Delta opioid receptor (DOR): A novel target for reprogramming tumor-associated macrophage (TAM) immunosuppressive phenotype to overcome acquired resistance and enhance the effectiveness of cancer immunotherapies

Abstract

Abstract Studies show that opioids, both endogenous and exogenous, can affect the function of macrophages, effecting their proliferation, phagocytosis, secretion of cytokines, and polarization. However, the relative contribution of opioid receptors to macrophage functions has not yet been investigated TAMs and myeloid-derived suppressor cells (MDSCs) are the key immunosuppressive cells within the tumorigenic microenvironment (TME). Our lab has demonstrated that the DOR is highly expressed on tumor associated MDSCs and plays a functional role in MDSC immunosuppressive phenotype. In this study, we investigated the role of DOR in modulating functions of TAMs across their subtypes (M0, M1, M2). Given that macrophages possess remarkable plasticity between pro- and anti-tumorigenic phenotypes, elucidating DOR's role in TAM related immunosuppressive functions may provide translational relevance to overcome acquired resistance to cancer immunotherapies. Mouse Bone marrow derived cells (mBMDCs) were cultured with M-CSF for 7 days, then polarized into M1 and M2 phenotypes using LPS/IFN-γ and IL-4/IL-13, respectively. RAW264.7 were polarized to M1 and M2 using the same cytokine treatment as BMDMs. Multi-color flow cytometry was used to assess M1 and M2 immunophenotypes, ROS production, and iNOS expression. Arginase enzyme activity was measured by the conversion of arginine to urea using a colorimetric assay. T-cell suppression was evaluated through co-culture assays using CFSE-labeled T-cells and macrophages, with T-cell proliferation assessed by flow cytometry. Macrophage generation and polarization from the M0 to M1 and M2 phenotypes were validated and characterized by flow cytometry using established accepted methodologies. Polarization from M0 to M1 and M2 in both RAW264.7 and BMDMs led to an increase in DOR surface expression, which was higher in M1 than M2 polarized macrophages (P ≤ 0.05). Specifically, in RAW264.7 cells, DOR+ cells and mean fluorescence intensity (MFI) were: M0 (66% / 10,718), M1 (96% / 38,264), and M2 (57.29% / 8,896) (P ≤ 0.05). In BMDMs, corresponding values were: M0 (92.70% / 43,935), M1 (89.35% / 52,773), and M2 (74.24% / 41,970) (P ≤ 0.05). Ex vivo analysis of myeloid and lymphoid populations from B16F10 tumor-bearing mice revealed that DOR expression was highest in myeloid cells compared to lymphocytes. Notably, within tumor-associated macrophages (TAMs; CD45⁺CD11b⁺F4/80⁺), MHC-II⁺ M1-like TAMs exhibited higher DOR expression (80% DOR⁺) compared to CD206⁺ or CD163⁺ M2-like TAMs (40% DOR⁺) (P ≤ 0.05), thus validating the in vitro findings. Co-culture assays showed that BMDM1 had minimal effect on T-cell proliferation (<5% T-cell suppression), whereas BMDM2 potently suppressed T-cell expansion (~ 90% T-cell suppression). These results indicate that immunosuppressive activity is restricted to the M2 subtype. To investigate the immunosuppressive role of DOR in M2 macrophages, a T cell/BMDM2 co-culture assay was performed. Pharmacological inhibition of DOR using the selective antagonist TH-31 (N,N-(Me)₂-Dmt-Tic-NH₂) reversed BMDM2-mediated immunosuppression and enhanced T cell proliferation. Treatment of T-cells alone with TH-31 DOR antagonist did not alter their proliferation, indicating that its effect is specifically mediated through modulation of DOR signaling in BMDM2 cells. Additionally, TH-31 treatment decreased arginase enzyme activity in BMDM2. This suggests that TH-31's inhibition of arginase activity may contribute to improved T-cell proliferation. This study successfully characterized M1 and M2 macrophage polarization, revealing that DOR expression is enhanced in M1 and less so in M2 compared to M0 cells. These findings were validated in TAMs from B16F10 tumor-bearing mice, demonstrating DOR expression was higher in M1-and M2 phenotype TAMs versus other immune cell populations. Despite expressing higher level of DOR, BMDM1 did not exhibit immunosuppressive activity, suggesting that DOR may serve distinct functions in M1 and M2 macrophages. Studies further examining the role of DOR on M1and M2 macrophage functionality are ongoing. Critically, pharmacological DOR inhibition reversed M2-mediated immunosuppression and enhanced T-cell proliferation. These results expand our understanding of DOR's role beyond MDSCs to include M2 macrophages and the potential inhibition of DOR as a novel approach to modulating the immunosuppressive effect of these key cellular components in the TME.