Immature monocytic cells within tumors differentiate into immunosuppressive cells in resistant tumors to immunotherapy

Immune checkpoint inhibitors (ICIs) have improved outcomes in advanced cancers, yet resistance remains a major obsticle. Here, we investigated the role of myeloid cells in shaping the immunosuppressive tumor microenvironment that contributes to ICI resistance. Using mutagenized ICI-sensitive and resistant 4T1 breast cancer clones, we performed single-cell RNA sequencing to characterize immune cell populations post-ICI therapy. We identified monocytic dendritic progenitors (MDPs) and common monocytic progenitors (cMOPs) enriched in sensitive tumors, which may differentiate into immunosuppressive cells in resistant tumors. Analysis of public datasets confirmed the presence of MDP-cMOPs in tumors and blood of breast, lung, and colorectal cancer patients. We found high expression of CXCR4 and IL6R in MDP-cMOPs, and inhibiting these pathways blocked their recruitment and differentiation. Combined targeting of CXCR4 and IL6 pathway with ICI improved responses in resistant tumors, highlighting MDP-cMOPs as contributors to immunotherapy resistance and potential therapeutic targets. To investigate tumors responsive and resistant to immunotherapy, we used an experimental model of the 4T1 cell line, a triple negative breast carcinoma known for its resistance to immune checkpoint inhibitor (ICI) therapy. We exposed 4T1 cells to 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) and generated polyclonal cells that would respond to anti-PD1 (referred to as 4T1m) as opposed to the resistant (parental) clone (referred to as 4T1p). The cells were then implanted into the mammary fat pad of mice and subsequently treated with anti-PD1 or IgG control. At the endpoint (following two weeks of treatment), both 4T1p and 4T1m tumor-bearing mice treated with anti-PD1, were harvested and processed into single-cell suspensions for further analysis. Evetually, we isolated cells from anti-PD1-treated 4T1p and 4T1m tumors based on the GR-1 marker.