Research ArticleCancer

Immunological mechanisms of the antitumor effects of supplemental oxygenation

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Science Translational Medicine  04 Mar 2015:
Vol. 7, Issue 277, pp. 277ra30
DOI: 10.1126/scitranslmed.aaa1260
  • Fig. 1. Respiratory hyperoxia promotes tumor regression and survival and decreases metastasis.

    (A) Respiratory hyperoxia promotes tumor regression in mice with 11-day established MCA205 pulmonary tumors. Mice were placed in chambers with 60% oxygen after 11 days of tumor growth (identified as 60% O2*; P = 0.001), and lungs were harvested at day 21. Stronger regression was observed when mice were placed in 60% oxygen units immediately after tumor inoculation (identified as 60% O2; P = 0.0003) (n = 5 mice per group, averages represented as horizontal bars). (B) Hyperoxia-enhanced tumor regression in mice with B16 melanoma pulmonary tumors (n = 5 mice per group, averages represented as horizontal bars; P = 0.0001). (C) Respiratory hyperoxia leads to long-term survival in 40% of MCA205 tumor–bearing mice (n =5 mice per group; P = 0.009). (D) Respiratory hyperoxia strongly decreases spontaneous lung metastasis of orthotopically grown 4T1 breast tumors (n = 5 mice per group; P = 0.005). Balb/c mice were injected in the third mammary fat pad with 4T1 tumor cells. After tumors became palpable at day 7, mice were placed in either 21 or 60% oxygen until assay completion on day 28.

  • Fig. 2. Antitumor effects of respiratory hyperoxia require endogenous T and NK cells.

    (A) Tumor-regressing effects of hyperoxia are lost in γc/Rag-2−/− mice deficient in T and NK cells. MCA205 tumor–bearing wild-type (WT) or γc/Rag-2−/− mice were placed in 21 or 60% oxygen after tumor inoculation, and lung tumors were assessed after 21 days (n = 5 mice per group, averages represented as horizontal bars; P = 0.0001). (B) Hyperoxia-induced regression of MCA205 pulmonary tumors is mediated by CD4, CD8, and NK cells. Depletion of different T cell subsets or NK cells using mAbs 2 days before tumor inoculation impaired or completely abrogated the antitumor effects of 60% oxygen (n = 5 mice per group, averages represented as horizontal bars; *P = 0.001, **P = 0.0001, ***P < 0.0001). (C) Respiratory hyperoxia improves tumor regression in MCA205 tumor–bearing WT mice but does not significantly improve the therapeutic benefit of genetic elimination of A2AR (n = 5 mice per group, averages represented as horizontal bars; P = 0.002 and P = 0.77 for WT and A2AR−/−, respectively).

  • Fig. 3. Antitumor T cells avoid hypoxic areas of the TME.

    (A) Immunohistochemical demonstration of CD8 T cells (red) preferentially localized outside of hypoxic areas (green) of intradermal (left panel) and lung (right panel) TME. Tissue sections from 14-day established lung or intradermal MCA205 tumors were analyzed. Statistical comparison between hypoxic and normoxic locations of CD8 T cells seen in the representative images on the left (scale bar, 100 μm) is shown in the histogram on the right (dermal: n = 3 mice, P = 0.0002; lung: n = 3 mice, P = 0.01). (B) Respiratory hyperoxia decreases hypoxic exposure of CD4 and CD8 T cells in the lung TME and spleen of tumor-bearing mice. Lymphocytes were isolated from MCA205 tumor–bearing lungs or spleen of mice breathing 21 or 60% oxygen, and the mean fluorescence intensity (MFI) of Hypoxyprobe-labeled T cells was analyzed by flow cytometry (lung: n = 4 mice per group; CD8 P = 0.005, CD4 P = 0.003; spleen: n = 3 mice per group; CD8 P = 0.003, CD4 P = 0.03).

  • Fig. 4. Respiratory hyperoxia results in an immunopermissive TME.

    (A) Immunohistochemical demonstration of the enhanced infiltration of endogenous CD8 T cells into established MCA205 pulmonary tumors due to hyperoxic breathing (means ± SEM, P = 0.015; n = 3 mice per group; scale bar, 200 μm). (B) Hyperoxic breathing promotes the accumulation of highly activated endogenous CD8 T cells as shown by flow cytometric analysis of the pulmonary TME. Mice with 11-day established MCA205 pulmonary tumors were treated with respiratory hyperoxia for 4 days, and the number of CD8+, CD69+, and CD44+ cells was analyzed by flow cytometry (means ± SEM, *P = 0.04; n = 3 mice per group). (C) Respiratory hyperoxia increases the levels of immunostimulating cytokines and chemokines as detected using custom-made RT-PCR arrays to screen for changes in 94 different chemokines and cytokines. Mice with 11-day established MCA205 pulmonary tumors were treated with respiratory hyperoxia for 72 hours (means ± SEM, exact P values listed in table S1; n = 3 mice per group). (D) Respiratory hyperoxia decreases the levels of TGF-β in the lung TME (means ± SEM, P = 0.03; n = 3 mice per group). Inset: Immunoblot for TGF-β in lung tumors from mice breathing 21 and 60% oxygen. Mice with 11-day established MCA205 pulmonary tumors were treated with respiratory hyperoxia for 72 hours.

  • Fig. 5. Respiratory hyperoxia weakens immunosuppression by Tregs in the lung TME.

    (A) Left: Respiratory hyperoxia decreases the percentage of CD4+/CD25+/Foxp3+ Tregs in the lung TME (P = 0.03; n = 5 mice per group). Mice bearing 11-day established MCA205 pulmonary tumors were placed in either 21 or 60% oxygen for 72 hours. Right: The expression of Foxp3 was also reduced by respiratory hyperoxia. The average MFI was 2227 and 1572 in mice breathing 21% versus 60% oxygen, respectively (P = 5.82 × 10−5; n = 5 mice per group). (B and C) Left: Respiratory hyperoxia reduces the expression of CD39 (B) and CD73 (C) on Tregs in the TME (P = 0.02 and P = 0.05; n = 5 mice per group). Right: The following are the average MFIs from 21 and 60% oxygen, respectively: CD39 (6329, 4226; P = 0.03) and CD73 (17054, 15761; P = 0.02).

  • Fig. 6. Respiratory hyperoxia decreases exposure of Tregs to hypoxia and reduces expression of CTLA-4.

    (A) Respiratory hyperoxia reduces the expression of CTLA-4 by Tregs. The average MFI of CTLA-4 on Tregs was 4786 in mice breathing 21% O2 and 2684 in mice breathing 60% O2 (P = 0.01; n = 5 mice per group). (B) Respiratory hyperoxia reduces the exposure of Tregs to hypoxia in both the lung and the spleen of MCA205 tumor–bearing mice (lung: P = 0.0002, n = 4 mice per group; spleen: P = 0.001, n = 3 mice per group). (C) CTLA-4High Tregs in the lung TME were also HypoxyprobeHigh, reflecting in vivo exposure to deeper levels of hypoxia. Respiratory hyperoxia decreased the numbers of CTLA-4High Tregs compared to mice breathing 21% oxygen (P = 0.002; n = 4 mice per group).

  • Fig. 7. Respiratory hyperoxia improves tumor regression in preclinical models of immunotherapies.

    (A) Adoptive immunotherapy in combination with respiratory hyperoxia enabled the complete regression of 11-day established MCA205 pulmonary tumors. Mice identified as 60%* were placed in the 60% oxygen units the same day as adoptive T cell immunotherapy, whereas mice identified as 60% were placed in oxygen units for the duration of the assay (21 days) [n = 5 mice per group, averages represented as horizontal bars; P = 0.03 (60%*) and P = 0.01 (60%)]. (B) Hyperoxia facilitates the infiltration of adoptively transferred T cells into 11-day established pulmonary tumors. Left: Fluorescent micrographs (scale bar, 200 μm) of CFSE-labeled adoptively transferred T cells (green) in mice breathing 60 or 21% oxygen 48 hours after adoptive transfer. Right: Enumeration of tumor-infiltrating transferred T cells. The average infiltration from ~100 tumors was 249 cells/mm2 in control mice and 723 cells/mm2 in mice breathing 60% oxygen (n = 3 mice per group; means ± SEM, P = 0.01). (C) Breathing as low as 40% oxygen results in pulmonary tumor regression [n = 5 mice per group, averages represented as horizontal bars; P = 0.046 (40% O2) and P = 3 × 10−6 (60% O2)]. (D) Alternating between breathing 60 and 40% oxygen or 60 and 21% oxygen every 12 hours enables tumor regression compared to mice continuously breathing 21% oxygen (n = 5 mice per group, averages represented as horizontal bars; P = 0.001 and P = 0.01, respectively). Breathing 60% oxygen continuously (24 hours/day) causes the strongest antitumor activity. (E) Respiratory hyperoxia improves the outcome of dual CTLA-4/PD-1 blockade in preclinical studies of lung tumor rejection. Mice were inoculated with MCA205 tumor cells and given mAbs for CTLA-4/PD-1 intraperitoneally on days 3, 6, and 9 (500 μg). Mice were treated with respiratory hyperoxia from days 3 to 21 or maintained at 21% O2 until assay completion (day 21) (n = 5 mice per group, averages represented as horizontal bars; P = 0.04).

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/7/277/277ra30/DC1

    Materials and Methods

    Fig. S1. The tumor-regressing effects of respiratory hyperoxia are lost in cγ/Rag-2−/− mice.

    Fig. S2. ROS scavenger does not prevent the antitumor effects of respiratory hyperoxia.

    Fig. S3. Respiratory hyperoxia reverses hypoxia-adenosinergic inhibition of NK cells.

    Fig. S4. Respiratory hyperoxia does not further improve the activity of tumor-reactive A2AR−/− T cells.

    Fig. S5. CD8 and CD4 T cells avoid hypoxic TME.

    Fig. S6. Tregs with higher expression of CTLA-4 are more hypoxic.

    Fig. S7. CD8 T cells from TDLN are enriched after culture activation for adoptive transfer.

    Fig. S8. Breathing 60% oxygen increased IFN-γ production by CD8 T cells in the lung TME.

    Table S1. Immunostimulating cytokines/chemokines increased by respiratory hyperoxia.

    Table S2. Full list of primer sets in RT-PCR arrays.

  • Supplementary Material for:

    Immunological mechanisms of the antitumor effects of supplemental oxygenation

    Stephen M. Hatfield, Jorgen Kjaergaard, Dmitriy Lukashev, Taylor H. Schreiber, Bryan Belikoff, Robert Abbott, Shalini Sethumadhavan, Phaethon Philbrook, Kami Ko, Ryan Cannici, Molly Thayer, Scott Rodig, Jeffrey L. Kutok, Edwin K. Jackson, Barry Karger, Eckhard R. Podack, Akio Ohta, Michail V. Sitkovsky*

    *Corresponding author. E-mail: m.sitkovsky@neu.edu

    Published 4 March 2015, Sci. Transl. Med. 7, 277ra30 (2015)
    DOI: 10.1126/scitranslmed.aaa1260

    This PDF file includes:

    • Materials and Methods
      Fig. S1. The tumor-regressing effects of respiratory hyperoxia are lost in cγ/Rag-2−/− mice.
    • Fig. S2. ROS scavenger does not prevent the antitumor effects of respiratory hyperoxia.
    • Fig. S3. Respiratory hyperoxia reverses hypoxia-adenosinergic inhibition of NK cells.
    • Fig. S4. Respiratory hyperoxia does not further improve the activity of tumor-reactive A2AR−/− T cells.
    • Fig. S5. CD8 and CD4 T cells avoid hypoxic TME.
    • Fig. S6. Tregs with higher expression of CTLA-4 are more hypoxic.
    • Fig. S7. CD8 T cells from TDLN are enriched after culture activation for adoptive transfer.
    • Fig. S8. Breathing 60% oxygen increased IFN-γ production by CD8 T cells in the lung TME.
    • Table S1. Immunostimulating cytokines/chemokines increased by respiratory hyperoxia.
    • Table S2. Full list of primer sets in RT-PCR arrays.

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