Human Cancer Research Research Articles

Zebrafish xenograft model of human lung cancer for studying the function of LINC00152 in cell proliferation and invasion

BackgroundNumerous studies have shown that long noncoding RNAs play important roles in human cancer progression. Although zebrafish xenografts have recently become a novel in vivo model for human cancer research, whether such models can be used to study the function of long noncoding RNAs remains unknown.MethodsIn vitro studies validated the roles of LINC00152 in the proliferation and invasion of lung cancer cells. In vivo studies of zebrafish xenografts also confirmed these roles of LINC00152. In vivo confocal imaging was used to more accurately evaluate the function of LINC00152 in cell proliferation and migration. Pharmacological experiments were further performed to study the potential ability of LINC00152 downregulation combined with an EGFR inhibitor to treat tumors in cultured cells and the zebrafish xenograft model.ResultsSilencing of LINC00152 suppressed cell proliferation and invasion in SPCA1 and A549 lung cancer cell lines in vitro. In the zebrafish xenograft model, knockdown of LINC00152 reduced the proliferation and migration of lung cancer cells, as indicated by the two imaging methods at different magnifications. Moreover, the knockdown of LINC00152 enhanced the inhibition effect of afatinib for lung cancer progression in cultured cells and the zebrafish xenograft model.ConclusionOur study reveals the oncogenic roles and potential for LINC00152 to be a target for tumor treatment in lung cancer using zebrafish xenograft models, and the findings suggest that this model could be used for functional and application studies of human long noncoding RNAs in tumor biology.

Comprehensive fundamental somatic variant calling and quality management strategies for human cancer genomes.

Next-generation sequencing (NGS) technology has revolutionised human cancer research, particularly via detection of genomic variants with its ultra-high-throughput sequencing and increasing affordability. However, the inundation of rich cancer genomics data has resulted in significant challenges in its exploration and translation into biological insights. One of the difficulties in cancer genome sequencing is software selection. Currently, multiple tools are widely used to process NGS data in four stages: raw sequence data pre-processing and quality control (QC), sequence alignment, variant calling and annotation and visualisation. However, the differences between these NGS tools, including their installation, merits, drawbacks and application, have not been fully appreciated. Therefore, a systematic review of the functionality and performance of NGS tools is required to provide cancer researchers with guidance on software and strategy selection. Another challenge is the multidimensional QC of sequencing data because QC can not only report varied sequence data characteristics but also reveal deviations in diverse features and is essential for a meaningful and successful study. However, monitoring of QC metrics in specific steps including alignment and variant calling is neglected in certain pipelines such as the 'Best Practices Workflows' in GATK. In this review, we investigated the most widely used software for the fundamental analysis and QC of cancer genome sequencing data and provided instructions for selecting the most appropriate software and pipelines to ensure precise and efficient conclusions. We further discussed the prospects and new research directions for cancer genomics.

Detection of miR-155-5p and imaging lung cancer for early diagnosis: in vitro and in vivo study

PurposeCurrently, the routine screening program has insufficient capacity for the early diagnosis of lung cancer. Therefore, a type of chitosan-molecular beacon (CS-MB) probe was developed to recognize the miR-155-5p and image the lung cancer cells for the early diagnosis.MethodsBased on the molecular beacon (MB) technology and nanotechnology, the CS-MB probe was synthesized self-assembly. There are four types of cells—three kinds of animal models and one type of histopathological sections of human lung cancer were utilized as models, including A549, SPC-A1, H446 lung cancer cells, tumor-initiating cells (TICs), subcutaneous and lung xenografts mice, and lox-stop-lox(LSL) K-ras G12D transgenic mice. The transgenic mice dynamically displayed the process from normal lung tissues to atypical hyperplasia, adenoma, carcinoma in situ, and adenocarcinoma. The different miR-155-5p expression levels in these cells and models were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The CS-MB probe was used to recognize the miR-155-5p and image the lung cancer cells by confocal microscopy in vitro and by living imaging system in vivo.ResultsThe CS-MB probe could be used to recognize the miR-155-5p and image the lung cancer cells significantly in these cells and models. The fluorescence intensity trends detected by the CS-MB probe were similar to the expression levels trends of miR-155 tested by qRT-PCR. Moreover, the fluorescence intensity showed an increasing trend with the tumor progression in the transgenic mice model, and the occurrence and development of lung cancer were dynamically monitored by the differen fluorescence intensity. In addition, the miR-155-5p in human lung cancer tissues could be detected by the miR-155-5p MB.ConclusionBoth in vivo and in vitro experiments demonstrated that the CS-MB probe could be utilized to recognize the miR-155-5p and image the lung cancer cells. It provided a novel experimental and theoretical basis for the early diagnosis of the disease. Also, the histopathological sections of human lung cancer research laid the foundation for subsequent preclinical studies. In addition, different MBs could be designed to detect other miRNAs for the early diagnosis of other tumors.

Patient-derived organoids as a potential tool for clinical decision in advanced gallbladder cancer.

e16717 Background: Human gallbladder cancer research lacks in vitro models that can recapitulate the pathophysiology of the original tumor and normal epithelia. Organoids cultures emulate the tumor heterogeneity and have been successively used as translational models to evaluate clinical response to anticancer drug. Here, we established and characterized patients derived organoids culture (PDOs) from human gallbladder cancer patients. Methods: Fresh tissues obtained from gallbladder cancer patients who underwent cholecystectomy were digested with Collagenase/Dispase, filtered and then suspension cells were mixed with Matrigel (1:1) for organoids establishment. Cultures were maintained at 37°C and 5% CO2. The organoids were characterized through histopathology evaluation, immunohistochemistry, and ultra-deed targeted sequencing (TruSeq protocol, Illumina, TSACP; 25 genes). In addition, we evaluated the response of organoids to gemcitabine, cisplatin and Fluorouracil (5-FU), using the CellTiter-Glo 3D luminescence assay. Results: We have successfully established PDOs cultures from six patients with gallbladder adenocarcinoma with a 60% efficiency (6/10). The PDOs recapitulated the histological features and maintained the expression of CK19, Ki67, P53 as compared with the epithelia of primary tumors. Moreover, the PDOs maintained the mutational status of major driver genes, including TP53 and PIK3CA. Finally, tumor organoids showed differential response to standard chemotherapy treatment. GBC-PDO2 and GBC-PDO4 showed high resistant to gemcitabine (IC50 > 10μM), which suggests that the personalized treatment is necessary for generate better response. Conclusions: the established gallbladder tumor organoids exhibited similar histological and genomic features in relation to their original epithelia. PDOs can be considered a valuable tool for future research aimed to understand gallbladder cancer biology and for developing personalized-medicine approaches for advanced gallbladder cancer patients. (Funding Source: FONDECYT #1171463, FONDECYT #1170893 and Fondef IT16I10051).

Naturally-Occurring Canine Mammary Tumors as a Translational Model for Human Breast Cancer.

Despite extensive research over many decades, human breast cancer remains a major worldwide health concern. Advances in pre-clinical and clinical research has led to significant improvements in recent years in how we manage breast cancer patients. Although survival rates of patients suffering from localized disease has improved significantly, the prognosis for patients diagnosed with metastatic disease remains poor with 5-year survival rates at only 25%. In vitro studies using immortalized cell lines and in vivo mouse models, typically using xenografted cell lines or patient derived material, are commonly used to study breast cancer. Although these techniques have undoubtedly increased our molecular understanding of breast cancer, these research models have significant limitations and have contributed to the high attrition rates seen in cancer drug discovery. It is estimated that only 3–6% of drugs that show promise in these pre-clinical models will reach clinical use. Models that can reproduce human breast cancer more accurately are needed if significant advances are to be achieved in improving cancer drug research, treatment outcomes, and prognosis. Canine mammary tumors are a naturally-occurring heterogenous group of cancers that have several features in common with human breast cancer. These similarities include etiology, signaling pathway activation and histological classification. In this review article we discuss the use of naturally-occurring canine mammary tumors as a translational animal model for human breast cancer research.

Optimization of internal reference genes for qPCR in human pancreatic cancer research.

BackgroundPancreatic cancer (PC) has been becoming a common cancer with high mortality and quantitative real-time polymerase chain reaction (qPCR) is one of the best choices for researching gene expression. Internal reference genes, such as actin beta (ACTB) and glyceraldehyde-3-phosphatide hydrogenase (GAPDH) have long been used in relative quantification analysis. But evidence shows that some internal reference genes expression may vary in different tissues, cell lines and different conditions. The present study aimed to find more stable internal reference gene for qPCR experiment in PC.MethodsTotal RNA of human PC tissues were prepared using TRIZOL reagent. qPCR was performed using FastStart Universal SYBR Green Master to reflects the expression of target genes. Normfinder and geNorm were used to analyze the stability of chosen internal reference genesResultsAccording to the results of NormFinder and geNorm, eukaryotic translation initiation factor 2B subunit alpha (EIF2B1) and importin 8 (IPO8) were the same most stable internal reference genes in PCs and non-neoplastic tissues. In addition, EIF2B1 and IPO8 remained the most stable internal reference genes only in PCs. Using a normalization factor NF2 by geNorm as reference, the normalized GAPDH and ACTB expression levels were obviously up-regulated by 3.29- and 2.23-fold change, meanwhile ribosomal protein S17 (RPS17) were down-regulated by 0.77-fold change in PCs comparing with corresponding adjacent tissues.ConclusionsThe use of the combination of EIF2B1 and IPO8 would provide more stable results in differential expression analysis and prognostic analysis of PC.

Identification of a potentially functional circRNA\u2013miRNA\u2013mRNA regulatory network for investigating pathogenesis and providing possible biomarkers of bladder cancer

BackgroundCircular RNAs (circRNAs) have received considerable attention in human cancer research. However, many circRNAs remain to be detected. In our study, we determined novel circRNAs and investigated their effects on bladder cancer (BCa).MethodsMicroarray dataset GSE92675 was downloaded from Gene Expression Omnibus (GEO). Then, we combined computational biology with quantitative real-time polymerase chain reaction (qRT-PCR) to select related circRNAs in BCa. The selected circRNA–microRNA (miRNA)–messenger RNA (mRNA) regulatory subnetwork was determined by Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses.ResultsThe regulatory network constructed from the microarray dataset (GSE92675) contained 49 differentially expressed circRNAs (DECs). GO and KEGG analyses showed that the MAPK and PI3K–AKT signaling pathways were statistically significant. On the basis of qRT-PCR and the degree value calculated by the cytoHubba plugin of Cytoscape, hsa_circ_0011385 was finally confirmed. The subnetwork around hsa_circ_0011385 was constructed. In addition, we created a protein–protein interaction (PPI) network composed of 67 nodes and 274 edges after removing independent nodes. GO and KEGG analyses showed that hubgenes were involved in cell cycle activities. Moreover, they could be regulated by miRNAs and play an eventful role in BCa pathogenesis.ConclusionsWe proposed a novel circRNA–miRNA–mRNA network related to BCa pathogenesis. This network might be a new molecular biomarker and could be used to develop potential treatment strategies for BCa.

Cancer-Related Gene Signature Selection Based on Boosted Regression for Multilayer Perceptron

Gene expression profiling is a useful technique for analyzing cellular function, and gene expression profiles are widely studied in human cancer research. Gene expression data usually consist of a very large number of features and a relatively small number of samples, and extracting a small number of important features from these data is a major challenge of gene expression-based analysis in cancer research. In this paper, we propose an embedded feature selection algorithm using boosted linear regression-based feature selection. The boosting technique is applied to derive the ensemble feature selector and improve the performance of linear regression-based feature selection. The proposed feature selection algorithm, called boosted regression-based feature selection for the multilayer perceptron (BREG-MLP), repeats the boosted feature selection process to extract the smallest feature subset while maintaining good classification performance. We apply the proposed BREG-MLP to some human cancer-related gene expression data sets for the purpose of extracting important features, and we confirm that BREG-MLP offers improved performance compared to single regression-based feature selection methods.

General Introduction—The Background to Human Papillomavirus and Cancer Research

General Introduction—The Background to Human Papillomavirus and Cancer Research

Characterization of a new glioblastoma cell line, GB-val4, with unusual TP53 mutation.

A novel cell line derived from a human glioblastoma (GB), named GB-val4, has been established and characterized. GB-val4 cells were hyperdiploid, with many numerical and structural chromosomal rearrangements. The cell line did not show mutations in IDH1/IDH2 genes or EGFR amplification, but it presented two missense mutations in TP53, which imply a very low p53 protein activity within the cell line. Cells also had gain of TP73 copies, hypermethylation of APC, CASP8 and RASSF1, increased expression of ARF1, CDH1 and NF-κB and decreased expression of CDKN2A. Tumorigenity was demonstrated by transplant of GB-val4 cells into athymic nude mice, where solid tumors were grown. Interestingly, a high percentage of GB-val4 cells presented expression of GSC markers CD133 or CD44. These GSC markers were increased in neurosphere cultures, which better mimic solid tumor conditions and maintain the genetic features of the tumor cells. In this study, we aimed to define the characteristics of this novel cell line and its applications in human cancer research. With its genetic features and a poor p53 activity, GB-val4 cells resemble GB tumors. Moreover, the important presence of GSCs in adherent cultures and especially in neurosphere cultures makes GB-val4 an attractive tool to study cancer stem cells, deepen in the knowledge the molecular pathways of GB and develop new therapeutic strategies for patients with these tumors.

DNA damage and repair measured by comet assay in cancer patients

The last decade witnessed an increase in the use of comet assay for DNA damage monitoring in cancer patients and controls. Apart from case-control studies, reports described the determination of DNA damage prior to (baseline value) and after chemo-/radiotherapy, the treatment resulted in significantly elevated DNA damage. However, studies on DNA damage as a factor reflecting cancer prognosis and therapy prediction are scarce. In most cases, DNA damage was analysed in surrogate tissues. The data on DNA damage are available for 17 types of cancer. The reviewed data unambiguously pinpoint the usefulness of the comet assay in human cancer research due to its sensitivity and cost-effectiveness in evaluating DNA damage associated with the disease and with the treatment.DNA repair capacity (DRC) represents a complex marker for functional evaluation of multigene DNA repair processes in cancer onset with future prospects in personalized prevention and/or cancer treatment. A comparison between studies and more general conclusions are precluded by a variable design of the studies and a lack of standard protocol for both DNA damage and DRC determination. Since cancer is a heterogeneous complex disease, numerous points have to be considered: a) DNA damage and DRC measured in surrogate/target tissues, b) changes in the levels of DNA damage and DRC may be a cause or a consequence of the disease, c) changes in DRC alter sensitivity of tumour cells to antineoplastic drugs, d) one time point-sampling of patients provides insufficient information on the role of DNA damage and its repair in carcinogenesis. Finally, systemic cancer therapy is targeted at DNA damage and its repair. A proper understanding of these processes is a key precondition for the optimisation of therapy regimens, prediction of therapeutic response and prognosis in cancer patients.

Conventionally used reference genes are not outstanding for normalization of gene expression in human cancer research

BackgroundThe selection of reference genes is essential for quantifying gene expression. Theoretically they should be expressed stably and not regulated by experimental or pathological conditions. However, identification and validation of reference genes for human cancer research are still being regarded as a critical point, because cancerous tissues often represent genetic instability and heterogeneity. Recent pan-cancer studies have demonstrated the importance of the appropriate selection of reference genes for use as internal controls for the normalization of gene expression; however, no stably expressed, consensus reference genes valid for a range of different human cancers have yet been identified.ResultsIn the present study, we used large-scale cancer gene expression datasets from The Cancer Genome Atlas (TCGA) database, which contains 10,028 (9,364 cancerous and 664 normal) samples from 32 different cancer types, to confirm that the expression of the most commonly used reference genes is not consistent across a range of cancer types. Furthermore, we identified 38 novel candidate reference genes for the normalization of gene expression, independent of cancer type. These genes were found to be highly expressed and highly connected to relevant gene networks, and to be enriched in transcription-translation regulation processes. The expression stability of the newly identified reference genes across 29 cancerous and matched normal tissues were validated via quantitative reverse transcription PCR (RT-qPCR).ConclusionsWe reveal that most commonly used reference genes in current cancer studies cannot be appropriate to serve as representative control genes for quantifying cancer-related gene expression levels, and propose in this study three potential reference genes (HNRNPL, PCBP1, and RER1) to be the most stably expressed across various cancerous and normal human tissues.

Effect of dihydroartemisinin on epithelial-to-mesenchymal transition in canine mammary tumour cells

Epithelial-to-mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal properties, generating metastases. Dihydroartemisinin (DHA) exhibits potent anti-cancer activities. CMTs is a potential suitable model for studies in human breast cancer research. In the present study, we separated DHA-untreated cells from CMTs cells to be a control group, the rest of the CMTs cells treated by DHA which were composed of three concentrations 5,10 as well as 20 μM. After that we cultivate the cells severally under the condition of 24, 48 and 72 h at 37 °C. CMTs cells were evaluated by Cell viability assay, Wound healing assay, Invasion assay and RT-PCR analysis for the expression of Slug, ZEB1, ZEB2 and Twist. Our results showed that DHA increased the inhibitory rate of CMTs cell and restrain TGF-β1-induced migration and invasion of cells in a time and concentration reliant manner efficaciously. Our result also show DHA inhibits the expression of Slug, ZEB1, ZEB2 and Twist at the mRNA in vitro, the media concentration DHA (10 μM) decreased the expression level of the Slug, ZEB1, ZEB2 mRNA significantly. In conclusion, DHA inhibits canine mammary tumours cells migration and invasiveness by regulating the expression of EMT-related genes.

Establishment of a drug-resistant human cervical cancer cell line and research on its drug-resistance.

To investigate the differences and international connections between the human cervical cancer cell line (HeLa cells) and the Taxol-resistant HeLa cell line (HeLa/Taxol). As parental cells, HeLa cells were cultured in stepwise escalating concentration of Taxol from 0.01 μg/ml (11.7 × 10-9 mol/L) to 0.5 μg/ml (585 × 10-9 mol/L). The drug resistance of HeLa/Taxol cells was detected by methyl-thiazolyl-tetrazolium assay. Real time-polymerase chain reaction (RT-PCR) was conducted to detect the messenger RNA levels of drug resistance genes and apoptosis-related genes. The proteins levels were detected through immunofluorescence and Western blot. Compared with parental HeLa cells, HeLa/Taxol with Taxol resistance had the following biological characteristics: first, they had a lower growth velocity; second, the expression of P-glycoprotein and glutathione S-transferases was significantly increased; Third, the expression of antiapoptotic protein Bcl-2 and apoptosis inhibitor protein survivin was prominently increased. The drug-resistance in HeLa/Taxol is mainly associated with the high expression of multidrug resistance genes, antiapoptotic protein Bcl-2, and apoptosis inhibitor protein survivin as an important reason for the failure of chemotherapy of tumor tissue.

Establishment and characterization of a new triple-negative canine mammary cancer cell line.

Canine mammary tumor (CMT) has always been an ideal animal model for human breast cancer (HBC) research, however, there is a lack of various established CMT cell lines corresponding to HBC cell lines. This study was designed to establish a new type of CMT cell line. The primary tumor, CMT-7364, was identified as the intraductal papillary carcinoma, and showed negative immunoreactivity to estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor-2 (HER-2) by immunohistochemistry (IHC) analysis. The CMT-7364 cell line from this primary tumor also shows a negative immunoreactivity to ER, PR, and HER-2, and was negative to epithelial cell markers and positive to mesenchymal cell markers by immunocytochemistry (ICC) analysis. This cell line, which has been stably cultured for more than 115 passages, and was characterized by epithelial origin with the expression of the epithelial antigen by ICC analysis and invasion ability by transwell analysis. In vivo, tumor mass and metastases in the lung were found after inoculating the CMT-7364 cells in the nude mice model, and the immune-complete mice model respectively. The tissues from the xenograft tumor were also negative to ER, PR, and HER-2 by IHC analysis. Thus, a novel triple negative canine mammary cancer cell line, CMT-7364, was successfully established, which could be used as a promising model for the research of immunotherapy and Epithelial-Mesenchymal Transition (EMT) mechanism of the triple-negative breast cancer both in canine and human.

Management of stage III benign mammary gland tumor in a 10-year-old female dog

A 10-year-old female German shepherd dog was presented with a large pendulous mass which was located in the area of the right inguinal mammary gland extending from M5–M2 with no marked lymphadenomegaly. The mass has progressively increased in size without regression over a period of 1 year. Radiography revealed a radiodense mass of tissue with no apparent metastasis to regional lymph nodes, no pulmonary metastases, and no abdominal viscera metastases. The tumor was staged as T3N1M0 after diagnostic clinical and radiological examinations based on WHO standards for tumor stage. Unilateral radical mastectomy with total mammary gland extirpation was performed and all glands from M5 to M1 on the right side were removed. The extirpated mass was 24 × 8 cm in size, weighed 2.25 kg, and was very firm. The tumor was lobular and was surrounded with thick band of fibrous capsule. Histology revealed numerous chondroblasts in the lacunae with moderate basophilic cytoplasm. These cells were monomorphic and pleomorphic (> 75% pleomorphic). There was anisocytosis and anisokaryosis. A good short-term clinical outcome was observed after surgical treatment and chemotherapy with tamoxifen and prednisolone for 21 days. Following clinical examinations, no pulmonary metastasis or recurrence of tumoral tissues was observed during 1-year post-surgery. Also, because the pathology of mammary gland tumors in dogs is similar to that of humans, it is strongly advocated that dogs are considered as natural animal model of human breast cancer research especially for testing new drugs and development of prophylactic and therapeutic measures.

Application of stable nitrogen isotopes in human colorectal cancer research

Application of stable nitrogen isotopes in human colorectal cancer research

Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.

Human liver cancer research currently lacks in vitro models that faithfully recapitulate the pathophysiology of the original tumour. We recently described a novel, near-physiological organoid culture system, where primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here, we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumours. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumour, allowing discrimination between different tumour tissues and subtypes, even after long term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumourogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug screening testing and lead to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized medicine approaches for the disease.

Application of Cancer Cell Reprogramming Technology to Human Cancer Research.

The cancer stem cell (CSC) hypothesis is an evolving concept of oncogenesis that has recently gained wide acceptance. By definition, CSCs exhibit continuous proliferation and self-renewal, and they have been proposed to play significant roles in oncogenesis, tumor growth, metastasis, chemoresistance, and cancer recurrence. The reprogramming of cancer cells using induced pluripotent stem cell (iPSC) technology is a potential strategy for the identification of CSC-related oncogenes and tumor-suppressor genes. This technology has some advantages for studying the interactions between CSC-related genes and the cancer microenvironment. This approach may also provide a useful platform for studying the mechanisms of CSCs underlying cancer initiation and progression. The present review summarizes the recent advances in cancer cell reprogramming using iPSC technology and discusses its potential clinical use and related drug screening.

Yin-yang regulating effects of cancer-associated genes, proteins, and cells: An ancient Chinese concept in vogue in modern cancer research.

Great achievements have been made in human cancer research, but most of this research is focused on conditions at the microscopic rather than the systemic level. Recent studies have increasingly cited the ancient Chinese theory of yin-yang in an effort to expand beyond the microscopic level. Various cancer-associated genes and proteins such as mitogen-activated protein kinase (MAPK), p38, p53, c-Myc, tumor necrosis factor (TNF)-α, NF-κB, Cyclin D1, and cyclin-dependent kinase (CDK) and cells such as T cells, B cells, macrophages, neutrophils, and fibroblasts have been reported to regulate various types of cancers in a yin-yang manner. These studies have brought the theory of yin-yang into vogue in cancer research worldwide.