Research ArticleCancer

Intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade

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Science Translational Medicine  03 Jan 2018:
Vol. 10, Issue 422, eaam7577
DOI: 10.1126/scitranslmed.aam7577
  • Fig. 1 Intravenous delivery of reovirus to primary and secondary brain tumors.

    (A) Representative immunohistochemistry (IHC) and in situ hybridization (ISH) trial and control patient tumor sections stained for reovirus σ3 protein (brown, top) and reovirus RNA (blue, bottom). The “control breast met” is a metastasis to the brain from a breast cancer primary. Arrows point to examples of positive cells/positive areas of tissue. Scale bars, 20 μm. HGG, high-grade glioma. (B) Trial and control patient tumor immunogold-TEM images for reovirus σ3 protein (arrows). CRC, colorectal cancer. Scale bars, 200 nm. (C) Quantitative reverse transcription polymerase chain reaction for reovirus σ3 gene, using whole tumor RNA. Data indicate femtograms (fg) reovirus RNA per microgram of whole tumor RNA. Histogram shows the mean of triplicate samples, and error bars indicate SD. (D) Representative immunofluorescence (IF) of trial patient tumor sections showing staining for reovirus RNA (blue), reovirus σ3 protein (red), and their coexpression (yellow). Scale bars, 80 μm.

  • Fig. 2 Correlation of reovirus RNA/protein with proliferating tumor cells.

    (A) Trial patient IHC tumor sections stained for Ki67 (brown) with indicated percentages of cells positive for Ki67 and reovirus σ3 protein (from table S3), showing examples of tumors with high reovirus σ3 staining (top) and no reovirus σ3 protein staining (bottom). Scale bars, 40 μm. GBM, glioblastoma multiforme. (B) Scatter plot and line of best fit, correlating the percentages of tumor cells positive by IHC for reovirus RNA or σ3 protein and for Ki67. (C) Representative tumor sections derived from trial patient 9 (high Ki67; top), trial patient 1 (intermediate Ki67; middle), and trial patient 4 (low Ki67; bottom), showing IF staining for reovirus RNA (blue), Ki67 (red), or their coexpression (yellow, arrows). Scale bars, 40 μm. (D) Representative trial patient tumor IF staining for tubulin (fluorescent red), reovirus σ3 protein (fluorescent green), and their coexpression (yellow). Nuclear counterstaining is blue. Scale bars, 40 μm (top and bottom) and 80 μm (middle).

  • Fig. 3 Tumor immune cell infiltration.

    (A) Fold change in cell-surface intercellular adhesion molecule (ICAM) expression on CD4 and CD8 T cells from trial patients’ peripheral blood. (B) Trial and control patient IHC tumor sections stained for CD3 (brown). Scale bars, 20 μm. “V” indicates blood vessel. (C) Trial and control patient IHC tumor sections stained for CD8 (brown). Scale bars, 20 μm.

  • Fig. 4 Expression of cleaved caspase 3, PD-L1, and PD-1 in HGGs after reovirus treatment.

    (A) Representative trial and control patient HGG sections stained for cleaved caspase 3 (brown) by IHC. Scale bars, 60 μm. (B) Representative trial and control patient HGG sections stained by IHC for programmed death ligand 1 (PD-L1) (brown). Scale bars, 30 μm. (C) Representative (one of three samples tested) flow cytometry for PD-L1 on GBM tumor-infiltrating lymphocytes (TILs) (bottom) or peripheral blood mononuclear cells (PBMCs) (top) derived from the same patient, after stimulation for 48 hours using 1 plaque-forming unit per cell reovirus. PBS, phosphate-buffered saline; Ab, antibody. (D) Representative trial and control patient HGG sections stained by IHC for programmed cell death protein 1 (PD-1) (brown). Scale bars, 30 μm. (E) Representative flow cytometry for PD-L1 on GBM1 cells after stimulation with combinations of purified interferon (IFN)–α/IFN-β/IFN-γ for 24 hours, each at 100 pg/ml. (F) Representative flow cytometry for PD-L1 on GBM1 cells after stimulation with ex vivo HGG-derived conditioned medium (CM) or reovirus-conditioned medium (RCM) for 24 hours (at a concentration of 1:4 of CM to native medium). (G) Representative flow cytometry for PD-L1 on GBM1 cells after stimulation using PBMC-derived CM or RCM for 24 hours (at a concentration of 1:4 of CM to native medium) with blockade of IFN-α+β/γ/α+β+γ or equivalent isotype controls.

  • Fig. 5 Combination intravenous reovirus and checkpoint inhibition in an orthotopic syngeneic brain tumor model.

    C57/BL6 reovirus-vaccinated mice (22) were injected with GL261 cells intracranially on day 1 and treated using combinations of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus intravenous reovirus and/or anti–PD-1 antibody. (A) Kaplan-Meier survival plot, with Mantel-Cox comparison of survival curves: control versus anti–PD-1 (P = 0.4617), control versus GM-CSF/reovirus (P = 0.0012), control versus GM-CSF/reovirus + anti–PD-1 (P < 0.0001), GM-CSF/reovirus versus GM-CSF/reovirus + anti–PD-1 (P < 0.0001), and anti–PD-1 versus GM-CSF/reovirus + anti–PD-1 (P < 0.0001). (B) Representative brain tumor hematoxylin and eosin–stained sections from PBS-treated and GM-CSF/reovirus-treated mice. Black arrows mark vascular endothelial cells; white arrows mark lymphocytes. Scale bars, 30 μm. (C) Flow cytometry quantification of CD3+ CD4+ IFN-γ+ or CD3+ CD8+ IFN-γ+ TILs from PBS-treated or GM-CSF/reovirus-treated mice. Graph shows the mean ± SD of four samples.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/422/eaam7577/DC1

    Fig. S1. Selective intravenous delivery of reovirus to intracranial melanoma in immunocompetent mice.

    Fig. S2. Reovirus carriage by WBC subsets and changes in serum IFN-α.

    Fig. S3. Secondary antibody–only control immunogold-TEM images from trial patient brain tumors.

    Fig. S4. Reovirus protein expression in endothelial cells.

    Fig. S5. Negative controls for reovirus coexpression.

    Fig. S6. Correlation of reovirus RNA/protein with time between infusion and surgery.

    Fig. S7. CD68 tumor-infiltrating cells.

    Fig. S8. Expression of cleaved caspase 3 and PD-L1 in brain tumor metastases after reovirus stimulation.

    Fig. S9. In vitro expression of PD-L1 and PD-1 in human-derived cell lines and healthy donor PBMCs after reovirus stimulation.

    Fig. S10. In vitro expression of PD-L1 on GBM1 cells after purified IFN or CM stimulation.

    Table S1. Participant baseline clinical characteristics, grade 3 or 4 adverse events, and survival after reovirus infusion.

    Table S2. Change in plasma inflammatory cytokines and chemokines after intravenous reovirus infusion.

    Table S3. Presence of reovirus protein and RNA in resected brain tumors.

    Table S4. Ki67, cleaved caspase 3, immune cell infiltration, and PD-1/PD-L1 expression in resected trial and control brain tumors.

    Table S5. RNA-seq expression data (provided as an Excel file).

    Table S6. Enriched biological processes within genes differentially expressed between control and trial GBM tumors.

  • Supplementary Material for:

    Intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade

    Adel Samson,* Karen J. Scott, David Taggart, Emma J. West, Erica Wilson, Gerard J. Nuovo, Simon Thomson, Robert Corns, Ryan K. Mathew, Martin J. Fuller, Timothy J. Kottke, Jill M. Thompson, Elizabeth J. Ilett, Julia V. Cockle, Philip van Hille, Gnanamurthy Sivakumar, Euan S. Polson, Samantha J. Turnbull, Elizabeth S. Appleton, Gemma Migneco, Ailsa S. Rose, Matthew C. Coffey, Deborah A. Beirne, Fiona J. Collinson, Christy Ralph, D. Alan Anthoney, Christopher J. Twelves, Andrew J. Furness, Sergio A. Quezada, Heiko Wurdak, Fiona Errington-Mais, Hardev Pandha, Kevin J. Harrington, Peter J. Selby, Richard G. Vile, Stephen D. Griffin, Lucy F. Stead, Susan C. Short,* Alan A. Melcher*

    *Corresponding author. Email: a.samson{at}leeds.ac.uk (A.S.); alan.melcher{at}icr.ac.uk (A.A.M.); s.c.short{at}leeds.ac.uk (S.C.S.)

    Published 3 January 2018, Sci. Transl. Med. 10, eaam7577 (2018)
    DOI: 10.1126/scitranslmed.aam7577

    This PDF file includes:

    • Fig. S1. Selective intravenous delivery of reovirus to intracranial melanoma in immunocompetent mice.
    • Fig. S2. Reovirus carriage by WBC subsets and changes in serum IFN-α.
    • Fig. S3. Secondary antibody–only control immunogold-TEM images from trial patient brain tumors.
    • Fig. S4. Reovirus protein expression in endothelial cells.
    • Fig. S5. Negative controls for reovirus coexpression.
    • Fig. S6. Correlation of reovirus RNA/protein with time between infusion and surgery.
    • Fig. S7. CD68 tumor-infiltrating cells.
    • Fig. S8. Expression of cleaved caspase 3 and PD-L1 in brain tumor metastases after reovirus stimulation.
    • Fig. S9. In vitro expression of PD-L1 and PD-1 in human-derived cell lines and healthy donor PBMCs after reovirus stimulation.
    • Fig. S10. In vitro expression of PD-L1 on GBM1 cells after purified IFN or CM stimulation.
    • Table S1. Participant baseline clinical characteristics, grade 3 or 4 adverse events, and survival after reovirus infusion.
    • Table S2. Change in plasma inflammatory cytokines and chemokines after intravenous reovirus infusion.
    • Table S3. Presence of reovirus protein and RNA in resected brain tumors.
    • Table S4. Ki67, cleaved caspase 3, immune cell infiltration, and PD-1/PD-L1 expression in resected trial and control brain tumors.
    • Table S6. Enriched biological processes within genes differentially expressed between control and trial GBM tumors.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S5 RNA-seq expression data (provided as an Excel file).

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