Tumor derived exosomal ENTPD2 impair CD8+ T cell function in colon cancer through ATP-adenosine metabolism reprogramming.

BACKGROUND: Extracellular ATP-AMP-adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell-derived ATPases in the development and progression of colon cancer. METHODS: Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues. RESULTS: We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells. CONCLUSION: Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP-adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.

HIF-1α Mediates Immunosuppression and Chemoresistance in Colorectal Cancer by Inhibiting CXCL9, -10 and -11.

Uncertainty exists regarding the mechanisms by which hypoxia-inducible factors (HIFs) control CD8+T-cell migration into tumor microenvironments. Here, we found that HIF-1α knockdown or overexpression resulted in increased or decreased CXCL9, -10, and -11 expression in vitro, respectively. Gene Set Variation Analysis revealed that elevated HIF-1α levels correlated with a poor prognosis, severe pathological stage, and an absence of CD8+ T cells in the tumor microenvironment in colorectal cancer (CRC) patients. HIF-1α was inversely associated with pathways beneficial to anti-tumor immunotherapy and cytokine/chemokine function. In vivo, inhibiting HIF-1α or its upstream regulator BIRC2 significantly suppressed tumor growth and promoted CD8+ T-cell infiltration. CXCR3 neutralizing antibodies reversed these effects, implicating the involvement of CXCL9, -10, and -11/CXCR3 axis. The presence of HIF-1α weakened the upregulation of CXCL9, -10, and -11 by bleomycin and doxorubicin. Combining HIF-1α inhibition with bleomycin promoted CD8+ T-cell infiltration and tumor suppression in vivo. Moreover, doxorubicin could upregulate CXCL9, -10 and -11 by suppressing HIF-1α. Our findings highlight the potential of HIF-1α inhibition to improve CRC microenvironments and increase chemotherapy sensitivity.

Low-dose radiotherapy combined with dual PD-L1 and VEGFA blockade elicits antitumor response in hepatocellular carcinoma mediated by activated intratumoral CD8+ exhausted-like T cells.

Atezolizumab (anti-PD-L1) combined with bevacizumab (anti-VEGFA) is the first-line immunotherapy for advanced hepatocellular carcinoma (HCC), but the number of patients who benefit from this regimen remains limited. Here, we combine dual PD-L1 and VEGFA blockade (DPVB) with low-dose radiotherapy (LDRT), which rapidly inflames tumors, rendering them vulnerable to immunotherapy. The combinatorial therapy exhibits superior antitumor efficacy mediated by CD8+ T cells in various preclinical HCC models. Treatment efficacy relies upon mobilizing exhausted-like CD8+ T cells (CD8+ Tex) with effector function and cytolytic capacity. Mechanistically, LDRT sensitizes tumors to DPVB by recruiting stem-like CD8+ Tpex, the progenitor exhausted CD8+ T cells, from draining lymph nodes (dLNs) into the tumor via the CXCL10/CXCR3 axis. Together, these results further support the rationale for combining LDRT with atezolizumab and bevacizumab, and its clinical translation.

Targeting RPA promotes autophagic flux and the antitumor response to radiation in nasopharyngeal carcinoma.

BACKGROUND: Autophagy is involved in nasopharyngeal carcinoma (NPC) radioresistance. Replication protein A 1 (RPA1) and RPA3, substrates of the RPA complex, are potential therapeutic targets for reversing NPC radioresistance. Nevertheless, the role of RPA in autophagy is not adequately understood. This investigation was performed to reveal the cytotoxic mechanism of a pharmacologic RPA inhibitor (RPAi) in NPC cells and the underlying mechanism by which RPAi-mediated autophagy regulates NPC radiosensitivity. METHODS AND RESULTS: We characterized a potent RPAi (HAMNO) that was substantially correlated with radiosensitivity enhancement and proliferative inhibition of in vivo and in NPC cell lines in vitro. We show that the RPAi induced autophagy at multiple levels by inducing autophagic flux, AMPK/mTOR pathway activation, and autophagy-related gene transcription by decreasing glycolytic function. We hypothesized that RPA inhibition impaired glycolysis and increased NPC dependence on autophagy. We further demonstrated that combining autophagy inhibition with chloroquine (CQ) treatment or genetic inhibition of the autophagy regulator ATG5 and RPAi treatment was more effective than either approach alone in enhancing the antitumor response of NPC to radiation. CONCLUSIONS: Our study suggests that HAMNO is a potent RPAi that enhances radiosensitivity and induces autophagy in NPC cell lines by decreasing glycolytic function and activating autophagy-related genes. We suggest a novel treatment strategy in which pharmacological inhibitors that simultaneously disrupt RPA and autophagic processes improve NPC responsiveness to radiation.

Knockdown LIMP2 inhibits colorectal cancer cells migration, invasion, and metastasis.

Colorectal cancer (CRC) is a common malignancy worldwide nowadays and liver metastasis is the primary cause of death in patients with CRC. Although lysosomal integral membrane protein 2 (LIMP2) has been reported to play important roles in gastric cancer and prostate cancer, its role in CRC remains unclear. The aim of this study was to investigate the function of LIMP2 in CRC invasion and migration, along with the potential underlying molecular mechanisms. We found that LIMP2 levels were higher in CRC tissues compared to adjacent normal tissues. Kaplan-Meier survival analysis showed that high expression of LIMP2 was associated with worse prognosis in CRC patients. Knockdown of LIMP2 significantly inhibited invasion, migration, and wound healing abilities of CRC cells in vitro, and inhibited CRC liver metastasis in vivo. Additionally, LIMP2 knockdown inhibited autophagy in CRC. Therefore, LIMP2 plays an important role in CRC progression. High expression of LIMP2 was associated with worse prognosis in CRC patients. Knockdown LIMP2 can effectively inhibit CRC cell migration and invasion in vitro and prevent liver metastasis in vivo. These findings suggest that LIMP2 may serve as an independent prognostic factor and potential therapeutic target for CRC.

Jun-APOE-LRP1 axis promotes tumor metastasis in colorectal cancer.

Apolipoprotein E (apoE) has previously been reported to play vital roles in tumor progression. However, the impact of apoE on colorectal cancer (CRC) metastasis remains largely unexplored. This study aimed to investigate the role of apoE in CRC metastasis and to identify the transcription factor and receptor of apoE involved in regulation of CRC metastasis. Bioinformatic analyses were conducted to examine the expression pattern and prognosis of apolipoproteins. APOE-overexpressing cell lines were utilized to explore the effects of apoE on proliferation, migration and invasion of CRC cells. Additionally, the transcription factor and receptor of apoE were screened via bioinformatics, and further validated through knockdown experiments. We discovered that the mRNA levels of APOC1, APOC2, APOD and APOE were higher in lymphatic invasion group, and a higher apoE level indicated poorer overall survival and progression-free interval. In vitro studies demonstrated that APOE-overexpression did not affect proliferation but promoted the migration and invasion of CRC cells. We also reported that APOE-expression was modulated by the transcription factor Jun by activating the proximal promoter region of APOE, and APOE-overexpression reversed the metastasis suppression of JUN knockdown. Furthermore, bioinformatics analysis suggested an interaction between apoE and low-density lipoprotein receptor-related protein 1 (LRP1). LRP1 was highly expressed in both the lymphatic invasion group and the APOEHigh group. Additionally, we found that APOE-overexpression upregulated LRP1 protein levels, and LRP1 knockdown attenuated the metastasis-promoting function of APOE. Overall, our study suggests that the Jun-APOE-LRP1 axis contributes to tumor metastasis in CRC.

Evacetrapib Elicits Antitumor Effects on Colorectal Cancer by Inhibiting the Wnt/ß-Catenin Signaling Pathway and Activating the JNK Signaling Pathway.

Despite advances in colorectal cancer (CRC) treatment, most advanced CRC patients who experience disease progression after chemotherapy, targeted therapy, and immunotherapy face a situation in which there is no available medicine. Thus, new therapeutic drugs for CRC are urgently needed. Studies have shown that cholesteryl ester transfer protein (CETP) has a vital role in tumor development and is a possible target for CRC therapy. We found that Evacetrapib, a CETP inhibitor, suppressed CRC cell growth by inhibiting the Wnt/ß-catenin signaling pathway and activating the c-Jun NH2-terminal kinase (JNK) signaling pathway in CRC. Therefore, Evacetrapib displays an anti-cancer effect and is a possible option for treating CRC.

Potential roles of serum ATPase and AMPase in predicting diagnosis of colorectal cancer patients.

Colorectal cancer (CRC) is a common gastrointestinal cancer with high incidence and mortality rates. CRC may be associated with regulation of circulating nucleotides. This study aimed to evaluate the serum levels of nucleotide-metabolizing enzymes (ATPase and AMPase) in patients with CRC and to explore the clinical diagnostic value of these enzymes. The gene set variation analysis (GSVA) score of the ATP-adenosine signature was calculated using tumor samples from The Cancer Genome Atlas (TCGA). ATP-adenosine signaling plays a central role in CRC progression. A total of 135 subjects, including 87 patients with CRC and 48 healthy controls, were included. The serum levels of ATPase and AMPase in the CRC group were significantly higher than those in the control group (P < 0.05). Furthermore, ATP and AMP hydrolysis levels significantly increased in the advanced CRC group (P < 0.05). ATP and AMP hydrolysis was decreased by the ENTPDase inhibitors (POM-1 and ARL67156) and CD73 inhibitor (APCP). The sensitivities of ATPase and AMPase were 95.4% and 75.9%, respectively, which were higher than those of CEA (67.8%) and CA19-9 (72.4%). The specificities of ATPase and AMPase were 69.9% and 73.9%, respectively, which were higher than that of CA19-9 (47.8%). The combination of CEA, ATPase, and AMPase demonstrated high sensitivity (92.0%) and specificity (87.0%). Collectively, ATPase and AMPase activities are upregulated in CRC with considerable diagnostic significance. The combination of CEA, ATPase, and AMPase may provide a novel approach for CRC screening.

Pyrimethamine inhibits cell growth by inducing cell senescence and boosting CD8+ T-cell mediated cytotoxicity in colorectal cancer.

BACKGROUND: The emergence of nonresponse or resistance to traditional chemotherapeutic agents is one of the main challenges of colorectal cancer (CRC) therapies. Thus, novel therapeutic drugs that can improve the clinical outcomes of CRC patients are urgently needed. The purpose of this study was to investigate the effects and mechanisms of pyrimethamine in CRC. METHODS AND RESULTS: In this study, we assessed the role of pyrimethamine on CRC cell growth by cell counting kit-8 and colony formation assays. Cell cycle distribution and cellular senescence were determined by flow cytometry and senescence-associated ß-galactosidase staining respectively. RNA-seq analysis and western blotting were used to investigate the potential pathways of pyrimethamine in CRC development. Moreover, animal experiments were performed to evaluate the effect of pyrimethamine in vivo. Our results demonstrated that pyrimethamine could inhibit cell growth by inducing S phase arrest followed by cellular senescence in CRC cells, and the p38MAPK-p53 axis was probably involved in that effect. In addition, pyrimethamine could also boost CD8+ T-cell mediated cytotoxicity and exert antitumor activity in vivo. CONCLUSION: These results indicated that pyrimethamine may be a promising candidate agent for CRC treatment.

The storage stability of Bacillus subtilis spore displaying cysteine protease of Clonorchis sinensis and its effect on improving the gut microbiota of mice.

Bacillus subtilis (B. subtilis) spore can serve as an ideal vehicle for expressing heterologous antigens, and elicit specific immune responses by oral administration. In previous studies, we successfully constructed the recombinant B. subtilis spores expressing cysteine protease of Clonorchis sinensis (C. sinensis, B.s-CsCP), and confirmed that oral administration of B.s-CsCP could elicit good protective immune responses in mice. In this study, Gram staining was used to observe the morphology of B.s-CsCP in different form, and the storage of liquid spores and lyophilized spores at different temperatures was compared. The mice were orally immunized with three different doses of spores (2×108, 1×109, and 5×109 CFU/day) for three times in total at biweekly interval. Then, antibody levels of mice were measured, the safety of spores was evaluated, and the changes of gut microbiota after oral gavage of spores (1×109 dose) were investigated. Results showed that B. subtilis was a typical Gram-positive bacterium, and its spore had good resistance to chemical dye. Liquid B. subtilis spores resuspended in sterile water could be stored for a long time at 4 °C or below, while lyophilized spores could be well stored even at RT and better at lower temperatures. Oral administration of B. subtilis spores to mice could stimulate both local mucosal and systemic immune responses in a dose-dependent manner without toxic side effects. Besides, beneficial bacteria producing butyrate such as Odoribacter were increased, while potential pathogens such as Escherichia-Shigella were decreased in mice intestine. Therefore, our work further confirmed that B. subtilis spores expressing CsCP could be a promising oral vaccine against C. sinensis with the advantages of stability, safety, easy storage, and promotion of intestinal health.Key Points• Recombinant CsCP B. subtilis spores could be easily preserved in either liquid or freeze-dried state.• Oral immunization of recombinant spores in mice could increase both local and system immune levels in a dose-dependent manner.• Oral administration of recombinant spores increased the number of beneficial bacteria and reduced the number of harmful bacteria in the intestinal tract of mice.

CD8+CXCR5+T cells infiltrating hepatocellular carcinomas are activated and predictive of a better prognosis.

CD8+ T cells are thought to be the primary cytotoxic lymphocytes exerting antitumor effects. However, few studies have focused on the antitumor effects of CD8+ T cell-mediated humoral immunity or on interactions between CD8+ T cells and B cells in hepatocellular carcinoma (HCC). We found that the frequency of IL-21-producing CD8+CXCR5+ T cells was higher in HCC tumor tissue than in peritumoral tissue or peripheral blood from the same patients or in blood from healthy donors. Moreover, CD8+CXCR5+ T cells migrated in response to supernatants from primary HCC (HCC-SN) cells, and HCC-SN cells also powerfully induced CXCR5 expression in CD8+ T cells and IL-21 expression in CD8+CXCR5+ T cells. CD8+CXCR5+ T cells from HCC patients, but not those from healthy individuals, stimulated CD19+ B cells to differentiate into IgG-producing plasmablasts. These findings reveal that CD8+CXCR5+ T cells strongly infiltrate HCC tumors, and their infiltration is predictive of a better prognosis. Surprisingly, moreover, CD8+CXCR5+ T cells produced IL-21, which induced B cells to differentiate into IgG-producing plasmablasts and to play a key role in humoral immunity in HCC.

Tumor-derived exosomal HMGB1 fosters hepatocellular carcinoma immune evasion by promoting TIM-1+ regulatory B cell expansion.

BACKGROUND: Regulatory B (Breg) cells represent one of the B cell subsets that infiltrate solid tumors and exhibit distinct phenotypes in different tumor microenvironments. However, the phenotype, function and clinical relevance of Breg cells in human hepatocellular carcinoma (HCC) are presently unknown. METHODS: Flow cytometry analyses were performed to determine the levels, phenotypes and functions of TIM-1+Breg cells in samples from 51 patients with HCC. Kaplan-Meier plots for overall survival and disease-free survival were generated using the log-rank test. TIM-1+Breg cells and CD8+ T cells were isolated, stimulated and/or cultured in vitro for functional assays. Exosomes and B cells were isolated and cultured in vitro for TIM-1+Breg cell expansion assays. RESULTS: Patients with HCC showed a significantly higher TIM-1+Breg cell infiltration in their tumor tissue compared with the paired peritumoral tissue. The infiltrating TIM-1+Breg cells showed a CD5highCD24-CD27-/+CD38+/high phenotype, expressed high levels of the immunosuppressive cytokine IL-10 and exhibited strong suppressive activity against CD8+ T cells. B cells activated by tumor-derived exosomes strongly expressed TIM-1 protein and were equipped with suppressive activity against CD8+ T cells similar to TIM-1+Breg cells isolated from HCC tumor tissue. Moreover, the accumulation of TIM-1+Breg cells in tumors was associated with advanced disease stage, predicted early recurrence in HCC and reduced HCC patient survival. Exosome-derived HMGB1 activated B cells and promoted TIM-1+Breg cell expansion via the Toll like receptor (TLR) 2/4 and mitogen-activated protein kinase (MAPK) signaling pathways. CONCLUSIONS: Our results illuminate a novel mechanism of TIM-1+Breg cell-mediated immune escape in HCC and provide functional evidence for the use of these novel exosomal HMGB1-TLR2/4-MAPK pathways to prevent and to treat this immune tolerance feature of HCC.

Sequence analysis and characterization of pyruvate kinase from Clonorchis sinensis, a 53.1-kDa homopentamer, implicated immune protective efficacy against clonorchiasis.

BACKGROUND: Clonorchis sinensis, the causative agent of clonorchiasis, is classified as one of the most neglected tropical diseases and affects more than 15 million people globally. This hepatobiliary disease is highly associated with cholangiocarcinoma. As key molecules in the infectivity and subsistence of trematodes, glycolytic enzymes have been targets for drug and vaccine development. Clonorchis sinensis pyruvate kinase (CsPK), a crucial glycolytic enzyme, was characterized in this research. RESULTS: Differences were observed in the sequences and spatial structures of CsPK and PKs from humans, rats, mice and rabbits. CsPK possessed a characteristic active site signature (IKLIAKIENHEGV) and some unique sites but lacked the N-terminal domain. The predicted subunit molecular mass (Mr) of CsPK was 53.1 kDa. Recombinant CsPK (rCsPK) was a homopentamer with a Mr. of approximately 290 kDa by both native PAGE and gel filtration chromatography. Significant differences in the protein and mRNA levels of CsPK were observed among four life stages of C. sinensis (egg, adult worm, excysted metacercaria and metacercaria), suggesting that these developmental stages may be associated with diverse energy demands. CsPK was widely distributed in adult worms. Moreover, an intense Th1-biased immune response was persistently elicited in rats immunized with rCsPK. Also, rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CONCLUSIONS: The sequences and spatial structures, molecular mass, and expression profile of CsPK have been characterized. rCsPK was indicated to be a homopentamer. Rat anti-rCsPK sera suppressed C. sinensis adult subsistence both in vivo and in vitro. CsPK is worthy of further study as a promising target for drug and vaccine development.

Csseverin inhibits apoptosis through mitochondria-mediated pathways triggered by Ca2 + dyshomeostasis in hepatocarcinoma PLC cells.

BACKGROUND: Numerous experimental and epidemiological studies have demonstrated a link between Clonorchis sinensis (C. sinensis) infestation and cholangiocarcinoma (CCA) as well as hepatocellular carcinoma (HCC). The underlying molecular mechanism involved in the malignancy of CCA and HCC has not yet been addressed. Csseverin, a component of the excretory/secretory products of C. sinensis (CsESPs), was confirmed to cause obvious apoptotic inhibition in the human HCC cell line PLC. However, the antiapoptotic mechanism is unclear. In the present study, we investigated the cellular features of the antiapoptotic mechanism upon transfection of the Csseverin gene. METHODS: In the present study, we evaluated the effects of Csseverin gene overexpression on the apoptosis of PLC cells using an Annexin PE/7-AAD assay. Western blotting was applied to quantify the activation of caspase-3 and caspase-9, the mitochondrial translocation of Bax and the release of Cyt c upon Csseverin overexpression in PLC cells. Laser scanning confocal microscopy was used to analyze the changes of intracellular calcium. Fluorescence assay and immunofluorescence assays were performed to observe the changes of the mitochondrial permeability transition pore (MPTP). RESULTS: The overexpression of Csseverin in PLC cells showed apoptosis resistance after the induction of apoptosis. Additionally, the activation of caspase-3 and caspase-9 was specifically weakened in Csseverin overexpression PLC cells. The overexpression of Csseverin reduced the increase in intracellular free Ca2+, thereby inhibiting MPTP opening in PLC cells. Moreover, Bax mitochondrial translocation and the subsequent release of Cyt c were downregulated in apoptotic Csseverin overexpression PLC cells. CONCLUSIONS: The present findings suggest that Csseverin, a component of CsESPs, confers protection from human HCC cell apoptosis via the inactivation of membranous Ca2+ channels. Csseverin might be involved in the process of HCC through C. sinensis infestation in affected patients.

Clonorchis sinensis lysophospholipase A upregulates IL-25 expression in macrophages as a potential pathway to liver fibrosis.

BACKGROUND: Liver fibrosis is an excessive wound-healing reaction that requires the participation of inflammatory cells and hepatic stellate cells (HSCs). The pathogenesis of liver fibrosis caused by viruses and alcohol has been well characterized, but the molecular mechanisms underlying liver fibrosis induced by the liver fluke Clonorchis sinensis are poorly understood. Lysophospholipase A (LysoPLA), which deacylates lysophospholipids, plays a critical role in mediating the virulence and pathogenesis of parasites and fungi; however, the roles of C. sinensis lysophospholipase A (CsLysoPLA) in C. sinensis-induced liver fibrosis remain unknown. METHODS: A mouse macrophage cell line (RAW264.7) was cultured and treated with CsLysoPLA. IL-25 and members of its associated signaling pathway were detected by performing quantitative real-time PCR, Western blotting and immunofluorescent staining. A human hepatic stellate cell line (LX-2) was cultured and exposed to IL-25. LX-2 cell activation markers were examined via quantitative real-time PCR, Western blotting and immunofluorescent staining. Migration was analyzed in transwell plates. RESULTS: Treating RAW264.7 cells with CsLysoPLA significantly induced IL-25 expression. Elevated PKA, B-Raf, and ERK1/2 mRNA levels and phosphorylated B-Raf and ERK1/2 were detected in CsLysoPLA-stimulated RAW264.7 cells. The PKA inhibitor H-89 weakened B-Raf and ERK1/2 phosphorylation whereas the AKT activator SC79 attenuated ERK1/2 phosphorylation in RAW264.7 cells. Both H-89 and SC79 inhibited CsLysoPLA-induced IL-25 upregulation. In addition, stimulation of LX-2 cells with IL-25 upregulated the expression of mesenchymal cell markers, including α-smooth muscle actin (α-SMA) and collagen type I (Collagen-I), and promoted cell migration. CONCLUSIONS: CsLysoPLA activates HSCs by upregulating IL-25 in macrophages through the PKA-dependent B-Raf/ERK1/2 pathway and potentially promotes hepatic fibrosis during C. sinensis infection.

Clonorchis sinensis granulin: identification, immunolocalization, and function in promoting the metastasis of cholangiocarcinoma and hepatocellular carcinoma.

BACKGROUND: Long-term infections by Clonorchis sinensis are associated with cholangitis, cholecystitis, liver fibrosis, cirrhosis, and even liver cancer. Molecules from the worm play vital roles in disease progress. In the present study, we identified and explored molecular characterization of C. sinensis granulin (CsGRN), a growth factor-like protein from C. sinensis excretory/secretory products (CsESPs). METHODS: The encoding sequence and conserved domains of CsGRN were identified and analysed by bioinformatics tools. Recombinant CsGRN (rCsGRN) protein was expressed in Escherichia coli BL21 (DE3). The localisation of CsGRN in adult worms and Balb/c mice infected with C. sinensis was investigated by immunofluorescence and immunohistochemistry, respectively. Stable CsGRN-overexpressed cell lines of hepatoma cells (PLC-GRN cells) and cholangiocarcinoma cells (RBE-GRN cells) were constructed by transfection of eukaryotic expression plasmid of pEGFP-C1-CsGRN. The effects on cell migration and invasion of CsGRN were assessed through the wound-healing assay and transwell assay. The levels of matrix metalloproteinase 2 and 9 (MMP2 and MMP9) in PLC-GRN or RBE-GRN cells were detected by real-time PCR (qRT-PCR). The levels of E-cadherin, vimentin, N-cadherin, zona occludens proteins (ZO-1), ß-catenin, phosphorylated ERK (p-ERK) and phosphorylated AKT (p-AKT) were analysed by Western blotting. RESULTS: CsGRN, including the conserved GRN domains, was confirmed to be a member of the granulin family. CsGRN was identified as an ingredient of CsESPs. CsGRN was localised in the tegument and testes of the adult worm. Furthermore, it appeared in the cytoplasm of hepatocytes and biliary epithelium cells from infected Balb/c mouse. The enhancement of cell migration and invasion of PLC-GRN and RBE-GRN cells were observed. In addition, CsGRN upregulated the levels of vimentin, N-cadherin, ß-catenin, MMP2 and MMP9, while it downregulated the level of ZO-1 in PLC-GRN/RBE-GRN cells. In total proteins of liver tissue from rCsGRN immunised Balb/c mice, vimentin level decreased, while E-cadherin level increased when compared with the control groups. Meanwhile, the levels of p-ERK reached a peak at 4 weeks post immunisation and the level of p-AKT did at 2 weeks after immunisation. CONCLUSIONS: The encoding sequence and molecular characteristics of CsGRN were identified. As a member of granulin superfamily, CsGRN induced mesenchymal characteristics of PLC and RBE cells and was found to regulate the activities of the downstream molecules of the ERK and PI3K/AKT signalling pathways, which could contribute to the enhancement of cell migration and invasion.