Influence of Chain Length of Gradient and Block Copoly(2-oxazoline)s on Self-Assembly and Drug Encapsulation.

Amphiphilic gradient copolymers represent a promising alternative to extensively used block copolymers due to their facile one-step synthesis by statistical copolymerization of monomers of different reactivity. Herein, an in-depth analysis is provided of micelles based on amphiphilic gradient poly(2-oxazoline)s with different chain lengths to evaluate their potential for micellar drug delivery systems and compare them to the analogous diblock copolymer micelles. Size, morphology, and stability of self-assembled nanoparticles, loading of hydrophobic drug curcumin, as well as cytotoxicities of the prepared nanoformulations are examined using copoly(2-oxazoline)s with varying chain lengths and comonomer ratios. In addition to several interesting differences between the two copolymer architecture classes, such as more compact self-assembled structures with faster exchange dynamics for the gradient copolymers, it is concluded that gradient copolymers provide stable curcumin nanoformulations with comparable drug loadings to block copolymer systems and benefit from more straightforward copolymer synthesis. The study demonstrates the potential of amphiphilic gradient copolymers as a versatile platform for the synthesis of new polymer therapeutics.

In Vivo Kidney Allograft Endothelial Specific Scavengers for On-Site Inflammation Reduction under Antibody-Mediated Rejection.

Kidney transplantation is the most effective therapy for patients with end-stage renal disease. However, antibody-mediated rejection (ABMR) threatens long-term survival of renal grafts. Although ABMR can be controlled by donor-specific antibody clearance and B- or (and) plasma-cells inhibition, the treatment often causes severe side effects in patients. Therefore, there is need to explore site-specific scavengers. In this study, a nanovehicle carrying an anti-inflammatory drug is developed with complement component 4d targeting, a specific biomarker expressed on allograft endothelium under ABMR. Moreover, the nanovehicle is endowed with photothermal properties to control drug release. Analysis through systematic in vitro and in vivo toxicity, non-invasive targeted imaging, and in situ remote controlled drug release show the nanovehicle specifically targets allograft kidney endothelium, releases an anti-inflammatory drug, methylprednisolone, locally upon laser irradiation, and promotes recovery of injured endothelium, without affecting systemic inflammation or innate immune responses. This strategy has the potential for future clinical application in ABMR treatment.

Mineralization of pH-Sensitive Doxorubicin Prodrug in ZIF-8 to Enable Targeted Delivery to Solid Tumors.

The zeolitic imidazolate framework (ZIF-8), composed of zinc ion and dimethylimidazole, is widely used in drug delivery because of the easy fabrication process and the good biosafety. However, ZIF-8 suffers from low affinity to nonelectric-rich drugs and does not have surface functional groups. Here, to deliver doxorubicin (DOX) with ZIF-8 to specific target sites, DOX was first modified with a pH-sensitive linker containing two carboxyl groups to form the inactive prodrug CAD and subsequently seeded inside ZIF-8 by a 5 min mineralization process. CAD has high affinity to ZIF-8 because of the carboxyl groups and can anchor to the ZIF-8 surface to enable the surface modification with folic acid for tumor targeting. Moreover, the DOX release is precisely controlled by three steps of acidic pH response, with the dissociation of the FA layer, the breakdown of the ZIF-8 structure, and the cleavage of the pH-sensitive linker in prodrug. This novel "prodrug-ZIF-8" strategy has opened a new horizon in drug delivery.

MicroRNA-125b attenuates epithelial-mesenchymal transitions and targets stem-like liver cancer cells through small mothers against decapentaplegic 2 and 4.

UNLABELLED: Emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving treatment of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play important roles in HCC; however, the mechanisms by which miRNAs target the EMT and their therapeutic potential remains largely unknown. To better explore the roles of miRNAs in the EMT process, we established an EMT model in HCC cells by transforming growth factor beta 1 treatment and found that several tumor-related miRNAs were significantly decreased. Among these miRNAs, miR-125b expression was most strongly suppressed. We also found down-regulation of miR-125b in most HCC cells and clinical specimens, which correlated with cellular differentiation in HCC patients. We then demonstrated that miR-125b overexpression attenuated EMT phenotype in HCC cancer cells, whereas knockdown of miR-125b promoted the EMT phenotype in vitro and in vivo. Moreover, we found that miR-125b attenuated EMT-associated traits, including chemoresistance, migration, and stemness in HCC cells, and negatively correlated with EMT and cancer stem cell (CSC) marker expressions in HCC specimens. miR-125b overexpression could inhibit CSC generation and decrease tumor incidence in the mouse xenograft model. Mechanistically, our data revealed that miR-125b suppressed EMT and EMT-associated traits of HCC cells by targeting small mothers against decapentaplegic (SMAD)2 and 4. Most important, the therapeutic delivery of synthetic miR-125b mimics decreased the target molecule of CSC and inhibited metastasis in the mice model. These findings suggest a potential therapeutic treatment of miR-125b for liver cancer. CONCLUSION: miR-125b exerts inhibitory effects on EMT and EMT-associated traits in HCC by SMAD2 and 4. Ectopic expression of miR-125b provides a promising strategy to treat HCC.

Small molecule Me6TREN mobilizes hematopoietic stem/progenitor cells by activating MMP-9 expression and disrupting SDF-1/CXCR4 axis.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) from bone marrow into the blood circulation has been widely used for hematopoietic transplantation. However, the current methods of cytokine- or small-molecule-stimulated HSPC mobilization are far from satisfactory. New mobilizing agents are needed to increase the number of stem cells in peripheral blood for effective reconstitution of hematopoiesis. Here, we report that the molecule Me6TREN (Me6) can induce rapid mobilization of hematopoietic progenitor cells and that Me6 exhibits more significant effects than granulocyte colony-stimulating factor (G-CSF) or AMD3100. Me6 also mobilizes long-term repopulating cells, which successfully engraft and expand in a multilineage fashion in primary and secondary transplant recipients. Mechanistically, Me6 inhibits both the SDF-1α-induced migration and VLA-4-mediated adhesion of mouse and human hematopoietic cells. Me6 appears to mobilize HSPCs by activating MMP-9 expression and disrupting the SDF-1α/CXCR4 axis. Therefore, Me6 may become a new potent and efficacious mobilizing agent of HSPCs.

Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: role of the S100A4-miR155-SOCS1-MMP9 axis.

UNLABELLED: Cancer-associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer-associated MSCs (LC-MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC-MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC-MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC-MSCs compared with liver normal MSCs (LN-MSCs) from adjacent cancer-free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC-MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)-155 in HCC cells was significantly up-regulated by coculture with LC-MSCs and by S100A4 ectopic overexpression. The invasion-promoting effects of S100A4 were significantly attenuated by a miR-155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR-155 expression in HCC cells. We demonstrate that S100A4 secreted from LC-MSCs promotes the expression of miR-155, which mediates the down-regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. CONCLUSION: S100A4 secreted from LC-MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013).

Decoding human in vitro terminal erythropoiesis originating from umbilical cord blood mononuclear cells and pluripotent stem cells.

Ex vivo red blood cell (RBC) production generates unsatisfactory erythroid cells. A deep exploration into terminally differentiated cells is required to understand the impairments for RBC generation and the underlying mechanisms. Here, we mapped an atlas of terminally differentiated cells from umbilical cord blood mononuclear cells (UCBMN) and pluripotent stem cells (PSC) and observed their dynamic regulation of erythropoiesis at single-cell resolution. Interestingly, we detected a few progenitor cells and non-erythroid cells from both origins. In PSC-derived erythropoiesis (PSCE), the expression of haemoglobin switch regulators (BCL11A and ZBTB7A) were significantly absent, which could be the restraint for its adult globin expression. We also found that PSCE were less active in stress erythropoiesis than in UCBMN-derived erythropoiesis (UCBE), and explored an agonist of stress erythropoiesis gene, TRIB3, could enhance the expression of adult globin in PSCE. Compared with UCBE, there was a lower expression of epigenetic-related proteins (e.g., CASPASE 3 and UBE2O) and transcription factors (e.g., FOXO3 and TAL1) in PSCE, which might restrict PSCE's enucleation. Moreover, we characterized a subpopulation with high proliferation capacity marked by CD99high in colony-forming unit-erythroid cells. Inhibition of CD99 reduced the proliferation of PSC-derived cells and facilitated erythroid maturation. Furthermore, CD99-CD99 mediated the interaction between macrophages and erythroid cells, illustrating a mechanism by which macrophages participate in erythropoiesis. This study provided a reference for improving ex vivo RBC generation.

Nanoengineered Red Blood Cells Loaded with TMPRSS2 and Cathepsin L Inhibitors Block SARS-CoV-2 Pseudovirus Entry into Lung ACE2+ Cells.

The enzymatic activities of Furin, Transmembrane serine proteinase 2 (TMPRSS2), Cathepsin L (CTSL), and Angiotensin-converting enzyme 2 (ACE2) receptor binding are necessary for the entry of coronaviruses into host cells. Precise inhibition of these key proteases in ACE2+ lung cells during a viral infection cycle shall prevent viral Spike (S) protein activation and its fusion with a host cell membrane, consequently averting virus entry to the cells. In this study, dual-drug-combined (TMPRSS2 inhibitor Camostat and CTSL inhibitor E-64d) nanocarriers (NCs) are constructed conjugated with an anti-human ACE2 (hACE2) antibody and employ Red Blood Cell (RBC)-hitchhiking, termed "Nanoengineered RBCs," for targeting lung cells. The significant therapeutic efficacy of the dual-drug-loaded nanoengineered RBCs in pseudovirus-infected K18-hACE2 transgenic mice is reported. Notably, the modular nanoengineered RBCs (anti-receptor antibody+NCs+RBCs) precisely target key proteases of host cells in the lungs to block the entry of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), regardless of virus variations. These findings are anticipated to benefit the development of a series of novel and safe host-cell-protecting antiviral therapies.

MiR-526b-3p enhances sensitivity of head and neck squamous cell carcinoma cells to radiotherapy via suppressing exosomal LAMP3-mediated autophagy.

Lysosomal associated membrane protein 3 (LAMP3) has been reported to be a tumour promoter in multiple cancer types by modulating tumour cell autophagy. However, the potential mechanism of LAMP3 in radio-resistance of head and neck squamous cell carcinoma (HNSCC) remains unknown. Therefore, our current study aims to detect the impacts of LAMP3 on the resistance of HNSCC cells to radiotherapy and meanwhile explore its functional mechanism. Through RT-Qpcr examination, LAMP3 expression was identified to be expressed at a significantly high level in irradiation-resistant HNSCC cell lines compared with irradiation-sensitive HNSCC cell lines. Functional assays including CCK-8, colony formation and Transwell assays demonstrated that LAMP3 enhanced the radio-resistance through inducing autophagy to promote HNSCC cell growth. Furthermore, irradiation-resistant HNSCC cells could transfer exosomal LAMP3 to elevate LAMP3 expression in irradiation-sensitive HNSCC cells. Mechanistically, microRNA (miRNA) miR-526b-3p could inhibit LAMP3 expression so as to strengthen sensitivity of HNSCC cells to radiotherapy. In a word, exosomal LAMP3 expression promoted radioresistance of HNSCC cells via inducing autophagy, while this effect could be suppressed by miR-526b-3p in a targeted manner.

Porphyrin Centered Paclitaxel Tetrameric Prodrug Nanoassemblies as Tumor-Selective Theranostics for Synergized Breast Cancer Therapy.

Although having undergone decades of development, nanoparticulate drug delivery vehicles for efficient cancer therapy remain a challenge, confined by low drug loading, instability, and poor cancer tissue selectivity. A self-assembled prodrug, the combination of prodrug strategy and the self-assembly merits, represents a special chemical entity which spontaneously organizes into supramolecular composites with defined architecture, therefore also providing a strategy to develop new medications. Paclitaxel (PTX) is still among the most generally prescribed chemotherapeutics in oncology but is restricted by poor solubility. Although photodynamic therapy, with its noninvasive features and barely developed drug resistance, signifies an alternative tool to suppress life-threatening cancer, sole use hardly fulfills its potential. To this end, a reduction-activatable heterotetrameric prodrug with the photosensitizer is synthesized, then formulated into self-assembled nanoparticles (NPs) for tumor imaging and combined chemo- and photodynamic therapy. Coating the NPs with amphiphilic polymer distearylphosphatidylethanolamine-polyethylene glycol-arginine-glycine-aspartate (DSPE-PEG-RGD) offers high stability and enables cancer tissue targeting. The as-prepared NPs enlighten disease cells and reveal more potent cytotoxicity comparing to PTX and the photosensitizer alone. Furthermore, the NPs selectively accumulates into tumors and synergistically inhibits tumor proliferation with reduced side effects in mice.

Mesenchymal stem cells from primary breast cancer tissue promote cancer proliferation and enhance mammosphere formation partially via EGF/EGFR/Akt pathway.

Mesenchymal stem cells (MSCs) play a critical role in promoting cancer progression. However, it is not clear whether MSCs are located in breast cancer tissues and correlated with tumor proliferation. The aim of this study was to investigate the presence of MSCs in breast cancer tissues and evaluate their interactions with cancer cells. We successfully isolated and identified MSCs from primary breast cancer tissues. Breast cancer-associated MSCs (BC-MSCs) showed homogenous immunophenotype, and possessed tri-lineage differentiation potential (osteoblast, adipocyte, and chondrocyte). When co-transplanted with cancer cells in a xenograft model in vivo, BC-MSCs significantly increased the volume and weight of tumors. We observed that BC-MSCs stimulated mammosphere formation in the transwell co-culture system in vitro. This effect was significantly suppressed by the EGF receptor inhibitor. We verified that BC-MSCs could secrete EGF and activate cancer cell's EGF receptors. Furthermore, our data showed that EGF derived from BC-MSCs could promote mammosphere formation via the PI3K/Akt signaling pathway. Our results confirmed the presence of MSC in primary breast cancer tissues, and they could provide a favorable microenvironment for tumor cell growth in vivo, partially enhance mammosphere formation via the EGF/EGFR/Akt pathway.

Epimorphin promotes human hepatocellular carcinoma invasion and metastasis through activation of focal adhesion kinase/extracellular signal-regulated kinase/matrix metalloproteinase-9 axis.

UNLABELLED: The high incidence rate of hepatocellular carcinoma (HCC) is mainly the result of frequent metastasis and tumor recurrence. Unfortunately, the underlying molecular mechanisms driving HCC metastasis are still not fully understood. It has been demonstrated that tumor stroma cells contribute to primary tumor growth and metastasis. Within the HCC environment, activated hepatic stellate cells (HSCs) can release a number of molecules and enhance cancer cell proliferation and invasiveness in a paracrine manner. Here, for the first time, we demonstrate that epimorphin (EPM; also called syntaxin-2), an extracellular protein, is strongly elevated in activated HSCs within tumor stroma. We show that knockdown of EPM expression in HSCs substantially abolishes their effects on cancer cell invasion and metastasis. Ectopic expression of EPM in HCC cancer cells enhances their invasiveness; we demonstrate that the cells expressing EPM have markedly increased metastasis potential. Furthermore, EPM-mediated invasion and metastasis of cancer cells is found to require up-regulation of matrix metalloproteinase-9 (MMP-9) through the activation of focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) axis. CONCLUSION: Our results show that EPM, secreted by activated HSCs within HCC stroma, promotes invasion and metastasis of cancer cells by activating MMP-9 expression through the FAK-ERK pathway.

Synthesis, characterization and biocompatibility of guar gum-benzoic acid.

A novel chemical functionalization of guar gum (GG) by benzoic acid (BA) via nucleophilic substitution reaction in aqueous solution has been reported. BA moieties are chosen due to coordination chemistry of carboxylic acid moieties, hydrophobicity and intermolecular interaction of aromatic rings. The presence of conjugated BA on guar gum-benzoic acid (GG-BA) with grafting density of 5.5% is confirmed by 1H NMR. Amorphous GG-BA with irregular morphology has been studied by UV-Vis, FTIR, XRD, SEM, TEM, TGA, computational chemistry and contact angle measurement. GG-BA in a concentration range from 0 to 4000 µg mL-1 has good biocompatibility to mouse embryonic fibroblasts (MEF), human mammary epithelial cells (MCF-10A) after 48 and 72 h of treatment using WST-1 assay. GG-BA shows great potential for the development of biomaterials such as bioadhesives, hydrogels, and coacervates.

Microfluidic-assisted biomineralization of CRISPR/Cas9 in near-infrared responsive metal-organic frameworks for programmable gene-editing.

Ribonucleoprotein (RNP) based CRISPR/Cas9 gene-editing system shows great potential in biomedical applications. However, due to the large size, charged surface and high biological sensitivity of RNP, its efficient delivery with precise control remains highly challenging. Herein, a microfluidic-assisted metal-organic framework (MOF) based biomineralization strategy is designed and utilized for the efficient delivery and remote regulation of CRISPR/Cas9 RNP gene editing. The strategy is realized by biomimetic growing of thermo-responsive EuMOFs onto photothermal template Prussian blue (PB). The RNP is loaded during MOFs crystallization in microfluidic channels. By adjusting different microfluidic parameters, well-defined and comparable RNP encapsulated nanocarrier (PB@RNP-EuMOFs) are obtained with high loading efficiency (60%), remarkable RNP protection and NIR-stimulated release capacity. Upon laser exposure, the nanocarrier induces effective endosomal escape (4 h) and precise gene knockout of green fluorescent protein by 40% over 2 days. Moreover, the gene-editing activity can be programmed by tuning exposure times (42% for three times and 47% for four times), proving more controllable and inducible editing modality compared to control group without laser irradiation. This novel microfluidic-assisted MOFs biomineralization strategy thus offers an attractive route to optimize delivery systems and reduce off-target side effects by NIR-triggered remote control of CRISPR/Cas9 RNP, improving the potential for its highly efficient and precise therapeutic application.

A Functional Screening Identifies a New Organic Selenium Compound Targeting Cancer Stem Cells: Role of c-Myc Transcription Activity Inhibition in Liver Cancer.

Cancer stem cells (CSCs) are reported to play essential roles in chemoresistance and metastasis. Pathways regulating CSC self-renewal and proliferation, such as Hedgehog, Notch, Wnt/ß-catenin, TGF-ß, and Myc, may be potential therapeutic targets. Here, a functional screening from the focused library with 365 compounds is performed by a step-by-step strategy. Among these candidate molecules, phenyl-2-pyrimidinyl ketone 4-allyl-3-amino selenourea (CU27) is chosen for further identification because it proves to be the most effective compound over others on CSC inhibition. Through ingenuity pathway analysis, it is shown CU27 may inhibit CSC through a well-known stemness-related transcription factor c-Myc. Gene set enrichment analysis, dual-luciferase reporter assays, expression levels of typical c-Myc targets, molecular docking, surface plasmon resonance, immunoprecipitation, and chromatin immunoprecipitation are conducted. These results together suggest CU27 binds c-Myc bHLH/LZ domains, inhibits c-Myc-Max complex formation, and prevents its occupancy on target gene promoters. In mouse models, CU27 significantly sensitizes sorafenib-resistant tumor to sorafenib, reduces the primary tumor size, and inhibits CSC generation, showing a dramatic anti-metastasis potential. Taken together, CU27 exerts inhibitory effects on CSC and CSC-associated traits in hepatocellular carcinoma (HCC) via c-Myc transcription activity inhibition. CU27 may be a promising therapeutic to treat sorafenib-resistant HCC.

Role of epimorphin in bile duct formation of rat liver epithelial stem-like cells: involvement of small G protein RhoA and C/EBPß.

Epimorphin/syntaxin 2 is a high conserved and very abundant protein involved in epithelial morphogenesis in various organs. We have shown recently that epimorphin (EPM), a protein exclusively expressed on the surface of hepatic stellate cells and myofibroblasts of the liver, induces bile duct formation of hepatic stem-like cells (WB-F344 cells) in a putative biophysical way. Therefore, the aim of this study was to present some of the molecular mechanisms by which EPM mediates bile duct formation. We established a biliary differentiation model by co-culture of EPM-overexpressed mesenchymal cells (PT67(EPM)) with WB-F344 cells. Here, we showed that EPM could promote WB-F344 cells differentiation into bile duct-like structures. Biliary differentiation markers were also elevated by EPM including Yp, Cx43, aquaporin-1, CK19, and gamma glutamyl transpeptidase (GGT). Moreover, the signaling pathway of EPM was analyzed by focal adhesion kinase (FAK), extracellular regulated kinase 1/2 (ERK1/2), and RhoA Western blot. Also, a dominant negative (DN) RhoA-WB-F344 cell line (WB(RhoA-DN)) was constructed. We found that the levels of phosphorylation (p) of FAK and ERK1/2 were up-regulated by EPM. Most importantly, we also showed that RhoA is necessary for EPM-induced activation of FAK and ERK1/2 and bile duct formation. In addition, a dual luciferase-reporter assay and CHIP assay was performed to reveal that EPM regulates GGT IV and GGT V expression differentially, possibly mediated by C/EBPß. Taken together, these data demonstrated that EPM regulates bile duct formation of WB-F344 cells through effects on RhoA and C/EBPß, implicating a dual aspect of this morphoregulator in bile duct epithelial morphogenesis.

Nanotechnology-based delivery of CRISPR/Cas9 for cancer treatment.

CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9) is a potent technology for gene-editing. Owing to its high specificity and efficiency, CRISPR/Cas9 is extensity used for human diseases treatment, especially for cancer, which involves multiple genetic alterations. Different concepts of cancer treatment by CRISPR/Cas9 are established. However, significant challenges remain for its clinical applications. The greatest challenge for CRISPR/Cas9 therapy is how to safely and efficiently deliver it to target sites in vivo. Nanotechnology has greatly contributed to cancer drug delivery. Here, we present the action mechanisms of CRISPR/Cas9, its application in cancer therapy and especially focus on the nanotechnology-based delivery of CRISPR/Cas9 for cancer gene editing and immunotherapy to pave the way for its clinical translation. We detail the difficult barriers for CRISIR/Cas9 delivery in vivo and discuss the relative solutions for encapsulation, target delivery, controlled release, cellular internalization, and endosomal escape.

The splicing regulatory factor hnRNPU is a novel transcriptional target of c-Myc in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common liver cancer with high mortality. Here, we found that hnRNPU is overexpressed in HCC tissues and is correlated with the poor prognosis of HCC patients. Besides, hnRNPU is of high significance in regulating the proliferation, apoptosis, self-renewal, and tumorigenic potential of HCC cells. Mechanismly, c-Myc regulates hnRNPU expression at the transcriptional level, and meanwhile, hnRNPU stabilizes the mRNA of c-MYC. We found that the hnRNPU and c-Myc regulatory loop exerts a synergistic effect on the proliferation and self-renewal of HCC, and promotes the HCC progression. Taken together, hnRNPU functions as a novel transcriptional target of c-Myc and promotes HCC progression, which may become a promising target for the treatment of c-Myc-driven HCC.

Integrative single-cell transcriptome analysis reveals a subpopulation of fibroblasts associated with favorable prognosis of liver cancer patients.

Single-cell transcriptome analysis has provided detailed insights into the ecosystem of liver cancer. However, the changes of the cellular and molecular components of liver tumors in comparison with normal livers have not been described at single-cell level. Here, we performed an integrative single-cell analysis of both normal livers and liver cancers. Principal component analysis was firstly performed to delineate the cell lineages in liver tissues. Differential gene expression within major cell types were then analyzed between tumor and normal samples, thus resolved the cell type-specific molecular alterations in liver cancer development. Moreover, a comparison between liver cancer derived versus normal liver derived cell components revealed that two subpopulations of fibroblasts were exclusively expanded in liver cancer tissues. By further defining subpopulation-specific gene signatures, characterizing their spatial distribution in tumor tissues and investigating their clinical significance, we found that the SPARCL1 positive fibroblasts, representing a group of tumor vessel associated fibroblasts, were related to reduced vascular invasion and prolonged survival of liver cancer patients. Through establishing an in-vitro endothelial-to-mesenchymal transition model, we verified the conversion of the fetal liver sinusoidal endothelial cells into the fibroblast-like cells, demonstrating a possible endothelial cell origination of the SPARCL1 positive fibroblasts. Our study provides new insights into the cell atlas alteration, especially the expanded fibroblasts in liver cancers.

RhoA promotes differentiation of WB-F344 cells into the biliary lineage.

When cultured on Matrigel, liver precursor epithelium WB-F344 cells could be induced to differentiate into biliary cells in which RhoA expression was upregulated. To further investigate the role of RhoA in WB cell differentiation initiated by Matrigel treatment, we constructed constitutively active RhoA-expressing vectors and stably transfected them into WB-F344 cells. Accompanying upregulation of biliary lineage markers and morphological changes, cells with ectopically active RhoA expression were found to form bile-duct-like structures even without Matrigel treatment. Besides, ROCK inhibitor Y27632 treatment eliminated luminal morphogenesis. F-actin cytoplasmic staining further verified that the RhoA-ROCK signal pathway was involved in differentiation of WB cells into the biliary lineage. In conclusion, our results suggested that the RhoA-ROCK-stress fibre system plays an obligatory role in Matrigel-induced WB-F344 cell luminal morphogenesis and further differentiation.