Reprogramming tumour-associated macrophages from immune suppressive to inflammatory state by Checkpoint kinase 1 inhibitor combination treatment

Background: Tumour-associated macrophages (TAMs) play critical roles within the tumour microenvironment regulating immune evasion and therapeutic response. Previously, we have shown that the combination of Checkpoint kinase 1 inhibitor (CHK1i) with a subclinical dose of hydroxyurea (LDHU) reprograms the tumour immune microenvironment to a pro-inflammatory status. Methods: We investigated a tumour-restricted Fcgr4 (Cd16.2) expressing macrophage population in multiple murine tumour models and the impact of CHK1i+LDHU on this population, using conventional and imaging flow cytometry as well as single-cell sequencing. Results: Transcriptional profiling using CITE-seq and single-cell RNA sequencing reveals that Fcgr4 TAMs closely resemble Fcgr4- TAMs but display modest enrichment of interferon-associated and inflammatory gene programs, consistent with a functionally biased state rather than a distinct lineage. Importantly, we show that a highly tumour selective CHK1i+LDHU therapy shifts TAMs toward a more inflammatory phenotype while preserving dominant immunosuppressive features. Depletion of CSF1R macrophages enhanced CD8 T cell activation without influencing tumour growth but significantly augmented therapeutic efficacy of CHK1i+LDHU. Conclusion: Together, these findings define a novel TAM population and establish how targeted therapy reshapes, but does not fully overcome, TAM-mediated immune regulation.