The role of tumour microenvironment: a new vision for cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a relatively rare malignant and lethal tumour derived from bile duct epithelium and the morbidity is now increasing worldwide. This disease is difficult to diagnose at its inchoate stage and has poor prognosis. Therefore, a clear understanding of pathogenesis and major influencing factors is the key to develop effective therapeutic methods for CCA. In previous studies, canonical correlation analysis has demonstrated that tumour microenvironment plays an intricate role in the progression of various types of cancers including CCA. CCA tumour microenvironment is a dynamic environment consisting of authoritative tumour stromal cells and extracellular matrix where tumour stromal cells and cancer cells can thrive. CCA stromal cells include immune and non‐immune cells, such as inflammatory cells, endothelial cells, fibroblasts, and macrophages. Likewise, CCA tumour microenvironment contains abundant proliferative factors and can significantly impact the behaviour of cancer cells. Through abominably intricate interactions with CCA cells, CCA tumour microenvironment plays an important role in promoting tumour proliferation, accelerating neovascularization, facilitating tumour invasion, and preventing tumour cells from organismal immune reactions and apoptosis. This review summarizes the recent research progress regarding the connection between tumour behaviours and tumour stromal cells in CCA, as well as the mechanism underlying the effect of tumour stromal cells on the growth of CCA. A thorough understanding of the relationship between CCA and tumour stromal cells can shed some light on the development of new therapeutic methods for treating CCA.

disease. [2][3][4] CCA is a devastating and aggressive disease that has dismal outcomes due to its late clinical presentation and stubborn resistance to chemotherapy. Surgical treatment is currently the first clinical choice for treating CCA, 1 but the treatment efficiency is low, yielding a poor prognosis and a low 5-year survival rate of 23.7% and the recurrence rate is high. 5 In accordance with previous research, tumour cells are dedicated to build their own favourable context by incorporating extracellular matrix, stromal cells that secret tumour-related mediators, and tumour angiogenesis that provides more blood supply for tumour growth. Hence, tumour microenvironment promotes proliferation of tumour cells, assists tumours to escape from anti-tumour immune reactions, and enhances the resistance of tumour cells to treatment. 6 A study by Leyva-Illades et al.
showed that CCA cells can promote formation of surrounding connective tissue under the support from an abundant tumour microenvironment, and this process contributes prominently to therapeutic resistance of CCA. 7 [11][12][13] In support of the notion that Wnt signalling promotes CCA tumourigenesis, Liu et al. confirmed that activated GSK-3β acts as an important mediator in the inhibition of CCA cells based on experimental studies. 14 In addition, a number of chemicals were found to have anti-CCA effects via suppressing the Wnt/β-catenin signalling pathway, indirectly confirming the role of this pathway in tumourigenesis of CCA. 9,15 Another crucial signalling pathway contributing to CCA is nuclear factor kappa B (NF-κB) signalling pathway. NF-κB, which binds to its inhibitor IκB in unstimulated cells, is activated when IκB is phosphorylated by the protein kinase of IκBs (IKK) or ubiquitnated by SCF-E3 and degraded by protease. Activated NF-κB enters the nucleus and binds to DNA to induce the transcription of target genes, thus modulating the growth and development of CCA. 16 Performed with a series of in vitro experiments, Srikoon et al. revealed that the inhibition of NF-κB signalling pathway impedes CCA metastasis and migration via suppressing the transcription of its target genes that express intercellular cell adhesion molecules in CCA cell lines. 17 What's more, a growing number of substances have been found to inhibit CCA by blocking NF-κB signalling pathway like Magnolol, 18 Berberine, 19 Caffeic acid phenethyl ester, 20 and beta-eudesmo. 21 The above evidence indirectly demonstrates the importance of NF-κB signalling pathway in the development of CCA. The notch signalling pathway also plays an important role in CCA progression. Binding of notch ligands and receptors induce the shear of notch protein, and then the generated notch intracellular domain (NICD) enters the nucleus to form a complex with transcription factor CLS (a kind of DNA-binding protein), 22 which activates the expression of CCAinducing target gene. 23 DSL family members act as notch ligands, of which Jagged 1 is one of the most significant CCA-associated notch ligands, 24 and Notch2 receptors have a close relationship with CCA. 25 The activation of notch signalling together with the inactivation of tumour suppressive gene p53 induced CCA tumourigenesis, growth, 26 aggressiveness, and malignant transformation. Claperon et al. found that the epidermal growth factor receptor (EGFR) pathway also contributes to the invasion, metastasis, and development of CCA. 27 EGFRs on cell membrane can bind with their ligands such as transforming growth factor α (TGF-α) to activate protein kinases to promote CCA growth and the overexpression of EGFRs can be effective prognostic factors for intrahepatic CCA. 28 In addition, activated EGFRs can regulate a few intracellular signalling pathways to influence cholangiocarcinogenesis. The downstream pathways contain the Ras2/Raf2/mitogen activated protein kinase (MAPK) signalling pathway, phosphatidyl inositol 3-kinase/threonine kinase (PI3K/AKT) signalling pathway and signal transducers, and activator of transcription (STAT) signalling pathway. 29 The molecules involved in the pathways mentioned above are mostly released from or affected by the surrounding tumour microenvironment  tumour-associated macrophages), tumour associated fibroblasts that promote proliferation of tumour cells, and inflammatory cells that might contribute to tumourigenesis. 30 Non-immune cells mainly F I G U R E 1 The pathways participate in tumourigenesis of CCA. A, Extracellular WNT glycoproteins appear and bind to Frizzled receptors and the co-receptors LRP5 (low-density lipoprotein receptor-related protein 5) and LRP6, DVL inhibit the destruction complex that made of APC, GSK3β and AXIN and results in the accumulation of β-catenin in cell cytoplasm. β-catenin enters nucleus and combines with TCF/LEF transcription factors to regulate the expression of target genes. Then, influencing the CCA. B, NF-kB integrates with inhibiting factor IkB in the stationary state. After IkBs are phosphorylated by IKK or ubiquitnated by SCF-E3 and degraded by protease, NF-kB is activated and entering nucleus to induce the transcription of target genes. C, DSL family bind to notch receptors and stimulate the notch protein shear. And the generated NICD turns into nucleus to combine with transcription factor CLS. The compound activates the expression of target genes to induce CCA (D) EGFRs combine with EGFR ligands to activate protein kinases. Thymidine kinase is phosphorylated to stimulate Ras2/Raf2/MAPK signalling pathway, PI3K/AKT pathway and JAK/STAT pathway that play important roles in CCA carcinogenesis and breast tumour models. 35 As for CCA, hypoxia modulates the genes expression to influence the production of proteins which are associated with cell cycle, apoptosis, cellular movement in CCA cells.
For instance, hypoxia upregulates proteins participating in tumour proliferation, such as trefoil factor-1 (TFF1), metalloprotease 12 (ADAM12), integrin-alpha 5 (ITGA5) and baculoviral IAP repeat-F I G U R E 2 Work model of the impact of tumour stromal cells on CCA. Endothelial cells promote CCA angiogenesis through pathway A (release inflammatory cytokines) and B (express COX-1 protein). Fibroblasts induce CCA invasion via pathway A (produce periostin), B (ITGα5β1/PI3K/AKT pathway) and C (express SDF-1), stimulate CCA metastasis through pathway A (express SDF-1) and promote proliferation of CCA by means of pathway A (express α-SMA) and B (produce SDF-1). Epithelial cells promote CCA invasion via pathway A (express low level of EpCAM) and B (epithelial-mesenchymal transition), induce CCA proliferation through pathway A (secret synemin) and stimulate CCA metastasis by means of pathway A (epithelial-mesenchymal transition). HSCs induce the angiogenesis, invasion and metastasis via pathway A (Hedgehog signalling) and B (produce TGF-β, PDGF and nuclear factor-Kappa B). Macrophages stimulate invasion and metastasis of CCA via pathway A (express MMP-9), induce CCA proliferation through Wnt signalling pathway. Cancer stem cells induce CCA proliferation by means of pathway A (produce signalling mediators) containing 5 (BIRC5/surviving), and downregulates factors related to cell adhesion, such as uridine 5′-monophosphate synthase (UMPS) and S100 calcium binding protein P (S100P), thus promoting CCA invasion and proliferation. 36 And the invasion of CCA also can be enhanced by hypoxia through hepatocyte growth factor receptor (Met)/extracellular signalregulated kinase (ERK) pathway. 37 In addition, hypoxia induces expressions of hypoxic-responsive proteins (especially HIF-1α), which are related to low CCA survival rate and poor prognosis of CCA. 38,39

| Exosomes
Exosome, a crucial part of tumour microenvironment which contains microRNAs, DNA fragments, and proteins, serve as vital communicators among cancer cells, immunocytes, and tumour microenvironment to modulate tumour growth. 40

| Fibroblasts
Through genetic lineage tracing and transplantation assays, Rinkevich et al. confirmed that fibroblasts are related to the formation of cancer stroma. 62 Fibroblasts are recruited to the CCA tumourigenic region and transformed into "active fibroblasts" or CCA associated fibroblasts which make up a large proportion of CCA stromal cells.

| Epithelial cells and mesenchymal stem cells
Metastasis, the main characteristic of carcinoma, is a major factor deciding the cancer-associated mortality in patients and related to EMT whose main participators are epithelial cells and MSCs. Epithelial cell extrusion involving the sphingosine-1-phosphatesphingosine-1-phosphate receptor 2 pathway enables the detachment of tumour cells from their primary sites, prompting the metastatic process. 104 MSCs recruited from the bone marrow and adjacent tissue by cytokines and chemokines are integrated into tumour stroma, and then become tumour-derived MSCs modulating tumourigenesis and tumour progression. 105 MSCs include two distinct phenotypes, MSC1 and MSC2, with different effects on cancer cells. MSC2 enhances tumour growth and metastasis while MSC1 does not. 106 MSCs can enhance tumour progression via the VEGF pathway and the secretion of CCL5, but their effects on tumour vary under different conditions. 107,108 For example, they promote the growth of breast cancer through the WNT signalling. 109 In a xenograft ovarian carcinoma model, Spaeth et al. discovered MSCs have multipotential capacity to differentiate into carcinoma associated fibroblasts that contribute to tumour progression and angiogenesis. 110 EMT induces the epithelial cells to undergo morphological changes, gives the cell metastasis and invasion ability and relates to the formation of circulating tumour cells which contributes to the tumour invasion and metastasis. 111 CCA is a malignancy that occurs in epithelium and in some cases the proliferation of bile duct epithelial cells accelerates CCA cells proliferation by overexpressing synemin (an intermediate filament protein in vascular cells and hepatic stellate cells, is over expressed in inflammation and fibrosis). 112 On the other hand, epithelial cells can occur EMT under the influence of miR-21 and Kruppel-like factor 4 via the Akt and extracellular signal-regulated kinase (ERK) 1/2 pathway, thus impacting the migration and growth of CCA. 113 Additionally, EMT augments the invasion and metastasis of CCA cells that expedites the progression of carcinoma 114,115 and it can be induced and adjusted by atypical protein kinase C-iota (aPKC-ι), 116 EGF/ EGFR axis 27 and protein tyrosine phosphatase PTP4A1. 117

| Hepatic stellate cells
HSCs are in Disse separation and glued to liver sinusoidal endothelial cells. During fibrosis and cirrhosis, HSCs can differentiate into myofibroblasts, 118 and the latter has been proven a crucial player in promoting CCA growth and progression via releasing α-SMA and EGFR signalling. 119 HSCs can be activated by numerous factors (cytokines and chemokines) and signalling pathways, 32 including NF-κ B, TGF-β, PDGF, and Hedgehog signalling that translates signal between CCA cells and HSCs. 120 Clonorchis sinensis can produce protein complex (clonorchis sinensis ferritin heavy chain CsFHC) to activate hepatic stellate cells in hepatic fibrosis and inflammation 121,122 and accelerate CCA through relevant mediators. 123 What's more, HSCs can increase the expression of CXCL5 in CCA cells by secreting IL-β to enhance the connection between CCA cells and CAFs that influences the progression of CCA. 124 The interaction between HSCs and Angiotensin (Ang) II induces tumour proliferation via Ang II/Ang II type 1 receptor (AT-1) axis. 125

| Cancer stem cells
Cancer stem cells (CSCs) are a group of cells that have the ability of self-renewal, hyperproliferation, and differentiating into tumour cells.
It has already been confirmed that the existence of CSCs in CCA. 126 Laminin-332, exists in CCA CSCs matrix, builds a circumstance of chemoresistance and quiescence for CCA. 127 Low expression of CD274 in CCA cells can influence the interaction between CSCs and tumour cells to increase tumourigenesis and help cancer cells escape from the immune reaction. 128 80 Similarly, other tumour stromal cells in CCA microenvironment can become drug target for cancer therapy, too. More comprehensive and complete mechanisms of the interaction between CCA and tumour microenvironment need to be explored and perfected and it will be the basis for our search for a more effective and accurate treatment for CCA. New treatment regimens combined with old treatments (Surgical resection, chemotherapy, radiotherapy, and transplantation therapy) will offer new hope for the treatment of CCA.

CONFLI CTS OF INTEREST
The authors confirm that there are no conflicts of interest.