AIMP3 inhibits cell growth and metastasis of lung adenocarcinoma through activating a miR-96-5p-AIMP3-p53 axis

Aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3) is a tumour suppressor, however, the roles of AIMP3 in non-small cell lung cancer (NSCLC) are not explored yet. Here, we reported that AIMP3 significantly inhibited the cell growth and metastasis of NSCLC (lung adenocarcinoma) in vitro and in vivo. We have firstly identified that AIMP3 was down-regulated in human NSCLC tissues compared with adjacent normal lung tissues using immunohistochemistry and western blot assays. Overexpression of AIMP3 markedly suppressed the proliferation and migration of cancer cells in a p53-dependent manner. Furthermore, we observed that AIMP3 significantly suppressed tumour growth and metastasis of A549 cells in xenograft nude mice. Mechanically, we identified that AIMP3 was a direct target of miR-96-5p, and we also observed that there was a negative correlation between AIMP3 and miR-96-5p expression in paired NSCLC clinic samples. Ectopic miR-96-5p expression promoted the proliferation and migration of cancer cells in vitro and tumour growth and metastasis in vivo which partially depended on AIMP3. Taken together, our results demonstrated that the axis of miR-96-5p-AIMP3-p53 played an important role in lung adenocarcinoma, which may provide a new strategy for the diagnosis and treatment of NSCLC.     

Development of an orthotopic human pancreatic cancer xenograft model using ultrasound guided injection of cells

Mice have been employed as models of cancer for over a century, providing significant advances in our understanding of this multifaceted family of diseases. In particular, orthotopic tumor xenograft mouse models are emerging as the preference for cancer research due to increased clinical relevance over subcutaneous mouse models. In the current study, we developed orthotopic pancreatic cancer xenograft models in mice by a minimally invasive method, ultrasound guided injection (USGI) comparable to highly invasive surgical orthotopic injection (SOI) methods. This optimized method prevented injection complications such as recoil of cells through the injection canal or leakage of cells out of the pancreas into the peritoneal cavity. Tumor growth was monitored in vivo and quantified by ultrasound imaging weekly, tumors were also detected by in vivo fluorescence imaging using a tumor targeted molecular probe. The mean tumor volumes for the USGI and SOI models after 2 weeks of tumor growth were 205 mm(3) and 178 mm(3) respectively. By USGI of human pancreatic cancer cell lines, human orthotopic pancreatic cancer xenografts were established. Based on ultrasound imaging, the orthotopic human pancreatic cancer xenograft take rate was 100% for both human pancreatic cancer cell lines used, MiaPaCa-2 and Su86.86, with mean tumor volumes of 28 mm(3)and 30 mm(3). We demonstrated that this USGI method is feasible, reproducible, facile, minimally invasive and improved compared to the highly-invasive SOI method for establishing orthotopic pancreatic tumor xenograft models suitable for molecular imaging.     

LJH-OS人骨肉瘤裸鼠原位移植模型的建立及其生物学特性

用人成骨肉瘤细胞系LJH- OS的传代移植瘤组织作为移植材料,进行胫骨原位移植及皮下移植。结果发现胫骨原位移植的潜伏期较短,生长快。皮下移植瘤呈局限性膨胀性生长,有不完整的纤维包膜,未见肺转移,观察7 周无明显消瘦;而胫骨原位移植瘤浸润基层,无纤维包膜,且发生肺转移,7 周时有明显消瘦。原位移植的裸鼠血清ALP水平高于皮下移植者。说明裸鼠胫骨微环境较皮下组织更适于人骨肉瘤的浸润及转移表达,裸鼠胫骨原位移植模型的恶性生物学行为更接近临床骨肉瘤患者的实际情况,该原位移植模型的建立为骨肉瘤的研究提供了良好的实验模型。     

Haishengsu, a Protein from Shellfish Tegillarca L. granosa, Inhibits the Growth and the Activity of Matrix Metalloproteinases-2 and -9 in Human Lung Carcinoma

Haishengsu (HSS) is a seashell protein extracted from Tegillarca L. granosa, a type of Malaysian shellfish. Previous in vitro studies showed that HSS might possess biological anticancer activity. In this combined in vitro and in vivo study, we investigated the inhibitory effects of HSS on tumor growth, invasion, and metastasis using human lung carcinoma cell lines A549 and NCI-H292, both intensely positive for matrix metalloproteinases-2 (MMP-2) and MMP-9. HSS significantly inhibited the proliferation of A549 and NCI-H292 as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The transwell chamber assay showed that HSS effectively blocked the invasion and migration of the carcinoma cells through the reconstituted extracellular matrix (Matrigel). Gelatin zymography analysis revealed that the secretion and activity of MMP-2 and MMP-9 in the supernatants of the cultured cells A549 and NCI-H292 were decreased after treatment with HSS. The levels of MMP-2 and MMP-9 in these cancer cells were further examined by Western blot assay in which a significant decrease of MMP-2 and MMP-9 was observed in A549 and NCI-H292 cells after 24 h of exposure to HSS. The anticancer activity of HSS was verified in a mouse model in which HSS delayed the growth of A549 xenografts after 3 weeks of oral administration. Inhibition of MMP-2 and MMP-9 expression was also demonstrated in the A549 xenografts as determined by Western blot analysis. These results suggest that HSS is a novel seashell protein that cannot only inhibit tumor growth but also prevent tumor invasion and metastasis through suppressing the activity of MMP-2 and MMP-9.     

三株荧光素酶标记的小鼠乳腺癌细胞在小鼠体内生长及转移的比较

目的采用活体成像技术比较三株荧光素酶标记的小鼠乳腺癌细胞在小鼠体内生长及转移情况,为研究肿瘤转移提供理想的动物模型以及活体分析方法。方法以荧光素酶（luciferase,Luc）作为报告基因导入小鼠乳腺癌细胞4T1、66c14和4TO7中,经G418筛选获得稳定表达荧光素酶的细胞克隆并扩大培养。标记细胞稀释成1×107cells/mL,取0.1 mL进行乳腺原位及尾静脉接种BALB/c小鼠,制作小鼠乳腺原位和尾静脉移植瘤模型,比较三株细胞在小鼠体内生长及转移情况。结果获得稳定表达荧光素酶基因的细胞克隆,将Luc标记的4T1、66c14、4TO7细胞对BALB/c小鼠乳腺原位接种后7 d,均有肿瘤生长,接种后28 d,4T1细胞乳腺原位移植瘤最大,66c14细胞瘤体次之,4TO7细胞瘤体最小;接种后35 d,三株细胞乳腺原位移植瘤大小较一致,但4T1和66c14原位移植瘤均发生转移,其中4T1细胞较66c14细胞转移严重,而4TO7细胞未见转移;接种后42 d,三株细胞乳腺原位移植瘤大小无明显差别,而4T1和66c14细胞随天数的增加,移植瘤转移程度逐渐严重,4T1较66c14细胞转移更严重,呈广泛性转移,4TO7细胞仍未见转移。将Luc标记的4T1、66c14、4TO7细胞对BALB/c小鼠尾静脉接种后7 d,小动物活体成像发现小鼠肺部均能检测到荧光,其中4T1细胞接种的小鼠肺部荧光信号最强,且小鼠陆续死亡;4TO7细胞接种小鼠肺部荧光信号次之;66c14细胞接种小鼠肺部荧光信号最弱。尾静脉接种后14 d,4TO7和66c14细胞随着观察天数的增加,转移程度逐渐严重,4TO7细胞接种小鼠肺部荧光信号较66c14细胞强且小鼠陆续死亡。结论乳腺原位自发转移模型较尾静脉转移模型更真实反应了肿瘤细胞在体的转移特性,且能完整地呈现肿瘤转移的全过程,可作为研究肿瘤转移的最理想模型。     

Self-renewing Pten-/- TP53-/- protospheres produce metastatic adenocarcinoma cell lines with multipotent progenitor activity

Prostate cancers of luminal adenocarcinoma histology display a range of clinical behaviors. Although most prostate cancers are slow-growing and indolent, a proportion is aggressive, developing metastasis and resistance to androgen deprivation treatment. One hypothesis is that a portion of aggressive cancers initiate from stem-like, androgen-independent tumor-propagating cells. Here we demonstrate the in vitro creation of a mouse cell line, selected for growth as self-renewing stem/progenitor cells, which manifests many in vivo properties of aggressive prostate cancer. Normal mouse prostate epithelium containing floxed Pten and TP53 alleles was subjected to CRE-mediated deletion in vitro followed by serial propagation as protospheres. A polyclonal cell line was established from dissociated protospheres and subsequently a clonal daughter line was derived. Both lines demonstrate a mature luminal phenotype in vitro. The established lines contain a stable minor population of progenitor cells with protosphere-forming ability and multi-lineage differentiation capacity. Both lines formed orthotopic adenocarcinoma tumors with metastatic potential to lung. Intracardiac inoculation resulted in brain and lung metastasis, while intra-tibial injection induced osteoblastic bone formation, recapitulating the bone metastatic phenotype of human prostate cancer. The cells showed androgen receptor dependent growth in vitro. Importantly, in vivo, the deprivation of androgens from established orthotopic tumors resulted in tumor regression and eventually castration-resistant growth. These data suggest that transformed prostate progenitor cells preferentially differentiate toward luminal cells and recapitulate many characteristics of the human disease.     

人胰腺癌裸鼠异种移植瘤模型的生物发光成像和超声成像比较

目的利用荧光素酶基因标记的人胰腺癌细胞株Capan-2建立胰腺癌裸鼠移植模型,评价生物发光和小动物超声成像在移植瘤模型建立中的作用。方法将表达荧光素酶基因的真核表达载体转入人胰腺癌细胞Capan-2,将1×106人胰腺癌细胞悬液分别接种于裸鼠胰腺和右后肢皮下,使其成瘤。生物发光成像和小动物超声成像系统观察肿瘤的生长情况。结果肿瘤细胞原位移植成功率为75%,皮下移植成功率为100%。生物发光成像系统在肿瘤细胞原位接种第7天,可以观察到肿瘤发光;小动物超声成像系统在肿瘤细胞皮下接种第7天,可以测量肿瘤的大小,但在肿瘤细胞原位接种的第7天不能测量肿瘤的大小。另外肿瘤细胞在裸鼠皮下生长的速度比原位生长速度快3倍左右。结论生物发光成像系统更适用于肿瘤早期监测,为深入研究胰腺癌的发生发展、侵袭转移机制提供理想工具。     

Bioluminescent orthotopic mouse models of human localized non-small cell lung cancer: feasibility and identification of circulating tumour cells

Background

Preclinical models of non-small cell lung cancer (NSCLC) require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC.

Methods

Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models.

Results

Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours.

Conclusions

Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.     

Bimodal paramagnetic and fluorescent liposomes for cellular and tumor magnetic resonance imaging

A novel bimodal fluorescent and paramagnetic liposome is described for cellular labeling. In this study, we show the synthesis of a novel gadolinium lipid, Gd.DOTA.DSA, designed for liposomal cell labeling and tumor imaging. Liposome formulations consisting of this lipid were optimized in order to allow for maximum cellular entry, and the optimized formulation was used to label HeLa cells in vitro. The efficiency of this novel bimodal Gd-liposome formulation for cell labeling was demonstrated using both fluorescence microscopy and magnetic resonance imaging (MRI). The uptake of Gd-liposomes into cells induced a marked reduction in their MRI T 1 relaxation times. Fluorescence microscopy provided concomitant proof of uptake and revealed liposome internalization into the cell cytosol. The optimized formulation was also found to exhibit minimal cytotoxicity and was shown to have capacity for plasmid DNA (pDNA) transfection. A further second novel neutral bimodal Gd-liposome is described for the labeling of xenograft tumors in vivo utilizing the enhanced permeation and retention effect (EPR). Balb/c nude mice were inoculated with IGROV-1 cells, and the resulting tumor was imaged by MRI using these in vivo Gd-liposomes formulated with low charge and a poly(ethylene glycol) (PEG) calyx for long systemic circulation. These Gd-liposomes which were less than 100 nm in size were shown to accumulate in tumor tissue by MRI, and this was also verified by fluorescence microscopy of histology samples. Our in vivo tumor imaging results demonstrate the effectiveness of MRI to observe passive targeting of long-term circulating liposomes to tumors in real time, and allow for MRI directed therapy, wherein the delivery of therapeutic genes and drugs to tumor sites can be monitored while therapeutic effects on tumor mass and/or size may be simultaneously observed, quantitated, and correlated.     

Comprehensive Evaluation of the Anti-Angiogenic and Anti-Neoplastic Effects of Endostar on Liver Cancer through Optical Molecular Imaging

Molecular imaging enables non-invasive monitoring of tumor growth, progression, and drug treatment response, and it has become an important tool to promote biological studies in recent years. In this study, we comprehensively evaluated the in vivo anti-angiogenic and anti-neoplastic effects of Endostar on liver cancer based on the optical molecular imaging systems including micro-computer tomography (Micro-CT), bioluminescence molecular imaging (BLI) and fluorescence molecular tomography (FMT). Firefly luciferase (fLuc) and green fluorescent protein (GFP) dual labeled human hepatocellular carcinoma cells (HCC-LM3-fLuc-GFP cells) were used to establish the subcutaneous and orthotopic liver tumor model. After the tumor cells were implanted 14∼18 days, Endostar (5 mg/kg/day) was administered through an intravenous tail vein injection for continuous 14 days. The computer tomography angiography (CTA) and BLI were carried out for the subcutaneous tumor model. FMT was executed for the orthotopic tumor model. The CTA data showed that tumor vessel formation and the peritumoral vasculature of subcutaneous tumor in the Endostar treatment group was significantly inhibited compared to the control group. The BLI data exhibited the obvious tumor inhibition day 8 post-treatment. The FMT detected the tumor suppression effects of Endostar as early as day 4 post-treatment and measured the tumor location. The above data confirmed the effects of Endostar on anti-angiogenesis and tumor suppression on liver cancer. Our system combined CTA, BLI, and FMT to offer more comprehensive information about the effects of Endostar on the suppression of vessel and tumor formation. Optical molecular imaging system enabled the non-invasive and reliable assessment of anti-tumor drug efficacy on liver cancer.     

The ARF tumor suppressor inhibits tumor cell colonization independent of p53 in a novel mouse model of pancreatic ductal adenocarcinoma metastasis

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.     

活体生物发光与荧光成像技术在干细胞研究中的应用

活体生物发光与荧光成像技术是近年发展起来的新兴技术,以其操作简便、灵敏度高、创伤性小在生命科学研究中有着较大的优势,目前已被广泛应用于基因标记、细胞凋亡、免疫细胞研究、肿瘤转移等诸多领域,尤其在新兴的干细胞研究方面更是发挥着不可替代的作用。综述了活体生物发光与荧光成像技术的原理、优势、应用范围及发展前景,特别对近年来该技术在胚胎干细胞的肝向分化、人造血干细胞重建小鼠造血系统、神经祖细胞治疗中枢神经系统肿瘤等方面的应用做了详细介绍。     

In Vivo Bioluminescent Imaging of Irradiated Orthotopic Pancreatic Cancer Xenografts in Nonobese Diabetic-Severe Combined Immunodeficient Mice: A Novel Method for Targeting and Assaying Efficacy of Ionizing Radiation

Adenocarcinoma of the pancreas is a lethal malignancy, and better models to study tumor behavior in vivo are needed for the development ofmore effective therapeutics. Ionizing radiation is a treatment modality that is commonly used in the clinical setting, in particular, for locally confined disease; however, good model systems to study the effect of ionizing radiation in orthotopic tumors have not been established. In an attempt to create clinically relevant models for studying treatments directed against pancreatic cancer, we have defined a methodology to measure the effect of varying doses of radiation in established human pancreatic cancer orthotopic xenografts using two different pancreatic cancer cell lines (Panc-1 and BXPC3) infected with a lentiviral vector expressing CMV promoter-driven luciferase to allow bioluminescence imaging of live animals in real time. Quantifiable photon emission from luciferase signaling in vivo correlated well with actual tumor growth. Bioluminescence imaging of the established pancreatic xenografts was used to direct delivery of radiation to the orthotopic tumors and minimize off-target adverse effects. Growth delay was observed with schedules in the range of 7.5 Gy in five fractions to 10 Gy in four fractions, whereas doses 3 Gy or higher produced toxic adverse effects. In conclusion, we describe a model in which the effects of ionizing radiation, alone or in combination with other therapeutics, in orthotopic xenografts, can be studied.     

Protein arginine methyltransferase 5 is essential for growth of lung cancer cells

PRMT5 (protein arginine methyltransferase 5) is an enzyme that catalyses transfer of methyl groups from S-adenosyl methionine to the arginine residues of histones or non-histone proteins and is involved in a variety of cellular processes. Although it is highly expressed in some tumours, its direct role in cancer growth has not been fully investigated. In the present study, in human lung tissue samples we found that PRMT5 was highly expressed in lung cancer cells, whereas its expression was not detectable in benign lung tissues. Silencing PRMT5 expression strongly inhibited proliferation of lung adenocarcinoma A549 cells in tissue culture, and silencing PRMT5 expression in A549 cells also abolished growth of lung A549 xenografts in mice. In vitro and in vivo studies showed that the cell growth arrest induced by loss of PRMT5 expression was partially attributable to down-regulation of fibroblast growth factor receptor signalling. These results suggest that PRMT5 and its methyltransferase activity is essential for proliferation of lung cancer cells and may serve as a novel target for the treatment of lung cancer.     

GFP标记的肿瘤生长和转移的整体荧光成像

Fugene 6脂质体介导pEGFP-C1转染人源肺癌细胞(SPC-A1),经G418抗性筛选和96孔板有限稀释获得稳定高表达GFP的单克隆细胞株SPC-A1-EGFP。裸鼠腹腔注射SPC-A1-EGFP细胞建立自发转移模型;裸鼠尾静脉注射SPC-A1-EGFP细胞建立实验转移模型。利用整体光学成像系统(wllole-body optical imaging system)对荷瘤鼠整体荧光成像。结果表明,整体光学成像系统可实时非侵入监测腹腔肿瘤生长和扩散过程,通过胸腔皮瓣窗chest—wall skin-flap window)可低侵入检测肺转移。该研究为在体监测原位移植瘤的自发转移和发现抗肿瘤新药物提供了良好实验平台。     

miR-146b Reverses epithelial-mesenchymal transition via targeting PTP1B in cisplatin-resistance human lung adenocarcinoma cells

Epithelial-mesenchymal transformation (EMT) is associated with drug resistance in human lung adenocarcinoma cells, but its specific mechanism has not been clarified. In this study, we investigated the effect of miRNA-146b on EMT in cisplatin (DDP) resistant human lung adenocarcinoma cells and the corresponding mechanism. Cisplatin resistant (CR) human lung adenocarcinoma cells (A549/DDP and H1299/DDP) were established, and the EMT characteristics and invasion and metastasis ability of CR cells were determined by tumor cell-related biological behavior experiments. The role of miR-146b in EMT of CR cells was determined by in vitro functional test. The targeted binding of miR-146b to protein tyrosine phosphatase 1B (PTP1B) was verified by biological information and double luciferin gene reporting experiments. The effect of miR-146b on tumor growth and EMT phenotype in vivo was investigated by establishing the xenotransplantation mouse model. Compared with the control group, H1299/DDP and A549/DDP cells showed the enhanced EMT phenotypes, invasion and migration ability. Besides, miR-146b was lowly expressed in H1299/DDP and A549/DDP cells. More importantly, overexpressed miR-146b could specifically bind to PTP1B, thus inhibiting the EMT process and ultimately reducing CR in H1299/DDP and A549/DDP cells. Finally, overexpressed miR-146b observably inhibited tumor growth in xenograft model mice and inhibited the EMT phenotype of A549/DDP cells in vivo by regulating the expressions of EMT-related proteins. Overexpressed miR-146b could reverse the EMT phenotype of CR lung adenocarcinoma cells by targeting PTP1B, providing new therapeutic directions for CR of lung adenocarcinoma cells.     

Nuclear architecture supports integration of physiological regulatory signals for transcription of cell growth and tissue-specific genes during osteoblast differentiation

Metastatic model of human tumor xenografts have been developed using orthotopic transplantation of histologically intact tissue (onplantation) of lung, stomach, colon, pancreatic, prostate and bladder carcinomas. These models represent the entire process of the metastasis, consisting of local tumor growth, vascular and lymphatic invasion at the local site, flow in the vessels and lymphatic, extravasation at the metastatic organs, and seeding and growth at relevant metastatic sites. Orthotopically transplanted human small-cell lung carcinoma displayed a different chemosensitivity pattern compared with the subcutaneous transplanted model, suggesting different pharmacodynamics between the orthotopic lung and the ectopic subcutaneous sites. The intact-tissue orthotopic-onplantation model seems to be useful to study the mechanism of metastasis for discovery of antimetastatic agents and for the patient tumors and for this treatment design.