E312: Macrophage function over phenotype determines sensitization to immunotherapy

AXL tyrosine kinase (AXL) serves a myriad of vital roles on a molecular level from proliferation to immune regulation; however, it has become implicated in ICB resistance when adherrrantly expressed. While AXL expression has been associated with immuno-suppressive M2 macrophage phenotype, AXL-directed macrophage function is less well defined. Our group explored the mechanism of macrophage- dependent augmentation of ICB response with AXL inhibition in melanoma.

Methods:

ICB-resistant AXL+ metastatic melanoma cells were used both in vitro and in vivo mouse models. Multiple aspects of AXL function within the tumor and macrophage phenotype switching were characterized in vitro by phagocytosis or efferocytosis assay, cytokine expression, PD-L1 expression and tumor behavior with Gas6 ligand stimulation or AXL inhibition by multiple AXL inhibition strategies. We investigated macrophage-specific impact of AXL inhibition in vivo in combination with ICB through overall tumor burden, flow cytometry, and  immune time-resolved-resonance energy transfer (iFRET).

Results:

In AXL+ melanoma cells and macrophage co-culture, M1 macrophages had both higher phagocytic and efferocytotic behaviors with loss of phagocytosis by M2. Gas6 stimulation, however, impacted M1 behavior with augmentation of tumor digestion. Migration of SKMEL-24 and YUMM1.7 melanoma increased with stimulation by Gas6 or M2 secreted factors but was impaired by AXL inhibition or M1 cytokines (p< 0.05). Cytokine profiling reveals specific increases in inflammatory cytokines with the addition of AXL inhibition to M2 macrophages and indicates secreted factors determine functional behavior over M1/M2 classification alone. In vivo, AXL inhibition reduced AXL+ tumor growth in our murine model and sensitized them to ICB. After depleting macrophages in a subset of mice using anti-CSFR, AXL antagonism showed a significant decrease in the proportion of M2 macrophages in tumor (p< 0.05). iFRET analysis showed a low PD-1/PD-L1 interaction in ICB-resistant tumors with increased engagement in the setting of AXL inhibition. IL-33R also has a relationship with AXL-related PD-1 expression on macrophages.

 

Conclusions:

AXL significantly lowers the immunogenicity and increases metastatic potential of melanoma, contributing to ICB resistance. Inhibition of AXL leads to a decrease in protumorigenic behaviors in AXL+ melanoma related to fundamental changes in innate immune function in the tumor microenvironment, validating further exploration of both macrophage-directed and AXL inhibition strategies in ICB-resistant melanoma.