Multiplexed Imaging Mass Cytometry Analysis Characterizes the Vascular Niche in Pancreatic Cancer.

Oncogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) is driven by complex interactions between the neoplastic component and the tumor microenvironment (TME), which includes immune, stromal, and parenchymal cells. In particular, most PDACs are characterized by a hypovascular and hypoxic environment that alters tumor cell behavior and limits the efficacy of chemotherapy and immunotherapy. Characterization of the spatial features of the vascular niche could advance our understanding of inter- and intra-tumoral heterogeneity in PDAC. Here, we investigated the vascular microenvironment of PDAC by applying imaging mass cytometry using a 26-antibody panel on 35 regions of interest (ROIs) across 9 patients, capturing over 140,000 single cells. The approach distinguished major cell types, including multiple populations of lymphoid and myeloid cells, endocrine cells, ductal cells, stromal cells, and endothelial cells. Evaluation of cellular neighborhoods identified 10 distinct spatial domains, including multiple immune and tumor-enriched environments as well as the vascular niche. Focused analysis revealed differential interactions between immune populations and the vasculature and identified distinct spatial domains wherein tumor cell proliferation occurs. Importantly, the vascular niche was closely associated with a population of CD44-expressing macrophages enriched for a pro-angiogenic gene signature. Together, this study provides insights into the spatial heterogeneity of PDAC and suggests a role for CD44-expressing macrophages in shaping the vascular niche.

Cellular reprogramming in vivo initiated by SOX4 pioneer factor activity.

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that SOX4 is sufficient to initiate hepatobiliary metaplasia in the adult mouse liver, closely mimicking metaplasia initiated by toxic damage to the liver. In lineage-traced cells, we assessed the timing of SOX4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, SOX4 directly binds to and closes hepatocyte regulatory sequences via an overlapping motif with HNF4A, a hepatocyte master regulatory transcription factor. Subsequently, SOX4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy in pancreatic ductal adenocarcinoma.

Acquired resistance to immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we find that resistance is reproducibly associated with an epithelial-to-mesenchymal transition (EMT), with EMT-transcription factors ZEB1 and SNAIL functioning as master genetic and epigenetic regulators of this effect. Acquired resistance in this model is not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, resistance is due to a tumor cell-intrinsic defect in T-cell killing. Molecularly, EMT leads to the epigenetic and transcriptional silencing of interferon regulatory factor 6 (Irf6), rendering tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings indicate that acquired resistance to immunotherapy may be mediated by programs distinct from those governing primary resistance, including plasticity programs that render tumor cells impervious to T-cell killing.

Plasticity-induced repression of Irf6 underlies acquired resistance to cancer immunotherapy.

Acquired resistance to immune checkpoint immunotherapy remains a critical yet incompletely understood biological mechanism. Here, using a mouse model of pancreatic ductal adenocarcinoma (PDAC) to study tumor relapse following immunotherapy-induced responses, we found that tumors underwent an epithelial-to-mesenchymal transition (EMT) that resulted in reduced sensitivity to T cell-mediated killing. EMT-transcription factors (EMT-TFs) ZEB1 and SNAIL function as master genetic and epigenetic regulators of this tumor-intrinsic effect. Acquired resistance was not due to immunosuppression in the tumor immune microenvironment, disruptions in the antigen presentation machinery, or altered expression of immune checkpoints. Rather, EMT was associated with epigenetic and transcriptional silencing of interferon regulatory factor 6 (Irf6), which renders tumor cells less sensitive to the pro-apoptotic effects of TNF-α. These findings show how resistance to immunotherapy in PDAC can be acquired through plasticity programs that render tumor cells impervious to T cell killing.

Evidence for in vitro extensive proliferation of adult hepatocytes and biliary epithelial cells.

Over the last several years, a method has emerged that endows adult hepatocytes with in vitro proliferative capacity, producing chemically induced liver progenitors (CLiPs). However, there is a growing controversy regarding the origin of these cells. Here, we provide lineage tracing-based evidence that adult hepatocytes acquire proliferative capacity in vitro using rat and mouse models. Unexpectedly, we also found that the CLiP method allows biliary epithelial cells to acquire extensive proliferative capacity. Interestingly, after long-term culture, hepatocyte-derived cells (hepCLiPs) and biliary epithelial cell-derived cells (bilCLiPs) become similar in their gene expression patterns, and they both exhibit differentiation capacity to form hepatocyte-like cells. Finally, we provide evidence that hepCLiPs can repopulate injured mouse livers, reinforcing our earlier argument that CLiPs can be a cell source for liver regenerative medicine. This study advances our understanding of the origin of CLiPs and motivates the application of this technique in liver regenerative medicine.

Physiological reprogramming in vivo mediated by Sox4 pioneer factor activity.

Tissue damage elicits cell fate switching through a process called metaplasia, but how the starting cell fate is silenced and the new cell fate is activated has not been investigated in animals. In cell culture, pioneer transcription factors mediate "reprogramming" by opening new chromatin sites for expression that can attract transcription factors from the starting cell's enhancers. Here we report that Sox4 is sufficient to initiate hepatobiliary metaplasia in the adult liver. In lineage-traced cells, we assessed the timing of Sox4-mediated opening of enhancer chromatin versus enhancer decommissioning. Initially, Sox4 directly binds to and closes hepatocyte regulatory sequences via a motif it overlaps with Hnf4a, a hepatocyte master regulator. Subsequently, Sox4 exerts pioneer factor activity to open biliary regulatory sequences. The results delineate a hierarchy by which gene networks become reprogrammed under physiological conditions, providing deeper insight into the basis for cell fate transitions in animals.

Rapid in vivo multiplexed editing (RIME) of the adult mouse liver.

BACKGROUND AND AIMS: Assessing mammalian gene function in vivo has traditionally relied on manipulation of the mouse genome in embryonic stem cells or perizygotic embryos. These approaches are time-consuming and require extensive breeding when simultaneous mutations in multiple genes is desired. The aim of this study is to introduce a rapid in vivo multiplexed editing (RIME) method and provide proof of concept of this system. APPROACH AND RESULTS: RIME, a system wherein CRISPR/caspase 9 technology, paired with adeno-associated viruses (AAVs), permits the inactivation of one or more genes in the adult mouse liver. The method is quick, requiring as little as 1 month from conceptualization to knockout, and highly efficient, enabling editing in >95% of target cells. To highlight its use, we used this system to inactivate, alone or in combination, genes with functions spanning metabolism, mitosis, mitochondrial maintenance, and cell proliferation. CONCLUSIONS: RIME enables the rapid, efficient, and inexpensive analysis of multiple genes in the mouse liver in vivo .

Bioengineering of a CLiP-derived tubular biliary-duct-like structure for bile transport in vitro.

The integration of a bile drainage structure into engineered liver tissues is an important issue in the advancement of liver regenerative medicine. Primary biliary cells, which play a vital role in bile metabolite accumulation, are challenging to obtain in vitro because of their low density in the liver. In contrast, large amounts of purified hepatocytes can be easily acquired from rodents. The in vitro chemically induced liver progenitors (CLiPs) from primary mature hepatocytes offer a platform to produce biliary cells abundantly. Here, we generated a functional CLiP-derived tubular bile duct-like structure using the chemical conversion technology. We obtained an integrated tubule-hepatocyte tissue via the direct coculture of hepatocytes on the established tubular biliary-duct-like structure. This integrated tubule-hepatocyte tissue was able to transport the bile, as quantified by the cholyl-lysyl-fluorescein assay, which was not observed in the un-cocultured structure or in the biliary cell monolayer. Furthermore, this in vitro integrated tubule-hepatocyte tissue exhibited an upregulation of hepatic marker genes. Together, these findings demonstrated the efficiency of the CLiP-derived tubular biliary-duct-like structures regarding the accumulation and transport of bile.

Dynamic Transcriptional and Epigenetic Changes Drive Cellular Plasticity in the Liver.

BACKGROUND AND AIMS: Following liver injury, a fraction of hepatocytes adopt features of biliary epithelial cells (BECs) in a process known as biliary reprogramming. The aim of this study was to elucidate the molecular events accompanying this dramatic shift in cellular identity. APPROACH AND RESULTS: We applied the techniques of bulk RNA-sequencing (RNA-seq), single-cell RNA-seq, and assay for transposase-accessible chromatin with high-throughput sequencing to define the epigenetic and transcriptional changes associated with biliary reprogramming. In addition, we examined the role of TGF-ß signaling by profiling cells undergoing reprogramming in mice with hepatocyte-specific deletion in the downstream TGF-ß signaling component mothers against decapentaplegic homolog 4 (Smad4). Biliary reprogramming followed a stereotyped pattern of altered gene expression consisting of robust induction of biliary genes and weaker repression of hepatocyte genes. These changes in gene expression were accompanied by corresponding modifications at the chromatin level. Although some reprogrammed cells had molecular features of "fully differentiated" BECs, most lacked some biliary characteristics and retained some hepatocyte characteristics. Surprisingly, single-cell analysis of Smad4 mutant mice revealed a dramatic increase in reprogramming. CONCLUSION: Hepatocytes undergo widespread chromatin and transcriptional changes during biliary reprogramming, resulting in epigenetic and gene expression profiles that are similar to, but distinct from, native BECs. Reprogramming involves a progressive accumulation of biliary molecular features without discrete intermediates. Paradoxically, canonical TGF-ß signaling through Smad4 appears to constrain biliary reprogramming, indicating that TGF-ß can either promote or inhibit biliary differentiation depending on which downstream components of the pathway are engaged. This work has implications for the formation of BECs and bile ducts in the adult liver.

Differentiation of chemically induced liver progenitor cells to cholangiocytes: Investigation of the optimal conditions.

Chemically induced liver progenitor (CLiP) cells, converted in vitro from mature hepatocytes, possess the bipotentiality to differentiate into both hepatocytes and cholangiocytes. Here, we aimed to investigate the optimal conditions for bile duct (BD) induction from rat CLiPs. A two-step induction protocol was used for the differentiation of cholangiocytes. We investigated the effects of passage number, preincubation times, Matrigel, and mouse embryonic fibroblast (MEF) feeder cells on the induction of cholangiocytes. Earlier passages of CLiPs were better for BD induction compared with stable CLiPs. Extending the preincubation time of CLiPs before induction delayed the formation of the BD. Matrigel provided cells with space to form three-dimensional (3D) structures, but the long-term use of Matrigel from the induction step did not benefit the differentiation of CLiPs to cholangiocytes. MEF feeder cells, through the Jag/Notch pathway, affected BD formation and function, as well as gene and protein expression. CLiPs were a good cell source for cholangiocyte differentiation under appropriate conditions and may offer a key vehicle for the study of cholangiopathies in vitro.

Transcriptomic Dissection of Hepatocyte Heterogeneity: Linking Ploidy, Zonation, and Stem/Progenitor Cell Characteristics.

BACKGROUND & AIMS: There is a long-standing debate regarding the biological significance of polyploidy in hepatocytes. Recent studies have provided increasing evidence that hepatocytes with different ploidy statuses behave differently in a context-dependent manner (eg, susceptibility to oncogenesis, regenerative ability after injury, and in vitro proliferative capacity). However, their overall transcriptomic differences in a physiological context is not known. METHODS: By using microarray transcriptome analysis, we investigated the heterogeneity of hepatocyte populations with different ploidy statuses. Moreover, by using single-cell quantitative reverse-transcription polymerase chain reaction (scPCR) analysis, we investigated the intrapopulational transcriptome heterogeneity of 2c and 4c hepatocytes. RESULTS: Microarray analysis showed that cell cycle-related genes were enriched in 8c hepatocytes, which is in line with the established notion that polyploidy is formed via cell division failure. Surprisingly, in contrast to the general consensus that 2c hepatocytes reside in the periportal region, in our bulk transcriptome and scPCR analyses, the 2c hepatocytes consistently showed pericentral hepatocyte-enriched characteristics. In addition, scPCR analysis identified a subpopulation within the 2c hepatocytes that co-express the liver progenitor cell markers Axin2, Prom1, and Lgr5, implying the potential biological relevance of this subpopulation. CONCLUSIONS: This study provides new insights into hepatocyte heterogeneity, namely 2c hepatocytes are preferentially localized to the pericentral region, and a subpopulation of 2c hepatocytes show liver progenitor cell-like features in terms of liver progenitor cell marker expression (Axin2, Prom1, and Lgr5).

Development of Bifunctional Three-Dimensional Cysts from Chemically Induced Liver Progenitors.

Chemically induced liver progenitors (CLiPs) have promising applications in liver regenerative medicine. Three-dimensional (3D) structures generated from liver progenitor cells possess wide applications in cell transplantation, disease model, and drug testing. Here, we report on the spontaneous formation of 3D cystic structures comprising maturing rat CLiPs on gelatin-coated dishes. Our 3D cysts contained Alb+/+CK19+/- and Ck19+/+Alb+/- cells. These cell types gradually diverged into specialized mature cells, as demonstrated by the expression of mature biliary markers (Cftr, Ae2, and Aqp1) and hepatic markers (Alb and Mrp2). The 3D cysts also expressed functional multidrug resistance protein 1 (Mdr1), as indicated by epithelial efflux of rhodamine. Furthermore, we observed bile canaliculi functions between hepatocytes and cholyl-lysyl-fluorescein extrusions, indicating that the functional characteristics of 3D cysts and active bile salt export pump (Bsep) transporters were intact. Thus, our study revealed a natural characteristic of rat CLiPs to spontaneously form 3D cystic structures accompanied with cell maturation in vitro, offering a platform for studies of liver development and drug screening.

Generation of human hepatic progenitor cells with regenerative and metabolic capacities from primary hepatocytes.

Hepatocytes are regarded as the only effective cell source for cell transplantation to treat liver diseases; however, their availability is limited due to a donor shortage. Thus, a novel cell source must be developed. We recently reported that mature rodent hepatocytes can be reprogrammed into progenitor-like cells with a repopulative capacity using small molecule inhibitors. Here, we demonstrate that hepatic progenitor cells can be obtained from human infant hepatocytes using the same strategy. These cells, named human chemically induced liver progenitors (hCLiPs), had a significant repopulative capacity in injured mouse livers following transplantation. hCLiPs redifferentiated into mature hepatocytes in vitro upon treatment with hepatic maturation-inducing factors. These redifferentiated cells exhibited cytochrome P450 (CYP) enzymatic activities in response to CYP-inducing molecules and these activities were comparable with those in primary human hepatocytes. These findings will facilitate liver cell transplantation therapy and drug discovery studies.

One of the most successful treatments for liver disease is transplanting a donor liver into a patient. But demands for donor livers far outstrips supply. A promising alternative could be, rather than replacing the whole organ, to transplant patients with individual liver cells called hepatocytes. These cells can then move into the liver, replace damaged cells, and help support the organ. However, hepatocytes are also in short supply, as despite the liver's amazing regenerative abilities, these cells struggle to divide outside of the body. Improving how these cells multiply, could therefore help more people receive hepatocyte transplants. In 2017, researchers found a way to convert mouse and rat hepatocytes into cells that could divide more rapidly using a cocktail of three small molecules. These 'chemically induced liver progenitors', or CLiPs for short, were able to mature into working hepatocytes and support injured mouse livers. But, discoveries made in rats and mice are not always applicable to humans. Now, Katsuda et al. ­ including some of the researchers involved in the 2017 work ­ have set out to investigate whether CLiPs can also be made from human cells, and if so, whether these cells can be used for hepatocyte transplantations. Using a similar cocktail of molecules, Katsuda et al. managed to convert infant human hepatocytes into CLiPs. As with the rodent cells, these human CLiPs were able to turn back into mature, working liver cells. When transplanted into mice with genetic liver diseases, the human CLiPs moved into the liver and became part of the organ. These transplanted cells were able to reconstruct the liver tissue of diseased mice, and in some cases, replaced more than 90% of the liver's damaged cells. Developing human CLiP technology could provide a new way to support people on the waiting list for liver transplantation. But there are some obstacles still to overcome. At present the technique only works with hepatocytes from infant donors. The next step is to improve the method so that it works with liver cells donated by adults.

Chemically Induced Liver Progenitors (CLiPs): A Novel Cell Source for Hepatocytes and Biliary Epithelial Cells.

Bipotent liver progenitor cells (LPCs) are promising cell sources for cell transplantation therapy in hepatic disorders as well as biliary dysfunctions. Using a cocktail of small molecules, we recently reported a novel approach to generate bipotent LPCs, named chemically induced liver progenitors (CLiPs), from adult rat hepatocytes. In this chapter, we describe a detailed protocol for the induction of rat CLiPs. We first describe the method to isolate primary rat hepatocytes and then describe how to induce CLiPs from the hepatocytes. In addition, we describe methods to induce the generated CLiPs to differentiate into hepatocytes and biliary epithelial cells.

Generation of Hepatic Organoids with Biliary Structures.

Incorporation of bile drainage system into engineered liver tissue is an important issue to advance liver regenerative medicine. Our group reported that three-dimensional (3D) coculture of fetal liver cells (FLCs) and adult rat biliary epithelial cells (BECs) allows reconstruction of hepatic spheroids that possess bile ductular structures. In this chapter, we describe the detailed protocol to isolate FLCs and BECs and to generate the spheroids with bile drainage system using these two types of primary cells.

Extracellular vesicles and encapusulated miRNAs as emerging cancer biomarkers for novel liquid biopsy.

Over the past several years, considerable focus has been placed on the need for 'biomarkers'. However, traditional biomarkers, such as CEA or CA19-9, for gastrointestinal cancer do not provide sufficient sensitivity and specificity for diagnosing cancer. Moreover, these biomarkers cannot provide information regarding the individual variability of patients. Recently, extracellular vesicles (EVs) and extracellular microRNA (miRNA) have shown potential in cancer diagnosis because tumor cells have been shown to release EVs and miRNAs, which mirror their cellular origin, into circulation. Therefore, detection of tumor-specific EVs and extracellular miRNA in body fluids from cancer patients could serve as a non-invasive liquid biopsy for cancer diagnosis and monitoring. This review explores the potential contribution of liquid biopsy using EVs and extracellular miRNA to diagnosis and monitor cancer, including an assessment of prognosis and early detection of disease recurrence in patients with cancer.

Generation of Chemically Induced Liver Progenitors (CLiPs) from Rat Adult Hepatocytes.

Primary mature hepatocytes (MHs) or their progenitor cells are candidate cell sources for cell transplantation therapy in severe liver diseases. However, stable culture of these cells or generation of equivalent cells from pluripotent stem cells has been limited. Using a cocktail of small molecules that we previously found useful in stable culture of multiple types of stem/progenitor cells, we recently established a novel method to generate bipotent liver progenitor cells, named chemically induced liver progenitors (CLiPs), from adult rat MHs. Here, we describe a detailed protocol for the induction of rat CLiPs. We first describe the method to isolate primary rat MHs and then describe how to induce CLiPs from these MHs. In addition, we describe a method to evaluate the bipotentiality of generated CLiPs to differentiate into hepatocytes and biliary epithelial cells. We also describe how to establish stable CLiPs through long-term culture with detailed example data. Primary CLiPs can be generated within 2 weeks, and stable CLiPs, which undergo 10 passages, can be established within 2.5-4 months with batch-to-batch variability.

Mesenchymal stem cell-derived extracellular vesicles: a glimmer of hope in treating Alzheimer's disease.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular plaques resulting from the accumulation of beta-amyloid peptide (Aß). To date, a definitive cure for this disease is still lacking as the currently approved drugs used are mainly symptomatic treatments. The revolutionary discovery of extracellular vesicles (EVs) has shed new light on the development of disease-modifying treatments for AD, owing to their potential in delivering the therapeutic agents to the brain. The feasibility of harnessing EVs for clinical applications is highly dependent on the donor cell, which determines the intrinsic properties of EVs. The merit of mesenchymal stem cells (MSCs) as therapeutic delivery vehicles, and the proven therapeutic effects of the EVs derived from these cells, make researchers esteem MSCs as ideal producers of EVs. Therefore, MSC-derived EVs (MSC-EVs) emerge to be an appealing therapeutic delivery approach for the treatment of AD. Here, we discuss perspectives on the therapeutic strategies using MSC-EVs to treat AD and the associated challenges in clinical application.

Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity.

A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated in vitro and whether such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, can convert rat and mouse MHs in vitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus, our study advances the goals of liver regenerative medicine.

In vitro reconstitution of breast cancer heterogeneity with multipotent cancer stem cells using small molecules.

A small fraction of tumor cells are thought to possess the potential for both multiple-lineage differentiation and self-renewal, which underlies the cancer stem cell hypothesis. However, the differentiation mechanisms of these cells have not been elucidated due to a lack of appropriate culture methods. Here, we established a culture condition for maintaining multipotent tumor cells from rat breast tumors using 4 small molecules. Cultured tumor cells in this condition retained their intrinsic myoepithelial features, expressing p63 and CK14 and vimentin. In a xenograft model, the p63-expressing cells formed epithelial tumors containing glandular, squamous and sebaceous compartments. Upon withdrawal of the small molecules, p63 and CK14 expression was lost, with concurrent increase in expression of mesenchymal markers. These transited cells acquired drug resistance and invasiveness and showed massive sarcomatoid tumorigenicity. Epithelial features could not be recovered by re-exposure to the small molecules in the transited cells. Here, we have identified multipotent cancer cells within primary mammary tumors and demonstrated that their plasticity is maintained by the small molecules.