for 9 weeks posttreatment before tumor recurred and the mouse died after 84 days. The last mouse in this group also showed complete tumor regression but died because of intracranial hemorrhage due to an unknown reason at 60 days posttreatment. One mouse in the HI-MV–treated group suffered excessive bleeding and died after virus injection, hence was excluded from the analysis.

Fig. 4.

Fig. 4

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Measles virus increases survival of CSCG tumor-bearing immune-competent mice. (A) Animals were implanted with 2 × 105 cells in the caudate putamen and tumor growth was measured by luciferase bioluminescence intensity (BLI). Animals were treated with a single intratumoral injection of PBS, 1.5 × 105 heat-inactivated virus (HI-MV), or 1.5 × 105 live MV-GFP. The tumor burden in the PBS group increased with time, whereas treatment with MV-GFP caused a significant decrease in the BLI or complete regression of the tumor. (B) Kaplan–Meier survival curve of treatment groups. A median survival of 32 and 34 days was observed for animals treated with PBS or HI-MV (log-rank test P = 0.53), respectively. Treatment with MV-GFP resulted in complete tumor regression and significant prolonged survival of animals compared with treatments with PBS (P = 0.0018) or HI-MV (P = 0.0027).

In the brains of mice treated with either PBS or HI-MV, hematoxylin and eosin (H&E) sections revealed large primitive neuroepithelial tumors composed of densely packed small, round, undifferentiated cells with mild nuclear pleomorphism, numerous mitotic figures, and scattered apoptotic bodies. In contrast, no tumor cells were evident in MV-treated mice brains (Fig. 5A).

Fig. 5.

Fig. 5

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CSCG cells form Group 3 medulloblastoma tumors in mice brain. (A) H&E staining of post-autopsy mouse brains from each treatment group. A large field of view by tile scan was captured with a 5× objective (left panel, scale bar = 500 μm), and a zoomed-in image was taken with a 40× objective (right panel, scale bar = 20 μm) with the Zeiss AxioImager M1 microscope. MV-GFP treatment resulted in complete abolition of tumor, whereas a large area of densely packed undifferentiated tumor cells was observed in PBS and HI-MV-treated mice. (B) Western blotting exhibited significantly increased expression of NPR3 and GABRA5, molecular markers of Group 3 medulloblastoma, in CSCG cells compared with CS cells. Also, no sign of increased apoptosis was observed in CSCG cells in vitro. (C) Immunohistochemistry of the brains from tumor-bearing animals showed positive staining for NPR3 and GABRA5 in the tumor cells. A zoomed-in area of staining captured at 40× is shown in insets. The adjacent normal brain tissues (NT) were negative for these markers. Scale bar = 50 μm.

On the molecular level, western blotting demonstrated that CSCG tumors express higher levels of the well-established Group 3 medulloblastoma markers, NPR3 and GABRA5, compared with CS cells (Fig. 5B). Immunohistochemistry of brain tissues exhibited high expression of NPR3 and GABRA5 in CSCG tumor cells, whereas the nontumor (NT) regions remained negative for these markers (Fig. 5C). Also, we did not observe any sign of increased apoptosis in Myc-transformed CSCG cells (Fig. 5B). These results suggest that CSCG cells grow and form tumors in the brains of immune-competent mice and are killed by MV in vivo, and that the CSCG tumors represent the highly aggressive Group 3 subtype of medulloblastoma.

Discussion

The preclinical efficacy of oncolytic MV against medulloblastoma15,16 and many other cancer types28 has been shown, but those studies were done in immune-compromised xenograft models. Given that anti-measles immunity is prevalent in the human population, results from immunodeficient models may not translate to human disease. The lack of any immune-competent murine model has been an obstacle to investigating the effect of the immune system on MV antitumor therapy of medulloblastoma. The biosafety of intracerebral and intrathecal injection of Edmonston vaccine strain of measles virus has been extensively evaluated in preclinical models including transgenic measles-sensitive mice and rhesus macaques.29–32 No evidence of systemic clinical or biochemical toxicity, behavioral symptoms, or neurotoxicity was observed in these studies, which laid the foundation for investigation of modified MVs in several clinical trials in both solid and hematological cancers.33

Among the 4 molecularly distinct subtypes of medulloblastoma, Group 3 tumors have the worst prognosis.24 The overexpression of MYC and GFI1/GFI1B has been reported as driver events in these highly aggressive tumors.25 In this paper, we report the successful manufacture of a murine cell line that mimics Group 3 medulloblastoma and is permissive for the infection and replication of oncolytic MV. In addition, this cell line forms rapidly growing tumors in the mouse brain. These tumors can be effectively treated with MV in these immune-competent mice.

The starting cells for the CSCG cell line are cerebellar stem cells isolated from transgenic mice expressing human CD46, the receptor for the Edmonston strain of measles virus in humans.34 Thus, by using these transgenic mice, we overcame the first barrier to growth of MV in murine cells. The P-gene of MV also encodes for 2 nonstructural proteins, V and C proteins. The V protein of MV binds to and inactivates the Stat1/Stat2 complex in human cells.21,35,36 Interferon signaling activates this complex, and MV requires prevention of complex activity for replication. Because the measles virus V protein does not bind to murine Stat1/Stat2, MV does not replicate well in murine cells, because the complex remains active. To overcome this barrier, we transduced cells with the SVC. This protein has the same function as the V protein of MV, but it does bind to and inactivate the murine Stat1/Stat2 complex.22,23 Therefore, the cell line reported here is infectable by MV, as it expresses the human MV receptor and is permissive for replication of MV, as the activity of the Stat1/Stat2 complex is prevented by SVC. This cell line and the model presented here can be used to examine the interaction of MV and the intact immune system. We recognize that the expression of the SVC protein may induce an immune response in host mice. This will be investigated in our ongoing studies of the effect of the immune system on MV therapy in this model.

Pei et al and Kawauchi et al reported that overexpression of Myc in TP53-deficient mice or in mice with dominant negative p53 protein forms tumors with a high degree of similarity to human Group 3 medulloblastoma in terms of gene expression profile and molecular pathology.37,38 However, Tabori et al showed that in clinical samples none of the TP53-mutated tumors showed MYC amplification and that tumors with TP53 mutation presented with aggressive but locally recurrent disease.39 Though mutation or deficiency of p53 is associated with a negative prognosis, these alterations are almost exclusively present in the sonic hedgehog and Wnt subtypes of medulloblastoma.24,40–42 In comparison, the CSCG cell line presented here has intact p53 and forms rapidly growing tumors with elevated expression of NPR3 and GABRA5 (Fig. 5B, C), molecular markers of Group 3 medulloblastoma.43,44 Pei et al reported that cell lines with overexpression of Myc had high rates of apoptosis and, thus, were not tumorigenic in mice.37 However, CSCG cells do not show increased signs of apoptosis compared with parental cells that expresses SVC, but not Myc and Gfi1b proteins (Fig. 5B).

To study the pathogenesis and antitumor efficacy of MV oncolysis, transgenic mouse strains have been developed with either organ-specific or ubiquitous expression of human CD46 receptor and/or inhibition of the innate interferon pathway.45 However, in most of these models, MV failed to grow in the brain.46 Therefore, studies in immune-competent murine models have been limited to using retargeted MVs armed with a cytotoxic prodrug-convertase with cyclophosphamide immunosuppression in a subcutaneous colon carcinoma model,47 or using a CD20-retargeted MV encoding antibodies against cytotoxic T lymphocyte antigen 4 and programmed death ligand 1 in a melanoma model.48 However, the replication of retargeted virus was limited in murine cells, and the efficacy was dependent on the therapeutic genes in the engineered viral genome.

The CSCG murine medulloblastoma cell line described in this paper allows efficient intracellular replication of MV due to SVC-mediated inactivation of the interferon pathway. Experiments are ongoing to assess the antitumor efficacy of MV on CSCG tumors in measles-naive and measles-immune animals. This model is well suited to examine the positive or negative effects of the intact immune system on the therapeutic efficacy of MV. In human patients, leptomeningeal dissemination of tumor cells is diagnosed at a higher rate in Group 3 and 4 medulloblastoma.49 However, in the CSCG model, we did not find evidence of leptomeningeal spread of tumor cells by bioluminescence imaging. This could be because tumor grew aggressively and animals died quickly after tumor cell implantation.

In conclusion, CSCG cells represent the first murine tumor cell line and tumor model of any tumor type that is permissive for MV replication and that grows in immune-competent mice. Importantly, the strategies used here are easily generalizable to other tumor types for which the driving mutations are known.

Funding

This project was supported by the startup grant from the Department of Neurological Surgery, and a Resource Allocation Program grant, awarded to Corey Raffel from UCSF.

Acknowledgment

We thank Robert Wechsler-Reya for providing retroviral plasmids of MycT58A and Gfi1b.

Conflict of interest statement. The authors do not have any conflicts to disclose.

References

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