**Background:** Macrophages can recognize and phagocytose tumour cells, but often fail to do so and may even support tumour growth. The liver, a major site for haematogenous metastasis, contains resident macrophages called Kupffer cells (KCs) that are highly phagocytic and may act as an innate immune barrier. The transcription factor ID3 is a lineage-determining factor for KCs, but its role in adult KC function and anti-tumour immunity was unknown. This study investigates whether ID3 endows KCs with anti-tumour activity and whether this property can be transferred to other macrophages.
**Methods:** The authors used multiple genetic mouse models to specifically deplete or modify KCs: Clec4f-cre-driven knockout of Csf1r, Spi1, or Id3; Clec4f-cre-driven expression of diphtheria toxin receptor (DTR) for inducible KC depletion; and Id3 germline knockout. Tumour models included orthotopic pancreatic injection (KPC cells), intraportal injection of various cancer lines (KPC, Pan02, B16F10, LLC1, MC38), and subcutaneous B16F10 melanoma. They performed RNA-seq, RT-qPCR, flow cytometry, immunofluorescence, intravital and time-lapse microscopy, CUT&RUN, and in vitro coculture assays. Human relevance was assessed using PDAC liver metastasis samples and hiPSC-derived macrophages.
**Key Results:**
- KC depletion (Clec4f-cre Csf1r f/f, Clec4f-cre Spi1 f/f, or DT-treated Clec4f-cre R26 LSL-DTR mice) dramatically increased liver and lung metastases after orthotopic KPC injection (100% of mice developed liver and lung metastases vs. ~50% in controls, Fig. 1c-e). Survival after intraportal KPC injection was reduced (Fig. 1g). KC depletion increased tumour cell numbers in the liver 24h post-injection by >3-fold (Fig. 1h) and liver tumour burden at 2 weeks by >5-fold (Fig. 1i,j). A subset of tumour cells (CD47bright CD9+ CD133+) with metastatic potential was increased ~10-fold in KC-deficient livers (Extended Data Fig. 1n).
- KCs were located exclusively around tumour nodules (Fig. 2a,b). They phagocytosed live tumour cells in vivo and in vitro (Fig. 2d-g). Around 50% of wild-type KCs engulfed at least one tumour cell in a 20h assay (Fig. 2g). KCs upregulated chemokines (CCL3, CCL4, CCL5) and cytokines (IL-12, IL-15, IL-18) at the tumour margin, correlating with enrichment of activated CD8+ T cells and NK cells (Fig. 2h-j).
- ID3 deletion in adult KCs (Clec4f-cre Id3 f/f) did not affect KC numbers, morphology, or peritumoural location, but completely abrogated their anti-tumour activity: liver and lung metastases were as large as in KC-depleted mice (Fig. 3g,h), and survival was similarly reduced (Fig. 3j). ID3-deficient KCs showed impaired phagocytosis (3-fold decrease at 24h, 5-fold decrease in vitro, Fig. 4f,g), reduced chemokine/cytokine expression (Fig. 4h), and reduced recruitment/activation of CD8+ T cells and NK cells (Fig. 4i-k).
- Mechanistically, ID3 deficiency led to increased expression of the inhibitory receptor SIRPA and decreased expression of the activating receptor dectin-1 (Fig. 4b-d). SIRPA blockade in ID3-deficient mice rescued phagocytosis, chemokine/cytokine expression, and lymphoid cell recruitment/activation (Fig. 5b-j). Conversely, dectin-1 blockade reduced phagocytosis and chemokine/cytokine production by wild-type KCs (Fig. 5e,f).
- CUT&RUN analysis showed that ID3 prevents binding of transcription factors E2A and ELK1 to the Sirpa promoter/enhancer regions (Fig. 5l). shRNA against E2A or ELK1 reduced Sirpa expression in ID3-deficient KCs (Fig. 5m). LPS further increased E2A/ELK1 binding and Sirpa expression in ID3-deficient but not wild-type KCs (Extended Data Fig. 8e-g).
- Ectopic expression of ID3 in mouse BMDMs or human hiPSC-Macs reduced SIRPA expression, increased dectin-1 expression, and conferred potent phagocytic activity against tumour cells (Fig. 6a,b). ID3-expressing hiPSC-Macs also produced chemokines/cytokines and their supernatants stimulated CD8+ T cell proliferation and IFNγ production by CD8+ T cells and NK cells (Fig. 6c-e).
- In vivo, intraportal injection of ID3-expressing BMDMs into Clec4f-cre Id3 f/f mice prevented liver tumour growth (Fig. 6f). In wild-type mice with LLC1 liver metastases, ID3-expressing BMDMs reduced tumour burden and improved survival (median survival ~5 weeks vs. <3 weeks for controls, Fig. 6g). In a subcutaneous B16F10 model, intratumoural injection of ID3-expressing BMDMs blocked tumour growth locally and recruited activated CD8+ T cells and NK cells (Fig. 6h).
- Human KCs in PDAC liver metastases also expressed ID3, were located peritumourally, contained tumour material, and expressed chemokines/cytokines (Extended Data Fig. 9c-e). scRNA-seq confirmed higher ID3 and lower SIRPA expression in human KCs compared to tumour-associated macrophages (Extended Data Fig. 9h).
**Clinical Implications:** This study identifies ID3 as a master regulator that enables macrophages to mount a potent, local anti-tumour response by orchestrating phagocytosis and lymphoid cell activation. The finding that ectopic ID3 expression can reprogram other macrophages (BMDMs, hiPSC-Macs) into potent anti-tumour effectors suggests a novel cellular immunotherapy approach. Engineering ID3-expressing macrophages could be a promising strategy for treating liver metastases and potentially other solid tumours. The conserved mechanism in human macrophages supports clinical translation.