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Casolino et al. Hepatoma Res 2021;7:76 https://dx.doi.org/10.20517/2394-5079.2021.79 Page 15 of 23

patients with BTC as part of the phase 2/3 INTR@PID BTC 055 trial (NCT04066491). However, due to
[103]
disappointing preliminary results, the trial has been recently discontinued .
Immune checkpoint inhibitors have also been tested in association with anti-angiogenic drugs. However,
[99]
results in BTCs were not satisfactory, in contrast with high efficacy observed in hepatocellular carcinoma .
Pembrolizumab plus ramucirumab, anti-vascular-endothelial growth factor, showed limited efficacy in
patients with previously treated aBTC, with a mOS of 6.4 months and mPFS of 1.6 months . Data from
[104]
the ongoing IMbrave-151 phase II trial are awaited to assess the role of the combination of CisGem
chemotherapy with Atezolizumab plus/minus Bevacizumab in the first-line setting (NCT04677504).

In conclusion, the efficacy and safety of immunotherapies for BTC are still unclear. ICIs monotherapy has
limited efficacy in unselected patients, while the combinations of ICIs and chemotherapies/targeted
therapies seem to achieve better results. However, further clinical trials are necessary to find the optimal
combination strategy and identify the predictive biomarkers for improved treatment selection. Table 4
shows a selection of ongoing clinical trials exploring ICIs.

Targeting the TME
For many tumour types, targeting the TME is an attractive therapeutic strategy in BTCs. High heterogeneity
of TME both at the molecular and the cellular level has been demonstrated through multi-omics studies,
and different clinically relevant subtypes of BTCs have been suggested based on TME specific
characteristics [72,105,106] . This increasing knowledge has led to the identification of possible therapeutic
strategies targeting several cell types and/or signalling pathways of the TME. In addition to treatments
targeting the immune cell infiltrate (already discussed above), many others addressed towards different
components of the TME have been recently proposed. In particular, their association with cytotoxic
[107]
chemotherapy and tumour cells targeted therapies represents a promising opportunity .
BTCs are characterised by significant desmoplastic reactions orchestrated by stromal cells, and different
types of immune cells infiltrate. The complex TME ecosystem is populated with a wide variety of cells,
including cancer cells, cancer-associated fibroblasts (CAFs), myeloid-derived suppressor cells, tumour-
associated macrophages (TAMs), TILs, tumour-associated neutrophils, natural killer cells, and many
others [108,109] . TME has a significant role in shaping a chemoresistant phenotype. On the one hand, it
constitutes a barrier that obstacles drug delivery; on the other, TME cells interact in dynamic crosstalk with
components of the extracellular matrix (ECM), soluble factors - including cytokines, chemokines, growth
factors - and biliary tumour cells to modulate chemo-resistance and drive tumour progression [110-112] . From a
therapeutic point, the most interesting cells are CAFs and TAMs.

CAFs are a heterogeneous group of cells characterised by the expression of alpha-smooth muscle actin and
platelet-derived growth factor receptor beta. They are the key protagonists in the TME of BTCs and have a
pivotal role in ECM composition and crosstalk with cancer cells through the secretion of TGF-β, stromal
cell‐derived factor 1, hepatocyte growth factor, connective tissue growth factor, epidermal growth factor and
platelet-derived growth factor . CAFs are first recruited by CCA tumour cells which subsequently exert
[108]
positive feedback and amplify their activity. Once activated, CAFs promote CCA progression.

TAMs and exerts a key role in cancer-related inflammation by promoting tumour-cell proliferation,
angiogenesis, matrix turnover and suppression of the adaptive immune response . TAMs create a
[113]
favourable niche by interacting with biliary cancer cells and other stromal cells via releasing multiple
protumour factors (including IL-4, IL-6 and IL-10, CCL17, CCL18, and MMP9) that ultimately sustain