The impact of initial tumor microenvironment on imaging phenotype.

Models of human cancer, to be useful, must replicate human disease with high fidelity. Our focus in this study is rat xenograft brain tumors as a model of human embedded cerebral tumors. A distinguishing signature of such tumors in humans, that of contrast-enhancement on imaging, is often not present when the human cells grow in rodents, despite the xenografts having nearly identical DNA signatures to the original tumor specimen. Although contrast enhancement was uniformly evident in all the human tumors from which the xenografts' cells were derived, we show that long-term contrast enhancement in the model tumors may be determined conditionally by the tumor microenvironment at the time of cell implantation. We demonstrate this phenomenon in one of two patient-derived orthotopic xenograft (PDOX) models using cancer stem-like cell (CSC)-enriched neurospheres from human tumor resection specimens, transplanted to groups of immune-compromised rats in the presence or absence of a collagen/fibrin scaffolding matrix, Matrigel. The rats were imaged by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and their brains were examined by histopathology. Targeted proteomics of the PDOX tumor specimens grown from CSC implanted with and without Matrigel showed that while the levels of the majority of proteins and post-translational modifications were comparable between contrast-enhancing and non-enhancing tumors, phosphorylation of Fox038 showed a differential expression. The results suggest key proteins determine contrast enhancement and suggest a path toward the development of better animal models of human glioma. Future work is needed to elucidate fully the molecular determinants of contrast-enhancement.

Angiopoietin-1 Promotes the Integrity of Neovascularization in the Subcutaneous Matrigel of Type 1 Diabetic Rats.

OBJECTIVE: This study aimed to investigate the effects of Ang-1 on neovascularization of diabetic organs by subcutaneous Matrigel angiogenesis model, established in type 1 diabetic rats. METHODS: Ang-1 adenoviral vector was constructed. The rat model was established by STZ and divided into four group. The Matrigel was inserted subcutaneously into the abdominal cavity of rats at 8 weeks, the treatment group was injected with Ang-1 adenovirus vector via tail vein, and the rats were sacrificed at 10 weeks. Neovascularization of Matrigel was observed with transmission electron microscopy. The marker of vascular endothelial cell and pericyte were detected by immunofluorescence. Immunohistochemical detection of the neovascular endothelial junction protein was performed. RT-PCR was used to determine protein expression of neovascular in Matrigel. RESULTS: Vascular cavity-like structure could be seen in subcutaneous Matrigel of diabetic rats, and the cavity was filled with a lot of red blood cells. Transmission electron microscopy showed that neovascular endothelial structure of the Matrigel was incomplete, while the Ang-1 treatment group had more vascular cavity-like structures, intact vascular endothelial structure, and reduced inflammatory cell infiltration in Matrigel. Additionally, the integrity of vascularization improved, and the marker of pericyte and the cell tight junctions protein was upregulated in Ang-1 treatment group. CONCLUSION: Hyperglycemia could induce pathological angiogenesis in subcutaneous Matrigel of diabetic rats, and Ang-1 could upregulate the expression of intercellular junction protein in subcutaneous Matrigel of diabetic rats and promote the integrity of neovascularization in the subcutaneous Matrigel of diabetic rats.

Laminin-511 Supplementation Enhances Stem Cell Localization With Suppression in the Decline of Cardiac Function in Acute Infarct Rats.

BACKGROUND: The extracellular matrix, in particular basement membrane components such as laminins (LMs), is essential for stem cell differentiation and self-renewal. LM511 and LM221 are the main extracellular matrix components of the epicardium, where stem cells were abundant. Here, we examined whether LMs affected the regeneration process by modulating stem cell activities. METHODS: In vitro, adhesive, and proliferative activities of mesenchymal stem cells (MSCs) were evaluated on LM511 and LM221. To examine the effects of LMs in vivo, we established an acute myocardial infarction model by ligation of the proximal part of the left anterior descending artery at the height of the left atrial appendage and then placed atelocollagen sheets with or without LM511 and LM221 over the anterolateral surface of the left ventricular wall. Four or 8 weeks later, cardiac function, histology, and cytokine expressions were analyzed. RESULTS: MSCs showed greater proliferation and adhesive properties on LM511 than on LM221. In vivo, at 4 weeks, isolectin B4-positive cells were significantly higher in the LM511-transplanted group than in the control group. Moreover, some isolectin B4-positive cells expressed both platelet-derived growth factor receptor α and CD90, suggesting that LM511 enhanced MSC recruitment and attachment at the implanted site. After 8 weeks, these cells were more abundant than at 4 weeks. Transplantation with LM511-conjugated sheets increased the expression of cardioprotective and angiogenic factors. CONCLUSIONS: Transplantation with LM511-conjugated sheets enhanced MSC localization to the implantation site and modulated stem cells activities, leading to angiogenesis in acute myocardial infarction rat models.

New model of proliferative vitreoretinopathy in rabbit for drug delivery and pharmacodynamic studies.

Blinding retinal diseases become more epidemic as the population ages. These diseases, such as diabetic retinopathy and macular edema, are of chronic nature and require protracted drug presence at the disease site. A sustained intravitreal porous silicon delivery system with dexamethasone (pSiO2-COO-DEX) was evaluated in a new rabbit model of proliferative vitreoretinopathy (PVR) in a real treatment design. In contrast to the pretreatment design model, pSiO2-COO-DEX was intravitreally injected into the eyes with active inflammation. Subretinal injection of vascular endothelial growth factor (VEGF) and Matrigel induced a late-onset vitreoretinal inflammation that gradually developed into PVR. This method mimics the human disease better than PVR induced by either intravitreal cell injection or trauma. The pSiO2-COO-DEX intervened eyes had minimal PVR, while balanced saline solution or free dexamethasone intervened eyes had significantly more PVR formation. In addition, adding VEGF to the Matrigel for subretinal injection induced greater inflammation and retinal neovascularization in comparison to only Matrigel injected under the medullary ray. Clinical and pathological examinations, including fundus fluorescein angiography and optical coherence tomography, confirmed these changes. In the current study, neither subretinal injection of Matrigel or subretinal injection of VEGF and Matrigel induced choroidal neovascularization. However, the current PVR model demonstrates a chronic course with moderate severity, which may be useful for drug screening studies.

Extracellular matrix and fibroblast injection produces pterygium-like lesion in rabbits

BACKGROUND: Translational research to develop pharmaceutical and surgical treatments for pterygium requires a reliable and easy to produce animal model. Extracellular matrix and fibroblast are important components of pterygium. The aim of this study was to analyze the effect of the subconjunctival injection of fibroblast cells (NIH3T3 cell line) and exogenous extracellular matrix in rabbits in producing a pterygium-like lesion. METHODS: Six 3-month-old white New Zealand rabbits were injected with 20,000 NIH3T3 cells and 5 µL of Matrigel in the right conjunctiva, and with only 5 µL of Matrigel in the left conjunctiva. The eyes were photographed under a magnification of 16× using a 12-megapixel digital camera attached to the microscope on day 1,3 and 7. Conjunctival vascularization was measured by analyzing images to measure red pixel saturation. Area of corneal and conjunctival fibrovascular tissue formation on the site of injection was assessed by analyzing the images on day 3 and 7 using area measurement software. Histopathologic characteristics were determined in the rabbit tissues and compared with a human primary pterygium. RESULTS: The two treatments promoted growth of conjunctival fibrovascular tissue at day 7. The red pixel saturation and area of fibrovascular tissue developed was significantly higher in right eyes (p < 0.05). Tissues from both treatments showed neovascularization in lesser extent to that observed in human pterygium. Acanthosis, stromal inflammation, and edema were found in tissues of both treatments. No elastosis was found in either treatment. CONCLUSIONS: Matrigel alone or in combination with NIH3T3 cells injected into the rabbits' conjunctiva can promote tissue growth with characteristics of human pterygium, including neovascularization, acanthosis, stromal inflammation, and edema. The combination of Matrigel with NIH3T3 cells seems to have an additive effect on the size and redness of the pterygium-like tissue developed.

Reconstitution of a Patterned Neural Tube from Single Mouse Embryonic Stem Cells.

The recapitulation of tissue development and patterning in three-dimensional (3D) culture is an important dimension of stem cell research. Here, we describe a 3D culture protocol in which single mouse ES cells embedded in Matrigel under neural induction conditions clonally form a lumen containing, oval-shaped epithelial structure within 3 days. By Day 7 an apicobasally polarized neuroepithelium with uniformly dorsal cell identity forms. Treatment with retinoic acid at Day 2 results in posteriorization and self-organization of dorsal-ventral neural tube patterning. Neural tube organoid growth is also supported by pure laminin gels as well as poly(ethylene glycol) (PEG)-based artificial extracellular matrix hydrogels, which can be fine-tuned for key microenvironment characteristics. The rapid generation of a simple, patterned tissue in well-defined culture conditions makes the neural tube organoid a tractable model for studying neural stem cell self-organization.

Induced Neural Differentiation of MMP-2 Cleaved (RADA)4 Drug Delivery Systems.

(RADA)4 self-assembling peptides (SAPs) are promising for neural nanoscaffolds with on-demand drug delivery capabilities due to their automated synthesis, in-situ assembly, and potential for interaction with and release of biomolecules. Neuroinflammation cued on-demand drug release, due to up-regulated proteases, may well be vital in the treatment of several neurological diseases. In these conditions, releasing neurotrophic growth factors (NTFs) could potentially lead to neuroprotection and neurogenesis. As such, (RADA)4 was made with the high and low activity matrix metalloproteinase 2 (MMP-2) cleaved sequences, GPQG+IASQ (CP1) and GPQG+PAGQ (CP2), the brain-derived NTF secretion stimulating peptide MVG (DP1) and the ciliary NTF analogue DGGL (DP2). PC-12 cell culture was performed to assess bioactive substrate cell adhesion and NTF specific neuronal differentiation. The laminin-derived IKVAV peptide, known for neural cell attachment and interaction, was tethered to (RADA)4-IKVAV and mixed in increasing increments with (RADA)4 for this purpose. With 1 nanomolar MMP-2 treatment, product formation was observed to increase over a three day period, with (RADA)4/(RADA)4-CP1/CP2 mixture, however there was little difference between groups. Smaller CP1/CP2 concentrations displayed comparable (RADA)4 nanoscale morphology to higher concentrations. Acetylcholine esterase and neural differentiation was observed over 3 days with 1 nM MMP-2 treatment according to the following makeup: 8/1/1 (RADA)4/(RADA)4-IKVAV/(RADA)4-CP1/CP2-DP1/DP2. Signalling gradually increased in all groups, and neurite outgrowth was visible after three days.

Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation In vitro.

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin-1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin-coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co-localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair.

Nanofiber-based hydrogels with extracellular matrix-based synthetic peptides for the prevention of capsular opacification.

Nanofiber-based hydrogels (nanogels) with different, covalently bound peptides were used as an extracellular environment for lens epithelial cells (LECs) in order to modulate the capsular opacification (CO) response after lens surgery in a porcine eye model. Lenses were divided into 15 groups (n = 4 per group), the lens content was removed and the empty capsules were refilled with nanogel without peptides and nanogels with 13 combinations of 5 different peptides: two laminin-derived, two fibronectin-derived, and one collagen IV-derived peptide representing cell adhesion motifs. A control group of 4 lenses was refilled with hyaluronan. After refilling, lenses were extracted from the porcine eye and cultured for three weeks. LECs were assessed for morphology and alpha smooth muscle actin (αSMA) expression using confocal laser scanning microscopy. Compared to hyaluronan controls, lenses filled with nanogel had less CO formation, indicated by a lower αSMA expression (P = 0.004). Microscopy showed differences in morphological cell response within the nanogel refilled groups. αSMA expression in these groups was highest in lenses refilled with nanogel without peptides (9.54 ± 11.29%). Overall, LEC transformation is reduced by the presence of nanogels and the response is improved even further by incorporation of extracellular matrix peptides representing adhesion motifs. Thus, nanomaterials targeting biological pathways, in our case interactions with integrin signaling, are a promising avenue toward reduction of CO. Further research is needed to optimize nanogel-peptide combinations that fully prevent CO.

In vivo Tube Assay: An Optimised Protocol of the Directed in vivo Angiogenesis Assay by Implementing Immunohistochemistry.

BACKGROUND: Angiogenesis, the formation of new blood vessels, is an essential process under physiological and pathological conditions. METHOD: Here, we improved the directed in vivo angiogenesis assay (DIVAA®) test, which is based on the usage of small Matrigel-filled tubes that are implanted into mice subcutaneously for a period of up to 15 days. The subsequent ex vivo assessment of neoangiogenesis within the silicon tubes is then achieved by fluorometry. RESULTS: We showed that the immunohistochemical quantification of the ingrowth of endothelial cells, based on CD31, was superior to the fluorometric quantification advised in the manufacturer's instructions. We optimised the explantation procedure, ensuring the complete recovery of the ingrown vessels. Using this modified protocol, we investigated the effect of the length of stay of the implanted tubes as well as of the concentration of the growth factors VEGF and FGF on the assay. CONCLUSION: Our improved protocol offered an effective and reliable alternative to the original assay, which is expected to facilitate in vivo research on angiogenesis and, thus, might drive the development of novel therapeutic agents.

Physical supports from liver cancer cells are essential for differentiation and remodeling of endothelial cells in a HepG2-HUVEC co-culture model.

Blood vessel remodeling is crucial in tumor growth. Growth factors released by tumor cells and endothelium-extracellular matrix interactions are highlighted in tumor angiogenesis, however the physical tumor-endothelium interactions are highly neglected. Here, we report that the physical supports from hepatocellular carcinoma, HepG2 cells, are essential for the differentiation and remodeling of endothelial cells. In a HepG2-HUVEC co-culture model, endothelial cells in direct contact with HepG2 cells could differentiate and form tubular structures similar to those plated on matrigel. By employing HepG2 cell sheet as a supportive layer, endothelial cells formed protrusions and sprouts above it. In separate experiments, fixed HepG2 cells could stimulate endothelial cells differentiation while the conditioned media could not, indicating that physical interactions between tumor and endothelial cells were indispensable. To further investigate the endothelium-remodeling mechanisms, the co-culture model was treated with inhibitors targeting different angiogenic signaling pathways. Inhibitors targeting focal adhesions effectively inhibited the differentiation of endothelial cells, while the growth factor receptor inhibitor displayed little effect. In conclusion, the co-culture model has provided evidences of the essential role of cancer cells in the differentiation and remodeling of endothelial cells, and is a potential platform for the discovery of new anti-angiogenic agents for liver cancer therapy.

Self-assembling nanofibers alter the processing of amyloid precursor protein in a transgenic mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is one of the most common dementia, which is not effectively cured to date. Amyloid-beta (Abeta) deposition cascade and disintegrity of brain extracellular matrix (ECM) scaffold attribute to the progress of AD. Thus, it maybe an effective way to treat AD by altering the processing of amyloid precursor protein (APP) and regaining the integrity of ECM. The peptide amphiphile (PA) with a laminin epitope isoleucine-lysine-valine-alanine-valine (IKVAV) (IKVAV-PA) can be trigged into ECM in vivo. In addition, IKVAV-PA could significantly improve cognitive impairment with remarkable increase of endoneurogensis in the hippocampus, as well as reduction of burden of amyloid plaque in the brain. METHODS: We used heterozygous AbetaPPswe/PS1dE9 double transgenic mice as the animal model of AD. After 1 week of initial stereotaxic administration into bilateral hippocampus, the mice were subjected to the Morris Water Maze (MWM) test. At the end of MWM test, immunohistochemical staining, Western blot and real-time polymerase chain reaction (PCR) were performed in mice. RESULTS: Here we showed that IKVAV-PA significantly improved cognitive impairment accompanying with reducing the burden of Abeta plaques, as well as the levels of soluble Abeta1-40 and Abeta1-42 in the cortex and hippocampus after 2 weeks of initial administration into bilateral hippocampus. Further examination demonstrated that IKVAV-PA also altered the processing of APP via inhibiting the gene expression of beta-secretase (BACE1), as well as improving the gene expression of insulin-degrading enzyme (IDE) and neprilysin (NEP). CONCLUSION: Our data suggest that IKVAV-PA may serve as an alternative therapeutic intervention for treating the learning and memory losses in AD.

Matrigel: from discovery and ECM mimicry to assays and models for cancer research.

The basement membrane is an important extracellular matrix that is found in all epithelial and endothelial tissues. It maintains tissue integrity, serves as a barrier to cells and to molecules, separates different tissue types, transduces mechanical signals, and has many biological functions that help to maintain tissue specificity. A well-defined soluble basement membrane extract, termed BME/Matrigel, prepared from an epithelial tumor is similar in content to authentic basement membrane, and forms a hydrogel at 24-37°C. It is used in vitro as a substrate for 3D cell culture, in suspension for spheroid culture, and for various assays, such as angiogenesis, invasion, and dormancy. In vivo, BME/Matrigel is used for angiogenesis assays and to promote xenograft and patient-derived biopsy take and growth. Studies have shown that both the stiffness of the BME/Matrigel and its components (i.e. chemical signals) are responsible for its activity with so many different cell types. BME/Matrigel has widespread use in assays and in models that improve our understanding of tumor biology and help define therapeutic approaches.

Establishment of an orthotopic pancreatic cancer mouse model: cells suspended and injected in Matrigel.

AIM: To establish an orthotopic mouse model of pancreatic cancer that mimics the pathological features of exocrine pancreatic adenocarcinoma. METHODS: Pan02 cells were suspended in low-temperature Matrigel and injected into the parenchyma of pancreatic tails of C57BL/6 mice, with cells suspended in phosphate buffered saline (PBS) serving as a control. Primary and implanted tumors were confirmed pathologically. The rate of tumor formation and intraperitoneal implantation in the two groups were compared at different time points after injection. Leakage and intra-abdominal dispersion of Matrigel and PBS, both dyed with methylene blue, were compared after injection into the parenchyma of the pancreas. We observed adherence and proliferation in Pan02 cells suspended in Matrigel in vitro. We also compared the pathological manifestation of this orthotopic pancreatic cancer model in the head and tails of the pancreas. The characteristics of the origin of epithelial cells and exocrine markers of established orthotopic pancreatic tumors were confirmed using immunohistochemistry. RESULTS: Diluted Matrigel could form a gel drip in the pancreatic parenchyma, effectively preventing leakage from the injection site and avoiding dispersion in the abdominal cavity. Pan02 cells were able to adhere to a dish, proliferate, and migrate in the gel drip. The tumor formation rate in the Matrigel group was 100% at both 2 and 3 wk after injection, whereas it was 25.0% and 37.5% in the PBS group at 2 and 3 wk, respectively (P < 0.05). The intraperitoneal tumor implantation rate was 75.0% in the PBS group after 3 wk of injection, while it was 12.5% in the Matrigel group (P < 0.05). Hepatoduodenal ligament and duodenal invasions with obstructive jaundice and upper digestive obstruction with mesenteric lymph node metastasis were observed in the pancreatic head group. In the pancreatic tail group, spleen and gastric invasion were dominant, leading to retroperitoneal lymph nodes metastasis. Positive immunohistochemical staining of cytokeratin and negative staining of vimentin and chromogranin A confirmed that the orthotopic pancreatic tumor injected with Pan02 cells suspended in Matrigel was of epithelial origin and expressed exocrine markers of cancer. CONCLUSION: This method of low-temperature Matrigel suspension and injection is effective for establishing an orthotopic mouse model of pancreatic cancer.

Facile bench-top fabrication of enclosed circular microchannels provides 3D confined structure for growth of prostate epithelial cells.

We present a simple bench-top method to fabricate enclosed circular channels for biological experiments. Fabricating the channels takes less than 2 hours by using glass capillaries of various diameters (from 100 µm up to 400 µm) as a mould in PDMS. The inner surface of microchannels prepared in this way was coated with a thin membrane of either Matrigel or a layer-by-layer polyelectrolyte to control cellular adhesion. The microchannels were then used as scaffolds for 3D-confined epithelial cell culture. To show that our device can be used with several epithelial cell types from exocrine glandular tissues, we performed our biological studies on adherent epithelial prostate cells (non-malignant RWPE-1 and invasive PC3) and also on breast (non-malignant MCF10A) cells We observed that in static conditions cells adhere and proliferate to form a confluent layer in channels of 150 µm in diameter and larger, whereas cellular viability decreases with decreasing diameter of the channel. Matrigel and PSS (poly (sodium 4-styrenesulphonate)) promote cell adhesion, whereas the cell proliferation rate was reduced on the PAH (poly (allylamine hydrochloride))-terminated surface. Moreover infusing channels with a continuous flow did not induce any cellular detachment. Our system is designed to simply grow cells in a microchannel structure and could be easily fabricated in any biological laboratory. It offers opportunities to grow epithelial cells that support the formation of a light. This system could be eventually used, for example, to collect cellular secretions, or study cell responses to graduated hypoxia conditions, to chemicals (drugs, siRNA, …) and/or physiological shear stress.

Local co-delivery of pancreatic islets and liposomal clodronate using injectable hydrogel to prevent acute immune reactions in a type 1 diabetes.

PURPOSE: The purpose of this study was to investigate the effect of locally delivered pancreatic islet with liposomal clodronate (Clodrosome®) as an immunoprotection agent for the treatment of type 1 diabetes. METHOD: The bio-distribution of liposomal clodronate in matrigel was checked by imaging analyzer. To verify the therapeutic efficacy of locally delivered islet with liposomal clodronate using injectable hydrogel, four groups of islet transplanted mice (n = 6 in each group) were prepared: 1) the islet group, 2) the islet-Clodrosome group, 3) the islet-Matrigel group, and 4) the islet-Matrigel-Clodrosome group. Immune cell migration and activation, and pro-inflammatory cytokine secretion was evaluated by immunohistochemistry staining and ELISA assay. RESULTS: Cy5.5 labeled liposomes remained in the matrigel for over 7 days. The median survival time of transplanted islets (Islet-Matrigel-Clodrosome group) was significantly increased (>60 days), compared to other groups. Locally delivered liposomal clodronate in matrigel effectively inhibited the activation of macrophages, immune cell migration and activation, and pro-inflammatory cytokine secretion from macrophages. CONCLUSIONS: Locally co-delivered pancreatic islets and liposomal clodronate using injectable hydrogel effectively cured type 1 diabetes. Especially, the inhibition of macrophage attack in the early stage after local delivery of islets was very important for the successful long-term survival of delivered islets.

Prostate specific membrane antigen produces pro-angiogenic laminin peptides downstream of matrix metalloprotease-2.

Prostate specific membrane antigen (PSMA) is a pro-angiogenic cell-surface protease that we previously demonstrated regulates blood vessel formation in a laminin and integrin ß1-dependent manner. Here, we examine the principal mechanism of PSMA activation of integrin ß1. We show that digesting laminin sequentially with recombinant matrix metalloprotease-2 (MMP-2) and PSMA generates small peptides that enhance endothelial cell adhesion and migration in vitro. We also provide evidence that these laminin peptides activate adhesion via integrin α6ß1 and focal adhesion kinase. Using an in vivo Matrigel implant assay, we show that these MMP/PSMA-derived laminin peptides also increase angiogenesis in vivo. Together, our results reveal a novel mechanism of PSMA activation of angiogenesis by processing laminin downstream of MMP-2.

Multiple metastases in a novel LNCaP model of human prostate cancer.

Metastasis is a frequent and lethal consequence of prostate cancer. Current treatments for metastasis are palliative only. Thus, experimental animal models of metastatic prostate cancer are required for investigations of its pathogenesis and for the development of treatment strategies; however, few models exist at present. In the present study, the LNCaP prostate cancer cell line was co-transfected with a PGK-luciferase-GFP lentivirual vector (LNCaP-luc). Repeated subcutaneous injections of LNCaP-luc cells with Matrigel in nude mice followed by isolation of the cells from tumors resulted in the generation of the LNCaP1-luc cell line. We used CCK-8 and Transwell migration assays, western blot analysis and polymerase chain reaction to detect differences in the characteristics between the LNCaP-luc and LNCaP1-luc cells, and used LNCaP cells to generate a mouse model of metastatic prostate cancer by intracardiac injection. Metastasis was evaluated by bioluminescence imaging, and histological and immunohistochemical staining. the characteristics of the LNCaP1-luc cells differed from those of LNCaP cells, and LNCaP1-luc cells showed increased cell proliferation, cell invasion, tumorigenicity and metastasis potential, and underwent epithelial-mesenchymal transition. In addition, the LNCaP1-luc cells induced multiple metastases in mice when injected into the left cardiac muscle.

Primary tumorectomy promotes angiogenesis and pulmonary metastasis in osteosarcoma-bearing nude mice.

PURPOSE: To investigate the effect of primary tumorectomy on angiogenesis and pulmonary metastasis in osteosarcoma-bearing nude mice. METHODS: Osteosarcoma was introduced to nude mice via subcutaneous injection of MG-63 cells. One hundred and eighty osteosarcoma-bearing mice were used equally in 3 parallel experiments. The effect of tumorectomy (TR) on the expression of vascular endothelial growth factor (VEGF) and endostatin was investigated by ELISA. Meanwhile, the effect on angiogenesis was evaluated by Matrigel plug assay, and pulmonary metastasis assessed by calculating the metastatic foci. Sham-operation (SO) and untreated (UT) groups served as controls. RESULTS: The VEGF (TR: 79.55 ± 7.82 pg/mL vs. SO: 110.01 ± 5.69 pg/mL, UT: 123.50 ± 10.41 pg/mL; p < 0.01) and endostatin (TR: 47.09 ± 6.22 ng/mL vs. SO: 117.64 ± 7.39 ng/mL, UT: 126.73 ± 6.55 ng/mL; p<0.01) were down-regulated significantly after tumorectomy, and angiogenesis was significantly promoted simultaneously. The incidence of pulmonary metastatic foci was 80.0% in the TR group, 40.0% in the SO group and 35.0% in the UT group. CONCLUSION: Primary tumorectomy can down-regulate the expression of VEGF and endostatin and promote angiogenesis which leads to the acceleration of pulmonary metastasis. These findings imply that anti-angiogenic treatment can be considered after primary tumorectomy.

Primary tumorectomy promotes angiogenesis and pulmonary metastasis in osteosarcoma-bearing nude mice

PURPOSE: To investigate the effect of primary tumorectomy on angiogenesis and pulmonary metastasis in osteosarcoma-bearing nude mice. METHODS: Osteosarcoma was introduced to nude mice via subcutaneous injection of MG-63 cells. One hundred and eighty osteosarcoma-bearing mice were used equally in 3 parallel experiments. The effect of tumorectomy (TR) on the expression of vascular endothelial growth factor (VEGF) and endostatin was investigated by ELISA. Meanwhile, the effect on angiogenesis was evaluated by Matrigel plug assay, and pulmonary metastasis assessed by calculating the metastatic foci. Sham-operation (SO) and untreated (UT) groups served as controls. RESULTS: The VEGF (TR: 79.55 ± 7.82 pg/mL vs. SO: 110.01 ± 5.69 pg/mL, UT: 123.50 ± 10.41 pg/mL; p < 0.01) and endostatin (TR: 47.09 ± 6.22 ng/mL vs. SO: 117.64 ± 7.39 ng/mL, UT: 126.73 ± 6.55 ng/mL; p<0.01) were down-regulated significantly after tumorectomy, and angiogenesis was significantly promoted simultaneously. The incidence of pulmonary metastatic foci was 80.0% in the TR group, 40.0% in the SO group and 35.0% in the UT group. CONCLUSION: Primary tumorectomy can down-regulate the expression of VEGF and endostatin and promote angiogenesis which leads to the acceleration of pulmonary metastasis. These findings imply that anti-angiogenic treatment can be considered after primary tumorectomy.