Researchers investigated this question because metastasis causes most cancer deaths, and the liver is a frequent site of spread for many tumor types, including colorectal, hepatocellular, and melanoma. Kupffer cells, the liver’s resident macrophages, help limit metastasis by phagocytosing disseminated tumor cells, but the tumor-intrinsic molecules that influence this process have remained poorly understood compared with signals affecting bone-marrow-derived macrophages.
Previous work had identified some Kupffer cell–specific factors, such as ID3 and Dectin-2, that regulate their phagocytic activity. Studies on other macrophages had mapped “eat me” and “don’t eat me” signals, including CD47 and CD24. The authors’ earlier genome-scale CRISPR–Cas9 knockout screen in vivo had shown enrichment of gRNAs targeting Slc38a4 in liver metastatic tissues, and separate studies had linked low SLC38A4 expression to worse prognosis in hepatocellular carcinoma (HCC) and colorectal cancer (CRC).
In the new experiments, the team used gain- and loss-of-function approaches in multiple mouse and human tumor cell lines (Hepa1-6 HCC, MC38 CRC, B16F10 melanoma, and human counterparts). They generated stable SLC38A4 overexpression or knockdown lines via lentivirus and injected the cells intrasplenically into immunocompetent C57BL/6 or T-cell-deficient nude mice to model liver metastasis. Metastatic burden was quantified by counting nodules and measuring lesion area on H&E-stained sections. Phagocytosis was assessed both in vitro (co-culture of CFSE-labeled tumor cells with isolated mouse Kupffer cells, measured by flow cytometry as CFSE+ F4/80+ cells) and in vivo (12 hours after intrasplenic injection, analyzing non-parenchymal liver cells). Kupffer cells were depleted with clodronate liposomes in some experiments to test dependency.
To probe mechanism, the researchers measured expression of known “don’t eat me” molecules, performed qPCR and Western blots for mRNA and protein levels, conducted luciferase promoter reporter assays, and used CUT&RUN to assess transcription factor binding. They also analyzed clinical samples by immunohistochemistry (IHC) on 70 HCC tissues (30 with liver micrometastases) and 57 CRC tissues, plus public TCGA and GEO datasets for RNA-level correlations. Experiments were performed with biological replicates (typically n=3 for in vitro, n=5–8 mice per group for in vivo).
Overexpression of SLC38A4 in tumor cells reduced the number and size of liver metastases across the tested lines, while knockdown increased metastatic burden. This effect occurred early (detectable by bioluminescence within 12 hours) and was independent of direct changes in tumor cell proliferation or T-cell activity. Depletion of Kupffer cells abolished the protective effect of SLC38A4 overexpression, indicating that the anti-metastatic activity depends on these macrophages.
SLC38A4 overexpression increased phagocytosis of tumor cells by Kupffer cells both in vitro and in vivo; knockdown decreased it. SLC38A4 reduced expression of the “don’t eat me” signal CD24 at mRNA and protein levels without altering CD47 or PD-L1. Manipulating CD24 levels (knockdown or overexpression) or using CD24-blocking antibodies showed that CD24 functionally mediates the phagocytosis effects: CD24 knockdown rescued the reduced phagocytosis caused by SLC38A4 loss, and CD24 overexpression blocked the gain from SLC38A4. SLC38A4 lowered MYC protein levels; MYC directly bound the CD24 promoter and activated its transcription. Reducing MYC mimicked SLC38A4’s effects on CD24 and phagocytosis, while MYC overexpression reversed them.
In patient tissues, lower SLC38A4 protein (by IHC) correlated with higher rates of liver metastasis in both HCC and CRC cohorts, along with higher MYC and CD24 levels (negative Spearman correlations). Similar patterns appeared in RNA data from public databases.
The researchers concluded that SLC38A4 on tumor cells suppresses liver metastasis by enhancing Kupffer cell phagocytosis through downregulation of CD24 via reduced MYC activity. “This study demonstrates that SLC38A4 promotes Kupffer cell phagocytosis and restricts tumor liver metastasis by suppressing CD24. The SLC38A4/MYC/CD24 axis represents a novel phagocytosis checkpoint for Kupffer cells and a potential therapeutic target for liver metastasis.”
The study is largely preclinical. It relies on intrasplenic injection models that deliver tumor cells directly to the liver, which may not fully replicate all steps of spontaneous metastasis from distant primary sites. Patient analyses are correlative, based on IHC from a modest number of samples (70 HCC, 57 CRC) without longitudinal outcome data. The work does not test therapeutic agents targeting the axis, nor does it address potential effects on other macrophage populations or long-term immune consequences of modulating SLC38A4 or CD24. Findings were consistent across several tumor types and models but remain to be validated in additional contexts.
References:
Renjie Wang, Sen Lin, Jie Li, Yong-Da Liu, Xing-Yi Lin, Yi-Qing Zhu, Wan-Peng Lu, Yan-Fang Tang, Xing-Peng Guo, Yu-Han Ai, Ting-Ting Xu, Mei Huang, Jinghan Wang, Xiao-Ting Zhu, Ji-Hang Yuan et al. SLC38A4 promotes Kupffer cell phagocytosis and suppresses tumor liver metastasis. Experimental & Molecular Medicine (2026).
