Research ArticleCancer

Neoadjuvant oncolytic virotherapy before surgery sensitizes triple-negative breast cancer to immune checkpoint therapy

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Science Translational Medicine  03 Jan 2018:
Vol. 10, Issue 422, eaao1641
DOI: 10.1126/scitranslmed.aao1641
  • Fig. 1 Localized treatment of TNBC cancers kills tumor cells and minimizes the metastatic burden.

    (A) Hematoxylin and eosin (H&E) and immunostaining showing the expression of Maraba proteins and cytokeratin-18 in a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) treated intratumorally with Maraba (n = 2). Scale bars, 200 μm. NV, no virus. (B to D) Quantification of average non-necrotic regions based on H&E staining obtained for the TNBC PDX (B), the luminal B breast cancer PDX (C), or 4T1 tumors (D) (n = 10; Mann-Whitney two-tailed U test, ***P < 0.005). PBS, phosphate-buffered saline. (E and F) Maraba was administered intratumorally to 4T1 tumors before resection, and the spontaneous lung metastases were visualized by histology (E) (n = 6; Mann-Whitney two-tailed U test, **P < 0.01) or India ink perfusion (F) (n = 5 for control and n = 6 for Maraba-treated mice; Mann-Whitney two-tailed U test, *P < 0.05). (G and H) The experiment was repeated in the EMT6 (G; n = 10) and E0771 (H; n = 10) tumor models.

  • Fig. 2 Maraba treatment results in complete responses in the window of opportunity setting.

    (A) Schematic representation of the treatment schedule used for the tumor rechallenge model. (B to D) Tumor growth and Kaplan-Meier survival curves obtained using 4T1 (B), EMT6 (C), or E0771 (D) cells in the tumor rechallenge model (n = 10 mice per group per experiment). Maraba treatments were administered intratumorally. NT, no treatment. (E and F) The same experiment as in (B) was repeated using intravenous delivery of Maraba virus (E) or immunocompromised CD-1 nude mice (F). (G) Primary EMT6 or 4T1 tumors were treated with Maraba or left untreated and were resected, and all animals were rechallenged with 4T1 tumors. The dotted lines indicate the time of Maraba treatment. Statistical analysis for tumor measurements: *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired multiple two-tailed t test). Statistical analysis for survival curves: *P < 0.05, **P < 0.01, ***P < 0.001 (Mantel-Cox test).

  • Fig. 3 Local Maraba treatment of TNBC tumors provides long-term systemic protection.

    (A) Time of death of the 4T1 tumor–bearing animals in the rechallenge model. The cause of death is color-coded. (B) Maraba-treated mice (n = 10) from 4T1 tumor rechallenge experiments that were complete responders 100 days or more after tumor seeding were rechallenged with 4T1 and EMT6 cells. The right graph shows the percentage of tumors that could be detected before the animals reached end point. Statistical analysis according to the multiple unpaired t test: **P < 0.01, ***P < 0.001.

  • Fig. 4 Maraba infection triggers inflammation.

    (A and B) Microarray analysis of 4T1 (A) or EMT6 (B) cells infected with Maraba or ultraviolet (UV)–inactivated Maraba. The heat map includes the top genes that were enriched more than fourfold as compared to uninfected cells. R1 and R2 represent distinct experimental replicates. (C and D) Top 10 gene ontology enrichments of the genes induced by Maraba in the 4T1 (C) or EMT6 (D) model. FDR, false discovery rate. (E) Western blot analysis of 4T1 and EMT6 cell lysates. The cells were transfected with different small interfering RNAs (siRNAs) for 24 hours and infected with Maraba at a multiplicity of infection of 1 for an additional 12 hours before sample collection. STAT1, signal transducer and activator of transcription 1; pSTAT1, phospho-STAT1; IRF3, interferon regulatory factor 3; pIRF3, phospho-IRF3; VSV, vesicular stomatitis virus. (F) The same samples were also analyzed by quantitative polymerase chain reaction (n = 4) for the expression of different genes. Statistical analysis by unpaired t test with Welch’s correction: *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 Maraba induces antitumor T cell immunity.

    (A) Migration of splenocytes induced by conditioned medium from uninfected or infected 4T1 or EMT6 cells (n = 4 experimental replicates). (B) The same experiment was performed in the 4T1 cell line in the presence of control or blocking antibodies (Ab) (n = 4 experimental replicates; unpaired two-tailed t test with Welch’s correction, *P < 0.05, **P < 0.01, and ***P < 0.001). (C and D) Percentage of cells within 4T1 tumors that stained positive for CD45 as quantified by flow cytometry 10 days (C) or 2 days (D) after Maraba treatment with and without CXCR3-blocking antibodies (n = 5 mice; unpaired two-tailed t test with Welch’s correction, **P < 0.01 and ***P < 0.001). (E) Immunohistological assessment of CD3+ cells in 4T1 tumors at the time of resection in the tumor rechallenge model. Scale bars, 100 μm. (F) Interferon-γ (IFN-γ) ELISPOT (enzyme-linked immunospot) analysis of splenocytes harvested 10 days after treatment from mice treated with Maraba for five consecutive days (n = 3 to 5 mice per group). Restim, ex vivo restimulation with cells. (G) The same experiment was repeated with or without blocking antibodies administered intraperitoneally (n = 5 mice per group; unpaired two-tailed t test, **P < 0.01 and ***P < 0.001). The cells were restimulated ex vivo with EMT6 or 4T1 cells. IFN-αR1, IFN-α receptor 1.

  • Fig. 6 Maraba treatment sensitizes 4T1 tumors to immune checkpoint blockade.

    (A) Cell surface programmed death ligand 1 (PD-L1) expression on 4T1, EMT6, and E0771 cells after an incubation with Maraba-infected 4T1 cell–conditioned medium with or without IFN-αR1–blocking antibody. MFV, mean fluorescence value; MRB, Maraba. (B) Mice with 4T1 tumors were treated intratumorally with Maraba (days 7 to 11) and sacrificed on day 20. The graph shows the percentage of T cells that were FOXP3+. Two-tailed unpaired t test, **P < 0.01. (C) Mice bearing 4T1 tumors were treated intratumorally with Maraba (days 7 to 11) followed by anti-cytotoxic T lymphocyte–associated protein 4 (CTLA4) and anti–PD-1 (programmed cell death protein 1) antibodies injected intraperitoneally on days 21, 23, 25, 27, and 29. The tumors were measured on day 31 (three separate experiments). The graph shows the volume of each tumor relative to the average volume of the tumors from the control group. Unpaired two-tailed t test, *P < 0.05, **P < 0.01, ***P < 0.001. ICI, immune checkpoint inhibitor. (D) Treatment schedule used in (E), (F), and (G). (E to G) Tumor growth and Kaplan-Meier survival analysis of 4T1 (E), EMT6 (F), or E0771 (G) tumor-bearing mice using the tumor rechallenge model. The dotted lines indicate the days of Maraba treatment. Unpaired multiple two-tailed t test, *, #, xP < 0.05, **,xxP < 0.01, ***,xxxP < 0.001. The symbol “*” denotes the difference between NT and Maraba + ICI. The symbol “#” denotes the difference between Maraba and Maraba + ICI. The symbol “x” denotes the difference between ICI and Maraba + ICI. For survival curves, **P < 0.01, ***P < 0.001 (Mantel-Cox test).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/422/eaao1641/DC1

    Fig. S1. Maraba efficiently infects and kills breast cancer PDXs and 4T1 tumors.

    Fig. S2. Maraba treatment shows limited efficacy in murine TNBC models.

    Fig. S3. Maraba treatment reduces lung metastases.

    Fig. S4. The survival benefit conferred by Maraba in the tumor rechallenge model requires replicating virus and is tumor-specific.

    Fig. S5. Local Maraba treatment provides systemic protection.

    Fig. S6. Local Maraba treatment does not protect against lung metastasis in immunocompromised mice.

    Fig. S7. Maraba infection induces the production of cytokines and chemokines.

    Fig. S8. Maraba infection activates NFκB.

    Fig. S9. Several chemokines are important for the chemoattractive activity of the Maraba-conditioned medium.

    Fig. S10. Viable immune cells extracted from tumors were analyzed by flow cytometry.

    Fig. S11. Maraba infection promotes T cell infiltration.

    Fig. S12. The combination of Maraba and ICIs improves tumor control.

  • Supplementary Material for:

    Neoadjuvant oncolytic virotherapy before surgery sensitizes triple-negative breast cancer to immune checkpoint therapy

    Marie-Claude Bourgeois-Daigneault,* Dominic Guy Roy, Amelia Sadie Aitken, Nader El Sayes, Nikolas Tim Martin, Oliver Varette, Theresa Falls, Lauren Elizabeth St-Germain, Adrian Pelin, Brian Dennis Lichty, David Francis Stojdl, Guy Ungerechts, Jean-Simon Diallo, John Cameron Bell*

    *Corresponding author. Email: mbourgeois{at}ohri.ca (M.-C.B.-D.); jbell{at}ohri.ca (J.C.B.)

    Published 3 January 2018, Sci. Transl. Med. 10, eaao1641 (2018)
    DOI: 10.1126/scitranslmed.aao1641

    This PDF file includes:

    • Fig. S1. Maraba efficiently infects and kills breast cancer PDXs and 4T1 tumors.
    • Fig. S2. Maraba treatment shows limited efficacy in murine TNBC models.
    • Fig. S3. Maraba treatment reduces lung metastases.
    • Fig. S4. The survival benefit conferred by Maraba in the tumor rechallenge model requires replicating virus and is tumor-specific.
    • Fig. S5. Local Maraba treatment provides systemic protection.
    • Fig. S6. Local Maraba treatment does not protect against lung metastasis in immunocompromised mice.
    • Fig. S7. Maraba infection induces the production of cytokines and chemokines.
    • Fig. S8. Maraba infection activates NFκB.
    • Fig. S9. Several chemokines are important for the chemoattractive activity of the Maraba-conditioned medium.
    • Fig. S10. Viable immune cells extracted from tumors were analyzed by flow cytometry.
    • Fig. S11. Maraba infection promotes T cell infiltration.
    • Fig. S12. The combination of Maraba and ICIs improves tumor control.

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