Hepatic stellate cells and extracellular matrix in hepatocellular carcinoma: more complicated than ever
Vinicio Carloni et al. / Liver International, Volume 34, Issue 6, pages 834–843, July 2014
BDL — bile duct ligation
CCl4 — carbon tetrachloride
CCN1 — matricellular protein cysteine-rich protein 61
CM — conditioned medium
ECM — extracellular matrix
HBV — hepatitis B virus
HCC — hepatocellular carcinoma
HCV — hepatitis C virus
HMGA1 — high-mobility group A1
HSC — hepatic stellate cells
p16INK4a — cyclin-dependent kinase inhibitor
PAI-1 — plasminogen activator inhibitor-1
PDGF — platelet-derived growth factor
TGF — transforming growth factor
α-SMA — alpha-smooth muscle actin
Гепатоцеллюлярная карцинома (HCC) является пятым по распространенности раком во всем мире согласно ежегодному инциденту и третьей основной причиной смерти от рака, а куративная терапия не опция для многих пациентов из-за лежащего в основе заболевания печени [1, 2]. HCC — следствие хронического некровоспаления, которое представляет основной элемент областей HCC, может индуцировать хромосомные мутации и, в конечном счете, злокачественную трансформацию пролиферирующих гепатоцитов.
В Западных странах, вирусный гепатит С (HCV), вирусный гепатит B (HBV), определенные метаболические заболевания печени, наследственный гемохроматоз, порфирия и тяжелое потребление алкоголя наблюдаются почти во всех случаях HCC, характеризующихся наличием цирроза, результатом терминального фиброза. Новой и хорошо признанной причиной терминальной болезни печени — цирроза в комбинации с HCC — является неалкогольная жировая болезнь печени, и особенно ее наиболее агрессивная форма, неалкогольный стеатогепатит .
Хотя инцидент HCC варьирует по этиологии, расовой и этнической принадлежности, полу, возрасту и географическому региону, наличие фиброза — общее звено каждого из этих рисков. Риск HCC может быть сокращен, устраняя начальное, воспалительное повреждение, но персистирующий фиброз сам по себе имеет канцерогенный риск . Следовательно, прояснение вирус-ассоциированных факторов у цирротического пациента с гепатитом могло бы остановить прогрессирование заболевания, но не будет снижать риск HCC [3, 5-7]. Поэтому, сложность HCC и лежащего в основе заболевания печени имеет основные импликации к национальным медицинским услугам и глобальному здоровью. Недавние эпидемиологические данные указывают, что в США и Европе уровень смертности для HCC увеличивается и показатели HCC инцидента и летальности, как ожидают, удвоятся в следующие 10-20 лет [8-10]. В этом отношении маркеры HCV инфекции найдены в 27-75% всех случаев HCC в Европе и США. Наличие этого резервуара HCV инфекции в общей популяции поэтому вызывает опасения относительно возможности нарастания инцидента цирроза и HCC в ближайшие годы на этих территориях [11-13].
Гепатоцеллюлярная карцинома и гетерогенность, следствия для лечения
Одной из основных причин затруднений в лечении HCC является гетерогенность. HCC, даже в одном узелке, представляет очень неоднородное заболевание, которое широко варьирует в клиническом исходе и ответе на терапию . Гетерогенность может объясняется фактом, что HCC — многоступенчатый процесс, и каждая из вышеупомянутых этиологий представляет сложные и различные совокупности хромосомных аберраций с генетическими и эпигенетическими альтерациями и молекулярными сигнальными путями, меняющимися в процессе длительного периода развития опухоли (10-20 лет), прогрессирования и метастазирования . Поэтому одна из самых сложных задач заключается в идентифицировании онкогенной аддикции в процессе развития HCC, подобно другим опухолям, что может направлять выявление новых и очень специфических препаратов для таргетирования HCC.
Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide by annual incidence and the third leading cause of cancer death, and curative therapy is not an option for many patients because of underlying liver disease [1, 2]. HCC originates from chronic necroinflammation which is a key element of HCC occurrence that can induce chromosomal mutations and eventually malignant transformation of proliferating hepatocytes.
In Western countries, hepatitis C virus (HCV) infection, hepatitis B virus (HBV) infection, certain metabolic liver diseases, hereditary haemochromatosis, porphyria and heavy alcohol intake are found in almost all HCC cases characterized by the presence of cirrhosis, the result of end-stage fibrosis. A new and increasingly recognized cause of end-stage liver disease, that is cirrhosis in combination with HCC, is nonalcoholic fatty liver disease, and particularly its more aggressive form, nonalcoholic steatohepatitis .
Nonetheless, the incidence of HCC varies by aetiology, race, ethnicity, gender, age and geographical region, the presence of fibrosis is a common link among each of these risks. The risk of HCC may be reduced by abrogating the initial, inflammatory insult, but increasing evidence suggests that persistent fibrosis confers its own carcinogenic risk . Hence, clearing viral-related factors in a cirrhotic patient with hepatitis might halt progression of the disease, but will not reduce the risk of HCC [3, 5-7]. Therefore, the complexity of HCC and the underlying liver disease has major implications to national hospital services and global health. Recent epidemiological data indicate that, in the USA and Europe, the mortality rate for HCC is increasing and the incidence- and mortality rates of HCC are expected to double over the next 10–20 years [8-10]. In this regard, markers of HCV infection are found in 27–75% of all HCC cases in Europe and the USA. This reservoir of HCV infection in the general population therefore raises concerns about the possibility that there is likely to be an increase in the incidence of cirrhosis and HCC in the forthcoming years in these areas [11-13].
Hepatocellular carcinoma and heterogeneity, consequences for treatment
One of the major reasons concerning the difficulty to treat HCC is the heterogeneity. It is established that HCC, even in the same nodule, is a very heterogeneous disease that differs widely in clinical outcome and in response to therapy . The heterogeneity can be explained by the fact that HCC is a multistep process and each of the above-mentioned aetiologies represents complex and different constellations of chromosomal aberrations with genetic and epigenetic alterations and molecular signalling pathways that are changing during the long time course of tumour development (10–20 years), tumour progression and metastasis . Therefore, one of the biggest challenges is to identify the oncogene addiction loops in the process of HCC tumour development as has been the case with other cancers that could drive the identification of new and very specific drugs to target HCC.
Significant improvements have been made in screening and subdividing HCC by using the so-called ‘-omics signatures’ which are directed to identify genomic signatures and to characterize and categorize HCCs into prediction, phenotype, function and molecular target signatures according to their utilities and properties as is reviewed by Woo et al., . For example, an unsupervised transcriptome analysis subdivided HCC into six subgroups which were associated with clinical and genetic characteristics. Moreover, this analysis showed that 50% of the tumours were characterized by wingless-related integration site and protein kinase B pathway activation which inhibition may be a potential new therapeutic target . Wurmbach et al., have analysed, in a stepwise manner, the carcinogenic process from patients with HCV infection towards cirrhosis and dysplasia, very early HCC, HCC and metastatic tumour formation identifying gene signatures associated and characteristic for each stage of progress . A 186-gene expression signature has been shown to be specific for not only the overall death in HCV patients with surgically treated HCC but also showed to be a sensitive predictor of future risk of poor outcome in patients with newly diagnosed HCV-related early-stage cirrhosis without HCC at the time of diagnosis. Therefore, this 186-signature allows to stratify HCV-related Child-Pugh class A cirrhotic patients into prognostic subgroups thus enabling the identification of patients at risk of HCC development at an early time-point in their lifespan [18, 19]. Moreover, Divella et al. demonstrated that tumour heterogeneity is closely linked with inflammation such as for example in the presence of viral HCV/HBV and confirmed that the presence of viral infections increased the risk for HCC development. Indeed, a significant correlation was found between the frequency of the 4G allele and genetic variability of 4G/5G Serpin1/PAI-1 (plasminogen activator inhibitor-1) polymorphisms in HCC patients and this was compared to patients with and without HCV/HCB. The data demonstrated the presence of two distinct pathogenic mechanisms in HCC which depended on the viral infection aetiology .
Nonetheless, all the major efforts made so far to unravel the complex molecular working mechanisms leading to HCC development, HCC treatment is still characterized by a high rate of drugs and chemotherapy resistance and is also limited by the failure of adjuvant/neoadjuvant chemotherapy to target disseminated tumour cells giving rise to late tumour recurrence and metastases [21, 22]. Most common applications to treat HCC, and the choice of treatment depend on the location of the tumour, tumour size and the presence of multiple tumours which will/can be treated according to evidence-based treatment approaches such as the Barcelona Clinic Liver Cancer classification [23, 24].
Until today, therapeutic interventions in HCC treatment can be divided into curative approaches such as tumour resection, liver transplantation, local radiofrequency ablation and high-intensity focused ultrasound thermal ablation and palliative interventions such as transarterial chemoembolization. These interventions can be combined with the administration of small molecular multityrosine kinase inhibitors such as sorafenib or sunitinib which are characterized by a broad inhibitory profile against rapidly accelerated fibrosarcoma kinases, vascular endothelial growth factor receptor 2 and 3 and platelet-derived growth factor (PDGF) receptor. However, patients with advanced HCC treated with Sorafenib have improved overall survival and a delay of tumour progression of only 3 months [25, 26]. Other ‘new’ drugs such as multikinase inhibitors sunitinib, erlotinib or brivanib have all failed in clinical studies .
Several studies have demonstrated that targeting exclusively the tumour cells is uneffective for the survival of the patients and suggested that the tumour microenvironment and stromal cells affect the tumour cell behaviour thus indicating a major pathogenic role for the diseased liver microenvironment or ‘field effect’ in HCC development (Fig. 1) . Indeed, recent studies have demonstrated the importance of the tumour microenvironment during HCC progression, which can predict tumour recurrence, metastasis and patient survival [18, 29]. These data indicate that the tumour microenvironment actively favours the selection and expansion of cellular clones that are more likely to survive and adapt to the changes induced by stromal cells . This led to new objectives and a new era in the search for antifibrogenic compounds  to be used in combination with anticancer therapy to treat HCC. Moreover, anticancer drugs can have antifibrotic effects such as shown for Sorafenib .
Figure 1. It is established that hepatocellular carcinoma (HCC), even in the same nodule, is a very heterogeneous disease that differs widely in clinical outcome and in response to therapy. During HCC development, the tumour cells and activated hepatic stellate cells (HSC) are accompanied by carcinoma-associated fibroblasts, myofibroblasts and new activated HSC, tumour-associated macrophages, tumour-associated endothelial cells, pericytes, dendritic cells and stem/progenitor cells; generally called tumour stromal cells each able to be a potential target. The degree of influence that each of these cells has on the tumour cells, and vice versa, is an area of significant active study.
Unresolved question in hepatocellular carcinoma: do tumour microenvironment and stromal cells matter?
Tumour and tumour microenvironment
Hepatocellular carcinoma develops and progresses when fully embedded in a surrounding microenvironment that favours its growth. HCC develops in 90% of cases during the clinical course of chronic-inflammatory liver diseases leading to cirrhosis and is regarded as a result of different environmental risk factors each involving different genetic, epigenetic- and chromosomal alterations and gene mutations. This results in altered molecular pathways with cell proliferation as the most important mechanism of liver cancer progression . This further suggests that chronic changes in the liver microenvironment including neoangiogenesis and the development of a fibro-inflammatory stroma contribute to HCC progression. These primary changes, because of a specific aetiological-diseased liver, add up for the molecular/mechanistical support towards HCC development and this might be an additional factor to explain heterogeneity in HCC. During HCC development, the tumour cells and activated hepatic stellate cells (HSC) are accompanied by other tumour stromal cells such as cancer-associated fibroblasts, immune cells and myofibroblasts. The latter originate from different fibroblast populations and differentiate into alpha-smooth muscle actin (α-SMA) positive myofibroblasts (Fig. 2) (this topic is extensively reviewed elsewhere) [34-38]. Each of these stromal cell types is able to be a potential target. For example, carcinoma-associated fibroblasts  myofibroblasts and activated HSC , tumour-associated macrophages [41, 42], tumour-associated endothelial cells [43, 44], pericytes , dendritic cells  and stem/progenitor cells [47, 48]. The degree of influence that each of these cells has on the tumour cells, and vice versa, is an area of significant active study (reviewed extensively elsewhere) [29, 49, 50].
Figure 2. Immunohistochemistry for α-SMA (alpha-smooth muscle actin, a marker for cells with smooth muscle differentiation, including myofibroblasts and HSCs) and CD34 (marker for endothelial cells) in a human HCC with acinar growth pattern (A–D) and trabecular growth pattern (E–H). α-SMA positive cells (A, E; ×4) and (C, G; ×20) are primarily in or around the capillarized sinusoids. Long cytoplasmic processes can be observed. There is a continuous immunohistochemical positivity of the endothelial lining of the capillarized sinusoids marked by CD34 (B, F; ×4) and (D, H; ×20).
Tumour microenvironment extracellular matrix and tension
In the microenvironment, stromal cells produce an altered extracellular matrix (ECM) that provides biochemical and mechanical cues to the tumour cells and surrounding tumour microenvironment. Several of the ECM components such as collagens, laminins, fibronectin, glycosaminoglycans and proteoglycans interact directly and indirectly with HCC cells and the stroma cell types, thereby changing phenotype and function of HCC and stroma cells. These changes are sensed by the cell with the help of stress sensors such as integrins that form on the inner side of the plasma membrane major protein complexes that regulate specific signalling pathways to respond to the stimuli . The mechanical stress arisen from changes in ECM will impose an ‘outside-in’ signalling linking the actin cytoskeleton to the microenvironment by increasing intracellular contractile forces regulating signalling pathways fundamental to determine cell phenotype. In response to ‘outside–in’ stimuli, the anchored cells can pull on the ECM by the presence of increased ECM adhesions and focal adhesions i.e. the so-called mechanical regulators required for the ‘inside-out’ signalling . Thus, the enhanced production of ECM and ECM reorganization can lead to a mechanically stiff microenvironment that can promote proliferation, invasion and can also change the gene expression of the different stromal cell types and cancer cells hence enhancing tumour progression. Shrader et al., have demonstrated that increased matrix stiffness promotes proliferation and chemotherapeutic resistance. Interestingly, Shrader et al. showed that a soft environment induces reversible cellular dormancy and stem cell characteristics in HCC . Changes in adhesion capacity and the increased expression of known important mediators of mechanotransduction such as integrins in human HCC tumour tissue coincide with an upregulation of integrin-associated focal adhesions (FAK) in the surrounding parenchyma. Furthermore, a positive correlation was found between ECM mechanical stiffness and integrin B1 expression and was correlated with Edmondson pathological grade, encapsulation, metastasis and HBV infection . Many studies have investigated the effect of changing transmembrane receptors/mechanical regulators-sensors on HCC cell migration, invasion and improved the knowledge on mechanistical insights into the complex interaction between a changed ECM and the intracellular signalling pathways in stroma cells that will favour HCC development and progression .
Do differences in aetiology affect changes in extracellular matrix composition during hepatocellular carcinoma initiation and progression?
One might ask the question whether the ECM composition differs between different aetiologies and if so whether HCC progresses only by the overall changes in ECM composition or very oversimplified that just one ECM compound need to be quantitatively present at a sufficient cut-off level to stimulate HCC initiation/growth. Previous immunohistochemical studies indicated that the distribution and staining patterns of ECM components such as distribution of fibronectin, laminin and collagen IV in HCC reflect the differentiation of the tumour, with differentiated tumours showing a relatively intact basement membrane whereas poorly differentiated tumours are marked with a sharply defective basement membrane composition . Similar findings were demonstrated when comparing small vs. large liver cell dysplasia indicating that liver cell dysplasia was characterized by an abnormal tenascin and type IV collagen expression i.e. small vs. large liver cell dysplasia thus reflecting the defective ECM pattern previously observed in HCC . Fang et al. have combined conventional immunohistochemistry and quantum dot-based multiplex imaging to simultaneously detect the tumour cells and major tumour ECM such as collagen type IV, LOX and tumour angiogenesis components. The authors revealed that type IV collagen degradation and repatterning of the ECM is a continuous process that allows the HCC tumour cells to invade .
Very recently, it was Lai et al. which compared in a qualitative and quantitative manner possible changes in ECM and associated receptor proteomes by using two mouse models relevant to human HCC. The PDGF-C transgenic and phosphatase and tensin homologue null mice [57, 58] are two models with similar features in the disease progression of fibrosis and steatohepatitis towards HCC. In this study, liver tissues were collected at different disease stages in the two mouse models, and by employing mass-spectrometry-based profiling, the investigators characterized changes in the liver proteome occurring in fibrotic, steatotic tissue and tumour tissues. Twenty-six collagen-encoding genes were identified with the expression at the protein level for 16 collagens. Moreover, important modifications at the post-transcriptional protein level for six collagens and lysine hydroxylation modifications for 14 collagens were observed. Very interestingly, this approach identified new ECM and integrin components, proposed novel ECM–tumour cell networks that are common but also very specific depending on the mouse model used which is relevant for the classification, diagnosis, prevention or treatment of HCC . Overall from these studies, it is becoming clear that certain ECM components such as the fibril-forming collagens types I and III are predominant in abundance in HCC in comparison to the other collagens which goes along with an increased and extensive lysine hydroxylation modification. Consequently, the dynamic changes in ECM that occur during tumour initiation and progression affect the tumour and stromal cells and contribute to a continuous matrix remodelling and changes in ECM stiffening. Of note, the high increase in fibrillar collagens such as collagen type I and III observed in these HCC-related studies, are already the predominant collagens during the dynamic process of liver fibrogenesis as being used as serum markers for the assessment of liver fibrosis i.e. the enhanced liver fibrosis test (ELF) (60). This further emphasizes the use of antifibrogenic compounds in parallel with anticancer therapy. ECM components are not the only elements affecting HCC initiation and progression since a vast amount of evidence show the existence of tumour cell-derived secreted extracellular membrane vesicles, also called exosomes, oncosomes or shed microvesicles, as carriers containing multifaceted molecular information such as signalling proteins and genetic material, with high pleiotropic biological effects implicated in physiological and pathological events including tumour development and metastasis . Besides these extracellular membrane vesicles which contribute to the ECM structure, a group of structurally diverse, ECM-associated glycoproteins, secreted by tumour and adjacent stromal cells, the so-called matricellular proteins, gain slowly interest in the process of liver tumour development and in the priming of the metastatic niche [62-64].
Cross-talk between tumour cells and the principal producing extracellular matrix cells: hepatic stellate cells
Hepatic stellate cells are liver sinusoidal resident vitamin A-storing cells and are considered as the most relevant profibrogenic cell type operating in chronic liver diseases [65-67]. During the process of liver injury, these cells undergo phenotypic transformation from ‘quiescent’ cells into ‘activated’ cells characterized by proliferation, contractility, increased synthesis and secretion of ECM, altered matrix protease activity and promitogenic cytokines. The HSC are marked by an increased secretion/responsiveness towards several soluble mediators which are all features of profibrogenic and proinflammatory cells (extensively reviewed elsewhere, [40, 66, 68-71]).
As mentioned before, several in-depth studies have shown that during the process of chronic liver disease changes in specific protein families of ECM such as collagens, laminins and integrins give rise to the formation of new and specific networks with their specific ligands and receptors. These events add up to the complexity of the microenvironment that forms a support for the increased intrinsic communication between the tumour cells and stromal cells through paracrine- and endocrine secretion of growth factors and exosomes, matricellular proteins and vice versa, this stimulates stromal cells to prepare the ‘ideal’ microenvironment to promote tumour growth.
One major player in the formation of the ideal tumour cell microenvironment is the activated HSC. Already activated by the initial, chronic stimulus, HSC express and secrete higher levels of collagens (specifically fibrillar collagen type I, II and collagen type III), forming thick and highly cross-linked collagen bundles. The cells develop a contractile apparatus, therefore gaining the ability to infiltrate the stroma of liver tumours, to localize around tumour sinusoids, fibrous septa and capsules. This indicates that besides playing a key role in fibrosis, HSC are of equal importance during HCC development and progression. Several elegant studies showed that coculturing of hepatocytes and LX2 cells (a spontaneous immortalized human HSC cell line ), results in a bidirectional cross-talk with LX2 cells promoting HCC proliferation, migration and inducing an inflammatory reaction [73, 74]. Simultaneous in vivo implantation of human HSC and HCC cells subcutaneously in nude mice promotes tumour growth, invasiveness and inhibits necrosis . Several studies have shown that the transforming growth factor-β/PDGF paracrine axis is crucial during the tumour-stroma cross-talk [14, 74] with the existence of a tumour-stroma gene signature that reveals a strong prognostic capacity for immune responses, ECM remodelling mechanism and angiogenic activity . The proangiogenic role of activated HSC during fibrosis and during new microvasculature in tumour formation plays a key role that further exacerbates the disease and contributes to the overall formation and dynamic reorganization of the tumour and stromal cells, and the ECM microenvironment [76, 77].
One way of investigating the effect of HSC on different cancer cell signalling pathways and functions is exposing the cancer cells to the conditioned medium (CM) collected from activated HSC being cultured for 24/48 h in serum-free medium [75, 78-80]. This CM contains cytokines, growth factors, ECM and soluble factors produced and secreted by the HSC. Investigating the content of the CM will identify possible key players and their function in the interaction between HSC and HCC. Still, one important aspect is the full characterization and the comparison of the CM from in vitro-activated HSC obtained from patients having different aetiology liver diseases. This would further define whether or not different secreted key players are important depending on the aetiology and HCC development. Indeed, the concept of using the secreted proteins to investigate important tumour stromal signalling pathways or to identify possible tumour-specific biomarkers has been further shown by Srisomsap et al. By analysing and identifying the proteins secreted by cholangiocarcinoma cells and HCC cells, the authors have identified lipocalin 2 as a specific marker in human cholangiocarcinoma . The screening of the secretome of four different HCC cell lines showed that each cell line had remarkably specific clusters of secreted proteins, ranging from metabolism, cytoskeleton/mobility, protein synthesis and degradation, transport binding proteins, DNA replication, gene regulation, cell cycle and signalling transduction pathways depending on which HCC cell line was investigated . Therefore, this could be a possible important contributing factor to the heterogeneity of HCC.
Overall because of the complexity of HCC, in vivo and in vitro mechanistic evidence is still not well documented. Limitations in these studies or major obstacles of being translational towards clinical studies is the usage of cell lines vs. primary isolated HSC, usage of nude mouse models, and tumour formation by dorsal subcutaneous injections without respecting the normal immunological and microenvironment of HCC i.e. the liver.
The relevance of in vitro-activated hepatic stellate cells vs. in vivo-activated hepatic stellate cells and ‘deactivated’ – hepatic stellate cells in hepatocellular carcinoma
For more than two decades, investigators isolated human and rodent hepatic stellate cells to study the underlying primary molecular signalling pathways of fibrogenesis. The reason to use the in vitro model of HSC activation was because the primary quiescent HSC, representing the normal HSC found in a healthy liver, spontaneously ‘transdifferentiate’ into an activated myofibroblast-like cell when cultured on plastic. This in vitro model is well established to investigate and analyse the importance of proteins and genes during fibrogenesis. Even more, as we and other investigators have demonstrated this set-up of experiments is useful to investigate and compare the possible antifibrogenic effects of new compounds between quiescent HSC, transdifferentiated HSC and activated HSC [71, 82] which was extrapolated many times into in vivo models . Recently, De Minicis et al., using a gene expression microarrays analysis approach have determined that there are clear differences between in vitro-activated HSC and in vivo-activated HSC derived from two different models of HSC activation [carbon tetrachloride (CCl4) injection and bile duct ligation (BDL) model]. Thus, these findings favour the use of in vivo-activated HSC isolated from a fibrotic liver over in vitro-activated HSC , with in vivo-activated HSC reflecting more the specific microenvironment of HSC in the fibrotic liver. This model has been adapted by other investigators wishing to explore, in a more detailed manner, the interplay of HSC and their original microenvironment [85, 86]. Indeed, this concept has gained more attention and has been used to compare the behaviour of in vitro-activated with in vivo-activated HSC in different animal models/chronic diseases. Nevertheless, the classical set-up of using quiescent HSC isolated from normal, healthy liver is not completely forgotten for several reasons including its use to identify molecular mechanisms and possible changes between quiescent HSC and activated HSC between in vitro- and in vivo-activated HSC and/or to exclude the effect of a possible antifibrogenic compound on quiescent HSC or to determine the specific target cell i.e. quiescent vs. activated HSC [87-89].
The removal of the aetiological source of chronic injury in patients and in rodent models coincides with liver fibrosis regression [90-92]. Therefore, investigators have studied, more in depth, the possible consequences and fate of the HSC during chronic liver diseases. Recent elegant studies have shown for the first time that reversal of activated HSC towards a more quiescent phenotype, the so-called ‘inactivated or deactivated’ HSC, contributes to the termination of liver fibrogenesis [93, 94]. To do so, Troeger et al. have used single-cell quantitative polymerase chain reaction and genetic tracking in transgenic mice. Vimentin, an established marker of HSC activation was used in a tamoxifen-inducible Vimentin-CreER model and VIM-mGFP was measured and traced by genetic tracking. A six-fold to 16-fold induction of vimentin in liver and in isolated HSC was observed in mice after CCl4 injections. These data indicated that HSC activation occurs at an early time-point and that activated HSC gain the ability to migrate as differences in localization were observed i.e. peri-sinusoidal towards fibrotic septa. Furthermore, after cessation of liver injury, 40–45% of myofibroblasts/HSC in liver sections and HSC isolations remained in a ‘priming’ state i.e. between quiescence and activated HSC and showed a higher sensitivity towards fibrogenic stimuli. Of note, in this study the possibility of mesenchymal–epithelial transition was excluded since hepatocytes or cholangiocytes were not fluorescent marked after liver injury . Similar and additional data were obtained in another study, in which different animal models and various genetically labelled markers were used . Kiselleva et al. have demonstrated that (i) regression of liver fibrosis coincide with activated HSC/myofibroblasts (α-SMA + Coll + cells) undergoing apoptosis and (ii) activated HSC are the major source of liver CCl4-induced myofibroblast population (Vitamin A + Coll+). Furthermore, in vitro and in vivo data demonstrated that the so-called ‘inactivated HSC’ have a similar phenotype as the quiescent HSC i.e. inactivated HSC express desmin, glial fibrillary acidic protein and synemin, but lack α-SMA expression. These cells showed an increased response to a second fibrogenic stimulus in comparison to quiescent HSC i.e. being primed. Moreover, inactivated HSC isolated after 1 month of recovery, followed by adoptive transfer into a liver exposed to CCl4 injury, showed to be fully integrated into the liver scar tissue and this is in contrast to quiescent HSC . These in vivo studies increase our knowledge about the role of HSC during liver fibrosis and especially fibrosis regression. These data indirectly might indicate the importance of targeting not only activated HSC but also genes to induce inactivation in stromal HSC as well HCC during anticancer therapy. Near future will reveal the participation of the different HSC phenotypes during the process of HCC formation. Several questions need to be answered such as (i) presence and importance of the inactivated/deactivated HSC during the tumour stromal cross-talk vs. in vivo-activated HSC and (ii) whether this population of inactivated/deactivated HSC promotes tumour development, progression and whether these cells should be targeted. Overall, these studies can lead to new objectives and to a possible new era in the search for antifibrogenic compounds to be used in combination with anticancer therapy to treat HCC. To date, no efficient antifibrogenic therapy exists .
Hepatic stellate cells senescence in hepatocellular carcinoma
Cellular senescence is a stable arrest of proliferation initiated by a variety of molecular triggers and has been viewed as favourable to correct DNA damage and to arrest abnormal excessive cell cycle into a permanent cell cycle arrest. Schnabl et al. have employed microarray analysis to compare gene expression in human culture-activated HSC vs. human senescent HSC and found a different expression in cell cycle and proliferation-related genes. Not only cell proliferation was changed by senescence but senescent human HSC showed to be less fibrogenic, contain an increased pro-inflammatory phenotype and underwent apoptosis as a consequence . Interestingly, matricellular protein cysteine-rich protein 61 (CCN1), a matricellular protein, accumulates in hepatocytes of human cirrhotic livers and when secreted triggers senescence in activated HSC and portal fibroblasts. In this way, and by engaging integrin alpha6beta1, several reactive oxygen species are formed thereby inducing an antifibrotic gene expression . These data indicated that senescence of activated HSC might help the reversal of liver fibrosis. Moreover, Krizhanovsky et al. have confirmed these findings and demonstrated that senescence of activated HSC limits liver fibrosis in a CCl4-induced liver fibrosis model. Senescent positive cells were located along the fibrotic scar in human and mouse livers and were positively stained for p53 and HMGA1 (high-mobility group A1) (senescence markers) in combination with HSC markers desmin and α-SMA. Whereas all α-SMA positive cells accumulated SA-beta-gal activity which indicated the participation of all myofibroblast cell types, but no other liver cells. Interestingly, immune cells were found in the proximity of the senescent HSC in vivo and authors showed that the Natural Killer cells (NK cells) selectively clear the senescent HSC . The NK-induced clearance of senescent HSC is because of granule exocytosis and not to death receptor-mediated apoptosis as Sagiv et al. later reported . Klein et al. have shown that in BDL-induced fibrotic rats treated with atorvastatin, a statin previously shown to be beneficial for the treatment of hepatic fibrosis and portal hypertension, induced senescence in activated hepatic stellate cells. More importantly, senescence was only induced when rats were treated after 5 weeks of BDL and not 3 weeks. Senescence markers (p21, beta-galactosidase) in combination with mouse HSC marker desmin and myofibroblasts marker α-SMA showed that the higher rate of senescent cells detected after 5 weeks BDL vs. 3 weeks BDL was because of the fact that livers have more advanced fibrosis and, consequently, more myofibroblasts. This indicates that the induction of senescence in activated HSC and myofibroblasts by chemical treatment such as atorvastatin is favourable during full established fibrogenesis .
Previously cellular senescence was thought to be a mechanism, a barrier, to tumourigenesis by inducing stable cell cycle arrest. More recently, it has been shown that senescent cells can produce pro-inflammatory signalling proteins that promote tumour growth, the so-called senescence-associated secretory phenotype. Yoshimoto et al. showed the importance of senescence in activated HSC during obesity. HCC was induced in p21-p-luciferase (p21Waf1/Cip1) bearing neonatal mice, treated with 7,12-dimethylbenz(a)anthracene, a chemical carcinogen, in combination with a high-fat diet. This model induced HCC without liver fibrosis indicating that the origin of senescent HSC was not caused by liver fibrosis. Activated HSC, detected by α-SMA and desmin positivity, in the vicinity of cancer cells, were positive for p21Waf1/Cip1 and p16INK4a (cyclin-dependent kinase inhibitor), but lack proliferation marker such as Ki67 and hepatocyte growth factor (senescence markers) with an increased expression of IL6, Gro-α and CXCL9 .
Overall when the senescence machinery is intact, the senescence of HSC or hepatic myofibroblasts in general, restricts progression of fibrosis, whereas during chronic liver disease when the senescence machinery becomes ‘hijacked’, it triggers proliferation and transformation of hepatocytes, thus helping/promoting tumour formation and affecting the tumour microenvironment.
Despite major improvements, many current HCC therapies have potential harmful side effects and are marked by a low overall survival rate. Liver cancer progresses in a large percentage of cases during the clinical course of chronic fibro-inflammatory liver diseases leading to cirrhosis. Therefore, HCC development is regarded as the result of different environmental risk factors each involving different genetic, epigenetic- and chromosomal alterations and gene mutations. Recent investigations further demonstrated a high impact of the dynamic stromal tumour microenvironment on the cancer cells, indicating that in the future liver cancer research should switch its focus more on investigating the relationship between tumour microenvironment and tumour development. Identifying these altered mechanisms regulating the bidirectional cross-talk between HCC and HCC microenvironment components may improve our knowledge and is crucial for the design of novel molecular targets.
Several issues should be taken into consideration such as (i) resistance to treatment which remains the major challenge for targeted therapy, (ii) developing a new strategy to combine chemotherapy regimens with different agents aimed at specifically targeting not only tumour cells but also profibrotic stromal cells, (iii) the different subgroups of HCC should be further classified according to the genomics/proteomics and possible secretomics which will reveal new and potential new targets. Identifying new targets and their agents will lead to a more personalized therapy.
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