# Icaritin eliminates tumor-associated macrophages via STX16-dependent extracellular vesicle delivery of autophagosomes from hepatocellular carcinoma cells

## TL;DR

Icaritin fights liver cancer by reducing harmful macrophages through a chain of metabolic, epigenetic, and autophagy-related processes.

## Contribution

Icaritin's novel mechanism targeting ALDOB, STX16, and STAT3 to eliminate M2 TAMs is revealed.

## Key findings

- Icaritin suppresses ALDOB to reduce lactate and H3 lactylation on the STX16 promoter.
- STX16 deficiency causes autophagosome accumulation and EV-mediated macrophage death.
- Icaritin inhibits tumor growth and M2 TAM infiltration in vivo via the ALDOB/STX16/autophagy/STAT3 axis.

## Abstract

To elucidate the mechanism by which icaritin—a novel agent for hepatocellular carcinoma (HCC)—remodels the tumor microenvironment (TME) by inhibiting HCC cell metabolism-mediated M2 polarization of tumor-associated macrophages (TAMs).

Integrative approaches spanning in vitro Transwell cocultures, RNA-seq, LC3-based autophagy tracing, STX16 gene edition, and orthotopic xenografts mechanistically dissected the affective and mechanism of icaritin in remodeling TME.

Our results indicate that icaritin transcriptionally suppresses ALDOB in HCC cells to reduce lactate production. Consequently, the lactylation of histone H3 at lysine 9 and lysine 18 (H3K9/H3K18la) on the STX16 promoter is diminished, thereby ablating STX16 transcription. STX16 deficiency blocks autophagolysosome biogenesis, leading to the accumulation of autophagosomes in HCC cells. These autophagosomes are subsequently delivered to macrophages via extracellular vesicle (EVs) and then triggers autophagic cell death and p62-guided STAT3 destruction within the macrophages, thereby eliminating M2 TAMs and reprograming the tumor immune microenvironment. In vivo validation confirmed icaritin suppressed tumor growth and M2 macrophage infiltration via the ALDOB/STX16/autophagy/STAT3 axis.

These results indicate that by orchestrating a novel "metabolism-epigenetics-autophagy-EVs" cascade to eliminate TAMs, icaritin targets ALDOB, STX16, and STAT3, revealing key nodes for therapeutic intervention.

The online version contains supplementary material available at 10.1186/s13046-026-03671-0.