Oxygen-carrying nanoemulsions and respiratory hyperoxia eliminate tumor hypoxia–induced immunosuppression
Katarina Halpin-Veszeleiova, Michael P. Mallouh, Lucy M. Williamson, Ashley C. Apro, Nuria R. Botticello-Romero, Camille Bahr, Maureen Shin, Kelly M. Ward, Laura Rosenberg, Vladimir B. Ritov, Michail V. Sitkovsky, Edwin K. Jackson, Bruce D. Spiess, Stephen M. Hatfield
Katarina Halpin-Veszeleiova, Michael P. Mallouh, Lucy M. Williamson, Ashley C. Apro, Nuria R. Botticello-Romero, Camille Bahr, Maureen Shin, Kelly M. Ward, Laura Rosenberg, Vladimir B. Ritov, Michail V. Sitkovsky, Edwin K. Jackson, Bruce D. Spiess, Stephen M. Hatfield
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Research Article Immunology Oncology

Oxygen-carrying nanoemulsions and respiratory hyperoxia eliminate tumor hypoxia–induced immunosuppression

Abstract

Hypoxia/hypoxia-inducible factor 1α–driven immunosuppressive transcription and cAMP-elevating signaling through A2A adenosine receptors (A2ARs) represent a major tumor-protecting pathway that enables immune evasion. Recent promising clinical outcomes due to the blockade of the adenosine-generating enzyme CD73 and A2AR in patients refractory to all other therapies have confirmed the importance of targeting hypoxia-adenosinergic signaling. We report a feasible approach to target the upstream stage of hypoxia-adenosinergic immunosuppression using an oxygen-carrying nanoemulsion (perfluorocarbon blood substitute). We show that oxygenation agent therapy (a) eliminates tumor hypoxia, (b) improves efficacy of endogenously developed and adoptively transferred T cells, and thereby (c) promotes regression of tumors in different anatomical locations. We show that both T cells and NK cells avoid hypoxic tumor areas and that reversal of hypoxia by oxygenation agent therapy increases intratumoral infiltration of activated T cells and NK cells due to reprogramming of the tumor microenvironment (TME). Thus, repurposing oxygenation agents in combination with supplemental oxygen may improve current cancer immunotherapies by preventing hypoxia-adenosinergic suppression, promoting immune cell infiltration and enhancing effector responses. These data also suggest that pretreating patients with oxygenation agent therapy may reprogram the TME from immunosuppressive to immune-permissive prior to adoptive cell therapy, or other forms of immunotherapy.

Authors

Katarina Halpin-Veszeleiova, Michael P. Mallouh, Lucy M. Williamson, Ashley C. Apro, Nuria R. Botticello-Romero, Camille Bahr, Maureen Shin, Kelly M. Ward, Laura Rosenberg, Vladimir B. Ritov, Michail V. Sitkovsky, Edwin K. Jackson, Bruce D. Spiess, Stephen M. Hatfield

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Figure 3

Oxygenation agent therapy reprograms the TME toward immune-permission.

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Oxygenation agent therapy reprograms the TME toward immune-permission. (...
(A) Antitumor T cells and NK cells avoid hypoxic regions of solid tumors. MCA205 intradermal tumors were isolated and 5-μm frozen tissue sections were prepared. Fixed sections were fluorescently labeled with anti-Hypoxyprobe, anti-CD8, and anti-NK1.1 antibodies to determine infiltration of immune cells into the hypoxic (green) or nonhypoxic regions (dark blue, DAPI). Left panels: Representative fluorescence images demonstrating that CD8+ T cells (top, light blue) and NK cells (bottom, red) avoid hypoxic regions (green). Right panels: Quantification of CD8+ T cell (top) and NK cell (bottom) infiltration into hypoxic versus nonhypoxic areas. A total of 60 different regions from the TME across 15 tumors were analyzed using CellSens software (Olympus) and results are shown in the bar graphs (right). ****P < 0.0001 by unpaired t test. Data presented as mean ± SEM. Scale bar: 50 μm (original magnification, ×10). (B–D) C57BL/6 mice with 8-day-established MCA205 intradermal tumors received daily administration (i.v., 15 mL/kg) of oxygenated Perflubron for 72 hours while breathing 60% O2 (respiratory hyperoxia) or 21% O2 (normoxia). Mice were injected i.p. with Hypoxyprobe-1 (80 mg/kg) 1 hour before sacrifice to determine levels of intratumoral hypoxia. After sacrifice, tumors were isolated and divided for analysis via immunofluorescence or flow cytometry. (B) Left: Quantitative analysis using CellSens software (Olympus) of 5-μm-thick frozen tumor sections correlating the number of infiltrating NK cells and CD8+ cells per tumor. Each dot represents 1 tumor (n = 18). Dotted line represents 95% CI. Right: Bar graph depicting the relationship between NK cells (red) and CD8+ cells (blue) and levels of intratumoral hypoxia. Each bar represents 1 tumor and corresponding numbers for infiltration and hypoxic regions (n = 18). TILs, tumor-infiltrating lymphocytes. (C) Quantitation of the numbers CD8+CD25+ (left) and NK+CD25+ (right) cells from tumors from mice treated with oxygenation agent therapy versus control. P values were calculated using 1-way ANOVA with Tukey’s post hoc test for multiple comparisons. *P < 0.05. Data are presented as mean ± SEM, n = 5. (D) Pearson’s correlation analyses between the percentage of hypoxic regions in the TME and the number of infiltrating CD8+ T cells (left) or NK cells (right). Each dot represents 1 tumor (n = 18).