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Page 6 of 16 Cadamuro et al. Hepatoma Res 2022;8:11 https://dx.doi.org/10.20517/2394-5079.2021.140

growth factor, and stromal cell-derived factor (SDF)-1 [12,30,31] . Notably, SDF-1 is only weakly expressed by
fibroblasts in the peritumoral area, but it is highly expressed and secreted by CAFs. Strong evidence for a
role of CAFs in promoting CCA aggressiveness was demonstrated in a study in which a syngeneic rat model
of CCA was treated with navitoclax (a small BH3-mimetic compound) to induce selective CAF depletion,
thus suppressing tumor growth and improving host survival . The relationship between CAF and other
[26]
components of the TME, such as inflammatory cells and vessels, is very complex, and it is mediated by a
series of growth factors [vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)],
TGFβ, cytokines, and chemokines [monocyte chemoattractant protein (MCP-1) and C-X-C motif ligand
(CXCL) 12 and 14] and MMPs that promote tumor growth and spread, modifying the matrix, attracting the
precursors of vascular and lymphatic vascular cells, and favoring an immunosuppressive
[15]
microenvironment .
Endothelial cells
An important but still little studied element in the TME of CCA is its lymphatic vascular bed. Lymphatic
metastatic spread occurs early during the course of CCA progression and often precludes curative surgical
approaches. Notably, lymphatic endothelial cells are more represented in the TRS than blood endothelial
cells and are localized in close proximity to CAFs . Their involvement in the progression and metastatic
[15]
spread of CCA seems to be due to the ability of both neoplastic and stromal cells to secrete
lymphangiogenic growth factors (including VEGF-C, VEGF-D, and angiopoietins) [32,33] . Lymphatic vessels
have large fenestrations that make them more permeable to the passage of immune cells and they secrete
many chemokines [e.g., C-C motif ligand (CCL) 21] that promote the intravasation of macrophages and
other inflammatory cells [32,34] . It has been shown that increased lymphatic density is associated with a worse
prognosis and reduced disease-free and overall survival [35,36] .

Innate immune cells
Tumor-associated macrophages
Two macrophage populations coexist in the TME of CCA: the liver resident macrophages (or Kupffer cells),
and TAMs. On the contrary to the non-tumoral tissue, populated more by the M1 (or classically activated)
subtype, which exert proinflammatory effects and defend the organism from invasion of pathogens, TAMs
+
+
are mostly of the M2 (or alternatively activated) type and derive from circulating CD14 CD16 monocytes
that are usually involved in tissue repair and remodeling, angiogenesis, and matrix deposition . TAMs play
[37]
an active role in suppressing T cell activation and proliferation, in the promotion of angiogenesis, in the
induction of tissue remodeling, and in stimulating apoptosis of M1 macrophages, which, however, contrast
the neoplastic cells [38,39] . Whereas CAFs are spread over the entire tumor mass, TAMs are mainly located at
the tumor invasive front, putatively recruited by neoplastic cells. CCA cells in fact secrete interleukins (IL) -
6, -13, and -34, TGFβ, and osteoactivin, all molecules able to recruit monocytes and stimulate their M2
transdifferentiation [40,41] . Conversely, CAFs support TAM recruitment to the tumoral microenvironment by
secreting CCL2 and colony stimulating factor 1 (CSF1) . Many other secreted chemotactic mediators such
[42]
as the cytokines IL-1β, IL-4, IL-10, and IL-16, CCL3, CCL4, and CXCL12 play a supportive role [16,43] .

TAMs can act as a trophic cell population for neoplastic cells by secreting IL6, tumor necrosis factor (TNF)
α, TGFβ, and VEGF and activating cyclooxygenase-2 and WNT/β-catenin signaling [44-47] . TAM-secreted
[48]
VEGF can also stimulate neoangiogenesis, thus contributing to CCA metastasis . Moreover, TAMs are the
main source of metalloproteases, in particular MMP-9, which, by degrading the matrix, favors metastasis .
[39]
Finally, TAMs can attract immunosuppressive cells, such as TANs and MDSCs, through the secretion of
different soluble mediators (IL-4, IL-8, IL-10, CCL2, CCL17, and CCL22) to generate an
[49]
immunosuppressive environment that favors the malignant behavior of CCA .