Delta opioid receptor (DOR) expression on myeloid-derived suppressor cells (MDSCs) represents a novel target to overcome resistance to immune checkpoint inhibitors (ICIs)

Abstract

Abstract Several studies suggest an association between opioid use for cancer pain and shorter survival, with research in animals indicating that opioids may promote angiogenesis, tumor growth, and metastasis. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that contribute significantly to creating an immunosuppressive tumor microenvironment (TME), suppressing anti-tumor immune responses, and contribute significantly to both angiogenesis and tumor metastasis. We asked whether pro-tumorigenic effects of opioids may be mediated through a specific opioid receptor on MDSCs. To address this, we investigated whether the delta opioid receptor (DOR) is expressed on and plays a role in the immunosuppressive functionality of MDSCs. We demonstrate that the DOR is highly expressed on MDSCs from tumors. In addition, antagonism of DOR with a specific inhibitor modulated a variety of direct and indirect MDSC-mediated immunosuppressive factors and reversed T cell suppression, suggesting the DOR may be a novel target to reprogram MDSC induced immunosuppression in the TME. In vitro, both bone marrow-derived MDSCs from C57BL/6 and BALB/c were generated with a combination of GM-CSF, G-CSF, and IL-6 exhibited robust expression of DOR, with over 50% of the cells positive by flow measurement using a DOR specific antibody. These MDSCs exhibited hallmark suppressive functions, including reactive oxygen species (ROS) production and inhibiting CD4+ and CD8⁺ T cell proliferation. More importantly, treatment of MDSCs with TH-31, ([N, N-(Me)2-Dmt-Tic-NH2), a highly specific DOR antagonist, reduced ROS production and the expression of inducible nitric oxide synthase (iNOS) and arginase 1, key enzymes involved in MDSC-mediated suppression compared to MDSCs without TH-31. We further analyzed MDSCs from bone marrow, spleen, and tumors (B16F10 melanoma-bearing C57BL/6 mice). Both polymorphonuclear (PMN-MDSCs) and monocytic (M-MDSCs) subsets were detected across all tissues. Tumor-infiltrating PMN-MDSCs showed significantly elevated DOR expression compared to bone marrow or spleen (49.47±14.14%, 3.50±1.56%, 9.61±2.36%, p≤0.001, respectively), with a similar trend observed for M-MDSCs (66.8±5.40%, 0.89±0.42%, 7.29±2.62%, p≤0.001, respectively). Simultaneously, these tumor-infiltrating PMN-MDSCs exhibited significantly higher reactive oxygen species (ROS) production compared to bone marrow or spleen derived (100±23.00%, 4.53±52.12%, 3.32±48.27%, p=<0.01, respectively ) and total MDSC arginase activity significantly higher compared to bone marrow or spleen derived (100±4.75%, 16.82±2.00%, 38.64±0.967%, p=<0.05, respectively ). Functional assays confirmed that MDSCs effectively suppressed T cell proliferation in a MDSC: T ratio dependent manner. To assess the translational relevance of these findings, we evaluated MDSCs isolated from the bone marrow of a patient with aplastic anemia who progressed to large granular lymphocyte (LGL) leukemia. Patient-derived MDSCs suppressed CD4⁺ T cell proliferation in vitro. Treatment of MDSCs (CD33⁺-enriched MDSCs) with TH-31 for 48 hours, followed by co-culture with healthy donor T cells (MDSC:T ratios 1:2 or 1:4), resulting in the complete reversal of MDSC inhibition of T cell proliferation. Together, these preliminary findings demonstrate that DOR may represent a single target of intervention to reprogram multiple functional pathways coupled to MDSC induced immunosuppression. Additional data on the effects of DOR ligands on MDSCs migration, and on the levels and expressions of key functional molecules modulating MDSC activity will be available at the date of the meeting.