Multi-dimensional single-cell characterization revealed suppressive immune microenvironment in AFP-positive hepatocellular carcinoma

Alpha-fetoprotein (AFP)-secreting hepatocellular carcinoma (HCC), which accounts for similar to 75% of HCCs, is more aggressive with a worse prognosis than those without AFP production. The mechanism through which the interaction between tumors and the microenvironment leads to distinct phenotypes is not yet clear. Therefore, our study aims to identify the characteristic features and potential treatment targets of AFP-negative HCC (ANHC) and AFP-positive HCC (APHC). We utilized single-cell RNA sequencing to analyze 6 ANHC, 6 APHC, and 4 adjacent normal tissues. Integrated multi-omics analysis together with survival analysis were also performed. Further validation was conducted via cytometry time-of-flight on 30 HCCs and multiplex immunohistochemistry on additional 59 HCCs. Our data showed that the genes related to antigen processing and interferon-gamma response were abundant in tumor cells of APHC. Meanwhile, APHC was associated with multifaceted immune distortion, including exhaustion of diverse T cell subpopulations, and the accumulation of tumor-associated macrophages (TAMs). Notably, TAM-SPP1(+) was highly enriched in APHC, as was its receptor CD44 on T cells and tumor cells. Targeting the Spp1-Cd44 axis restored T cell function in vitro and significantly reduced tumor burden when treated with either anti-Spp1 or anti-Cd44 antibody alone or in combination with anti-Pd-1 antibody in the mouse model. Furthermore, elevated IL6 and TGF-beta 1 signaling contributed to the enrichment of TAM-SPP1(+) in APHC. In conclusion, this study uncovered a highly suppressive microenvironment in APHC and highlighted the role of TAM-SPP1(+) in regulating the immune microenvironment, thereby revealing the SPP1-CD44 axis as a promising target for achieving a more favorable immune response in APHC treatment.

Automated summary

This study aimed to identify the characteristics and potential treatment targets of AFP-negative HCC (ANHC) and AFP-positive HCC (APHC). Single-cell RNA sequencing was used to analyze tumor cells from ANHC, APHC, and adjacent normal tissues. The study found that APHC was associated with immune distortion and the accumulation of tumor-associated macrophages (TAMs) enriched with TAM-SPP1(+). Targeting the Spp1-Cd44 axis improved T cell function and reduced tumor burden in APHC treatment.