Sustained macrophage reprogramming is required for CD8 T cell-dependent long-term tumor resolution

Tumor-associated macrophages (TAMs) exhibit a dual role in tumor progression and antitumor immunity. However, understanding the functional states and molecular mechanisms of antitumor TAMs remains a challenge. Here, we show that combined TLR3 and CD40 agonists (myeloid cell treatment, MCT) reprogram TAMs to adopt a protective antitumor phenotype in an orthotopic mouse breast cancer model, leading to tumor regression. Single-cell RNA sequencing of TAMs from different tumor stages and post-MCT treatment revealed a transient antitumor TAM phenotype, present at 12h post-MCT, characterized by markers such as iNOS and CD38, which was replaced by TAMs co-expressing tumor-limiting and promoting features by 72h post-MCT. Maintenance of antitumor TAMs required repeated MCT administrations, and reactive oxygen species and TNF-a were pivotal molecular mechanisms in TAM-mediated tumor control. Importantly, repeated MCT promoted the activation of CD8 T cells and long-term tumor eradication. Our findings uncover the vulnerability of transient TAM reprogramming which can be overcome by repeated MCT administrations to sustain efficient immune responses. Overall design: We analyzed TAMs at 12h and 72h post-MCT to capture early dynamics and later stages of tumor regression. For the 72h time point, we selected responders with a tumor size ratio (T3/T0) close or inferior to 1. To differentiate between protumor and antitumor inflammation originating from TAMs, we collected tumor-infiltrating myeloid cells from mice with progressing tumors (PT), defined as those exceeding 500 mm³ in volume. Each group (NT, MCT-72h, PT) included a minimum of three samples from distinct biological replicates, except for MCT-12h, which had two biological replicates. To integrate the four groups and mitigate potential batch effects, we included an NT sample in each FACS-sorting session. A permissive gating approach, including all live CD45+CD19-CD3-NK1.1- cells was used to sort all myeloid cell subsets. Isolated immune cells were processed using 10X Genomics chromium technology to prepare libraries for single-cell RNA-sequencing.