Research ArticleCancer

High-dose vitamin C enhances cancer immunotherapy

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Science Translational Medicine  26 Feb 2020:
Vol. 12, Issue 532, eaay8707
DOI: 10.1126/scitranslmed.aay8707
  • Fig. 1 VitC delays tumor growth in immunocompetent syngeneic mice.

    (A) The indicated cell lines were injected orthotopically (100,000 cells for TS/A and 4T1 models, 50% Matrigel) or subcutaneously (500,000 cells for CT26, MC38, B16, and PDAC models) in immunocompetent syngeneic mice. VitC (4 g/kg) was administered daily by intraperitoneal injections, and treatment was started when tumor volume reached around 100 mm3 (indicated by the black arrow). (B) In parallel, mouse tumor cells were injected in immunocompromised NOD-SCID mice, and treatment was conducted in the same setting as indicated in (A). Every experimental group was composed at least of seven mice. Every experiment in (A) was performed at least twice. Data and error bars indicate mean ± SEM. P values were calculated by two-tailed unpaired Student’s t test. n.s., not significantly different; Ctrl, control.

  • Fig. 2 VitC affects tumor growth in a T cell–dependent manner.

    (A and B) Flow cytometry analysis of IFN-γ release on CD4 (A) and CD8 (B) spleen-derived lymphocytes isolated from untreated and VitC-treated mice injected orthotopically with TS/A cancer cells. Spleens were harvested 30 days after tumor cell injection, and T lymphocytes were stimulated in vitro. Percentages were calculated relative to CD4 and CD8 live events. The indicated cell percentages were gated on CD45+ live, CD4+/CD8+, and IFN-γ (500,000 events were taken for each sample). (C) Depletion of CD4 T cells and (D) depletion of CD8 T cells in the indicated cell models. Mice were treated with anti-CD4 (αCD4)– or anti-CD8 (αCD8)–depleting mAbs (400 μg per mouse at day 0 and then 100 μg per mouse at day 1, day 2, and every 3 days through the entire course of the experiment). Control mice were administered the isotype antibody. (E) Adoptive T cell transfer was performed according to the indicated experimental design. (F and G) Adoptive cell transfer of untreated and VitC-treated CD4 T cells (F) or CD8 T cells (G) isolated from the spleens of immunocompetent mice and infused into NOD-SCID mice orthotopically injected with TS/A; CTRL indicates tumor growth in NOD-SCID without T cell administration. (H) To test the effect of VitC on CD8 T cells in the absence of CD4 lymphocytes, immunocompetent mice were pretreated with a depleting CD4 T cell antibody or isotype antibody (as a control) and then administered VitC. CD8 T cells isolated from these immunocompetent mice were injected into the tail vein of immunocompromised NOD-SCID mice bearing orthotopic TS/A tumors (n = 4). Black arrows indicate the time points of T cell tail vein infusion. Five million T cells per injection were administered to each mouse. Every experimental group was composed of at least of six mice, with the exception of adoptive cell transfer experiments, which were composed of four mice per group. Every experiment was performed at least twice except for those shown in (A), (B), (D), and (H). Data and error bars indicate mean ± SEM. P values were calculated using two-tailed unpaired Student’s t test for (A) and (B); one-way analysis of variance (ANOVA) for all other panels at the indicated time points.

  • Fig. 3 The efficacy of ICT is enhanced by VitC.

    (A) PDAC pancreatic cancer cells were injected subcutaneously (500,000 cells) into syngeneic mice that were treated with VitC, ICT, or their combination. (B) 4T1 breast cancer cells were injected orthotopically (100,000 cells, 50% Matrigel) in syngeneic mice that were treated with VitC, ICT, or their combination. (C) TS/A breast cancer cells were injected orthotopically (100,000 cells, 50% Matrigel) in syngeneic mice that were treated with VitC, ICT, or their combination. (D) Tumor relapse-free survival of mice treated with VitC, ICT, or their combination and followed for over a year. Two independent experiments performed on a total of n = 13 mice are shown in survival curves. The vertical black arrows indicate the time point at which mice were rechallenged with live tumor cells. (E) CT26 colorectal cancer cells were injected subcutaneously (500,000 cells) in syngeneic mice. (F) Tumor relapse-free survival of mice treated with VitC, ICT, or their combination and followed up to a year. Two independent experiments performed on a total of n = 13 mice are shown in survival curves. The vertical black arrow indicates the time point at which mice were rechallenged with live tumor cells. VitC (4 g/kg) was administered intraperitoneally 5 days per week starting when tumors reached a volume around 100 mm3 in the TS/A, 4T1, and PDAC models. VitC treatment started when tumor volume was around 800 to 1000 mm3 in the CT26 model. Anti–CTLA-4 (200 μg per mouse) and anti–PD-1 (250 μg per mouse) were given at the time points indicated by the dashed vertical lines in the graphs. In combinatorial treatments, VitC was administered starting with the first cycle of immunotherapy. Every experimental group was composed of at least five mice. Every experiment was performed twice except for those shown in (A) and (B). Data and error bars indicate mean ± SEM. Statistical analysis used one-way ANOVA for tumor growth comparison at the indicated time points and log-rank test (Mantel-Cox) for survival analysis.

  • Fig. 4 VitC in combination with ICT enhances infiltration and activation of T lymphocytes.

    TS/A orthotopic tumors were explanted and analyzed for immune infiltration. (A) Immunofluorescence analysis of CD4 and CD8 tumor-infiltrating lymphocytes. Maximum projection of a 10-image stack along the z axis. Scale bar is representative of 75 μm. (B) Quantification of CD4 and CD8 T cells from (A). T cell counts per high-power field (HPF) from six different mice. (C and D) TS/A orthotopic tumors were explanted, single cell–suspended, and analyzed by flow cytometry. Staining for memory/effector markers on CD4 (C) and CD8 (D) T cells. The fraction of positive cells was calculated on CD4+ and CD8+ live events, respectively (500,000 events were acquired for each sample). Individual values are presented, and error bars indicate ±SD. P values were calculated using nonparametric analysis for (B) and (C); one-way ANOVA for (D).

  • Fig. 5 Addition of VitC to individual immune checkpoint inhibitors induces remission in MMRd tumors.

    (A) MLH1-WT and MLH1-KO cells were first subcutaneously injected (500,000 cells) in immunocompromised mice (shown in fig. S12) according to the indicated experimental design. (B) Small fragments of untreated tumors bearing the indicated MLH1 genotype were transplanted into immunocompetent syngeneic mice; VitC (4 g/kg) was administered by intraperitoneal injection 5 days per week, starting when tumors reached a volume around 150 to 200 mm3 (black arrow) to ensure tumor engraftment. (C) Percentage of mice [animals from the experiment shown in (A)] whose tumor volume was less than 500 mm3, which we set as an arbitrary end point. (D) In the same setting as in (A), MLH1-KO tumors were transplanted into immunocompetent syngeneic mice and treated with ICT and VitC (4 g/kg) starting at a tumor volume of 800 to 1000 mm3. Anti–CTLA-4 (200 μg per mouse) and anti–PD-1 (250 μg per mouse) were given at the time points indicated by the dashed vertical lines in the graphs. (E) Tumor relapse-free survival of mice treated with VitC, immune checkpoint inhibitors, or their combination shown in (C). The black arrows indicate tumor rechallenge with the same cancer cells. Every experimental group was composed of at least five mice. Every experiment was performed twice except for the models shown in (B) and (C). Data and error bars indicate mean ± SEM. Statistical analysis used two-tailed unpaired Student’s t test for (B); one-way ANOVA for (D) at the indicated time points. Survival analysis in (C) and (E) used log-rank test (Mantel-Cox) analysis.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/532/eaay8707/DC1

    Fig. S1. VitC quantification in plasma.

    Fig. S2. High doses of VitC are required for maximal antiproliferative effects in murine breast tumors.

    Fig. S3. Administration of antioxidant NAC does not impair VitC anticancer effect.

    Fig. S4. Flow cytometry for IFN-γ in spleen-derived T lymphocytes.

    Fig. S5. Isolation of T cells for adoptive T cell transfer.

    Fig. S6. Depletion of CD4 T cells for adoptive cell transfer experiments.

    Fig. S7. Tumor growth of individual mice treated as shown in Fig. 3 (A and B).

    Fig. S8. Tumor growth of individual mice treated as shown in Fig. 3 (C to E).

    Fig. S9. Modulation of tumor immune infiltration induced by VitC.

    Fig. S10. Flow cytometry on CD44 and CD69 T cell markers on CD4 T lymphocytes.

    Fig. S11. Representative CD44- and CD69-positive events on CD8 T lymphocytes by flow cytometry.

    Fig. S12. VitC effect on growth of MMR-deficient tumors in NOD-SCID mice.

    Fig. S13. Tumor growth of individual mice bearing MLH1-KO tumors and treated with ICT and VitC as shown in Fig. 5D.

    Fig. S14. Tumor volume variations since treatment start of TS/A MLH1-KO and CT26 MLH1-KO tumors treated with ICT and VitC.

    Data file S1. Tumor measurements of experiments in Fig. 1.

    Data file S2. Tumor measurements of experiments in Fig. 2.

    Data file S3. Tumor measurements of experiments in Fig. 3.

    Data file S4. Immuno-phenotiping of CD4 and CD8 T cells.

    Data file S5. Tumor measurements of experiments in Fig. 5.

    Data file S6. Plasma VitC analysis.

    Data file S7. Tumor measurements of experiments in figs. S2 and S3.

    Data file S8. Tumor measurements of experiments in fig. S6.

    Data file S9. Tumor measurements of experiments in fig. S8.

    Data file S10. Tumor measurements of experiments in fig. S12.

  • The PDF file includes:

    • Fig. S1. VitC quantification in plasma.
    • Fig. S2. High doses of VitC are required for maximal antiproliferative effects in murine breast tumors.
    • Fig. S3. Administration of antioxidant NAC does not impair VitC anticancer effect.
    • Fig. S4. Flow cytometry for IFN-γ in spleen-derived T lymphocytes.
    • Fig. S5. Isolation of T cells for adoptive T cell transfer.
    • Fig. S6. Depletion of CD4 T cells for adoptive cell transfer experiments.
    • Fig. S7. Tumor growth of individual mice treated as shown in Fig. 3 (A and B).
    • Fig. S8. Tumor growth of individual mice treated as shown in Fig. 3 (C to E).
    • Fig. S9. Modulation of tumor immune infiltration induced by VitC.
    • Fig. S10. Flow cytometry on CD44 and CD69 T cell markers on CD4 T lymphocytes.
    • Fig. S11. Representative CD44- and CD69-positive events on CD8 T lymphocytes by flow cytometry.
    • Fig. S12. VitC effect on growth of MMR-deficient tumors in NOD-SCID mice.
    • Fig. S13. Tumor growth of individual mice bearing MLH1-KO tumors and treated with ICT and VitC as shown in Fig. 5D.
    • Fig. S14. Tumor volume variations since treatment start of TS/A MLH1-KO and CT26 MLH1-KO tumors treated with ICT and VitC.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Tumor measurements of experiments in Fig. 1.
    • Data file S2 (Microsoft Excel format). Tumor measurements of experiments in Fig. 2.
    • Data file S3 (Microsoft Excel format). Tumor measurements of experiments in Fig. 3.
    • Data file S4 (Microsoft Excel format). Immuno-phenotiping of CD4 and CD8 T cells.
    • Data file S5 (Microsoft Excel format). Tumor measurements of experiments in Fig. 5.
    • Data file S6 (Microsoft Excel format). Plasma VitC analysis.
    • Data file S7 (Microsoft Excel format). Tumor measurements of experiments in figs. S2 and S3.
    • Data file S8 (Microsoft Excel format). Tumor measurements of experiments in fig. S6.
    • Data file S9 (Microsoft Excel format). Tumor measurements of experiments in fig. S8.
    • Data file S10 (Microsoft Excel format). Tumor measurements of experiments in fig. S12.

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