The Spatial Impact of a Western Diet in Enriching Galectin-1-Regulated Rho, ECM, and SASP Signaling in a Novel MASH-HCC Mouse Model

Academic Background

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, and metabolic dysfunction-associated steatohepatitis (MASH) is one of the primary triggers of HCC. With the prevalence of the Western diet (WD), the incidence of MASH continues to rise, but there are currently no effective drugs to prevent MASH, and treatment options for HCC are very limited. Galectin-1 (Gal-1) is a biomarker for HCC and plays a crucial role in hepatocarcinogenesis. Previous studies have shown that silencing Gal-1 can effectively treat mouse HCC, but the impact of WD on Gal-1 signaling in the progression from MASH to HCC remains unclear. This study aims to fill this knowledge gap by exploring how WD affects the tumor microenvironment of HCC through Gal-1 signaling.

Source of the Paper

This paper was co-authored by Tahereh Setayesh, Ying Hu, Farzam Vaziri, Dongguang Wei, and Yu-Jui Yvonne Wan, all from the University of California, Davis. The paper was published in 2024 in the journal Biomarker Research, titled The spatial impact of a western diet in enriching galectin-1-regulated Rho, ECM, and SASP signaling in a novel MASH-HCC mouse model.

Research Process

1. Establishment of the MASH-HCC Mouse Model

The study first established a novel MASH-HCC mouse model. FVB/N male mice were fed a WD (21.2% fat, 34% sucrose, 0.2% cholesterol) after weaning for five months. In the experimental group, mice received hydrodynamic injections of plasmids containing myr-Akt1 and N-RasV12 two months before euthanasia to induce HCC. Control mice were fed a healthy diet.

2. Silencing and Overexpression of Gal-1

To study the role of Gal-1, the study used adeno-associated virus (AAV9) to silence or overexpress Gal-1. Gal-1 siRNA was administered via intravenous injection, with scramble-AAV9 used as the control. Gal-1 overexpression was achieved using AAV9-Gal-1, with AAV9 without an insert as the control.

3. Histology and Multiplex Immunohistochemistry (IHC)

Liver tissues were fixed and stained with hematoxylin and eosin (H&E), and multiplex IHC was used to detect markers such as E-cadherin, N-cadherin, Gal-1, and F4/80. Fluorescence signals were scanned using the Vectra 3 automated quantitative pathology imaging system, and signal unmixing was performed using INFORM software.

4. RNA Extraction, Sequencing, and Analysis

RNA was extracted from healthy liver, HCC, and MASH-HCC samples for cDNA synthesis and RNA sequencing. Differentially expressed genes were analyzed using Reactome, and pathway enrichment analysis was performed using GSEA and iDEP.

5. Spatial Transcriptomics and Protein Analysis

The GeoMx DSP technology was used for whole transcriptome sequencing and protein analysis of liver sections. Regions of interest (ROIs) inside the tumor, at the tumor margin, and outside the tumor were selected for data acquisition, and data analysis was performed using Nanostring’s GeoMx NGS pipeline software.

6. Cell Deconvolution Analysis

SpatialDecon GeoScript was used for cell deconvolution analysis to determine the abundance of immune cells in the tumor microenvironment.

Main Results

1. Morphological Characteristics of MASH-HCC

The study found that the livers of MASH-HCC mice exhibited significant steatosis, fibrosis, and tumor formation. Compared to WD-induced metabolic dysfunction-associated fatty liver disease (MAFLD), MASH-HCC had a significantly higher hepatic collagen concentration.

2. Molecular Impact of WD on Healthy Liver and HCC

WD intake significantly activated pathways such as Rho signaling, extracellular matrix (ECM) remodeling, and senescence-associated secretory phenotype (SASP). Compared to healthy livers, MAFLD mice showed significant enrichment of ECM, collagen formation, and degradation pathways.

3. Molecular Differences Between MASH-HCC and HCC

Compared to healthy livers, MASH-HCC showed significant enrichment of ECM, cytokine signaling pathways, and SASP. Additionally, MASH-HCC exhibited significant enrichment of Rho GTPase effectors, ECM remodeling, and cell cycle pathways in the tumor interior and margin regions.

4. Spatial Effects of Gal-1

Silencing Gal-1 significantly reduced the tumor burden in MASH-HCC mice and downregulated immune and ECM regulatory pathways at the tumor margin. Overexpression of Gal-1 enhanced Rho GTPase signaling and SASP.

5. Impact of WD on Gal-1-Mediated Signaling

WD intake and Gal-1 overexpression jointly upregulated Rho GTPase effectors and ECM degradation pathways, while silencing Gal-1 downregulated these pathways.

Conclusion

This study reveals the extensive impact of WD on the HCC tumor microenvironment through Gal-1-mediated signaling pathways, including Rho signaling, ECM remodeling, and SASP. These changes not only promote hepatocarcinogenesis but may also affect the outcomes of HCC immunotherapy. The findings provide new insights into the role of WD in HCC and offer potential targets for future treatment strategies.

Research Highlights

1. Novel MASH-HCC Mouse Model: This study is the first to establish a human-relevant MASH-HCC mouse model, providing an important tool for studying the impact of WD on HCC.
2. Spatial Transcriptomics Technology: Through spatial transcriptomics, the study revealed the spatially specific effects of WD on the tumor microenvironment.
3. Key Role of Gal-1: The study confirmed the critical role of Gal-1 in regulating Rho signaling, ECM remodeling, and SASP, offering new directions for targeted therapies.

Research Significance

This study not only fills the knowledge gap regarding the role of WD in Gal-1 signaling in MASH-HCC but also provides a theoretical foundation for developing new HCC treatment strategies. By revealing the complex impact of WD on the tumor microenvironment, the study offers important references for future dietary interventions and immunotherapies.