Myeloid cells in cancer immunity: Intravital characterization at single-cell level

Keehoon Jung

Department of Anatomy and Cell Biology, Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, Korea

Our goal is to better understand the immune microenvironment in cancers utilizing advanced methodologies including in vivo imaging and single-cell genomics. In particular, we aim to unveil the immense

potential of myeloid populations in cancers, which is an emerging field. Real-time intravital multi-photon microscopy provides insight into dynamic cell migration and intercellular communication. We reveal the

heterogeneity of myeloid cells through single-cell RNA sequencing in both pre-clinical and clinical specimens. This approach will provide novel avenues for immunotherapy of cancers by means of identifying promising targets in tumor immunity. Specifically, we have uncovered an immunosuppressive role for non-classical Ly6C low monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following the VEGF signaling blockade, resulting in recruitment of CX3CR1 + Ly6C low monocytes into the tumor. We also found that treatment with VEGF-A reduced expression of CX3CL1 in endothelial cells. Intravital microscopy revealed that CX3CR1 is critical for Ly6C low monocyte transmigration across the endothelium in tumors. Moreover, Ly6C low monocytes recruit Ly6G + neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6C low monocyte or

Ly6G + neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, we developed a gene therapy using a nanoparticle formulated with a siRNA against CX3CL1,

which reduced Ly6C low monocyte recruitment and improved outcome of anti-VEGFR2 therapy. Taken together, we identified immunosuppressive non-classical Ly6C low monocytes as key players in tumor resistance to anti-angiogenic therapy in cancers. We also revealed molecular mechanisms underlying anti-angiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies, proven by state-of-the-art in vivo imaging modalities.