• Hepatocellular carcinoma
• cell line
• metastasis
• primary culture
• xenograft

• Humans
• Carcinoma, Hepatocellular/genetics
• Cell Count
• Cell Line
• Liver Neoplasms/genetics
• Sorafenib

Several recent studies have confirmed that intratumoural microorganisms can affect the occurrence and development of hepatocellular carcinoma (HCC); however, their role in tumor progression remains unclear. Hence, there is a need for further research on the role of intratumoural microorganisms in HCC.

To investigate the changes in intratumoural microorganisms in HCC and the effect of Propionibacterium on HCC progression.

HCC and normal liver tissue specimens were subjected to fluorescence in situ hybridization (FISH). After performing 16S rRNA sequencing on HCC and peritumoral tissues to analyze the differences between the two groups. Propionibacterium was cocultured with HCC cells in vitro. Changes in cell proliferation and migration capacity were evaluated. The expression of NF-κB pathway related proteins in tumor cells was compared. The orthotopic liver implantation model and the subcutaneous xenograft model were constructed. liver tissues and subcutaneous tumors were collected 2 weeks later.

FISH demonstrated the presence of microorganisms in HCC and normal liver tissues. 16S rRNA sequencing revealed an abundance of Lysobacter, Lachnospiraceae, Pseudomonas, and Lactobacillus in HCC tissues. The distribution and abundance of Propionibacterium showed differences between HCC and peritumoral tissues (P < 0.05). In vitro studies demonstrated that Propionibacterium and its metabolite propionic acid (PA) inhibited the proliferation and migration of HCC cells (P < 0.05). The expression of the proteins in NF-κB signaling pathway also decreased in HCC cells (P < 0.05).

Microorganisms in HCC and normal liver tissues displayed significant disparities. The PA-producing bacterium Propionibacterium in HCC exerts an effect on the NF-κB pathway, thereby affecting the biological behavior of HCC.

• Hepatocellular carcinoma
• Intratumoural microbiome
• 16S rRNA sequencing
• Propionibacterium

• in vitro investigating
• orthotopic transplantation
• patient-derived HCC cell lines
• therapeutic testing

• Humans
• Animals
• Mice
• Carcinoma, Hepatocellular/drug therapy
• Drug Evaluation, Preclinical
• Liver Neoplasms/drug therapy
• Transplantation, Heterologous
• Cell Line

• KA5390/2-1 Deutsche Forschungsgemeinschaft

• Cell line
• Liver cancer
• Passage
• Primary culture
• Short tandem repeat

• Animals
• Humans
• Carcinoma, Hepatocellular/pathology
• Cell Line, Tumor
• Karyotyping
• Liver Neoplasms/pathology

According to the World Health Organization, more than 250 million people worldwide are chronically infected with the hepatitis B virus, and almost 800.000 patients die annually of mediated liver disorders. Therefore, adequate biological test systems are needed that could fully simulate the course of chronic hepatitis B virus infection, including in patients with hepatocellular carcinoma.

In this study, we will assess the effectiveness of existing protocols for isolation and cultivation of primary cells derived from patients with hepatocellular carcinoma in terms of the yield of viable cells and their ability to replicate the hepatitis B virus using isolation and cultivation methods for adhesive primary cells, flow cytometry and quantitative polymerase chain reaction. Another part of our study will be devoted to evaluating the effectiveness of hepatocellular carcinoma grafting methods to obtain patient-derived heterotopic and orthotopic xenograft mouse avatars using animal X-ray irradiation and surgery procedures and in vivo fluorescent signals visualization and measurements. Our study will be completed by histological methods.

This will be the first extensive comparative study of the main modern methods and protocols for isolation and cultivation primary hepatocellular carcinoma cells and tumor engraftment to the mice. All protocols will be optimized and characterized using the: (1) efficiency of the method for isolation cells from removed hepatocellular carcinoma in terms of their quantity and viability; (2) efficiency of the primary cell cultivation protocol in terms of the rate of monolayer formation and hepatitis B virus replication; (3) efficiency of the grafting method in terms of the growth rate and the possibility of hepatitis B virus persistence and replication in mice. The most effective methods will be recommended for use in translational biomedical research.

• 2D versus 3D cell culture
• isolation of the cell lines
• methods for cell line characterisation
• primary cancer cell lines
• tumour dissociation

• CAFs
• EGFR inhibitors
• cancer cell models
• genomic profile
• penile cancer
• protein expression
• translatomic profile

To establish a primary human gastric cancer cell line.

Fresh gastric cancer tissue samples were separated into a cell suspension, and DMEM/F12 medium containing 10% foetal bovine serum was used for primary culture and subculture. The morphology of the cells was observed under a light microscope, and the cell growth curve was plotted. A soft agar colony formation assay was used to detect the colony formation ability of the cell line. Immunohistochemical methods were used to detect cytokeratin, vimentin and Ki-67, the chromosome G banding method was used to analyse the karyotype of the cells, and the tumourigenic ability of the cells was detected by subcutaneous inoculation of BALB/C nude mice.

We established a gastric cancer cell line from a 68-year-old male patient. This gastric cancer cell line was named XGC-1 and had a doubling time of approximately 48 h. The cell line displayed strong colony formation ability and tumourigenicity in BALB/C nude mice and had complicated chromosomal abnormalities. When nutrients were insufficient, the cells shed and floated in the medium, but adherent growth was observed in nutrient-rich conditions.

The XGC-1 cell line will be useful for future studies of gastric cancer development, progression, metastasis and therapy.

• Cell line
• Primary culture
• Stomach neoplasm
• XGC-1

Background: With recorded under-performance of current standard therapeutic strategies as highlighted by high rates of post-treatment (resection or local ablation) recurrence, resistance to chemotherapy, poor overall survival, and an increasing global incidence, hepatocellular carcinoma (HCC) constitutes a medical challenge. Accumulating evidence implicates the presence of HCC stem cells (HCC-SCs) in HCC development, drug-resistance, recurrence, and progression. Therefore, treatment strategies targeting both HCC-SCs and non-CSCs are essential. Methods: Recently, there has been an increasing suggestion of MALAT1 oncogenic activity in HCC; however, its role in HCC stemness remains unexplored. Herein, we investigated the probable role of MALAT1 in the SCs-like phenotype of HCC and explored likely molecular mechanisms by which MALAT1 modulates HCC-SCs-like and metastatic phenotypes. Results: We showed that relative to normal, cirrhotic, or dysplastic liver conditions, MALAT1 was aberrantly expressed in HCC, similar to its overexpression in Huh7, Mahlavu, and SK-Hep1 HCC cells lines, compared to the normal liver cell line THLE-2. We also demonstrated a positive correlation between MALAT1 expression and poor cell differentiation status in HCC using RNAscope. Interestingly, we demonstrated that shRNA-mediated silencing of MALAT1 concomitantly downregulated the expression levels of β-catenin, Stat3, c-Myc, CK19, vimentin, and Twist1 proteins, inhibited HCC oncogenicity, and significantly suppressed the HCC-SCs-related dye-effluxing potential of HCC cells and reduced their ALDH-1 activity, partially due to inhibited MALAT1-β-catenin interaction. Additionally, using TOP/FOP (TCL/LEF-Firefly luciferase) Flash, RT-PCR, and western blot assays, we showed that silencing MALAT1 downregulates β-catenin expression, dysregulates the canonical Wnt signaling pathway, and consequently attenuates HCC tumorsphere formation efficiency, with concurrent reduction in CD133+ and CD90+ HCC cell population, and inhibits tumor growth in SK-Hep1-bearing mice. Conclusions: Taken together, our data indicate that MALAT1/Wnt is a targetable molecular candidate, and the therapeutic targeting of MALAT1/Wnt may constitute a novel promising anticancer strategy for HCC treatment.

• HCC-SCs
• LncRNA MALAT1
• cancer recurrence
• drug resistance
• tumorspheres

This study aimed to develop a nude mouse model of orthotopic liver transplantation of HCCLM3 human hepatocellular carcinoma (HCC) cell xenografts and the use of imaging and histology to evaluate tumor development and progression.

HCCLM3 cells were injected subcutaneously into 25 healthy male athymic BALB/c (nu/nu) nude mice. The tumors that developed were transplanted into the liver of a new set of nude mice. After four weeks and six weeks, the mice were imaged using ultrasound (US), software-assisted contrast-enhanced ultrasound (CEUS), fluorodeoxyglucose-positron emission tomography (FDG-PET). Histology was performed on the liver and liver tumors, and included immunohistochemistry for vascular endothelial growth factor (VEGF), CD31, CD34, and α-smooth muscle actin (α-SMA).

The success rate for orthotopic tumor transplantation in the mouse liver was 90% (18/20). Liver tumors measured 11.8±2.6 mm in diameter and 525.9±250.8 mm3 in volume on the sixth week. CEUS showed rapid wash-in and washout in the liver tumors, and PET showed low tumor cell metabolism. Bone metastases were present in 45% (9/20) of mice in the sixth week. Immunohistochemistry showed positive expression for VEGF, CD31, CD34, and α-SMA.

The nude mouse orthotopic liver transplantation model of human HCC was shown to be a reliable model that has the potential for future research on the pathogenesis and progression of HCC and studies on drug development.

Unconventional prefoldin RNA polymerase II subunit 5 interactor (URI1) has emerged as an oncogenic driver in hepatocellular carcinoma (HCC). Although the hepatitis B virus (HBV) represents the most common etiology of HCC worldwide, it is unknown whether URI1 plays a role in HBV-related HCC (HCC-B). In the present study, we investigated URI1 expression and its underlying mechanism in HCC-B tissues and cell lines. URI1 gene-promoter activity was determined by a luciferase assay. Human HCC-B samples were used for a chromatin immunoprecipitation assay. We found that c-MYC induced URI1 expression and activated the URI1 promoter through the E-box in the promoter region while the HBx protein significantly enhanced it. The positivity of URI1 expression was significantly higher in HCC-B tumor tissues than in non-HBV-related HCC tumor tissues, suggesting that a specific mechanism underlies URI1 expression in HCC-B. In tumor tissues from HCC-B patients, a significantly higher level of c-MYC was recruited to the E-box than in non-tumor tissues. These results suggest that HBx and c-MYC are involved in URI1 expression in HCC-B. URI1 expression may play important roles in the development and progression of HCC-B because HBx and c-MYC are well-known oncogenic factors in the virus and host, respectively.

• E-box
• HBV-related hepatocellular carcinoma
• HBx
• URI1
• c-MYC

• Carcinoma, Hepatocellular/etiology
• Disease Susceptibility
• Genes, myc
• Hepatitis B/complications
• Hepatitis B/virology
• Hepatitis B virus/genetics
• Humans
• Liver Neoplasms/etiology
• Models, Biological
• Promoter Regions, Genetic
• Repressor Proteins/genetics
• Trans-Activators/genetics
• Transcriptional Activation
• Viral Regulatory and Accessory Proteins

The lack of response to pharmacological treatment constitutes a substantial limitation in the handling of patients with primary liver cancers (PLCs). The existence of active mechanisms of chemoresistance (MOCs) in hepatocellular carcinoma, cholangiocarcinoma, and hepatoblastoma hampers the usefulness of chemotherapy. A better understanding of MOCs is needed to develop strategies able to overcome drug refractoriness in PLCs. With this aim, several experimental models are commonly used. These include in vitro cell-free assays using subcellular systems; studies with primary cell cultures; cancer cell lines or heterologous expression systems; multicellular models, such as spheroids and organoids; and a variety of in vivo models in rodents, such as subcutaneous and orthotopic tumor xenografts or chemically or genetically induced liver carcinogenesis. Novel methods to perform programmed genomic edition and more efficient techniques to isolate circulating microvesicles offer new opportunities for establishing useful experimental tools for understanding the resistance to chemotherapy in PLCs. In the present review, using three criteria for information organization: (1) level of research; (2) type of MOC; and (3) type of PLC, we have summarized the advantages and limitations of the armamentarium available in the field of pharmacological investigation of PLC chemoresistance.

• chemoresistance
• cholangiocarcinoma
• hepatoblastoma
• hepatocellular carcinoma
• multidrug resistance
• resistome

• Diethylnitrosamine
• Hepatocellular carcinoma
• Lactoferrin
• Protien kinase B
• Vascular endothelial growth factor

• Alanine Transaminase/blood
• Animals
• Anticarcinogenic Agents/pharmacology
• Aspartate Aminotransferases/blood
• Cell Proliferation/drug effects
• Chemoprevention
• Diethylnitrosamine
• Lactoferrin/pharmacology
• Liver/drug effects
• Liver/enzymology
• Liver/pathology
• Liver Function Tests
• Liver Neoplasms, Experimental/enzymology
• Liver Neoplasms, Experimental/pathology
• Liver Neoplasms, Experimental/prevention & control
• Male
• Proto-Oncogene Proteins c-akt/metabolism
• Rats, Wistar
• Signal Transduction/drug effects

• biological markers
• carcinoma, hepatocellular
• medical research

Insufficient delivery of systemically administered anticancer drugs to tumors can compromise therapeutic efficacy and develop drug delivery-based therapeutic resistance. Nanotherapeutics such as PEGylated liposomal doxorubicin (PLD) are designed to preferentially accumulate in tumors utilizing enhanced permeation and retention effect. However, their antitumor effects and resulting clinical outcomes are modest and heterogeneous among tumors. Here, we aimed to investigate whether the amount and efficacy of PLD delivered to tumors are tumor site dependent. We established orthotopic primary tumor or liver metastases models of murine breast cancer using 4 T1 cells. PLD showed significant therapeutic effects against tumors that grew in primary mammary sites but not in the liver. We found that differences in therapeutic efficacy were not because of the intrinsic biological resistance of cancer cells but rather were associated with tumor site-dependent differences in transport properties, such as the amount of PLD delivery, blood vessel function, relative vascular permeability, and mechanical pressure in tumors. Thus, transport properties in tumor is site dependent and can be used as phenotypic surrogate markers for tumor drug delivery and therapeutic efficacy.

• LOH of 16q
• Meta-analysis
• Survival
• Wilms’ tumor

• Chromosomes, Human, Pair 16/genetics
• Humans
• Kidney Neoplasms/genetics
• Kidney Neoplasms/mortality
• Loss of Heterozygosity
• Prognosis
• Survival Rate
• Wilms Tumor/genetics
• Wilms Tumor/mortality

• Biomarkers
• Cell Line HCS1220
• Colon cancer liver metastasis
• Karyotype analysis
• STR analysis

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and has poor prognosis. Specially, patients with HCC usually have poor tolerance of systemic chemotherapy, because HCCs develop from chronically damaged tissue that contains considerable inflammation, fibrosis, and cirrhosis. Since HCC exhibits highly heterogeneous molecular characteristics, a proper in vitro system is required for the study of HCC pathogenesis. To this end, we have established two new hepatitis B virus (HBV) DNA-secreting HCC cell lines from infected patients.

Based on these two new HCC cell lines, we have developed chemosensitivity assays for patient-derived multicellular tumor spheroids (MCTSs) in order to select optimized anti-cancer drugs to provide more informative data for clinical drug application. To monitor the effect of the interaction of cancer cells and stromal cells in MCTS, we used a 3D co-culture model with patient-derived HCC cells and stromal cells from human hepatic stellate cells, human fibroblasts, and human umbilical vein endothelial cells to facilitate screening for optimized cancer therapy.

To validate our system, we performed a comparison of chemosensitivity of the three culture systems, which are monolayer culture system, tumor spheroids, and MCTSs of patient-derived cells, to sorafenib, 5-fluorouracil, and cisplatin, as these compounds are typically standard therapy for advanced HCC in South Korea.

In summary, these findings suggest that the MCTS culture system is the best methodology for screening for optimized treatment for each patients with HCC, because tumor spheroids not only mirror the 3D cellular context of the tumors but also exhibit therapeutically relevant pathophysiological gradients and heterogeneity of in vivo tumors.

The online version of this article (10.1186/s13046-018-0752-0) contains supplementary material, which is available to authorized users.

• Hepatocellular carcinoma (HCC)
• MCTS-based chemosensitivity assays
• Multicellular tumor spheroids (MCTS)
• Optimized treatment

Granulin-epithelin precursor (GEP) has been demonstrated to confer enhanced cancer stem-like cell properties in hepatocellular carcinoma (HCC) cell line models in our previous studies. Here, we aimed to examine the GEP-expressing cells in relation to the stem cell related molecules and stem-like cell properties in the prospective HCC clinical cohort. GEP protein levels were significantly higher in HCCs than the paralleled non-tumor liver tissues, and associated with venous infiltration. GEP^high cells isolated from clinical HCC samples exhibited higher levels of stem cell marker CD133, pluripotency-associated signaling molecules β-catenin, Oct4, SOX2, Nanog, and chemodrug transporter ABCB5. In addition, GEP^high cells possessed preferential ability to form colonies and spheroids, and enhanced in vivo tumor-initiating ability while their xenografts were able to be serially subpassaged into secondary mouse recipients. Expression levels of GEP and pluripotency-associated genes were further examined in the retrospective HCC cohort and demonstrated significant correlation of GEP with β-catenin. Notably, HCC patients with high GEP and β-catenin levels demonstrated poor recurrence-free survival. In summary, GEP-positive HCC cells directly isolated from clinical specimens showed β-catenin elevation and cancer stem-like cell properties.

• cancer stem cells
• granulin-epithelin precursor
• hepatocellular carcinoma
• β-catenin

• LOH 16q
• Wilms’ tumor
• meta-analysis
• relapse

• Dysplastic
• Pre-neoplastic
• Primary culture
• Transformed

• Cell line establishment
• Hepatocellular carcinoma
• Patient-derived xenograft

• canine cancer
• cell line panel
• comparative oncology
• doxorubicin
• drug sensitivity
• gene expression
• microRNA