Imaging the recruitment of cancer-associated fibroblasts by liver-metastatic colon cancer

The tumor microenvironment (TME) is critical for tumor growth and progression. However, the formation of the TME is largely unknown. This report demonstrates a color-coded imaging model in which the development of the TME can be visualized. In order to image the TME, a green fluorescent protein (GFP)-expressing mouse was used as the host which expresses GFP in all organs but not the parenchymal cells of the liver. Non-colored HCT-116 human colon cancer cells were injected in the spleen of GFP nude mice which led to the formation of experimental liver metastasis. TME formation resulting from the liver metastasis was observed using the Olympus OV100 small animal fluorescence imaging system. HCT-116 cells formed tumor colonies in the liver 28 days after cell transplantation to the spleen. GFP-expressing host cells were recruited by the metastatic tumors as visualized by fluorescence imaging. A desmin positive area increased around and within the liver metastasis over time, suggesting cancer-associated fibroblasts (CAFs) were recruited by the liver metastasis which have a role in tumor progression. The color-coded model of the TME enables its formation to be visualized at the cellular level in vivo, in real-time. This imaging model of the TME should lead to new visual targets in the TME.     

Stimulation of liver functions in hierarchical co-culture of bone marrow cells and hepatocytes

A hierarchial co-culture, in which rat hepatocytes and non-parenchymal liver cells (NPLCs) were separated by a collagen layer and which was designed to mimic the in vivo microenvironment, was carried out with the aim of developing a module for bio-artificial liver support. Compared with a monolayer co-culture and hepatocytes cultured alone in a monolayer, higher urea synthesis activity was maintained for 6 d in the hierarchical co-culture. When a rat hepatoma cell line H4-II-E-C3, which retains the induction of tyrosine aminotransferase (TAT), was co-cultured in a monolayer with NPLCs, dose-dependent stimulation of TAT induction was observed. In a hierarchical co-culture, NPLCs further stimulated TAT induction in H4-II-E-C3 cells. Since peritoneal macrophages could stimulate TAT induction in hepatocytes in both monolayer and hierarchical co-cultures, bone marrow cells, which can proliferate and differentiate into macrophages in vitro, were investigated as a possible substitute for NPLCs. Bone marrow cells isolated from rat femurs were cultivated in the presence of IL-3 and macrophage colony-stimulating factor (M-CSF), and co-cultured with hepatocytes. Urea synthesis and TAT induction of hepatocytes were stimulated in the co-culture. The co-culture of bone marrow and H4-II-E-C3 cells, both of which have proliferation ability in vitro, was also shown to be effective in stimulating liver functions. The hierarchical configuration, in which two cell types can communicate with the soluble factor(s) through a collagen layer, was found to be more effective than a monolayer in long-term co-culture.     

Content of dolichol and retinol in isolated rat non-parenchymal liver cells

The liver sinusoids, that are considered as a functional unit, harbour four types of sinusoidal cells (Ito, Kupffer, endothelial and pit cells). Dolichol content has been determined in many tissues and subcellular compartments, alteration has been reported in many types of liver injury, but until now no data are available on its content in every type of sinusoidal non-parenchymal liver cells. Dolichol and retinol metabolism might intersect in their traffic in biological membranes. Intercellular as well as intracellular exchange of retinoids is an essential element of important processes occurring in liver cells. It has been suggested that the role of dolichol, besides being a carrier of oligosaccharides in the biosynthesis of N-linked glycoproteins, may be to modify membrane fluidity and permeability, and facilitate fusion of membranes. Dolichol in the membrane is intercalated between the two halves of the phospholipid bilayer, but its exact disposition is not known and the movement and distribution of retinoid in membranes may vary with the geometry of the membranes. Therefore the aim of this study is to obtain a global understanding of the sinusoidal system regarding dolichol and retinol content in each type of isolated rat liver sinusoidal cell, in normal conditions and after vitamin A administration. The information that can be drawn from the present results is that with normal vitamin A status of the animal, the dolichol content is almost uniform in all liver cells. After vitamin A supplementation, a great increase of dolichol, together with the known increase of retinol, can be measured only in a subpopulation of the Ito cells, the Ito-1 subfraction. Therefore in the cells that are present in the hepatic sinusoid, different pools of dolichol may have separate functions. Because retinol traffic among cells, membranes and plasma still remains to be fully understood, roles of dolichol in the exchange of vitamin A among sinusoidal liver cells are discussed. © 1998 John Wiley & Sons, Ltd.     

Pluripotent plasticity of stem cells and liver repopulation

Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin of hepatic and/or extra‐hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self‐repair and replace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about a year. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, and sinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety of clinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential to provide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio‐artificial liver‐assisted devices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver. Copyright © 2010 John Wiley & Sons, Ltd.     

A delicate balance: TGF-beta and the tumor microenvironment

The activated form of TGF-beta is a known regulator of epithelial cell autonomous tumor initiation, progression, and metastasis. Recent studies have also indicated that TGF-beta mediates interactions between cancer cells and their local tumor microenvironment. Specifically, the loss of TGF-beta signaling in stromal components including fibroblasts and T-cells can result in an "activated" microenvironment that supports and even initiates transformation of adjacent epithelial cells. TGF-beta signaling in cancer can be regulated through mechanisms involving ligand activation and expression of essential components within the pathway including the receptors and downstream effectors. TGF-beta signaling in the tumor microenvironment significantly impacts carcinoma initiation, progression, and metastasis via epithelial cell autonomous and interdependent stromal-epithelial interactions in vivo.     

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.     

肝干细胞的几个生物学问题

对肝干细胞的认识,自卵圆细胞发现以来已经经历了五十多年。期间许多的研究提示和证明肝干细胞具有很好的生物医学应用前景。近年来,肝干细胞的概念已被广泛接受和认可,肝干细胞的生物学特性及其医学应用的探索也成为了研究的热点。目前在该领域中,主要以肝干细胞存在的位置、与其所处微环境的相互作用、肝干细胞的分子标记以及其分化特性等方面备受关注。肝干细胞的研究为肝脏疾病及其他相关疾病的治疗带来了新希望。然而,肝干细胞研究的过程中仍存在很多未解的问题,因此将肝干细胞研究引入临床应用则还需要更加深入的探索。     

Intercellular communication among liver cells in the perisinusoidal space of the injured liver: Pathophysiology and therapeutic directions

Hepatic stellate cells (HSCs) in the perisinusoidal space are surrounded by hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and other resident immune cells. In the normal liver, HSCs communicate with these cells to maintain normal liver functions. However, after chronic liver injury, injured hepatocytes release several proinflammatory mediators, reactive oxygen species, and damage-associated molecular patterns into the perisinusoidal space. Consequently, such alteration activates quiescent HSCs to acquire a myofibroblast-like phenotype and express high amounts of transforming growth factor-β1, angiopoietins, vascular endothelial growth factors, interleukins 6 and 8, fibril forming collagens, laminin, and E-cadherin. These phenotypic and functional transdifferentiation lead to hepatic fibrosis with a typical abnormal extracellular matrix synthesis and disorganization of the perisinusoidal space of the injured liver. Those changes provide a favorable environment that regulates tumor cell proliferation, migration, adhesion, and survival in the perisinusoidal space. Such tumor cells by releasing transforming growth factor-β1 and other cytokines, will, in turn, activate and deeply interact with HSCs via a bidirectional loop. Furthermore, hepatocellular carcinoma-derived mediators convert HSCs and macrophages into protumorigenic cell populations. Thus, the perisinusoidal space serves as a critical hub for activating HSCs and their interactions with other cell types, which cause a variety of liver diseases such as hepatic inflammation, fibrosis, cirrhosis, and their complications, such as portal hypertension and hepatocellular carcinoma. Therefore, targeting the crosstalk between activated HSCs and tumor cells/immune cells in the tumor microenvironment may also support a promising therapeutic strategy.     

Biophysical interactions between components of the tumor microenvironment promote metastasis

During metastasis, tumor cells need to adapt to their dynamic microenvironment and modify their mechanical properties in response to both chemical and mechanical stimulation. Physical interactions occur between cancer cells and the surrounding matrix including cell movements and cell shape alterations through the process of mechanotransduction. The latter describes the translation of external mechanical cues into intracellular biochemical signaling. Reorganization of both the cytoskeleton and the extracellular matrix (ECM) plays a critical role in these spreading steps. Migrating tumor cells show increased motility in order to cross the tumor microenvironment, migrate through ECM and reach the bloodstream to the metastatic site. There are specific factors affecting these processes, as well as the survival of circulating tumor cells (CTC) in the blood flow until they finally invade the secondary tissue to form metastasis. This review aims to study the mechanisms of metastasis from a biomechanical perspective and investigate cell migration, with a focus on the alterations in the cytoskeleton through this journey and the effect of biologic fluids on metastasis. Understanding of the biophysical mechanisms that promote tumor metastasis may contribute successful therapeutic approaches in the fight against cancer.     

Construction of pancreas–muscle–liver microphysiological system (MPS) for reproducing glucose metabolism

Although in vitro models are widely accepted experimental platforms, their physiological relevance is often severely limited. The limitation of current in vitro models is strongly manifested in case of diseases where multiple organs are involved, such as diabetes and metabolic syndrome. Microphysiological systems (MPS), also known as organ-on-a-chip technology, enable a closer approximation of the human organs and tissues, by recreating the tissue microenvironment. Multiorgan MPS, also known as multiorgan-on-a-chip or body-on-a-chip, offer the possibility of reproducing interactions between organs by connecting different organ modules. Here, we designed a three-organ MPS consisting of pancreas, muscle, and liver, to recapitulate glucose metabolism and homeostasis by constructing a mathematical model of glucose metabolism, based on experimental measurement of glucose uptake by muscle cells and insulin secretion by pancreas cells. A mathematical model was used to modify the MPS to improve the physiological relevance, and by adding the liver model in the mathematical model, physiological realistic glucose and insulin profiles were obtained. Our study may provide a methodological framework for developing multiorgan MPS for recapitulating the complex interaction between multiple organs.     

Claudin-2 promotes breast cancer liver metastasis by facilitating tumor cell interactions with hepatocytes

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.     

Liver transplantation of hepatic stem cells: potential use for treating liver diseases

Hepatic stem cells are an alternative means for repopulating the liver after various injuries instead of liver transplantation. The first step before use is to select stem cells that will be good candidates. This review discusses the different candidates including fetal progenitor bipotential hepatic stem cells; adult hepatocytes, which can be considered as unipotential committed stem cells; and oval cells, a type of nonparenchymal pluripotential hepatic stem cell. The advantages and disadvantages of each type of cell are discussed and several other possible alternatives, such as the use of hematopoietic stem cells are analyzed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bone marrow to liver: the blood of Prometheus

The existence of hepatic stem or progenitor cells has been controversial for decades, though it was presumed that if such cells existed, they would lie within the liver. There is now consensus, however, that not only do facultative hepatic stem cells exist within the liver, but also that cells from extra-hepatic sites, in particular the bone marrow, can contribute to hepatocyte and cholangiocyte regeneration. Despite confidence that engraftment of marrow cells in the liver occurs, the mechanistic details of this process remain poorly understood. Moreover, the physiological importance and therapeutic utility of this phenomenon remains controversial.     

A novel mouse model for liver metastasis of prostate cancer reveals dynamic tumour‐immune cell communication

ObjectivesIn contrast to extensive studies on bone metastasis in advanced prostate cancer (PCa), liver metastasis has been under‐researched so far. In order to decipher molecular and cellular mechanisms underpinning liver metastasis of advanced PCa, we develop a rapid and immune sufficient mouse model for liver metastasis of PCa via orthotopic injection of organoids from PbCre⁺; rb1^f/f;p53^f/f mice.Materials and MethodsPbCre⁺;rb1^f/f;p53^f/f and PbCre⁺;pten^f/f;p53^f/f mice were used to generate PCa organoid cultures in vitro. Immune sufficient liver metastasis models were established via orthotopic transplantation of organoids into the prostate of C57BL/6 mice. Immunofluorescent and immunohistochemical staining were performed to characterize the lineage profile in primary tumour and organoid‐derived tumour (ODT). The growth of niche‐labelling reporter infected ODT can be visualized by bioluminescent imaging system. Immune cells that communicated with tumour cells in the liver metastatic niche were determined by flow cytometry.ResultsA PCa liver metastasis model with full penetrance is established in immune‐intact mouse. This model reconstitutes the histological and lineage features of original tumours and reveals dynamic tumour‐immune cell communication in liver metastatic foci. Our results suggest that a lack of CD8⁺ T cell and an enrichment of CD163⁺ M2‐like macrophage as well as PD1⁺CD4⁺ T cell contribute to an immuno‐suppressive microenvironment of PCa liver metastasis.ConclusionsOur model can be served as a reliable tool for analysis of the molecular pathogenesis and tumour‐immune cell crosstalk in liver metastasis of PCa, and might be used as a valuable in vivo model for therapy development.     

Crosstalk between cancer‐associated fibroblasts and immune cells in cancer

Multiple studies have shown that cancer‐associated fibroblasts (CAFs) play an important role in tumour progression, including carcinogenesis, invasion, metastasis and the chemoresistance of cancer cells. Immune cells, including macrophages, natural killer cells, dendritic cells and T cells, play a dual role in the tumour microenvironment. Although increasing research has focused on studying interactions between distinct cells in the tumour microenvironment, the complex relationships between CAFs and immune cells remain unclear and need further study. Here, we summarize our current understanding of crosstalk between CAFs and immune cells, which may help clarify their diagnostic and therapeutic value in tumour progression.     

The organ microenvironment and cancer metastasis

Primary neoplasms are biologically heterogeneous and the process of metastasis consists of a series of sequential, selective steps that few cells can complete. The outcome of cancer metastasis depends on multiple interactions between metastatic cells and homeostatic mechanisms that are unique to one or another organ microenvironment. The specific organ microenvironment determines the extent of cancer cell proliferation, angiogenesis, invasion, and survival. Therapy of metastasis should therefore be targeted not only against tumor cells, but also against the host factors that contribute to and support the progressive growth and survival of metastatic cancer cells.     

间充质干细胞的来源及其对肝脏损伤及修复的研究进展

全球终末期肝病、肝衰竭的发病率和死亡率逐年升高,且目前肝移植是唯一疗效确切的治疗选择,但是,肝移植的使用受到肝源供体严重不足,长期存活率低,医疗费用昂贵等缺点使得原位肝移植的应用受限,绝大多数患者无法受益。为了克服肝脏器官短缺,干细胞替代治疗策略逐渐成为另一个肝病治疗的重要选择,干细胞治疗,特别是间充质干细胞（MSC）提供了一个新的肝病治疗选择。MSC是一群贴壁生长的成纤维细胞样细胞,由于MSC能够分化为多种类型的细胞,能够产生多种的细胞因子和生长因子,具有造血支持和免疫调节和抗炎功能,MSC被认为在再生医学领域具有重大的科学和实用价值。另外,由于MSC应用于治疗实验性肝损伤能明显提高动物存活率,明显改善肝功能。此外,一些临床前研究和临床研究也表明MSC对肝损伤性疾病具有显著地疗效。因此MSC在损伤性和退行性肝脏疾病的治疗具有广阔的应用前景。本文综述了MSC在肝损伤疾病治疗应用的进展,并对MSC在肝病治疗中的应用前景进行了展望。