Normal goat serum (ngs)

The three cell lines were seeded at 35,000 cells per well onto 15-mm glass slides in a 24-well plate and incubated at 37°C for 24 h. Cells were fixed at room temperature with 4% paraformaldehyde for 15 min and subsequently washed with phosphate-buffered saline (PBS) 3 times for 3 min each. Following this, cells were blocked with 5% normal goat serum (Boster Biological Technology, Ltd., Wuhan, China) for 30 min at room temperature. After thorough washing with PBS 3 times for 3 min each, the cells were incubated with anti-TROP2 monoclonal antibody (1:500; catalogue no. sc-376746; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) overnight at 4°C. Subsequent to 3 washes for 3 min each with PBS, the cells were stained with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin G (1:200; catalogue no. ZF-0312; Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) for 1 h at room temperature. Fluorescence-labeled TROP2 was observed and photographed under a fluorescent microscope.

Liver tissue were fixed with formaldehyde and hydrated with gradient ethanol as described in HE staining. Following citrate buffer antigen retrieval, slices were incubated with 3% deionized water for 20 min and then washed with PBS for 5 min, 3 times. Following blocking with 5–10% normal goat serum (Boster Biological Technology Co., Ltd.) for 25 min at room temperature, 4 µm thick slices were incubated with DPPIV primary antibody (cat. no. 10940_1_ap; 1:100; Proteintech group, Inc., Wuhan, Hubei) at 4°C overnight. Subsequently, samples were incubated with goat anti-rabbit secondary antibody (cat. no. GB23303; 1:200; Wuhan Goodbio Technology Co., Ltd., Wuhan, Hubei) for 50 min at room temperature. Following color development with 3,3′-diaminobenzidine reagent for 5 min at room temperature, the sections were mounted and observed under a light microscope (magnification, ×400; Nikou Corporation), and the appearance of a brown-yellow liver cell membrane indicated positive expression. A total of five non-overlapping fields of view were selected and the immunohistochemical scores (IHS) method (17 (link)) was used to score samples according to the percentage of positive cells and their staining intensity, and the results were analyzed with Image Pro-Plus version 6.0 (Media Cybernetics, Rockville, Maryland, USA).

The sections (4 µm thick) wereobtained from 10% formalin-fixed for 24 h and paraffin-embedded TMA blocks and used for IHC. The sections were washed in xylene to remove the paraffin and rehydrated in a graded alcohol series of 100, 100, 90, 80 and 70%, followed by PBS washing. Antigen retrieval was achieved by heating sections in EDTA antigen retrieval solution for 20 min at 99°C. Endogenous peroxidase activity was blocked using 3% H₂O₂/methanol for 20 min at 37°C. After blocking non-specific protein binding with 10% normal goat serum (Boster Biological Technology co.ltd) for 30 min at 37°C, the sections were incubated with primary antibodies against CXCL17 (1:50; cat. no. 422208; R&D Systems, Inc.) and CXCR8 (1:750; cat. no. DF4973; Affinity Biosciences), respectively, overnight at 4°C. After leaving at room temperature for 30 min and rinsing three times in PBS, the sections were incubated with secondary antibody (ready to use, Dako K8002, Agilent Technologies, Inc.) for 30 min at 37°C. The 3,3′-diaminobenzidine substrate was applied to the sections, which were then counterstained for 1 min at room temperature with haematoxylin, dehydrated and mounted. Negative controls were run in parallel and PBS was used instead of primary antibody.