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-α 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. 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.