Rebound Polarization of Monocyte-derived Tumor Macrophages Under Repeated Anti-CD40 Immunotherapy

To investigate TAM phenotypes and potential stress-induced feedback mechanism upon immunotherapy, we treated MC38 tumor-bearing C57BL/6 mice with a single dose of agonistic anti-CD40 or isotype control on day 11 (1× anti-CD40) or with repeated doses on days  11,  13, and  15 (3× anti-CD40). Twenty-four hours after the last injection, tumors were harvested for flow cytometry and single-cell RNA sequencing (scRNA-seq). A single anti-CD40 injection significantly elevated CD45+F4/80+ macrophage numbers. PCA on CD45+F4/80+ cell RNAseq data , followed by gene set enrichment analysis (GSEA) and transcription factor enrichment, revealed a pronounced transcriptional transformation. After the first round of immunotherapy, most macrophages shifted toward an IFN–STAT1–driven, immune-activated phenotype—reflecting a robust antitumor response. However, repeated anti-CD40 doses induced a rebound in macrophage gene expression, reverting toward the original “tumor-conditioned” state highlighted by strong Spp1 expression. This rebound raised questions about stress signals in the local tumor environment driving macrophage immunosuppression. Across all conditions, we detected a striking polarity along a phenotype gradient from Spp1+ immunocompromised TAMs towards immune-activated Cxcl9+ Cxcl10+ MHC class II macrophages. Overall design: Mice bearing subcutaneous (s.c.) MC38 tumors were treated intravenously (i.v.) with either isotype control (ctrl)l or agonistic anti-CD40 on day 11 (single injection; 1xCD40) or on days 11, 13, and 15 (multiple injections, 3xCD40). Twenty-four hours after the final injection, tumors were harvested, and CD45+F4/80+ cells were FACS-sorted for multiplexed scRNA-seq.