*2.4. TCS Promotes Infiltration of CD8+ T Cells into HCC*

TCS promoted CD8+ T-cell infiltration in cancer tissues [15]. Therefore, we investigated the level of the infiltration of CD8+ T cells in mouse HCC xenograft tissues. The number of CD8+ T cells in HCC tumor tissues increased with the doses of TCS (Figure 5A). Interestingly, enrichment of CD8+ T cells could be seen at the edge of tumor tissues (Figure 5B).

Chemokines are signaling molecules necessary for normal T-cell transport and function [31], and the interleukin family plays an important role in immune regulation and inflammatory responses [32]. In addition, the increased secretion of TNF-α and IFN-γ contributes to the antitumor interaction with T cells [33]. Therefore, mRNA expressions of chemokines CCL2, CCL17 and CCL22, as well as IL-6, IL-18, TNF-α and IFN-γ, in tumor tissues and H22 cells were detected by RT-qPCR. The results of RT-qPCR showed that the expression levels of CCL2, CCL22, TNF-α and IFN-γ in tumor tissues were significantly increased after TCS treatment. Additionally, CCL2, CCL17, CCL22 and TNF-α were also increased in H22 cells after TCS treatment (Figure S1). Furthermore, according to the results of RT-qPCR, four cytokines with significant differences, CCL2, CCL22, TNF-α and IFN-γ, were detected for protein levels by ELISA. The results of tumor tissue samples showed that the protein levels of chemokines CCL2 and CCL22, as well as TNF-α and IFN-γ, were significantly increased upon TCS treatment (Figure 5C). Serum levels of CCL22, TNF-α and IFN-γ were elevated after TCS treatment, although only IFN-γ statistically significantly increased (Figure 5D). In addition, the expression levels of chemokines CCL2 and CCL22 in HCC cell culture fluid were also significantly increased upon TCS treatment (Figure 5E).

These results suggested that TCS could enrich CD8+ T cells to tumor tissues and promote the expression of chemokines in HCC cells, which would enhance the anti-tumor immune response of the organism.

**Figure 5.** Effects of TCS on chemotactic enrichment. (**A**) Immunofluorescence method detected CD8-positive cells in the center of HCC tissues. Bar = 100 μm; (**B**) Immunofluorescence method detected CD8-positive cells at the edge of HCC tissues. Bar = 100 μm; (**C**) ELISA method detected the protein levels of CCL2, CCL22, TNF-α and IFN-γ in HCC tissues treated with different doses of TCS (0, 0.5, 1 and 2 μg/g); (**D**) ELISA method detected the protein levels of CCL2, CCL22, TNF-α and IFN-γ of mouse serum; (**E**) ELISA method detected the protein levels of CCL2, CCL22 and TNF-α in H22 HCC cells treated with 25 μg/g TCS after 24 h, 48 h and 72 h, GAPDH as the internal reference gene. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

#### *2.5. TCS Enhances the Expression of Granzyme B and M6PR*

GrzB, a serine proteinase released by cytotoxic T cells and NK cells, mediates cell apoptosis in target cells [34,35]. Since TCS could recruit CD8<sup>+</sup> T cells to xenograft tumor tissues, we next examined whether the expression and transportation of GrzB were affected by TCS. As expected, GrzB was elevated in HCC tissues in TCS-treated mice in a dosedependent manner (Figure 6A–C). The proportion of TUNEL+/GrzB+ cells in tumor tissues significantly increased as TCS dosages were raised (Figure 7A–C). We also confirmed that the alterations in the number of TUNEL+ and TUNEL+/GrzB+ cells were positively correlated with TCS dosages (Figure 7B,C). Our previous studies showed the translocation of GrzB from mannose-6-phosphate receptors (M6PR) to HCC cells was enhanced by TCS [23]. Therefore, we next examined the level of M6PR, both in cell lines and xenograft tumor tissues. We showed that TCS promoted the expression level of M6PR in tumor tissues and HCC cells (Figure 8C,D). As TCS dosages were augmented, the proportion of TUNEL+/M6PR+ cells in tumor tissues also significantly increased (Figure 8A,B). The number of M6PR and TUNEL double-positive cells showed a positive correlation with the TCS dose (Figure 8A,B). These data indicated that TCS could inhibit HCC growth by inducing the upregulation of GrzB and promote HCC cell apoptosis in vivo by encouraging M6PR to deliver GrzB into HCC cells.

**Figure 6.** Effects of TCS on GrzB expression in vivo. (**A**,**B**) Immunochemical method detected GrzB in hepatocellular carcinoma tissues. Bar = 100 μm; (**C**) Western blot method detected the level of GrzB in HCC tissues treated with different doses of TCS. \*, *p* < 0.05; \*\*, *p* < 0.01; \*\*\*, *p* < 0.001.

**Figure 7.** TCS promotes GrzB-induced apoptosis in hepatocellular carcinoma. (**A**) Immunochemical method detected TUNEL and GrzB in HCC tissues after TCS treatment. Bar = 100 μm; (**B**) Correlation analysis of GrzB positivity with the number of positive signals for TUNEL; and (**C**) Correlation analysis of the number of simultaneous positive signals for GrzB and TUNEL with the dose of TCS.

**Figure 8.** TCS promotes M6PR expression in vivo and in vitro. (**A**) Immunochemical method detected GrzB and M6PR in HCC tissues upon TCS treatment. Bar = 100 μm; (**B**) Correlation analysis of the number of simultaneous positive signals of GrzB and M6PR with the dose of TCS; (**C**) Western blot method detected the levels of M6PR in HCC tissues treated with different doses of TCS (0, 0.5, 1 and 2 μg/g); (**D**) Western blot method detected the levels of M6PR in H22 HCC cells treated with different doses of TCS (0, 12.5, 25 and 50 μg/mL). \*, *p* < 0.05; \*\*\*, *p* < 0.001.

#### **3. Discussion**

TCS is the major active ingredient of *Trichosanthes Kirilowii* [36]. Previous studies reported TCS alone showed an excellent inhibitory effect on cancer cell proliferation in vitro [9,23,37]. However, TCS alone had not significantly inhibited tumor growth in immunodeficient nude mice in vivo [23]. In the present study, we constructed tumor models in BALB/c mice with a functional immune system. We found that TCS not only activated caspase family proteins in tumor tissues, but also promoted T-cell immunity. Chemotactic enrichment of T cells in HCC tissues and elevated levels of GrzB were observed in vivo. This suggests that TCS has considerable promise as an immunotherapy tool and may increase the effectiveness of anti-tumor treatments.

TCS induced cell cycle block, autophagic death and caspase-mediated apoptosis in cancer cells [28,38]. Proper levels of autophagy remove damaged organelles from cells and contribute to the maintenance of normal cell survival, while inducing excessive autophagy causes cell death [39,40]. TCS induced ROS production in gastric cancer cells, which in turn promoted the autophagic death of gastric cancer cells [28]. However, in the present study, TCS barely promoted the autophagic death of HCC cells. Recently, Hu et al. detected by proteomics that TCS inhibited nuclear proliferation factors in human choriocarcinoma cell lines, mainly inducing caspase-mediated apoptosis in cancer cells [41]. Caspase 8 is a key protease in apoptosis caused by exogenous factors [42]. Caspase 9 is an endogenous apoptosis-related protease activated by damage to organelles, such as endoplasmic reticulum or mitochondria [43]. Both cleaved-caspase 8 and cleaved-caspase 9 activate caspase 3 to form cleaved-caspase 3, which in turn degrades the DNA repairassociated protein PARP and induces apoptosis in cancer cells [44,45]. The results of our study showed that apoptosis inhibitor (Z-VAD-FMK) was able to inhibit TCS-induced HCC cell death, and caspase proteins were significantly activated. Thus, TCS mainly activated caspase-mediated endogenous and exogenous apoptotic pathways to inhibit HCC cells.

TCS can regulate the immune functions of macrophages [46], DC cells [47] and T cells [48]. However, variable effects of TCS on the regulation of immune cell function have been observed in different diseases, such as physiological conditions [48], inflammation [46], HIV infection [49] and cancer [15]. Enhancement of the antitumor response of T cells would be one of the effective ways to inhibit HCC [50]. T cells can induce cancer cell death through both receptor and non-receptor mediated pathways [51]. TCS could enhance the immune effect of T cells against lung cancer through the receptor pathway by enhancing the expression of class I, restricted, T cell-associated molecules in CD8+ T cells [15]. A recent study showed that tonics containing the Chinese herb *Trichosanthes* significantly elevated the serum levels of cytokines, such as IFN-γ, IL-6 and TNF-α, which would be beneficial in enhancing the immune effect of the lymphocyte system [52]. Numerous immune subpopulations, including T cells, natural killer (NK) cells and B cells, can produce IFN-γ in the tumor microenvironment [53]. CD8+ cytotoxic T lymphocytes (CTL) are known to be the main producers of IFN-γ and one of the indicators of activation of the T-cell antitumor function [54]. Studies have shown that IFN-γ could induce apoptosis or scorch death of cancer cells through IFN-γ receptors on the surface of cancer cells [55,56]. TNF-α plays different roles in the pre-cancerous and cancer microenvironments. Despite a sustained inflammatory response possibly being detrimental to suppressing precancerous lesions, increased TNF-α in a tumor microenvironment could effectively activate TNFR1 to trigger cancer cell suppression [57,58]. Tumor-infiltrating lymphocytes are an important prognostic factor for cancer progression and a key player in cancer immunotherapy. CCL2, CCL17 and CCL22 are cytokines with a role in T-cell recruitment [59,60]. Despite the role of CCL2 in recruiting both cytotoxic T cells (CTL) and monocytes to tumor sites [61,62], studies have shown that enhancement of the CCL2/CCR2 axis [63] or inhibition of CCL2 nitration [64] in antitumor therapy significantly promotes T-cell infiltration and exerts antitumor effects. Binding of CCL22 with CCR4 enhances T-cell dendritic cell binding and increases CTL activation [65], while enhancing tumor cell responses to IFN-γ [66]. GrzB, the cytokine secreted by CD8+ T cells, has the greatest killing effect on cancer cells

by directly or indirectly activating caspases to inhibit cancer cell proliferation and induce apoptosis [67]. GrzB could be transported by M6PR into cancer cells [68]. Previous studies by our group demonstrated that the combination of TCS and GrzB in the treatment of nude mice implanted with HCC significantly inhibited the growth of HCC, and TCS promoted the transport of GrzB by M6PR into HCC cells [23]. In the present study, we examined the anti-tumor mechanisms of TCS by facilitating the xenograft tumor model within BALB/c mice with a functional immune system. We observed that CD8+ T cells were recruited into the HCC tissues in the TCS-treated group in a dose-dependent manner. The expression level of chemokines was elevated in HCC tissues, a favorable condition for T cells to kill HCC cells through the receptor or cytokine pathway. Additionally, TCS directly elevated GrzB levels in tumor tissues, which were significantly and positively correlated with apoptotic cancer cells. This implies that TCS can enhance T-cell anti-tumor immunity against HCC by encouraging T-cell enrichment, elevating the expression of chemokines in tumor cells and promoting their production of GrzB. These results provide supporting information for the study of TCS to enhance anti-tumor immunity.
