Advances in research on tumor microenvironment of hepatocellular carcinoma

Gao Li, Dong-Xin Tang, Feng-Xi Long, Zhu Yang,*

1Guiyang College of Traditional Chinese Medicine, Guiyang 550002, China.

Introduction

Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world, and the mortality rate of HCC patient’s ranks second among all cancers, and this trend is still rising. 75% of HCC patients worldwide are in East Asia, and more than half of the world's HCC patients are in China [1]. It is found that the tumor microenvironment plays a vital role during the occurrence and development of HCC. Thus, the research on the tumor microenvironment may provide a new strategy for HCC. The relationship between tumor microenvironment and HCC is now described.

Tumor microenvironment

The tumor microenvironment is a complex dynamic network structure, which mainly includes tumor cells,stromal cells and extracellular matrix [2]. The continuous reproduction and invasion of tumors may be considered as the constant change of the tumor microenvironment, including steady state destruction of the tumor microenvironment, and the abnormal expression of a large number of tumor-related factors.The most classic is Steven Paget's "soil and seed"theory [3], the theory described tumor cells as “seeds”,and treated tumor microenvironment as “soil”.Therefore, the tumor microenvironment is crucial for tumor growth.

Extracellular matrix and HCC

Extracellular matrix (ECM) is a macromolecule synthesized by animal cells and secreted outside the cell,distributed on the cell surface or between cells, mainly polysaccharides, proteins, and proteoglycans. The extracellular matrix plays a dual role in HCC. On the one hand, the extracellular matrix may provide survival signals and control the proliferation, differentiation, and metastasis of HCC; On the other hand, the reconstituted extracellular matrix can also promote the proliferation of HCC cells and inhibit the apoptosis and differentiation of HCC cells. [4]. Studies have shown that the remodeling of the extracellular matrix is a dynamic structure, and its functional diversification has different effects on tumors [5]. Some studies have also shown that the occurrence of HCC is closely related to tissue remodeling and matrix interaction in the microenvironment [6]. In addition to extracellular matrix remodeling, extracellular matrix hydrolases have important effects on invasion and metastasis of HCC,such as plasminogen activator and metalloproteinases,among which metalloproteins are the most critical [7].The proliferation and metastasis of HCC is an extremely complicated process, but it is worthy of recognition that this is closely related to the extracellular matrix.

Stromal cells and HCC

Stromal cells can regulate cell-to-cell contact in the tumor microenvironment, and change the state of surrounding normal tissues through a series of related factors, providing a very important condition for the malignant development and invasion and metastasis of tumors. However, some stromal cells have both a side that promotes tumor growth and a side that inhibits tumors.

Cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs) are mainly differentiated from mesenchymal cells and are widely present in the cell matrix. Hepatocarcinoma-associated fibroblasts can promote the development of HCC through its secreted cytokines and growth factors, such as TGF-β, HGF, FGF, and VEGF, IL-6 [8]. Studies have shown that IL-6 may activate mTOR signaling, and STAT3 pathway to promote the progression of HCC [9，10]. TGF-β is an important transforming growth factor in the tumor microenvironment, TGF-β promotes HCC growth and epithelial-mesenchymal transition (EMT).One of the mechanisms in the process is the activation of CAFs, which induces microenvironment changes[11]; CAFs can also produce MMPs to break down ECM and cadherin-based cancer adhesion junctions to further enhance tumor cell migration [12]. In addition,clinical studies have also shown that the driver turnover of HCC is associated with increased expression of tumor-associated fibroblasts [13].

Immune cells

Immune cells play a vital role in immunity in the tumor microenvironment. In the study of the tumor microenvironment, we focus on the relationship between immune cells and HCC, and the mechanism of immune escape in HCC cells. Tumor-associated macrophages (TAMs) are important immune cell populations in the microenvironment. In the early stage of tumors, TAMs may combine with CD4+ T cells to eliminate tumor senescent cells; however, in the cancer microenvironment, TAMs are mainly composed of M2-polarized tumor-associated macrophages, which secrete various tumor proliferation-promoting cytokines,such as IL-1β, IL-6, TGF-β, platelet-derived growth factor (PDGF), et al. are the tumors that construct the inflammatory microenvironment that facilitates their growth [14]; another aspect can be through the secretion of anti-inflammatory factors and blocking T cell detection points. Monocyte-macrophages express high levels of programmed death-1 (PD-1) and PD-1 receptor binding on CD8+ T cells, which may inhibit anti-tumor cytotoxic T cell responses [15]. In addition,TAMs may promote the recruitment of regulatory T cells and inhibit the activity of NK cells through Toll-like receptor (TLR) 4 and CD48/2B4 [16,17].Angiogenic factors such as VEGF, by binding to endothelial receptors flt and flt/KDR, promote angiogenesis and accelerate tumor growth [18]. T cells are also important immune cells in the microenvironment, such as CD8+ cytotoxic T lymphocytes (CTL), and their secreted interferon(IFN)-γ has an important killing effect on tumors.However, studies have found that in HCC tissues CD8+T cells are rare and Tregs are highly expressed. The high expression of Tregs may down-regulate the expression of CD80 and CD86 on antigen-presenting cells, and the secretion of TGF-β and IL-10 is effectively to inhibit T effector cells [19].Tumor-associated neutrophils are also involved in the process of HCC immunity, which can stimulate reactive oxygen species (ROS) and telomere DNA damage in hepatocytes, or secreting cytokines. Moreover, other functional molecules such as CCL2, HGF, Oncostatin M (OSM), et al., promote tumor progression [20].

Tumor-associated endothelial cells (TAECs)

TAECs, also known as tumor endothelial cells, are important cell members in the tumor microenvironment and have the effect of inducing tumor angiogenesis. The main source of TAECs is mainly proliferating from endothelial cells in tumor microvessels, or differentiates endothelial progenitors from peripheral systems, or transforms tumor cells TAECs are mainly involved in the neovascularization of HCC, but these neovascular and normal blood vessels have functional and structural differences, and there are also large gaps between endothelial cells, which often lead to increased exudation and interstitial hypertension. It also facilitates tumor cell penetration for metastasis. In the previous studies, it was also found that TAECs have stronger angiogenesis and anti-apoptosis ability than normal tissue-derived endothelial cells [21]; Studies have shown that TAECs may enhance the invasive ability of HCC cells by secreting cytokines such as IL-6, IL-8,MCP-1 and GRO-α, which are correlated with Akt,ERK1/2, STAT3, and NF-κB pathways [22]. In addition to the construction of neovascularization, TAECs may also promote tumor growth through the paracrine pathway, which secretes IL-6 and heparin-binding epidermal growth factor [23].

Tumor micr oenvironmental characteristics and HCC

In addition to the above correlations, the relationship between tumor microenvironment and HCC may be related to the characteristic stress of the tumor microenvironments such as hypoxia, acid environment,and interstitial hypertension [24].

Tumor microenvironment anoxic conditions

The tumor microenvironment generally has a hypoxic phenomenon [25]. The hypoxic condition in the tumor environment is not caused by a single factor. The mechanism of hypoxia is often related to the angiogenesis of tumor cells. In the hypoxic environment,tumor cells produce a large number of pro-angiogenic factors such as VEGF, PDGF, TNF, et al., the high expression of these factors greatly promoted the angiogenesis of tumors, but these neovascular and normal blood vessels are structurally abnormal, and the new blood vessels are not able to regulate blood flow very well. Therefore, excessive perfusion hypoxia is caused. The uneven distribution of tumor microvessels,the difference in the effective feeding distance between tumor microvessels and oxygen, causes oxygen to be transported to the required oxygen site [26]. In the environment of hypoxia, in order to ensure their own growth, tumor cells often change this state by a series of mechanisms, such as activation of hypoxia-inducible factor (HIF) and stabilization of PH value of carbonic anhydrase 9 [27].

Tumor microenvironment acid environment

In order to ensure their growth, proliferation, infiltration,and metastasis, tumor cells often form a micro-environment that is different from other normal tissues. The acid environment is one of the most important components. In the microenvironment of the tumor microenvironment, it is generally considered that the lactic acid produced by the glycolysis of the tumor and the tumor carbonic anhydrase are excess [28].Studies have shown that in the acidic microenvironment,acid-sensitive ion channel 1a (ASIC1a) can promote the proliferation of HCC cells in vitro and in vivo by activating β-catenin and accumulating nuclei, and is associated with poor prognosis of HCC [29]. The growth and metastasis of HCC are closely related to its acidic microenvironment. However, the mechanism still needs to be further clarified

Tumor microenvironmental interstitial hypertension

The pressure of normal tissue in the tumor microenvironment is lower than that of the tumor tissue.This pressure can be regulated by the lymphatic system,but there is no such system in the tumor cells, which leads to this specific pressure. In addition, the neovascularization of the tumor is different from the normal blood vessels, and the tumor blood vessels are in a hypertonic state, and the loss of the tumor lymphatic system leads to tumor interstitial hypertension. Studies have confirmed that extracellular pressure can drive the PKC-β-IKK-IκB-NF-κB pathway and stimulate tumor proliferation [30].

Correlation betw een r elated signalin g pathways and HCC in the tu mor microenvironment

With the continuous exploration of HCC signaling pathways, it is found that the progression of HCC has a great relationship with certain proteins and factors in many signaling pathways that mediate microenvironmental changes. Studies have found that Wnt/β-catenin signaling pathway can up-regulate the expression of cyclooxygenase-2 (COX2) to promote tumor angiogenesis, thereby promoting tumor metastasis [31], mediating the high expression of the β-catenin protein can promote the growth of HCC [32].In the Notch signaling pathway, cyclooxygenase-2(COX2) was found to be closely related to the invasion and metastasis of HCC [33]. It was also found that Fibroblasts (CAFs) secrete high levels of IL-6, and activation of Notch signaling by STAT3 and Tyr705 phosphorylation is closely related to hepatocarcinogenesis [34]. Studies have found that under the microenvironment of tumor hypoxia melatonin can inhibit the vascular activity of VEGF and inhibit the vascular activity through the regulation of Hif1α and STAT3 [35]. It was also found that MiRNA199a-3p may inhibit HCC by targeting the inhibition of VEGFR1, VEGFR2, HGF, and MMP2,and thus reducing angiogenesis [36]. Glycolysis and cell energy metabolism can produce a large number of acidic products. The acidic microenvironment is beneficial to the metastasis of HCC. AMPK can correct energy metabolism and reduce tumor metastasis by inhibiting the synthesis of glycogen and activating P53[37]. In addition, some pathways such as Rac1/JNK[38], TGF-β, ROR, et al. are closely related to the occurrence of HCC. In the microenvironment of HCC,the occurrence and development of tumors is not a single signal pathway involved, but also a variety of signaling pathways. However, based on the research of microenvironmental signaling pathways, understanding complex signal networks is our way to seek tumor personalization and new therapeutic measures [39].

Based on t umor microenvironment and HCC treatment

In the traditional tumor treatment process, the traditional treatment methods pay more attention to the tumor itself, and the treatment methods are relatively simple, such as surgical resection of the tumor,radiotherapy, and chemotherapy, et al. However, with the understanding of the tumor microenvironment, the treatment of tumors is changing with each passing day,and these new treatments are applied to the treatment of cancer. The comprehensive treatment of traditional tumors will achieve a better curative effect on tumor treatment.

Immunotherapy

A very important component of the tumor microenvironment is the presence of multiple immune cells and immune molecules [40]. In the new biological treatment of HCC, immunotherapy for HCC is one of the most popular, including ACT treatment, tumor vaccine, and immune checkpoint block treatment.Sorafenib used in clinical practice has found that its mechanism of prolonging the life of HCC patients is that Sorafenib relieves cell-intrinsic and cell-extrinsic inhibitions of effector T cells in the tumor microenvironment to augment antitumor immunity [41].Although there are not many immune preparations for liver cancer in tumor microenvironment immunotherapy,the treatment of liver cancer will achieve the better curative effect for the treatment of immunotherapy.

Inflammation treatment

A large number of inflammatory factors accumulate in the tumor environment, which provides conditions for tumor cells to escape immunity. In the study of HCC, it was found that the inflammatory mediator Cox-2 is closely related to the occurrence of HCC [42].Therefore, in the treatment of HCC, by blocking the inflammatory mediators, it can be considered as a breakthrough in the treatment of tumors. In modern clinical applications, anti-inflammatory drugs (such as COX-2 inhibitors, non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs, such as dexamethasone et al.) can reduce the incidence of tumors when administered as a prophylactic.

Nanometer interventional therapy

In the tumor microenvironment, the tumor is rich in blood vessels. Some scholars have used platelets to maintain the stability of tumor vasculature. The anti-platelet antibody (R300) and the chemotherapy drug doxorubicin (Dox) are designed as key components to target the tumor-destroying blood vessels. Tumor suppression and improved microenvironment provide new ideas [43].

Regulators for extracellular matr ix remodeling control signals

This inhibitor is primarily a chemical intervention for the microenvironment of HCC. Such as TGF-β inhibition, studies have reported that TGF-β inhibitors LY2109761, LY2157299 are effective in inhibiting the migration of HCC cells, and its mechanism is related to inhibition of TGF-β signaling, pSMAD2, E-cadherin,VEGF and CTGF dephosphorylation [44].

HCC microenvironment and traditional Chinese medicine

Chinese medicine has a very important role and characteristic advantages in improving tumor microenvironment. From the etiology, Chinese medicine believes that the occurrence of tumors is formed by the “Xu”, “Tan”, “Yu” and “Aidu” et al., in which the “Pi (spleen)” is considered to be its core. The various characteristics of the tumor microenvironment can be explained by the theory of traditional Chinese medicine. For example, the inflammatory and acidic environment in the tumor microenvironment can be considered to be related to “Tan” [45]; “Spleen”deficiency can be considered as hypoxia in the tumor microenvironment and "mitochondria are considered the spleen of Chinese medicine" [46]. In the understanding of improving the micro-environment of HCC, the theory of Chinese medicine has also played an essential role. For example, Professor Liu Jiaxiang's"Fuzheng Theory", Professor Zhou Zhongyu's "Cancer Poison Theory", Professor Li Zhong's "Cancer Status Theory", Zhou Yihan Professor "with tumor survival",and Professor Liu Shangyi "negative rule of law".Based on the understanding of the tumor microenvironment, it proposed in the change of tumor microenvironment, such as tonifying qi, strengthening blood circulation, clearing away heat and detoxifying.Modern research found that these footholds are highly compatible with direct inhibition of tumor proliferation,inhibition of new blood vessels, and improved immunity. For example, in the study of “Yiqi Huoxue Jiedu” method, it has been found that Chinese medicine has significant effects on neovascularization,improvement of hypoxia and inflammatory environment[47]. Under the method of “Fuzheng Buqi”, Chinese medicine has been proven to improve the surveillance function of immune cells and ameliorate immunosuppression [48]. The principle of clearing heat and detoxification is generally aimed at the inflammatory microenvironment. The use of rosmarinic acid and baicalin in the inhibition of HCC is associated with change inflammatory environment [49]. Of course,in the process of modern Chinese medicine, many Chinese medicine monomers and compounds are gradually being discovered in the mechanism of improving the microenvironment. For example,Professor Liu Shangyi makes good use of blood-activating drugs [50]. Some studies have found that it can inhibit the expression of COX-2, VEGF,inhibiting Wnt pathway, reducing Stat3 phosphorylation,and interfere with microenvironment through Notch-1[51]. Astragalus membranaceus can improve the microenvironment by inhibiting COX-2, VEGF, HIF-1α,MMPs, et al. [52]. Heat-clearing drugs such as radix sophorae flavescentis can down-regulate MMP-9 and MMP-2 expression and inhibit the NF-κB pathway[53].In the traditional Chinese medicine compound,stony Paeonia suffruticosa compound can inhibit the expression of TGF-β1, IL-4, IL-10 in a rat tumor model,inhibiting CD133+ expression, interfere with tumor inflammatory microenvironment and delays tumor progression in rat tumor model [54]. Feiyanning decoction can inhibit the expression of MMP-2, MMP-9,NF-κBp65, and improve the microenvironment by interfering with NF-κB signaling pathway [55]. Chinese medicine plays an important role in the intervention of tumor microenvironment.

Discussion

In summary, we elaborated the relationship between tumor microenvironment and HCC. At present, research on HCC microenvironment is mainly focused on inflammation, angiogenesis, immunity, and signaling pathways. The author believes that the microenvironment has formed before HCC. The formation of liver cancer is caused by long-term stimulation of the microenvironment, resulting in hepatic cell genome mutations and other induced liver cancer. Therefore, microenvironmental changes can be considered as the cause of inducing liver cancer, rather than that the microenvironment only exists in the development stage of liver cancer. Second, in the related research of microenvironment and HCC, we often choose single or two factors for research, but the microenvironment of HCC is a complex environment maintained by multi-factor interconnection, so multi-factor, multi-target, multi-signal is the focus of our future research. Third, advocating Chinese medicine intervention in the micro-environment, the author's point of view is mainly: (1). For the micro-environment it is an interrelated "net", modern clinical oncology drugs are basically aimed at its "net"target. The overall concept of TCM and syndrome differentiation can be integrated and individualized to diagnose and treat the microenvironment, and its curative effect is also significant. (2). Under the guidance of theoretical diversification, traditional Chinese medicine has diversified choices in the tumor microenvironment, which is one of the great features of traditional Chinese medicine. (3). Chinese medicine has better comprehensiveness in improving tumor microenvironment. In the treatment of HCC microenvironment or adjuvant therapy, in addition to improving the microenvironment itself, traditional Chinese medicine can also improve tumor-related complications. It has been reported that in the inflammatory and hypoxic environment. For example,in addition to anti-inflammatory effects, Peony and Licorice Decoction can also protect neurons from cancer pain [56]. (4). For Western medicine treatment,its drug adverse reactions, drug resistance are many difficulties involved in Chinese medicine to better treatment of tumors; (5). Chinese medicine treatments of tumors can significantly reduce the side effects and drug resistance of western medicine. HCC resections,chemoradiotherapy, liver transplantation, including targeted therapy are all therapies for the treatment of tumors, but the recurrence and survival results are not satisfactory. Gatenby [57] believes that "eliminating cancer will accelerate the emergence of drug resistance and tumor recurrence, thereby reducing the chances of survival of patients" and advocate controlling tumors.However, the two are not comprehensive. They should advocate prevention of cancer while preventing prevention. How to prevent it? Improve the microenvironment and survive with the tumor.

Conclusion

Based on the mechanisms of microenvironment and development of HCC, understanding the various factors involved in the microenvironment, the transformation of signaling pathways, and the characteristics of the microenvironment are essential for the study of HCC.Based on these characteristics of the microenvironment,it can provide more support for new drug preparations of tumors, and it cannot ignore the great role of traditional Chinese medicine in improving microenvironment treatment. Among the current research results, the research on mechanisms and other aspects is still not very clear, which requires further in-depth study.

Reference

1. Omata M, Cheng AL, Kokudo N, et al. Asia-Pacific clinical practice guidelines on the management of hepatocellular carcinoma: a 2017 update. Hepat Internat 2017, 11: 1-54.

2. Spano D, Zollo M. Tumor microenvironment: a main actor in the metastasis process. Clin Exper Metastasis 2012, 29: 381-395.

3. Fidler IJ. The pathogenesis of cancer metastasis:the 'seed and soil' hypothesis revisited. Nature Reviews Cancer 2003, 3: 453.

4. Wu XZ, ChenD, Xie GR. Extracellular matrix remodeling in hepatocellular carcinoma: effects of soil on seed? Med Hypotheses 2006, 66:1115-1120.

5. Yeldag, G, Rice A, Del Río Hernández, A.Chemoresistance and the self-maintaining tumor microenvironment. Cancers 2018, 10: 471.

6. Tu T, Budzinska MA, Maczurek AE, et al. Novel aspects of the liver microenvironment in hepatocellular carcinoma pathogenesis and development. Internat J Molecular Sciences 2014,15: 9422-9458.

7. Saxena S, Jain A, Rani V. MicroRNAs mediated MMP regulation: current diagnostic and therapeutic strategies for metabolic syndrome.Current Gene Therapy 2017, 17: 214-227.

8. Norio, K, Kenichiro, et al. Cancer-associated fibroblasts in hepatocellular carcinoma. World J gastroenterology 2016, 22: 6841.

9. Liu F, Zhang W, Yang F, et al.Interleukin-6-stimulated progranulin expression contributes to the malignancy of hepatocellular carcinoma cells by activating mTOR signaling. Sci Rep 2016, 6: 21260.

10. Grivennikov SI, Greten FR, Karin M. Immunity,inflammation, and cancer. Cell 2010, 140: 883-899.

11. Calon A, Lonardo E, Berenguerllergo A, et al.Stromal gene expression defines poor-prognosis subtypes in colorectal cancer. Nature Genetics 2015,47: 320-9.

12. Kang N, Shah VH, Urrutia R.Membrane-to-nucleus signals and epigenetic mechanisms for myofibroblastic activation and desmoplastic stroma: potential therapeutic targets for liver metastasis? Molecular Cancer Res 2015,13: 604-612.

13. Liu C, Liu L, Chen X, et al. LSD1 stimulates cancer-associated fibroblasts to drive Notch3-dependent self-renewal of liver cancer stem-like cells. Cancer Res 2017, 78: 938-949.

14. Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nature Med 2013, 19: 1423-1437.

15. Degroote H，Van DA, Geerts A，et al. Preclinical and clinical therapeutic strategies affecting tumor-associated macrophages in hepatocellular carcinoma. J Immunol Res 2018. Published online:https: //doi.org/10.1155/2018/7819520.

16. Yeung OW, Lo CM, Ling CC, et al. Alternatively activated (M2) macrophages promote tumor growth and invasiveness in hepatocellular carcinoma. J Hepatology 2015, 62: 607-616.

17. Wu Y, Kuang DM, Pan WD, et al.Monocyte/macrophage-elicited natural killer cell dysfunction in hepatocellular carcinoma is mediated by CD48/2B4 interactions. Hepatology 2013, 57: 1107-1116.

18. Belair DG, Miller MJ, Wang S, et al. Differential regulation of angiogenesis using degradable VEGF-binding microspheres. Biomaterials 2016,93: 27-37.

19. Ringelhan M, Pfister D, O’Connor T, et al. The immunology of hepatocellular carcinoma. Nature Immunol 2018, 19: 222–232.

20. Zhang Q, Lou Y, Bai XL, et al. Immunometabolism:a novel perspective of liver cancer microenvironment and its influence on tumor progression. World J Gastroenterology 2018, 24:3500-3512.

21. Hida K, Maishi N, Torii C, et al. Tumor angiogenesis-characteristics of tumor endothelial cells. Internat J Clin Oncol 2016, 21: 206-212.

22. Jian K, Lingqun K, Jinge K, et al. After insufficient radiofrequency ablation, tumor-associated endothelial cells exhibit enhanced angiogenesis and promote invasiveness of residual hepatocellular carcinoma. J Transl Med 2012, 10: 230-230.

23. Chouaib S, Kieda C, Benlalam H, et al. Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells,extracellular matrix and immune killer cells.Critical Rev Immun 2010, 30: 529.

24. Vaupel P. Tumor microenvironmental physiology and its implications for radiation oncology.Seminars Radiation Oncol 2004, 14: 198-206.

25. Jain RK. Antiangiogenesis strategies revisited:from starving tumors to alleviating hypoxia. Cancer Cell 2014, 26: 605-622.

26. Jain RK, Martin JD, Triantafyllos S. The role of mechanical forces in tumor growth and therapy.Annual Rev Biomedical Engineering 2014, 16:321-346.

27. Ivanova L, Zandberga E, Siliņa K, et al. Prognostic relevance of carbonic anhydrase IX expression is distinct in various subtypes of breast cancer and its silencing suppresses self-renewal capacity of breast cancer cells. Cancer Chemotherapy Pharmacol 2015, 75: 235-46.

28. Fukumura D, Jain RK. Tumor microvasculature and microenvironment: targets for anti-angiogenesis and normalization. Microvasc Res 2007, 74: 72-84.

29. Jin C, Yuan FL, Gu YL, et al. Over-expression of ASIC1a promotes proliferation via activation of the β-catenin/LEF-TCF axis and is associated with disease outcome in liver cancer. Oncotarget 2017, 8:25977-25988.

30. Basson MD, Zeng B, Downey C, et al. Increased extracellular pressure stimulates tumor proliferation by a mechanosensitive calcium channel and PKC‐β.Molec Oncol 2015, 9: 513-526.

31. Ke Y, Xin W, Zhang H, et al. The evolving roles of canonical WNT signaling in stem cells and tumorigenesis: implications in targeted cancer therapies. Labs invest 2015, 96: 116-136.

32. Pandit H, Li Y, Li X, et al. Enrichment of cancer stem cells via β-catenin contributing to the tumorigenesis of hepatocellular carcinoma. BMC Cancer 2018,18: 783.

33. Zhou L, Wang DS, Li QJ, et al. The down-regulation of notch1 inhibits the invasion and migration of hepatocellular carcinoma cells by inactivating the cyclooxygenase-2/Snail/E-cadherin pathway in vitro. Digestive Diseases & Sciences 2013, 58: 1016-1025.

34. Xiong S, Wang R, Chen Q, et al. Cancer-associated fibroblasts promote stem cell-like properties of hepatocellular carcinoma cells through IL-6/STAT3/Notch signaling. American J Cancer Res 2018, 8: 302.

35. Carbajopescador S, Ordoñez R, Benet M, et al.Inhibition of VEGF expression through blockade of Hif1α and STAT3 signalling mediates the anti-angiogenic effect of melatonin in HepG2 liver cancer cells. British J Cancer 2013, 109: 83-91.

36. Ghosh A, Dasgupta D, Ghosh A, et al.MiRNA199a-3p suppresses tumor growth,migration, invasion and angiogenesis in hepatocellular carcinoma by targeting VEGFA,VEGFR1, VEGFR2, HGF and MMP2. Cell Death& Disease 2017, 8: e2706.

37. Ha J, Guan KL, Kim J. AMPK and autophagy in glucose/glycogen metabolism. Molec Aspects Med 2015, 46: 46-62.

38. Zhao X, Fu J, Xu A, et al. Gankyrin drives malignant transformation of chronic liver damage-mediated fibrosis via the Rac1/JNK pathway. Cell Death & Disease 2015, 6: e1751.

39. Liu H, Kiseleva AA, Golemis EA. Ciliary signalling in cancer. Nature Reviews Cancer 2018,18: 511-524.

40. Barker HE, Paget JT, Khan AA, et al. The tumor microenvironment after radiotherapy:mechanisms of resistance and recurrence. Nature Rev Cancer 2015, 15: 409-425.

41. Chen ML, Yan BS, Lu WC, et al. Sorafenib relieves cell-intrinsic and cell-extrinsic inhibitions of effector T cells in tumor microenvironment to augment antitumor immunity. Internat J Cancer 2013, 134: 319-331.

42. Pikarsky E, Porat RM, Stein I, et al. NF-kappaB functions as a tumor promoter in inflammation-associated cancer. Nature 2004, 431:461-466.

43. Li S, Zhang Y, Wang J, et al.Nanoparticle-mediated local depletion of tumour-associated platelets disrupts vascular barriers and augments drug accumulation in tumours. Nature Biomed Engineering, 2017:667-679.

44. Dituri F, Mazzocca A, Fernando J, et al. Correction:differential inhibition of the TGF-β signaling pathway in HCC Cells using the small molecule inhibitor LY2157299 and the D10 monoclonal antibody against TGF-β receptor type II. Plos One 2016, 8: e67109.

45. Liu L, Zhang GB, Lou ZH, et al. Discussion on the relationship between tumor-associated fibroblasts and scorpion venom based on tumor microenvironment theory. Shizhen Guoyi Guoyao 2018,29(05): 1163-1165.

46. Chen MX, Zhang H. Cancer-specialist disease doctors confirmed the series of books. Changsha:Hunan Science Techn Press 2011: 441-468.

47. Liu J, Sun ZT, Liu W, et al. Analysis of the effect of Yiqi Huoxue Jiedu method on the microenvironment of lung cancer[J].Shanzhong Chin Med 2018,39: 1285-1287.

48. Hua HQ. Modern research on the treatment of malignant tumors with Fuzheng PeiBen medicine.Chin J Tradit Chin Med 2008, 33: 1094-1097.

49. Hu C, Cao W, Xu LP, et al. Study on the anti-hepatocarcinoma effect of heat-clearing and detoxifying traditional Chinese medicine monomer in vitro.Guangxi Med J 2016, 38: 85-88.

50. Yang Z. Liu Shangyi clinically used traditional Chinese medicine Baiwei selection. Beijing Science Press 2016: 155-156.

51. Wu J, Tang Q, Yang L, et al. Interplay of DNA methyltransferase 1 and EZH2 through inactivation of Stat3 contributes to β-elemene-inhibited growth of nasopharyngeal carcinoma cellS. Sci Rep 2017,7: 509.

52. Qian X, Xu YZ, Deng DH, et al. Research progress of natural drugs on neovascular microenvironment of gastric cancer. Chin J Tradit Chi Med 2017, 35:2302-2308.

53. Huang H, Du T, Xu G, et al. Matrine suppresses invasion of castration-resistant prostate cancer cells by downregulating MMP-2/9 via NF-κB signaling pathway. Internat J Oncol 2016, 50: 640.

54. Liu TS, Zhai K, Tan XN, et al. Effects of Tanghua Peony compound on the levels of TGF-β_1, IL-4 and IL-10 in serum of lung cancer rats. Hunan J Tradit Chin Med 2017,33: 129-131.

55. Si HL, Wang LF, Xu ZW, et al. Effects of Feiyanning granule on tumor-associated macrophages and tumor metastasis in Lewis lung cancer mice. J Shanghai Univ Tradit Chin Med 2016, 30: 47-52.

56. Ding SH, Yu FM. Analysis of the treatment of cancer pain with Shaoyao Gancao decoction.Yunnan J Tradit Chin Med 2016, 37: 53-54.

57. Gatenby RA. A change of strategy in the war on cancer. Nature 2009, 459: 508-509.