Advances in Molecular Mechanisms for Traditional Chinese Medicine Actions in Regulating Tumor Immune Responses

Traditional Chinese medicine (TCM) has been developed for thousands of years with its various biological activities. The interest in TCM in tumor prevention and treatment is rising with its synergistic effect on tumor cells and tumor immunosuppressive microenvironment (TIM). Characteristic of TCM fits well within the whole system and multi-target cancer treatment. Herein we discuss the underlying mechanisms of TCM actions in TIM via regulating immunosuppressive cells, including restoring the antigen presentation function of dendritic cells, enhancing NK cells-mediated killing activity, restraining the functions of myeloid cell-derived suppressor cells, and inhibiting cancer-associated fibroblasts. TCM also regulates tumor progression through enhancing immune response, preventing immune escape and inducing cell death of tumor cells, which triggers immune response in nearby cells. In addition, we discuss TCM in clinical applications and the advantages and disadvantages of TCM in cancer prevention and treatment, as well as current therapeutic challenges and strategies. It might be helpful for understanding the therapeutic potential of TCM for cancer in clinic.


INTRODUCTION
Tumor microenvironment (TME) plays a crucial role in the development and migration of tumor. Accumulating evidence show that cancer growth and metastasis occur as a result of disruption between cancer cells and the TME (Fidler, 2003). Immune cells in TME promote tumor progression developing a tumor immunosuppressive microenvironment (TIM), which releases immunosuppressive factors that alter the phenotype and function of immune cells. Some high risk factors in TME, including inflammatory cytokines, hyperosmosis, acid environment, and hypoxia promote TIM formation. Targeted organs can release various cytokines to induce angiogenesis, lymphangiogenesis and cell proliferation, resulting in metastasis formation. Meanwhile, tumor also regulates TME for their own survival. Therefore, we cannot simply consider one target or one pathway in tumor therapeutics. Tumor cells and TME should be taken into account as a whole system. Multi-target strategy has the potential to solve this existing problem.
Traditional Chinese medicine (TCM) is one of multi-target strategies for cancer therapeutics (Li and Lin, 2011) based on its overall adjustment treatment. It could suppress tumor growth and recurrence by inhibiting proliferation of cancer cells per se, and finetuning the homeostasis of TIM. Many Chinese herbs can reduce the toxicity of chemotherapy and radiotherapy, ultimately prolonging the overall survival time (Li et al., 2012). A number of studies elucidate the underlying mechanisms for TCM synergistic influence in mediating cancer cells as well as TME. Therefore, we here summarize advances in molecular mechanisms of TCM action on tumor, especially on regulating tumor immunity.

TCM COULD REGULATE TIM
TME is a complex system with multiple components, such as nontumor cells and extracellular matrix. Nontumor cells mainly consist of innate immune cells including macrophages, natural killer (NK) cells, dendritic cells (DCs), etc., acquired immune cells (T and B cells), myeloid-derived suppressor cells (MDSCs), fibroblasts (Egeblad et al., 2010). TME assists tumor cells to escape immune surveillance, and combines with extracellular matrix proteins and matrix-degrading enzymes to form TIM (Sun, 2015). TCM could reverse the inhibitory phenotype of immune cells, restore the function of innate immune cells (Table 1) and adaptive immune cells ( Table 2) in the TIM.

TCM Could Regulate Innate Immune Response
Since TCM could increase expression of inflammatory factors and decreases production of immunosuppressive cytokines, DCs can proliferate and differentiate normally, the ability of antigen presentation can be restored; the number of activated NK cells is increased; the number of M2 macrophages is decreased, and its ability of SLE (Yu et al., 2015;Li et al., 2017;Pang et al., 2017).
DCs are the main antigen-presenting cells (APCs) in innate and adaptive immunity; they present antigens to T cells to initiate the immune cycle at baseline (Chen and Mellman, 2013). DCs In vivo, a murine orthotopic mammary carcinoma resection model Could enhance efficiency of DC-based vaccine against metastasis of 4T1 mammary carcinoma and the improved survival in mice Increasing the expression of CD40, CD80 and CD86 in DCs and CD4 + and CD8 + T-cell proliferation (Chang et al., 2015)

NK cells YPF formula
In vivo, a LLCxenografted murine model Could inhibit the growth of LLC and prolong the survival of tumor-bearing mice Downregulating the protein levels of indoleamine 2,3-dioxygenase, TGF-b, and IL-10, which promoted tumor infiltration and killing capability of NK cells to LLC (Luo et al., 2016) Lupeol (TCM monomer) In vitro, gastric cancer cell lines BGC823, N87 and HGC27 Could inhibit the proliferation of gastric cancer cells Inducing the proliferation and promoting the killing power of NK cells through the upregulation of PFP, IFN-g and CD107a in NK cells (Wu et al., 2013) PSG-1 (TCM monomer) In vitro and in vivo, S-180 cells and a murine xenograft model of sarcoma Could inhibit S-180 cells in vitro and decrease sarcoma tumor weight and induce apoptosis in mice Increasing production of cytokines in Th1 cells and enhancing the cytotoxic activity of NK and CTL cells in mice by TLR4 (Yu et al., 2015) Macrophages BFD formula In vitro and in vivo, A549 and H1975 cells and murine xenograft models of NSCLC endocytose cell debris or dead tumor cells and then present tumor antigens to lymph nodes to activate T cells. However, in TIM, DCs are unable to proliferate and differentiate, leading to inhibit antigen presentation and activation of T cells (Gardner and Ruffell, 2016). Defective DCs have been found in various cancers, such as pancreatic cancer, non-small cell lung cancer (NSCLC), and hepatocellular carcinoma (HCC) and cervical squamous intraepithelial lesions (Gabrilovich, 2004;Bellone et al., 2006;Lee et al., 2006;Ormandy et al., 2006;Perrot et al., 2007). There is a TCM formula (SL), comprising Styphnolobium japonicum (L.) Schott [Fabaceae] and Lonicera japonica Thunb [Caprifoliaceae], which is traditionally used for melanoma treatment .
Using a mouse xenograft model of B16F10 melanoma, they found that an ethanolic extract of SL (SLE) could dramatically suppress tumor growth in melanoma-bearing mice, partially by inhibiting the activation of STAT3 and STAT3-targeted immunosuppressive cytokines, which involved in tumor growth and immune evasion. These anti-melanoma effects of SLE were also associated with increased recruitment of DCs to B16F10 melanoma tissues and mouse spleens to enhance tumor immune response (Liu et al., 2019 [Campanulaceae]) could increase the expression of CD40, CD80 and CD86 in DCs and CD4 + and CD8 + T-cell proliferation, resulting in the enhanced efficiency of DC-based vaccine against metastasis of 4T1 mammary carcinoma and the improved survival in mice (Chang et al., 2015). In addition to DCs, NK cells are also dysfunctional in TIM (Cekic et al., 2014). It has been reported that a typical Chinese herbal decoction Yu-Ping-Feng (YPF, contains A. mongholicus Bunge [Fabaceae]), Atractylodes macrocephala Koidz.
[Apiaceae]) could downregulate the protein levels of indoleamine 2,3-dioxygenase, TGF-b, and IL-10, which promoted tumor infiltration and killing capability of NK cells to LLC (Lewis lung cancer) in mice. Meanwhile, YPF also could inhibit the growth of LLC and prolong the survival of tumor-bearing mice (Luo et al., 2016). Lupeol is a natural secondary metabolite isolated and purified from Tamarindus indica L.
[Fabaceae] (Saleem, 2009). Lupeol could inhibit the proliferation of gastric cancer cell lines BGC823, N87 and HGC27 by inducing the proliferation of NK cells and promoting the killing power of NK cells in vitro. Further study showed Lupeol could upregulate the expression of PFP, IFN-g and CD107a in NK cells (Wu et al., 2013). Ganoderma atrum has been used for thousands of years as a traditional medicine. A polysaccharide is extracted from G. atrum, named as PSG-1. PSG-1 could inhibit S-180 cells in vitro, and decrease sarcoma tumor weight and induce apoptosis in mice. By using C3H/HeN (WT) and C3H/HeJ (TLR4-deficient) mice, they further found that PSG-1 could increase production of cytokines in Th1 cells, and enhance the cytotoxic activity of NK and CTL cells in WT, but not TLR4-deficient mice, suggesting that PSG-1mediated antitumor activity is likely dependent on TLR4 (Yu et al., 2015).
Macrophages exhibit different phenotypes upon different environmental stimuli, mainly including classically activated macrophages (M1) as well as alternatively activated macrophages (M2). While studies show that Tumor-associated macrophages (TAMs) are able to exhibit either phenotype, researchers tend to consider TAMs as M2-like macrophages. TAMs contribute to TIM through producing cytokines, proteases, chemokines and growth factors, as well as inducing the release of inhibitory immune checkpoint proteins in T cells (Siamon et al., 2015 [Campanulaceae] alleviated lung cancer-related symptoms in clinic. Another study in murine xenograft models of non-small cell lung cancer (NSCLC) using A549 and H1975 cells showed BFD could inhibit tumor growth and prolong the survival in mice by blocking the crosstalk between TAMs and cancer cells through decreasing IL-10 and PD-L1 in vitro and in vivo (Pang et al., 2017). In addition, baicalin, a major bioactive compound extracted from Scutellaria baicalensis Georgi [Lamiaceae] could inhibit tumor growth in a mouse orthotopic model of HCC by inducing the repolarization of TAM to M1-like phenotype and promoting the production of pro-inflammatory cytokines in tumor. These effects were associated with the induction of autophagy and activation of RelB/p52 (Tan et al., 2015).
MDSCs, one of main components in the TIM, are generated in the bone marrow, and migrated to peripheral lymphoid organs and tumor tissues and differentiate to DCs or macrophages . Recent studies showed that MDSCs in patients with breast cancer were positively associated with cancer progression (Guo et al., 2015). Shugan Jianpi ( . SGJP has an inhibitory effect on Gr-1 + CD11b + myeloid immunosuppressor cells in a murine model of 4T1 mammary cancer. Furthermore, SGJP could prevent MDSCsinduced IL-4, IL-13 and TGF-b expression and apoptosis of CD8 + T cells, and enhance inflammatory responses of NKT cells by JAK-STAT signaling (Guo et al., 2015).

TCM Could Regulate Adaptive Immune Response
In addition to innate immune cells, adaptive immune cells are also reversed by TCM. Regulatory T cells (Tregs) now have gotten more and more attention as their impact on inhibiting tumor-associated immune responses. Therefore, strategies targeting Tregs to improve adaptive immune response may be promising for cancer treatment (Chaudhary and Elkord, 2016 SYY has a therapeutic potential in multiple cancers Wang et al., 2015), including HCC (Xiong et al., 2010;Jia Q. A. et al., 2013). In addition, SYY also could inhibit tumor growth through Treg immunomodulation. In a murine xenograft model of liver cancer (Hepa1-6), SYY with moderate swimming (MS) could inhibit growth and lung metastasis of liver cancer and prolong mouse survival. Further study found that SYY and MS could increase the ratio of CD4 + to CD8 + , but reduce the proportion of Treg and TGF-b1 expression in spleen, peripheral blood as well as tumor tissue (Zhang Q. B. et al., 2016). In a murine xenograft model of hepatocarcinoma H22, administration of Polysaccharide from G. uralensis Fisch. ex DC.
[Fabaceae] (GP) could reduce the frequency of CD4 + CD25 + Tregs and Foxp3 expression, but increase the ratio of Th1/2 cytokines in serum, which partially contributes to GP-mediated inhibition of tumor growth (He et al., 2011).
B cells are critical in humoral immunity. Until recently, some studies have found B cells infiltration in solid tumors. However, the role of B cells in solid tumors is conflicting (Flynn et al., 2017;Kaplon and Dieu-Nosjean, 2018). Little is known about TCM in regarding B cell-mediated immune responses. Matrine is a medicinal herb derived from Sophora flavescens Aiton [Fabaceae]. One study identified the underlying mechanism for Matrine's anti-cancer effects in human acute lymphoblastic leukemia (ALL). Treatment of B cells from human ALL with Matrine could induce ROS generation and mitochondrial swelling, and cause a decline in mitochondrial membrane potential, thereby inducing apoptosis by upregulation of the proapoptotic protein Bax and downregulation of the anti-apoptotic protein Bcl-2 (Aghvami et al., 2018).

TCM COULD INHIBIT CANCER-ASSOCIATED FIBROBLASTS
Under physiological conditions, fibroblasts are able to secrete multiple cytokines, and their plasticity is high, which is essential for maintaining cell homeostasis and repairing tissue damage (Jacob et al., 2012). However, cells with similar morphology to myofibroblasts were found in the cell matrix of solid tumors, which are referred to as cancer-associated fibroblasts (CAFs) (Tremblay, 1979). CAFs can promote tumor invasion and migration by changing the phenotype of cancer cells. CAFs also promote tumor angiogenesis and affect the TME to facilitate cancer progression (Santi et al., 2018). Additionally, accumulating evidence have shown that CAFs mediate epithelialto-mesenchymal transition (EMT) in tumor cells (Christofori, 2006;Chen and Song, 2019). Therefore, strategies inhibiting

In vitro, prostate-CAFs
Could inhibit the growth of prostate-CAFs Induction of autophagy by increasing the activation of Beclin-1 and LC3 (Han S.Y. et al., 2016) CAFs are one of important approaches to suppress tumor progression (Table 3). Artemisinin (ART) is a chemical extract from Chinese herb Artemisia annua L. [Asteraceae], which has potent anti-malarial and anti-cancer activity. ART derivatives artesunate (ARS) and dihydroartemisinin (DHA) could reverse L-929 breast cancer cell-CAFs from activated to inactivated state through inhibition of TGF-b signaling, which resulted in a disruption of the interaction between tumor and TME. In vivo data also showed that ART derivatives also could suppress CAFs-induced growth and metastasis of breast cancer in an orthotopic model (Yao et al., 2018). Curcumin is a chemical component extracted from Curcuma longa L. [Zingiberaceae]. Studies have showed that curcumin has blood lipid lowering, antitumor, antiinflammatory, choleretic and anti-oxidant effects (Lestari and Indrayanto, 2014). Curcumin could inhibit EMT and metastasis of pancreatic cancer cells by inhibiting CAFs in a nude mouse model of pancreatic cancer Qu et al., 2018).
, a perennial herb of the lily family. PP-1 could selectively inhibit the growth of prostate-CAFs without affecting normal broblasts through induction of autophagy by increasing Beclin-1 and LC3, key autophagy-related proteins .

TCM COULD ENHANCE IMMUNE RESPONSE TOWARDS TUMOR CELLS
Immune response of tumor cells is critical for tumor progression. Furthermore, cell death can trigger immune responses of nearby cells through releasing damage-associated molecular patterns (DAMPs), including cytokines or chemokines (Lau et al., 2020). Accumulating evidence shows that TCM could enhance immune responses and protect from immune escape ( Table 4), and induce cell death of tumor cells ( Table 5).

TCM Could Increase Expression of Classic MHC Molecules
In general, MHC I presents tumor antigens to CD8 + T cells, also called cytotoxic T cells (CTLs), which kill tumor cells by cytolysis. In contrast, MHC II identifies tumor antigen peptides to CD4 + T Combination therapy with GQD and anti-PD-1 induced the frequency of CD8+ T cells in tumor tissues and peripheral blood. They also increased IFN-g and IL-2, but decreased PD-1. helper cells, which trigger cell-mediated immunity. Classical MHC molecules (I and II) enhance the interplay between tumor cells and NK cells or CTLs by identifying the tumor antigen (Guo et al., 2015). However, malignant and immune cells in TME may downregulate MHC I or II, but express nonclassical human leukocyte antigens (HLAs) including HLA-E/F/G that were associated with tumor cells escaping from T and NK cell-mediated recognition (Kochan et al., 2013). Exopolysaccharide (EPS) from an anamorph of Cordyceps sinensis could induce expression of MHC-II, CD40, CD80, and CD86 in dendritic cell sarcoma (DCS) cells, and enhance their ability of antigen uptake as well as secretion of IL-12 and TNF-a through decreasing p-JAK2 and p-STAT3 and increasing p-p65, suggesting that EPS may induce DCS cells to exhibit a mature phenotype, which is critical in initiating antitumor immunity (Song et al., 2013 [Campanulaceae]) could decrease the progression of cachexia and prolong the survival time in spleen-deficient liver cancer rat by increasing MHC I/II expression in liver tissues (Li et al., 2014).

TCM Could Inhibit PD-1/PD-L1 Signaling
Programmed cell death 1 (PD-1), as a currently well-known immune checkpoint, preferentially expressed on B and T cells, as well as other cells including DCs and monocytes (He et al., 2015). Under multiple physiological conditions, PD-1 modulates the activity of T cells in peripheral tissues by inducing inhibitory signals in immune system and maintains self-tolerance in inflammation or infection (Trivedi et al., 2015). However, PD-1/PD-L1 (ligand) pathway is utilized by the tumor cells to escape immunologic surveillance in the context of cancer (Bagley et al., 2015). Studies showed that PD-L1 was overexpressed in various tumor cells, including lung, ovarian and colon cancer (Keir et al., 2008). At present, there are accumulating TCM researches on PD-1/PD-L1. In addition to BFD that we mentioned above (Pang et al., 2017) [Euphorbiaceae], Scolopendra, Pheretima) also has effect on the PD-1/PD-L1 pathway in lung cancer. High concentration of QYSL could inhibit tumor growth by reducing PD-1 in spleen and PD-L1 in tumor in a murine xenograft model of LLC . [Fabaceae]) inhibited tumor growth in a murine xenograft model of CT26 CRC. Gut microbiota analysis revealed that GQD could modulate the gut microbiome composition. In particular, they found that combination therapy with GQD and anti-PD-1 induced the frequency of CD8 + T cells in tumor tissues as well as peripheral blood, and increased IFN-g and IL-2, but decreased PD-1. These data indicate that the combination therapy effectively restores T-cell functions by suppressing inhibitory checkpoints (Lv et al., 2019).

TCM Could Inhibit CSCs
Cancer stem cells (CSCs), a small population of cells, are critical in tumor development and drug resistance, resulting in metastasis and cancer relapse. CSCs hardly express molecules that favor immune response, such as HLA-DR. This is the reason that CSCs becomes a major clinical challenge in cancer treatment (Zeuner et al., 2014). Therefore, strategy targeting or inhibiting CSCs is a novel and promising approach for cancer therapeutics (Guo et al., 2015).
Accumulating evidence indicates that TCM could reduce CSCs. For instance, PZH (Abelmoschus moschatus Medik. [Malvaceae], Calculus bovis, Snake Gall and P. notoginseng (Burkill) F.H.Chen [Araliaceae]), a well-known TCM formula has been prescribed for hundreds of years in China, could reduce the population of the HT-29 CRC stem-like SP cells in a dose-dependent manner, and reduce the viability and sphereforming capacity of HT-29 SP cells. Mechanistically, PZH could inhibit ABCB1 and ABCG2 that are members of the ABC transporter superfamily contributing to the SP phenotype and multi-drug resistance (Wei et al., 2014). Trametes robiniophila Murr. (Huaier), which is a sandy beige mushroom from the trunk, has antitumor activity. Huaier could decrease the viabilities, numbers, and sizes of mammospheres. The clonogenicity of MCF7 breast cancer cell was impaired, along with less holoclones after Huaier exposure. Further, Huaier could decrease the proportion of cells expressing CD44 + /CD24 -, which exhibit cancer stem-like properties, and reduce the levels of stem cell markers including NANOG, NESTIN and OCT-4. Additionally, Huaier-mediated effect on CSCs was partially dependent on the hedgehog pathway (Wang et al., 2014). Another study showed that Huaier extract could inhibit the potential of spheroid formation and the population of aldehyde dehydrogenase (ALDH)−positive cell in primary CRC cells (T1 and T2 cells) by downregulating the Wnt/b−catenin pathway . Taken together, these findings provide experimental evidence that Huaier extract is a promising TCM for suppressing CSCs in multiple cancers.

Induction of Apoptosis
Induction of tumor cell death by apoptosis is a well-known antitumor strategy (Debatin, 2000;Xiu et al., 2015). Saponins are compounds of saponin and sugar, uronic acid or other organic acids, which are widely found in plants. The total saponins of A. mongholicus Bunge [Fabaceae] (AST) could induce the extrinsic apoptotic cascade and caused cell cycle arrest in HT-29 CRC cells by regulating both mTOR and ERK signaling pathways, of which inhibition of NF-kappaB is a critical latter event (Auyeung et al., 2010). Laminaria japonica is a traditional Oriental herbal medicine. Glycoprotein isolated from L. japonica (LJGP) exhibited anti-cancer activity in cultured human gastric carcinoma AGS cells. LJGP could inhibit tumor proliferation and induce apoptosis by upregulating pro-apoptotic Bax, and downregulating anti-apoptotic Bcl-2 and IAP family members, as well as activation of caspase-3/9. This was associated with downregulation of telomerase activity and prostaglandin E 2 synthesis by decreasing the levels of cyclooxygenase (COX)-2 (Ho et al., 2011).
Fas/FasL-mediated signaling is critical in regulation of cell death. Fas binds to its ligand FasL with FADD (Fas-related death domain structure protein), to form the DISC (death-inducing signaling complex), which induces activation of caspase-8 and then cleaves effector caspases to induce apoptotic cell death (Zhang Y. S. et al., 2016). Physiologically, Fas high FasL low cells combined with cytotoxic T cells (Fas low FasL high ) expressed FasL, leading to the activation of Fas receptor and triggering apoptosis of target-cells (Villa-Morales and Fernandez-Piqueras, 2012). However, tumor cells with high-expressed FasL and low or nonfunctional Fas bound to lymphocytes expressed Fas and abrogated immune responses (Reichmann, 2002)

Induction of Autophagy
Autophagy is a "self-eating" process to maintain homeostasis that removes potentially injurious intracellular components and proteins. Induction of autophagy in tumor cells is promising to prevent tumor progression. There are physical and functional complex interactions and regulations between autophagy and programmed cell death. Although autophagy often accompanies programmed cell death following multiple toxic insults, the requirement of autophagic machinery for execution of programmed cell death is highly contextual (Denton and Kumar, 2019). Bufalin, the active compound of cinobufacini, is derivated from bufadienolide and developed in anaesthetic, cardiotonic and cancer treatment (Gao et al., 2011). Bufalin could inhibit the proliferation of human hepatoma cells including Huh7, Hep3B and HA22T and regulated the cell death program in vitro. Further, bufalin could induce autophagy in hepatoma cells by increasing TNF, MAPK and autophagy-related genes such as BECN-1 and ATG8, and decreasing Bcl-2 and Bid (Hsu et al., 2013). Dihydroartemisinin (DHA) and its analogs have anticancer activity. A novel DHA derivative, DHA-37 could induce cell death in multiple human cancer cell lines, such as lung carcinoma A549 cells and gastric cancer SGC-7901 cells. Mechanistically, DHA-37 could trigger autophagic cell death (ACD) in A549 cells by activation of the MAPK signal and upregulation of high mobility group protein (HMGB1). Additionally, in a murine xenograft model of A549, DHA-37 could inhibit tumor growth, and increase p-ERK, p-P38, HMGB1, and LC3 in tumor tissue, which is consistent with in vitro data . The root of Platycodon grandiflorus (Jacq.) A.DC.
[Campanulaceae] (PG), a typical TCM herb, has anti-inflammatory  and anti-tumor (Kim et al., 2005) activity. The platycoside-containing butanol fraction of PG (PGB) has been reported to induce ACD of A549 cells by suppressing the AKT/mTOR pathway and activating the AMPK and MAPK pathways . Flavonoids (FOJ) and steroidal saponins (SSOJ) are the main active components of Ophiopogon japonicus (Thunb.) Ker Gawl.

Induction of Necroptosis
Necroptosis is a form of programmed cell death; the canonical pathway of activation requires activation of mixed lineage kinase domain-like (MLKL) by receptor-interacting protein kinase 1 (RIP1)-RIP3 and further oligomerization and translocation of MLKL to the plasma membrane (Wu et al., 2020). Recent studies show that TCM also exerts anti-cancer effects through induction of necroptosis. Resibufogenin could suppress tumor growth and metastasis in a murine xenograft model of CRC. Further study showed that the anti-tumor effect of resibufogenin was associated with induction of necroptosis through increasing RIP3 and pMLKL (Han et al., 2018). Shikonin, one of the active ingredients of Arnebia euchroma (Royle ex Benth.) I.M.Johnst.

Induction of Multiple Cell Death Pathways
Underlying mechanisms for the balance between programmed cell death including apoptosis and necroptosis, and autophagy are complex and contextual. With its characteristics, TCM has the synergistic effect on multiple cell death pathways. For example, S. barbata D.Don [Lamiaceae] (SB) is another typical TCM herb from the dried Labiatae plant. In addition to ER stress-, intrinsic mitochondrial-, P38/SIRT1-regulated cell apoptosis through G2/M phase arrest and extrinsic Fas/ FasL-mediated pathways, autophagy also was important in SB-induced cytotoxicity in CL1-5 lung cancer cells. In vivo, SB could suppress proliferation and angiogenesis, and increase apoptosis and autophagy in CL1-5 tumor-bearing mice . Furthermore, Hibiscus leaf polyphenolic (HLP) extract could induce cell apoptosis through increasing the caspases cleavages, Bcl-2, and Fas/FasL activation; it also could induce ACD by increasing ATG5, Beclin1, and LC3-II in A375 melanoma cells (Chiu et al., 2015). In addition, a novel strategy for improving the DC-based cancer vaccine was provided in cancer treatment. Shikonin could trigger RIP1and RIP3-dependent necroptosis in mouse stage IV mammary carcinoma 4T1-luc2 cells that is accompanied by enhanced autophagy. Shikonin-induced autophagy directly contributed to DAMP upregulation. Chloroquine-mediated inhibition of autophagy enhanced the surface DAMP activity and resulted in DC activation. In vivo study found that co-treatment of Shikonin with chloroquine stimulated DC activation and the derived vaccine efficacy in a murine model of mammary carcinoma (Lin et al., 2018).

CLINICAL APPLICATIONS
Many clinical randomized controlled trials have demonstrated that TCM could be beneficial as part of adjuvant chemotherapy in cancer treatment. YWKLF combined with chemotherapy prolonged the survival time compared to chemotherapy alone in a clinical trial of 123 patients with late stage gastric cancer (Li et al., 2008). Jianpi Bushen (JPBS) is used to invigorate the spleen and tonify the kidney. In a meta-analysis of randomized controlled trials, 26 studies with 3098 individuals were included to determine the effect of JPBS combined with chemotherapy for gastric cancer. JPBS treatment with regular chemotherapy improved the efficiency of clinical curative effect and quality of life (QOL) and immune response, and reduced side effects compared to chemotherapy alone . In a randomized controlled trial, herbal injection (Cinobufacini) and herbal decoction ( [Lamiaceae], and Ostreae concha) were used in a randomized controlled trial to explore TCM actions as maintenance therapy in 64 patients with advanced NSCLC. Results showed that TCM maintenance therapy improved QOL and had higher 1-year survival of patients compared to those in the chemotherapy group . In another randomized controlled trial of 106 patients with advanced NSCLC, TCM maintenance therapy (TCM decoction) improved 3-month progression-free survival (PFS) and QOL including physical well-being, emotional well-being and functional well-being .
In addition to NSCLC, a randomized controlled trial was conducted in 68 patients with HCC. Xiaoaiping (XAP), a TCM extracted from the roots of Marsdenia tenacissima (Roxb.) Moon [Apocynaceae], possessed antitumor activity and had been widely used in China for cancer treatment (Yu et al., 2019).
Results showed that compared with the control group, the XAP group had improved immediate therapeutic efficacy and QOL and prolonged the PFS in patients. Moreover, levels of CD3 + , CD4 + and CD4 + /CD8 + in the peripheral blood in the XAP group were higher than those in the control group (Huang Z. et al., 2013 , Foreknowledge) In a randomized controlled trial of 44 patients with advanced prostate cancer (APC), YJQR treatment with endocrine therapy for 6 months increased prostate specific antigen (PSA), free PSA (f-PSA) and hemoglobin (Hb) in serum. Meanwhile, they also improved QOL and immune function as well as reduced adverse reactions (Jia Y.J et al., 2013). Taken together, these findings suggest that TCM has the potential in either combination therapy with regular chemotherapy or maintenance therapy for cancer (Table 6). However, large sample randomized controlled trials and additional rigorously designed are further required to confirm the efficacy and safety for TCM applications in patients.

CONCLUSIONS AND CHALLENGES
Cancer remains a major threat to human life, which results in a huge healthcare burden. Most of therapeutic strategies targeting tumor cells ignore the importance of TIM. TIM plays a critical role in tumor development (Swartz et al., 2012). TIM includes abundant nontumor cell, stromal cells, extracellular matrix, cellular factors and chemokines, all of these promote tumor growth, invasion, and metastases, and protect tumor cells to escape from host immunity (Swartz et al., 2012). Therefore, understanding tumor cells and their interaction with TIM as a whole system is promising to have a better therapeutic effect.
Although the chemical composition in TCM formula is complex, this characteristic of TCM fits well with the treatment of TIM and tumor cells as a whole system. In contrast, western medicine has specific targets and strong effect; however, the toxic and side effects are also dramatic. such as dermatologic events associated with cixutumumab (Daher et al., 2011), severe cytotoxic with decitabine (Berdasco and Esteller, 2019). Levamisole hydrochloride was withdrawn due to its severe agranulocytosis. Most of the drugs can only slow disease progression, they are unable to achieve the goal of cure. In addition, the cost of developing anticancer drugs is huge, which makes it much harder to develop new drugs. Compared to western medicine, TCM has a milder effect and act synergistically on tumor-related multi-target groups. With the help of network pharmacology, TCM can better act on tumors and TIM and have a better anti-cancer effect. In conclusion, we summarized the underlying mechanisms by which TCM, including formula, medicinal herb and monomer (Figure 1), inhibits tumor cells and TIM, indicating that TCM suppresses tumor cells per se, as well as affects immune responses in TIM (Figure 2). More information regarding preparation of TCM and study conditions are shown in Supplemental tables. Additionally, TCM can enhance the therapeutic effect of radiotherapy and chemotherapy and reduce its toxicity, prolong lifetime. Therefore, in some cases, TCM may be an ideal treatment strategy for cancer due to its synergistic effect on tumor cells and TIM. However, the specific mechanism for effects of many TCM formulas on cancer still needs further to be investigated due to the chemical composition of TCM is very complicated.
Although TCM has good synergistic effects on TIM and cancer cells, many challenges remain. First, as we mentioned above, tumor cells interacted with TIM, however, the specific mechanism of the crosstalk between tumor cells and TIM is still not clear. Second, due to the complexity of TCM prescription, studies based on experiences limit the logic and repeatability, which remains an obstacle to draw certain conclusions. Third, safety of TCM is hard to guarantee because of lacking standardized and systemic operating procedures in processing TCM. Finally, clinical researches lack multi-center, randomized control, efficacy comparison, and large-samples. Therefore, an appropriate system, such as standardizing in vitro and in vivo models and complete quality control, should be developed to investigate TCM actions on multi-level and multi-channel.