Zika virus has oncolytic activity against glioblastoma stem cells

Abstract

Glioblastoma is a highly lethal brain cancer that frequently recurs in proximity to the original resection cavity. We explored the use of oncolytic virus therapy against glioblastoma with Zika virus (ZIKV), a flavivirus that induces cell death and differentiation of neural precursor cells in the developing fetus. ZIKV preferentially infected and killed glioblastoma stem cells (GSCs) relative to differentiated tumor progeny or normal neuronal cells. The effects against GSCs were not a general property of neurotropic flaviviruses, as West Nile virus indiscriminately killed both tumor and normal neural cells. ZIKV potently depleted patient-derived GSCs grown in culture and in organoids. Moreover, mice with glioblastoma survived substantially longer and at greater rates when the tumor was inoculated with a mouse-adapted strain of ZIKV. Our results suggest that ZIKV is an oncolytic virus that can preferentially target GSCs; thus, genetically modified strains that further optimize safety could have therapeutic efficacy for adult glioblastoma patients.

Figure 1.

ZIKV causes loss of GSC self-renewal and proliferation. (A and B) GSCs were uninfected (A) or infected (B) with ZIKV-Dakar (7 dpi). (C–F) GSCs uninfected (C and E) or infected (D and F) with ZIKV-Dakar for 48 h underwent immunofluorescence staining for ZIKV envelope protein (green) and DAPI (blue; C–F) with Sox2 (red; E and F). Brightfield and immunofluorescence images in A–F are representative of three independent experiments. (G and H) Relative cell number of paired GSCs (387, 3565, and 4121; G) and DGCs (H), infected with ZIKV-Dakar or ZIKV-Brazil, at an MOI of 5 for 7 d; all data were normalized to day 0. Data from G and H are from three independent experiments. (I) Sphere formation capacity of 387, 3565, and 4121 GSCs infected with indicated ZIKV strains or control. Data are from three independent experiments. (J–M) GSCs uninfected (J and L) or infected (K and M) with ZIKV-Dakar for 48 h underwent staining for ZIKV (green) and DAPI (blue) with Ki-67 (red; J and K) or AC3 (red; L and M). J–M are representative of three independent experiments. (N) At day 4, the frequency of Sox2⁺, Ki-67⁺, and AC3⁺ cells was measured by visual quantification in the three GSC lines with or without ZIKV infection. Data are derived from experiments performed in duplicate and pooled from three independent experiments. Error bars indicate SDs. Significance was analyzed with one-way ANOVA with Tukey’s multiple comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Bars: (A and B) 725 µm; (C–F and J–M) 100 µm.

Figure 2.

ZIKV infection causes loss of self-renewal and proliferation of human glioblastoma-derived organoids. (A–F) Brightfield images of GSC organoids after infection with ZIKV. GSCs were incubated in Matrigel for 3 d (A) or 3 wk (B). Organoids were infected with ZIKV-Brazil (C and E) or ZIKV-Dakar (D and F) 2 (C and D) or 4 (E and F) wk after infection. Images in A–F are representative of two independent experiments. (G) Organoid areas at 2 or 4 wk after ZIKV infection were determined for three GSC organoid models (387, 3565, and 4121). Data represent two independent experiments. (H–O) Representative images of uninfected control and ZIKV-Dakar-infected GSC organoids 2 wk after infection stained for ZIKV (green) and DAPI (blue; H–O) with Sox2 (red; H and I), AC3 (red; J and K), Ki-67 (red; L and M), or GFAP (red; N and O). Images are representative of three independent experiments. (P) Quantification of Sox2⁺, Ki-67⁺, AC3⁺, and GFAP⁺ subpopulations of DAPI⁺ cells; n = 6 organoids for each condition from two independent experiments. Values represent mean ± SD. Significance was analyzed with one-way ANOVA with Tukey’s multiple comparison test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Bars: (A–F) 725 µm; (H–O) 200 µm.

Figure 3.

ZIKV infects isolated human glioblastoma but not normal brain tissue slices. (A–C) Representative images from three independent experiments showing freshly resected glioblastoma after staining with H&E (A) or for Ki-67 (B) or GFAP (C). (D–R) Immunofluorescence staining of glioblastoma tissue uninfected (D–F) or infected with ZIKV-Dakar (G–L) or ZIKV-Brazil (M–R) after 7 d for ZIKV E protein (green) and DAPI (blue) with Sox2 (red; D, G, J, M, and P), Ki-67 (red; E, H, K, N, and Q), or GFAP (red; F, I, L, O, and R). D–R are representative images from three independent experiments. (S) Quantification of tumor cells infected with ZIKV (left) and ZIKV-infected cells that co-stain for Sox2, Ki-67, or GFAP (right). Values represent mean ± SD and are pooled from three independent experiments. (T–Z) Representative images showing freshly resected normal brain after staining with H&E (T) or for Ki-67 (U) or GFAP (V). (W–Z) Normal brain tissue uninfected (W and X) or infected with ZIKV-Dakar (Y and Z) after 7 d stained for ZIKV (green) and DAPI (blue) with NeuN (red; W and Y) or GFAP (red; X and Z). Images in W–Z are representative of three independent experiments. Significance was analyzed with one-way ANOVA with Tukey’s multiple comparison test (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). Bars: (A–I and R–Z) 100 µm.

Figure 4.

Mouse-adapted ZIKV-Dakar attenuates growth of mouse glioma cells and prolongs survival of mice with glioma in vivo. (A) Mouse glioma cells (C57BL/6 background: GL26, GL261, and CT-2A), microglial cells (BV2), and MS-DNCs were infected with the parental or mouse-adapted ZIKV-Dakar, and relative cell number was assessed over 1 wk, normalized to day 0. (B) Viral titer from supernatants of ZIKV-Dakar-infected cells (GL26, GL261, CT-2A, BV2, and MS-DNCs) was measured at 1 wk by FFA. (C–I) Mouse glioma model with GL261 and CT-2A. 1 wk after implantation, bioluminescence imaging (BLI; C) and H&E staining (D and E) demonstrating glioma. 3 wk after GL261 (F and G) and CT-2A (H and I) implantation without (F and H) or with mouse-adapted ZIKV-Dakar treatment (G and I). (J, left) Mice bearing GL261 glioma were treated with PBS (n = 15) or 10³ FFU of the mouse-adapted-ZIKV-Dakar (n = 18). (J, right) Mice bearing CT2A glioma were treated with PBS (n = 7) or 10³ FFU of the mouse-adapted ZIKV-Dakar (n = 8). (K) Mice bearing GL261 glioma were treated with PBS (n = 9) or 10⁵ FFU of the mouse-adapted ZIKV-Dakar (n = 9). (L–S) Immunofluorescence staining of GL261 glioma tumor–bearing mice at the endpoint after treatment with PBS control (L, N, and P) or 10³-FFU–adapted ZIKV-Dakar (M, O, Q, R, and S) for ZIKV (green) with DAPI (blue; L–Q), Sox2 (red; L, M, and R), GFAP (red; N and O), Ki-67 (red; P, Q, and S), and BrdU (blue; R and S). (T) At survival endpoint (J, left), quantification of cells infected with ZIKV; cells positive for BrdU and Ki-67 (left); and ZIKV-infected cells positive for Sox2, Ki-67, or BrdU (right). (U, left) Representative low- and high-power images of in situ hybridization staining for viral RNA in mice with CT2A glioma 2 wk after treatment with ZIKV-Dakar or PBS (representative of two experiments). Arrow indicates positive staining. (U, right) Representative high-power images of cleaved caspase-3 staining on the same tumors. In vitro experiments were pooled from three independent experiments performed in duplicate. Animal survival data were pooled from two independent experiments. Quantification of immunostaining was derived from six mice. Values represent mean ± SD. Significance was analyzed by one-way ANOVA with Tukey’s multiple comparison test for A and B and the log-rank test for J and K (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Bars: (D, F–I) 200 µm; (E; L–S; U, left; and U, right) 100 µm; (U, middle) 10 µm.

Figure 5.

ZIKV-E218A inhibits the growth of GSCs and has additive effects with TMZ. (A and B) GSCs were mock treated or incubated with parental ZIKV (MOI of 5), ZIKV-E218A (MOI of 5), TMZ (250 µM), or ZIKV-E218A (MOI of 5) and TMZ (250 µM) combined (E218AT). After 1 wk, GSC lines (387, 3565, and 4121) were assayed for relative cell number normalized to day 0 (A) and sphere formation (B). (C–F) Immunofluorescence staining of uninfected control (C and E) and ZIKV-E218A treated (D and F) GSCs on day 7 for Sox2 (red), DAPI (blue), and ZIKV-E218A (green). (G and H) Immunofluorescence staining of ZIKV-E218A-infected GSCs without (G) and with TMZ (250 µM; H) on day 7 for AC3 (red), DAPI (blue), and ZIKV-E218A (green). (I) Quantification of AC3⁺ apoptotic cells in three GSC lines treated with TMZ, ZIKV-E218A, or ZIKV-E218A combined with TMZ (E218AT). (J) Viral titer from supernatants of parental ZIKV-infected and ZIKV-E218A-infected GSCs over 1 wk measured by FFA. For every panel, data were pooled from three independent experiments performed in duplicate. Values represent mean ± SD. Significance was analyzed with one-way ANOVA with Tukey’s multiple comparison test (A, B, and I) and two-way ANOVA with the Bonferroni multiple comparison test (J; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Bars: (C–H) 100 µm.