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Department of Molecular Medicine, General Medicine Division, University-Hospital of Padua, Padua, ItalyLiver Center and Section of Digestive Diseases, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT
Intrahepatic cholangiocarcinoma (ICC) is the second most common liver malignancy. ICC typically features remarkable cellular heterogeneity and a dense stromal reaction. Therefore, a comprehensive understanding of cellular diversity and the interplay between malignant cells and niche cells is essential to elucidate the mechanisms driving ICC progression and to develop therapeutic approaches.
Even if the understanding of the disease has improved over the last decade, researchers and clinicians are still faced with an enigmatic and worrisome cancer, with marked heterogeneity making therapeutic choices difficult. Besides the well-established anatomical criteria, by which CCA is categorized into 3 different tumor types (intrahepatic, perihilar and distal), the multifaceted tumor microenvironment (TME) is another factor that contributes to the complexity of CCA. TME is an intricate ecosystem able to exchange a multitude of signals with the tumor counterpart, which may exert either pro- or anti-oncogenic functions.
Zhang et al. are the first to dissect intrahepatic CCA (iCCA) at the cellular level, and to demonstrate that indeed the CCA cellular landscape displays a strong diversity in the malignant, immune and stromal cells
(Fig. 1). Although based on a relatively small sample set (5 iCCAs, of which 3 tumors also included the matched ‘peritumoral’ tissue), this study drives the field forward by providing a single-cell resolution transcriptomic landscape of iCCA. Moreover, the authors identify a novel mechanism underpinning the deleterious interplay between the tumor and its microenvironment that can be harnessed for therapeutic targeting. In this study, Zhang et al. characterize distinct populations of immune and stromal cells, in particular tumor-infiltrating lymphocytes (TILs) and cancer-associated fibroblasts (CAFs) in the context of a wide inter- and intra-tumor heterogeneity that also involves malignant cholangiocytes, as anticipated by several studies performed in iCCA.
Within the tumor bulk, malignant cells form the core, and by definition, this population is classically regarded as heterogenous. Zhang et al. identified 4 different clusters in the tumor cell compartment of iCCA, harboring patient-specific somatic mutations, but also sharing common activated signatures, including IL-6, Wnt, transforming growth factor (TGF) and tumor necrosis factor (TNF) pathways. Furthermore, this study pinpoints novel pathways that could be relevant in CCA progression, in particular the serine peptidase inhibitor Kazal type-1 (SPINK1, also called TATI or PSTI). SPINK1 is a kinase inhibitor of premature trypsin activation in the pancreas, and its overexpression is associated with poor prognosis in iCCA. In vitro loss and gain function experiments indicate that SPINK1 contributes to stemness, cell invasion, and chemoresistance, which presumably involves a subset of tumor-promoting cells. Notably, the prognostic significance of SPINK1 overexpression has been reported in several cancers (lung, kidney, prostate, ovary, and breast) and determination of SPINK1 in serum has been proposed as a useful biomarker to identify patients at risk of a more aggressive disease.
Among TILs, CD4+ T regs are gaining special attention because of their strong immunosuppressive properties. T regs are mostly localized at the peritumoral region, where they dampen antitumor activity of NK and CD8+ cytotoxic T cells by secreting IL-10 and TGF-β1.
Moreover, overexpression of the transcription factor FoxP3, a distinct trait of their immunophenotype, upregulates cytotoxic T lymphocyte antigen-4 (CTLA-4), which inhibits CD8+ T cell activation by binding to CD80 expressed by antigen-presenting cells. Besides CTLA-4, Zhang et al. describe T regs expressing T cell Ig and ITIM domain (TIGIT), a coinhibitory receptor that synergizes with CTLA-4 to suppress antitumor immune responses. Accordingly, they report a persistent cytotoxic or activated phenotype of NK and T cells supported by enriched signaling related to hypoxia, apoptosis, interferon response and oxidative phosphorylation. However, TIGIT also mediates intercellular communication with malignant cholangiocytes that express the nectin-like protein, poliovirus receptor (PVR/CD155), which promotes tumor cell invasion, migration, proliferation, and immune escape to support tumor progression.
In iCCA, TME is characterized by an intense desmoplastic reaction sustained by a variety of stromal cells, laying within a rearranged and stiffer extracellular matrix (ECM). Several teams have already dissected the CCA microenvironment at tissue level by transcriptomics or proteomics,
and all have emphasized the significant contribution of CAFs. Zhang et al. demonstrate the existence of 5 CAF subpopulations in iCCA. To overcome the limited yield provided by single-cell analysis (only 1.6%) they turn to isolation of CAFs by a commonly used negative selection strategy. All CAF subsets express prototypal fibroblast genes, including ACTA2 (α-SMA at different levels across subpopulations), COL1a2 and PDGFRb. Of note, PDGFRb encodes the cognate receptor of platelet-derived growth factor-D (PDGF-D), which drives CAF recruitment and is secreted by the malignant cholangiocytes themselves.
Interestingly, the most abundant CAF subset (57.6%) displays a microvasculature signature (vCAF). Although CCA is regarded as a hypo-vascular tumor type, it contains an extensive lymphatic vasculature likely involved in the early dissemination to regional lymph nodes, a critical step that may preclude the curative effects of surgery.
Although not addressed by Zhang et al., it is tempting to speculate that the vCAF subset is actively involved in generating the lymphatic vascularization of iCCA, since they are localized to the tumor core and in microvascular regions closely aligned with malignant cells. Indeed, upon PDGF-D stimulation, CAFs secrete the vascular endothelial growth factors (VEGF)-A and -C, which promote tumor-associated lymphangiogenesis and the propensity of CCA cells to invade across the endothelial wall.
and can possibly be regarded as the cell source of vCAF in iCCA. Two CAF subclusters with low ACTA2 expression were identified, as previously showed in other cancers, one cluster is enriched in ECM proteins and collagen fibril organization (mCAF), the second cluster is related to inflammatory responses and complement activation (iCAF). The last 2 CAF clusters comprise a subpopulation of CAFs that either express the major histocompatibility complex II (MHC-II) genes and were termed antigen-presenting CAF (apCAF), or a subpopulation of CAFs expressing the epithelial markers (KRT19 and KRT8), designated the epithelial-to-mesenchymal transition (EMT)-like CAF (eCAF). This phenotypic diversity of CAFs matches the functional diversity that Zhang et al. have begun to unravel, starting with vCAF, the main CAF subpopulation identified. The authors explore the IL-6/IL-6R axis, a pro-invasive pathway in cholangiocytes that has been extensively studied in CCA. In the present study, the IL-6 axis represents a signature featuring malignant cholangiocyte subsets 1 and 2. Previously, IL-6 has been identified in the stroma of CCA
and is now assigned to the vCAF subset; it is upregulated when vCAFs come into contact with tumor cells. The mechanism by which malignant cells stimulate IL-6 in vCAFs revealed a contribution of exosomes containing miR-9-5p. Once produced by vCAFs, IL-6 targets tumor cells and increases their malignant nature through epigenetic alterations, specifically involving the polycomb group protein enhancer of zeste homolog 2 (EZH2), a type of histone methylation modification nuclear complex. Although the pleiotropic effects of EZH2 on proliferation, apoptosis, and angiogenesis in CCA are well recognized,
the authors show its involvement in fostering the cancer stem cell niche, as EZH2 inhibition by small molecules suppresses tumor sphere formation. Moreover, the authors provide further evidence supporting a ‘yin-yang’ cooperation between CAFs and tumor cells, as previously shown with the heparin-binding epidermal growth factor-like (HB-EGF)/TGF-β axis.
Taken together, the data outlined by Zhang et al. help to understand the multiple reasons behind the historical challenges in developing effective therapeutic strategies in iCCA. First, the common pathways found to be deregulated in tumor cholangiocytes are potentially druggable, reaching all malignant subsets (IL-6, Wnt, TGF, TNF), but unfortunately no inhibitor of these molecules has proven to be effective in humans despite rather promising preclinical studies.
Second, TIGIT targeting can fail given the presence of functional redundancy in the TME across coinhibitory receptor pathways that may compensate TIGIT deficiency. Third, as shown, CAFs are a hugely heterogeneous population, displaying distinctive phenotypic traits, which reflect specific functions (angiogenesis, immune modulation, ECM synthesis, EMT). This important concept is consistent with the conflicting results generated by CAF depletion in experimental models of desmoplastic epithelial cancers. In fact, an experimental rat model of syngeneic iCCA deprived of CAFs (through induction of apoptosis with the BH3 mimetic navitoclax) showed reduced primary tumor growth, tumor lymphatic vascularization, and metastasis to regional lymph nodes and to the peritoneum.
Conversely, in mouse models of pancreatic ductal adenocarcinoma, CAF depletion was associated with a more aggressive tumor phenotype and enhanced tumor spread, thus indicating the ambivalent roles of CAFs to either restrain or stimulate tumor growth.
These issues need to be tackled before relying on TME targeting as a powerful tool to fight iCCA invasiveness. Within this perspective, scRNA-seq may provide a valuable asset to capture the pronounced heterogeneity of iCCA at single-cell resolution and thus, lay the foundation to develop novel and effective personalized treatment approaches.
The authors received no financial support to produce this manuscript.
Manuscript preparation: LuFa and LaFo. Figure: LuFa. Review and editing: LuFa, JA and LaFo.
Conflict of interest
The authors declare no conflicts of interest that pertain to this work.