**4. Materials and Methods**

Recombinant TCS protein extraction and purification: The recombinant TCS plasmid was constructed by Tsingke Biotechnology Co., Ltd. (Guangzhou, China). The rTCS sequence was inserted between the EcoRI site and the XhoI site of pet-28a+. *E.coli* BL21 (DE3) was used to induce the expression of recombinant TCS protein (when OD600 = 0.60, add a final concentration of 2 mM IPTG). Centrifuge the prokaryotic expression induced bacterial solution at 4 ◦C, 12,000 rpm, 2 min; discard the supernatant; then, add the lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0); blow repeatedly; and perform ultrasonic lysis on ice (power: 30%, ultrasound: 5 s, stop: 10 s, total working time: 30 min); and then collect the supernatant at 4 ◦C, 12,000 rpm, 30 min. After the supernatant containing the target protein is filtered and sterilized, it is purified by the protein purifier (GE, AKTA purifier, Boston, MA, USA). The obtained recombinant TCS protein was freeze-dried and stored at −30 °C.

Cell culture: Mouse HCC cell line (H22) (Procell, CL-0341) was cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) medium containing 10% fetal bovine serum, 100 U/mL of penicillin and 100 μg/mL of streptomycin. HCC cells were cultured in a 5% CO2 incubator at a constant temperature of 37 ◦C.

HCC cell viability assay: H22 cells were inoculated in 96-well plates at a density of <sup>5</sup> × <sup>10</sup>3, with 100 <sup>μ</sup>L cell culture solution per well. For a concentration-dependent assay, TCS (0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL) was added to the treatment for 24 h, 48 h and 72 h. For a time-dependent assay, TCS (0, 25 μg/mL) was added to the treatment for 0 h, 12 h, 24 h, 36 h, 48 h, 60 h and 72 h. Cell viability was then determined by the method of CCK-8. Briefly, at the end of the intervention, 10 μL of CCK-8 solution was added to each well of the 96-well plate and mixed, and the reaction plates were incubated at 37 ◦C for 1–3 h. The absorbance of each well solution was measured, using the microplate reader (BioTek, Epoch, Winooski, VT, USA) at 450 nm wavelength detection light.

Calcein-AM/PI staining: Unspecific esterases present in living cells can metabolize Calcein-AM and emit green fluorescence. Propidium iodide (PI) will label the nuclei of dead cells. H22 cells were inoculated in 6-well plates at a concentration of 1 × 105/mL culture medium per well. Cells were treated with different doses of TCS, then Calcein-AM and PI were added and incubated for 5 min, and fluorescent pictures were collected under a confocal microscope. Cell death index calculation: percentage of dead cells = number of dead cells/total number of cells.

Antibodies: The antibody information in this study is shown in Table S1.

Reagents: Z-VAD-FMK (Beyotime, Nanjing, China, cat# C1202), Calcein-AM (MCE, Monmouth Junction, NJ, USA, cat# HY-D0041), PI (MCE, Monmouth Junction, NJ, USA, cat# HY-D0815), DAPI (Solarbio, Beijing, China, cat# D8200) DeadEnd™ Fluorometric TUNEL System (Beyotime, Nanjing, China, cat# C1088). IHC Reagent Kit (Solarbio, Beijing, China, cat# SP0021), Total Mrna Extraction Kit (Promega, Madison, WI, USA, cat# LS1040), Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen, Shanghai, China, cat# 11141ES60), GoldenstarTM RT6 cDNA Synthesis Kit (TSINGKE, Xi'an, China, cat# TSG302M), ELISA MAX™ Standard Set Mouse MCP-1 (BioLegend, San Diego, CA, USA, cat# 432701), ELISA MAX™Deluxe Set Mouse IFNγ (BioLegend, San Diego, CA, USA, cat# 430804), Mouse TNF-α ELISA kit (Jianglaibio, Shanghai, China, cat# JL10484), Mouse MDC/CCL22 ELISA kit (Jianglaibio, Shanghai, China, cat# JL11125), etc.

Apoptosis inhibition test assay: Z-VAD-FMK is a pan-caspase inhibitor that prevents the cleavage degradation of DNA repair enzyme PARP by Caspase family proteins [69]. H22 cells were inoculated in 6-well plates at a concentration of 1 × 105/mL culture medium per well. Z-VAD-FMK was intervened according to the following protocol: control (no TCS), TCS (12.5 μg/mL), TCS (25 μg/mL), TCS (50 μg/mL), TCS (12.5 μg/mL) + Z-VAD-FMK (40 μM), TCS (25 μg/mL) + Z-VAD-FMK (40 μM), TCS (50 μg/mL) + Z-VAD-FMK (40 μM) for 48 h of continuous intervention, and cells were analyzed by Calcein-AM/PI staining and immune-blotting for PARP-related proteins.

Xenograft tumor model: The procedure of the animal experiments performed in the study fulfilled the requirements of the ethical review committee. Male BALB/c mice (5 week-old, 20 ± 2 g, Guangdong Medical Laboratory Animal Center, Guangzhou) were housed in an SPF-grade environment with a 12 h light/dark cycle and maintained on free diets. Mice were injected subcutaneously with 5 × <sup>10</sup><sup>5</sup> H22 HCC cells in the right axilla. Beginning on the fifth day after implantation, mice with H22 HCC were randomly divided into four groups of five mice each and treated according to the following treatment protocols: phosphate-buffered saline (PBS) control group, 0.5 μg/g (TCS weight/mouse body weight) TCS group, 1 μg/g body weight TCS group, 2 μg/g body weight TCS group. Body weight TCS group. Control group was injected with 100 μL of PBS, and drug treatment was injected on the 4, 6, 8, 10, 12, 14 and 16 days after implantation of HCC cells. Tumor volume and body weight were measured, and tumor size was estimated according to the following formula: tumor volume = 0.5 × maximum diameter × shortest diameter × shortest diameter.

Immunohistochemistry (IHC) assay: Immunohistochemical staining of mouse tumor tissues was performed using the IHC kit and GrzB polyclonal antibody. Tumor tissues embedded in paraffin blocks were cut into 5 μm thick sections. The paraffin sections were dewaxed and hydrated, following 3% hydrogen peroxide treatment and antigen repair boiling in sodium citrate solution for 10 min. After incubation with normal goat serum at room temperature for 20 min, the tissue sections were incubated with anti-GrzB antibody (1:500) overnight at 4 ◦C. Then, samples were incubated with biotinylated goat anti-rabbit serum immunoglobulin G (IgG) antibody (1:100) for 30 min at 37 ◦C. After thorough washing, streptavidin-POD working solution was added and incubated for 30 min at 37 ◦C. Sections were counter-stained with hematoxylin. Morphological images were collected using an Olympus microscope. For immunofluorescence assay, tissue sections were incubated with antibodies against Ki67 (1:500), M6PR (1:500), GrzB (1:500) and CD8 (1:500) overnight at 4 ◦C. Samples were then incubated with appropriate FITC, Alexa Fluor 555 or Horseradish Peroxidase-conjugated secondary antibodies for 3 h. Nuclei were stained with DAPI or hematoxylin, followed by observation and image capture under a confocal microscope.

TUNEL apoptosis assay: Tumor tissues were examined using the Beyotime one-step TUNEL apoptosis assay kit. After dewaxing and hydration of the tumor tissues, the TUNEL staining procedure was performed, according to the manufacturer's instructions. The nuclei were stained with DAPI.

Western blot assay: Protein extraction and Western blot assay were performed, as described in the previous report [23]. The following antibodies were used for detection: anti-Caspase 3 (1:1000), anti-Caspase 8 (1:1000), anti-Caspase 9 (1:1000), anti-Cleavedcaspase 3 (1:1000), anti-Cleaved-caspase 8 (1:1000), anti-Cleaved-caspase 9 (1:1000), anti-GrzB (1:1000), anti-M6PR (1:1000), anti-LC3B (1:1000), anti-P62 (1:1000), anti-GAPDH

(1:5000). They were incubated overnight at 4 ◦C and then incubated with horseradish peroxidase (HRP)-conjugated counterpart secondary antibody (1:5000) at room temperature. Chemiluminescence was performed using ECL Ultra HRP substrate and photographed under the SAGECREATION ChemiMini™ Imaging System.

Quantitative real-time PCR: Total RNA from HCC cells and tumor tissues was extracted using the Total RNA Extraction Kit. Using the GoldenstarTM RT6 cDNA Synthesis Kit, mRNA was reversed into cDNA by reverse transcription procedure. Quantitative PCR amplification was performed using the Hifair III 1st Strand cDNA Synthesis Super Mix for qPCR in Quantstudio™ 7 Flex Real-Time PCR System (ABI). The expression of target genes was normalized against GAPDH using the 2−ΔΔCt assay. Primer oligos were synthesized by TSINGKE Biological Co., Ltd. (Beijing, China) and are listed in Table S2.

Enzyme linked immunosorbent assay (ELISA): Cell culture supernatants, mouse serum and tumor tissues were collected. Then, the levels of TNF-α, IFN-γ, CCL22 and CCL2 were assessed using ELISA kits, according to the manufacturer's instructions. Absorbance was measured using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

Statistical analysis: Statistical analyses were performed using SPSS 26.0 software (SPSS Inc., Chicago, IL, USA). The error bars in the graphical data represent means ± Standard Error of Mean (SEM). Three or more comparisons were compared using one-way ANOVA, followed by the least significant difference (LSD) test. Unpaired two-tailed Student's t-test was used to compare two sets of data. A value of *p* < 0.05 was considered a statistically significant difference between the data groups. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

#### **5. Conclusions**

In conclusion, the results of this study demonstrated that TCS inhibited HCC cells by activating caspase, recruiting CD8<sup>+</sup> T cells, enhancing the expression of chemokines and up-regulating M6PR genes to transport GrzB (Figure 9). This also indicates that TCS is a natural drug with great potential to enhance anti-tumor immunity, which is valuable for further in-depth pharmacological studies.

**Figure 9.** Diagram showing the molecular mechanism of TCS on anti-tumor activity. TCS stimulated the expression of chemokines CCL2, CCL17 and CCL22, which may encourage the enrichment of CD8+ T cells within HCC tissue. GrzB secreted by the T cells were transported into the HCC cells by cellular M6PR and facilitated cell apoptosis. TCS-induced caspases mediated apoptosis both in vivo and in vitro.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms24021416/s1.

**Author Contributions:** O.S., K.W. and C.L. designed this study. K.W., X.W. (Xiaona Wang), M.Z. (Minghuan Zhang), Z.Y. and Z.Z. performed the experiments and statistical analysis. K.W. and X.W. (Xiaona Wang) prepared figures and wrote the manuscript. K.Y.T., G.Z., F.G., M.Z. (Meiqi Zeng) and S.C.W.S. participated in the discussion and provided suggestions. X.W. (Xia Wang) and O.S. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was financially supported by the National Natural Science Foundation of China (81773939) and the Technology Foundation of Shenzhen City (JCYJ20210324094005015).

**Institutional Review Board Statement:** The animal study protocol was approved by the Animal Ethical and Welfare Committee of SZU (AEWXC-202200024 and 20220104).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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