4.10. Cell growth assay
The cell viability CCK8 assay (Beyotime, Shanghai, China) was used to measure cell viability after different treatments, as previously described (33). Briefly, cells were first seeded into a 96-well plate at a density of 5000 cells/well. After the different treatments, cells were subjected to 10 µL of CCK8 and incubated in the dark for 3 h at 37°C. Then, the optical density values at 450 nm were measured on a microplate spectrophotometer (Thermo Scientific, USA). For the colony formation assay after different treatments, cells were first seeded into a 6-well plate at a density of 1000 cells/well and routinely cultured for 14 days. The cells were subsequently fixed with 4% paraformaldehyde in PB buffer for 15 min and then stained with 0.1% crystal violet for 10 min. The colony formation images were captured on the camera.
4.11. In vitro caspase-3 assay
The caspase-3 activity of cells was quantified by flow cytometry analysis using the caspase-3 activity detection kit for live cells (Beyotime, Shanghai, China) in accordance with the manufacturer’s protocol (40). Briefly, after the different treatments, the cell culture medium was transferred to a suitable centrifuge tube, and the cells in the 6-well plate were collected using trypsin-EDTA solution (Boster Biological Technology Co. Ltd., CA, USA) and transferred to the same centrifuge tube. The cells were separated from the suspension by centrifugation at 1000 rpm for 5 min. After being carefully washed twice with PBS, the cells were incubated with 5 µM of GreenNuc Caspase-3 substrate in the dark for 30 min at 25°C. The cell samples were immediately analyzed on a flow cytometer (BD, CA, USA).
4.12. Cell apoptosis
The proportion of apoptotic cells was quantified by flow cytometry analysis using the Annexin V-FITC/PI Apoptosis Assay Kit (Boster Biological Technology Co. Ltd., CA, USA) Flow cytometry analysis was performed as previously described (33).
4.13. LysoTracker Green staining
After different treatments, cells were washed with fresh DMEM and incubated with 75 nM LysoTracker Green for 1 h at 37°C (Yeasen Biotech, Shanghai, China). Nuclei were stained with Hoechst 33258 (Servicebio, Wuhan, China), and images were captured with a fluorescence microscope.
4.14. Lysosomal membrane stability
Lysosomal membrane stability was tested using AO (Sigma-Aldrich, Shanghai, China). After different treatments, cells were incubated with AO solution (5 µg/mL) in complete medium for 15 min at 37°C, and images were captured with a fluorescence microscope.
4.15. Mitochondrial membrane potential measurement
The mitochondrial membrane potential (ΔΨm) of cells was detected with JC-1 from the mitochondrial membrane potential assay kit (Beyotime, Shanghai, China) in accordance with the manufacturer’s protocol. Briefly, after different treatments, cells in the 12-well plate were incubated with 500 µL of complete medium and 500 µL of JC-1 dyeing working fluid for 20 min at 37°C. After washing twice with precooled JC-1 staining buffer, the cells were observed and photographed under fluorescence microscopy.
4.16. Immunofluorescence analysis
Immunofluorescence analyses were conducted as previously described (
33). No cell-permeable fluid was used in the immunofluorescence procedures for proteins located on the surface of cell membranes. Images were captured with a fluorescence microscope, and relative quantitative fluorescence analysis was performed using ImageJ software.
4.17. Immunofluorescence co-staining analysis
Immunofluorescence co-staining was performed as previously described (
33). Briefly, the cells were incubated with mouse monoclonal PPT1 Ab (1:50) and rabbit polyclonal LAMP1 Ab (1:50) overnight at 4°C. Then, the cells were incubated with phycoerythrin PE-conjugated anti-mouse secondary antibody and FITC-conjugated rabbit secondary antibody (1:100, Boster Biological Technology Co., Ltd.) in the dark for 1 h. Nuclei were stained with Hoechst 33258 (Servicebio, Wuhan, China), and images were captured with confocal laser scanning microscopy. Immunofluorescence co-staining for PPT1 and Tom20 was performed using the same method. Fluorescence co-localization analysis was performed using ImageJ software (
41).
4.18. ELISA analysis
The respective media and supernatant of cells and tumor tissues after different treatments were collected for ELISA analysis of HMGB1, APT, IL-12, and IFN-γ using the appropriate ELISA kits and following the manufactures’ instructions.
4.19. In vivo DC maturation assays
To study the effects of DC661 treatment on DC maturation in vivo, a Hep 1–6 subcutaneous tumor model grown in C57BL/6 mice was used. When the tumors grew to about 300 mm3, the mice were treated by intraperitoneal injection of PBS (control) or DC661. The spleens of tumor-bearing mice were collected 7 d after the different treatments, and DCs were analyzed by flow cytometry. Mature DCs were defined as CD11c+CD80+CD86+ cells (29).
4.20. In vivo tumor immune microenvironment assays
To study the effects of DC661 treatment on the tumor immune microenvironment in vivo, a Hep 1–6 subcutaneous tumor model in C57BL/6 mice was used. When the tumors grew to about 300 mm
3, the mice were treated by intraperitoneal injection of PBS or DC661. Tumor tissues were collected 7 d after treatment and digested to form a single-cell suspension using a tumor digestion kit according to the manufacturer’s protocol. After lysing the red blood cells, a single-cell suspension of tumor tissue was analyzed by flow cytometry. Activated CD8
+ T cells were defined as CD3
+CD8
+CD69
+ cells, and activated CD4
+ T cells were defined as CD3
+CD4
+CD69
+ cells (
42). MDSCs were defined as CD45
+CD11b
+Gr-1
+ cells (
29).
4.21. In vivo drug treatment assay
All animal experiments were performed in compliance with the guidelines of the Animal Care Committee at Tongji Medical College, Huazhong University of Science and Technology (HUST, IORG number: IORG0003571), and the study was performed in accordance with the declaration of Helsinki. In total, 5 × 10
6 sorafenib-resistant Hep 1–6 cells were injected subcutaneously into the right front flanks of C57BL/6 mice. Once the tumors were established and reached approximately 300 mm
3, the mice were randomly divided into four groups (n = 18, in each group) treated with DMSO, sorafenib (30 mg/kg/d; TargetMol), DC661 (3 mg/kg/d; TargetMol), or sorafenib and DC661 combined. The mice were given sorafenib orally, while DC661 was administered intraperitoneally, both on a daily basis. Tumor dimensions were measured using a vernier caliper every 3 days and the volume calculated using the following formula: volume (cubic centimeters) = L × W × W × 0.5. The mice (n = 6, in each group) were treated for 21 days before sacrifice, at which point tumors were harvested and the volume and weight calculated. The survival times of the rest of the mice in each treatment group (n = 12) were recorded every other day.
4.22. Hematoxylin and eosin staining
Hematoxylin and eosin staining was performed as previously described (
33), and images were captured using optical microscopy (Olympus, Japan).
4.23. TUNEL assay
The TUNEL assay was performed as previously described (
33). DAPI was used to stain the nuclei, and images were captured by a fluorescence microscope.
4.24. Immunohistochemical analysis
Immunohistochemical staining was performed as previously described (33), and images were captured using optical microscopy (Olympus, Japan). Each tissue slice was assigned a score based on the proportion of stained cells (0 = 0%, 1 = 1–25%, 2 = 25–50%, 3 = 50–75%, 4 = 75–100%) and the intensity of the staining (0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining) as previously described (43).
4.25. Statistical analysis
The PPT1 expression levels in HCC tissues were distributed according to quartile. Survival analyses for PFS and OS were performed by utilizing the Kaplan–Meier method and log-rank test. A Cox proportional hazards model was performed to evaluate the relative risk factors associated with OS, with hazard ratios (HR) and 95% confidence intervals (95%CI) obtained for each variable. All quantitative data were analyzed using Student’s t-test or the Mann-Whitney U test wherever appropriate. The results are shown as the mean and standard deviation, and P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001). The statistical significance of the results was determined by GraphPad Prism.