CCA accounts for about 15% of all primary liver malignancies and is second after HCC in number of cases[131,132]. Indeed, CCA is a group of heterogeneous tumors arising at different levels of the biliary tree.
The most recent classification describes three different CCA subtypes looking at different anatomical regions: iCCA, peri-hilar CCA (pCCA) and distal CCA (dCCA)[133,134]. Several studies in the last two decades revealed that the incidence of these subtypes is vary. iCCA incidence has been increasing in contrast with the decrease of pCCA and dCCA incidence[135-137].
Different risk factors and survival rates seem to be related to each of them. Despite the etiologies remaining unclear in most cases of CCA, some risk factors are well established. For example, liver flukes (Opistorchis viverrini and Clonorchis sinensis) have been clearly associated with iCCA in East Asia. On the other hand, especially in Europe, primary sclerosing cholangitis (PSC) is a demonstrated risk factor for CCA, specifically correlated to pCCA variant. Viral hepatitis (HBV and HCV) has been identified as definitive risk factors more associated with iCCA than pCCA. Emerging role have been given to metabolic syndrome, alcohol and smoking[138-140].
Diagnosis is usually tardive due to its vague symptoms. Patients with iCCA are generally asymptomatic (20%-25% incidental finding), appearing tardively cachectic, with abdominal pain and fatigue. In contrast, pCCA most frequently manifests as painless jaundice. No proper biomarkers are available and diagnosis is a combination of clinical, radiological and unspecific histologic-biochemical markers. It has long been argued that a staging system for CCA needs to be found[133,141,142].
iCCA treatment is very dismal due to the well-known lack of response to the conventional chemotherapy[143,144]. Nowadays, surgical resection together with liver transplantation, for highly selected patients, are the only potentially curative treatments for iCCA, with median disease-free survival (DFS) duration of 12–36 mo. For advanced stage and unresectable iCCA, transarterial chemoembolization (TACE) is a treatment option considered to prolong OS in patients[145,146].
For patients with iCCA, due to the lack of effective curative strategies, understanding the molecular mechanism together with the TME interactions involved in tumoral progression and chemoresistance could open the possibility of developing new potential target therapies.
TME in CCA
CCA is characterized by a dense desmoplastic TME composed of high stromal cell infiltration together with immune cells from the adaptive and innate immune systems. In the last years, several studies have been focused on understanding the mechanism underlying the interplay between stromal cells and neoplastic cells in CCA.
In the liver, CCA CAFs have been shown to have multiple sources of origin, including from hepatic stellate cells, circulating bone marrow-derived precursor cells and portal fibroblasts[148,149].
In the tumoral milieu, CCA cells have been reported to be the main cellular component secreting PDGF-DD, which acts by binding to its receptor PDGFR-β expressed on CAFs, leading to their recruitment in the tumor site, together with other factors, such as TGFβ[150,151].
Once recruited and activated within the tumor, CAFs are able to enhance CCA growth and progression through the secretion of tumor matrix, providing the scaffold and the release of various soluble factors[152-154].
PDGF-BB is an important paracrine survival signal released by CAFs that influence tumor malignant phenotype. By binding to its cognate receptor, PDGFR-β on CCA cells, it leads to an intracellular signaling cascade able to protect tumor cells from TRAIL-induced apoptosis by activating Hedgehog signaling and sustaining TGFβ secretion[155,156].
Among cytokines, chemokines and growth factors released by CAFs, stromal cell-derived factor-1 (SDF)-1 and heparin-binding EGF-like growth factor (HB-EGF) have key roles in promoting proliferation, migration and invasion of CCA cells expressing their related receptors, CXCL4 and EGFR. In the meantime, the secretion of HB-EGF by CAFs is sustained by a TGFβ feedback release from CCA cells, upon EGFR activation[157-159].
This highlights the presence of multiple paracrine signals exchanged between CCA cells and CAFs. The latter promotes tumor cell proliferation and invasion and in turn actively recruited and activated by CCA in a self-perpetuating loop (Figure 4).
Figure 4 Role of cancer-associated fibroblasts in cholangiocarcinoma.
In the figure is shown the paracrine loop between tumor cells and cancer-associated fibroblasts, the main cellular component of cholangiocarcinoma tumor microenvironment (TME). Several survival and recruitment signals are exchanged within TME, leading to tumor growth and progression. CCA: Cholangiocarcinoma; TGFβ: Transforming growth factor beta; PDGF-β: Platelet-derived growth factor receptor beta; EGFR: Endothelial growth factor receptor.
Due to key role of stromal cells in CCA TME, in the last few years, several studies have been focused on targeting CAFs and their released factors[160-162]. The most recent study investigated the role of an FDA-approved anti-fibrotic drug called nintedanib. This tyrosine kinase inhibitor is able to inhibit fibroblast growth factor receptor (FGFR) and PDGFR, showing promising results in reduction of CCA growth and aggressiveness both in vitro and in vivo. The emerging important role of CAFs could be an attractive target to ameliorate the treatment of patients with CCA.
Together with CAFs, cells from the innate and adaptive immune systems have been found to infiltrate the TME and significantly sustain CCA malignant transformation. Among them, tumor-associated macrophages (TAMs) represent the most relevant primary immune cells.
Macrophages could be resident (KCs) or recruited from circulating monocytes via soluble factors, such as monocyte chemoattractant protein 1 (MCP-1/CCL2), released in the tumor milieu[151,164]. Once in the liver, they differentiate into tissue macrophages, acquiring specific subset and activation status according to the exposition of multiple signals in the TME.
Normally, macrophages could be polarized in two possible phenotypes: M1 (anti-tumorigenesis), which is activated by INF-g, and M2 (pro-tumorigenesis) in response to anti-inflammatory cytokines, such as IL-10, TGFβ and IL-4[166,167].
In CCA TME, studies revealed that either CAF or CCA cells are involved in TAM recruitment and inducement of a M2 phenotype via the STAT3 pathway. TAMs polarized as pro-tumorigenic, the most representative TAM in TME, have been shown to promote tumor progression and modulation of the surrounding microenvironment through the subsequent secretion of pro-inflammatory and tumor-promoting mediators (IL-6, IL-1, TGFβ, VEGF and PDGF)[147,168,169]. Indeed, increasing evidence has shown that, in iCCA, the higher number of M2 TAMs correlated with a poor prognosis[170-172].
In the last year, more attention has been paid to neutrophils, the most abundant of the white blood cells, which represent one of first lines of defense against invading pathogens. Different studies have revealed that neutrophils are recruited within tumors driven by CXCL5, a chemotactic cytokine. Tumor-associated neutrophils (TANs) and elevated pre-operative neutrophil to lymphocyte ratio (NLR) are associated with a poor prognosis in patients with iCCA[173-176].
Further studies are needed to deeper elucidate the role of TAMs and TANs in iCCA, in terms of possible use as predictive markers for patients undergoing surgical resection. As described for HCC, iCCA arises in a chronically inflamed liver as a consequence of bile duct injury and the related TME contributes to the development of an anti-tumor immune milieu.
CCA cells with CAFs and TAMs are able to secrete various factors, such as CCL2, recruiting and stimulating T regs. These release back IL-10 and TGFβ that inhibit cytotoxic T cells, suppressing the immune response[177,178]. Such mechanisms are similar and have already been described for HCC. Despite this, not much literature is present on TILs in iCCA, focusing mainly on immunohistochemistry analysis in terms of number and presence of CD4+ and CD8+ cells.
Briefly, CD4+ cells have been found to infiltrate tumor specimens. On the contrary, CD8+ cells are present at the tumor margin, correlating with prognosis. Patients with iCCA with low infiltration of cytotoxic T cells show a worse prognosis[179,180]. The presence of both TIL and PDL-1/PD-1 expression makes iCCA possibly suitable for immunological target therapies[181,182].
NK cells in iCCA
Less efforts have been made to unveil the specific role of NK cells in iCCA. However, several preclinical and clinical studies have assessed the activity of NK cells against iCCA. The use of in vitro cytokine-activated NK cells in combination with cetuximab, the mAb against EGFR, has shown benefits in a higher antibody-dependent cellular cytotoxicity response against human iCCA cell lines such as HuCCT-1 and OZ. Moreover, the multiple infusions of ex vivo-expanded human NK cells into iCCA xenograft mice (HuCCT-1 tumor-bearing nude mice) resulted in NK cell-mediated cytolytic response with inhibition of tumor growth.
Recently, an elevated intra-tumoral expression of CXCL9, an IFN-γ inducible chemokine, was associated with a large number of tumor-infiltrating NK cells, leading to favorable postoperative survival in patients with iCCA. Additionally, elevated expression of NKG2D ligands in human iCCA correlate with improved DFS and OS in patients. Although these findings hold promise, further studies are needed to investigate the role of NK cells in the pathogenesis of iCCA. In fact, similar to HCC, strategies with the aim of evading NK cell immunosurveillance in CCA have been reported. For instance, iCCA cells are able to induce apoptosis in NK cells, via the Fas/FasL pathway, and escape the inflammatory response by upregulating the antiapoptotic c-FLIP system. On the other hand, several nucleotide polymorphisms (SNPs) located within the NKG2D receptor gene (KLRK1) have been linked to impaired NK cell effector functions and higher risk of cancer.
Specifically, the development of CCA in patients with PSC have been associated with polymorphisms in the NKG2D gene, thus patients who are homozygous for the NKG2D alleles are likely to develop CCA. These data clearly support different roles and clinical impacts of NK cells in iCCA disease. However, it is still not clear how these activities are related to the specific blood circulating and liver resident NK cells.