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Various wet ball nanomilling-screening tools for poorly soluble APIs are available which differ in their milling principle, batch size and number of samples. Here, the transferability ...of results from screening (small to medium-scale) to pharmaceutical production (largescale) was investigated. Wet ball milling in a dual centrifuge (DC) (10–100 mg API, 40 samples in parallel) was used to identify stable nanoformulations. In addition different sized agitator bead mills were used for scale-up to industrial scales. DC-and small-scale agitator milling (AM) resulted in small and virtually identical API-particles. Additionally, similar API-particles were obtained using two different sized agitator bead mills (batch size 1.5 and 30 kg) and applying comparable specific grinding energies (SGE). The SGE used in the trials represents the grinding limit for this API-suspension. Using lower SGEs, AM results in larger API-particles. All used milling tools had no influence on the APIs crystal structure and wear of grinding media (Zr/Y) is low. The study confirmed the importance to choose the right formulation and process parameters, which positively affect grinding efficacy, particle size distribution and wear contamination. The excellent comparability of results obtained from DC-milling and AM significantly reduces the duration for successful and predictable formulation development.
Background & Aims Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. It is characterized by substantial tumor cell invasion and early-stage metastasis. We developed an in vivo ...model to analyze interactions between cancer and stromal cells during early stages of PDAC. Methods Human pancreatic adenocarcinoma cells were grafted onto the chick chorioallantoic membrane (CAM). Human and chicken GeneChips were used simultaneously to study gene regulation during PDAC cell invasion. Bioinformatic analysis was used to identify human orthologs and cell specificity of gene expression. The effects of netrin-1 encoded by NTN1 were investigated in adhesion, invasion, and apoptosis assays. The effects of NTN1 silencing with small interfering RNAs were investigated in PDAC cells in vivo. NTN1 expression was measured in human PDAC samples. Results PDAC cells rapidly invade the CAM stroma and remodel the CAM vasculature. Around 800 stromal genes were up-regulated by >2-fold; the angiogenesis regulators vascular endothelial growth factor D, thrombospondin 1, and CD151 were among the most highly regulated genes. Silencing of tumor cell NTN1 , which is up-regulated 4-fold in the PDAC model, inhibited tumor cell invasion in vivo. Netrin-1 conferred apoptosis resistance to tumor and endothelial cells in vitro, induced their invasion, and provided an adhesive substrate for tumor cells. NTN1 and its gene product are strongly overexpressed in human PDAC samples. Conclusions We developed a useful tool to study the invasive mechanisms of early-stage PDAC. Netrin-1 might be an important regulator of pancreatic tumor growth that functions in tumor and endothelial cells.
Because of the lack of suitable in vivo models of giant cell tumor of bone (GCT), little is known about its underlying fundamental pro-tumoral events, such as tumor growth, invasion, angiogenesis and ...metastasis. There is no existing cell line that contains all the cell and tissue tumor components of GCT and thus in vitro testing of anti-tumor agents on GCT is not possible. In this study we have characterized a new method of growing a GCT tumor on a chick chorio-allantoic membrane (CAM) for this purpose.
Fresh tumor tissue was obtained from 10 patients and homogenized. The suspension was grafted onto the CAM at day 10 of development. The growth process was monitored by daily observation and photo documentation using in vivo biomicroscopy. After 6 days, samples were fixed and further analyzed using standard histology (hematoxylin and eosin stains), Ki67 staining and fluorescence in situ hybridization (FISH).
The suspension of all 10 patients formed solid tumors when grafted on the CAM. In vivo microscopy and standard histology revealed a rich vascularization of the tumors. The tumors were composed of the typical components of GCT, including (CD51+/CD68+) multinucleated giant cells which were generally less numerous and contained fewer nuclei than in the original tumors. Ki67 staining revealed a very low proliferation rate. The FISH demonstrated that the tumors were composed of human cells interspersed with chick-derived capillaries.
A reliable protocol for grafting of human GCT onto the chick chorio-allantoic membrane is established. This is the first in vivo model for giant cell tumors of bone which opens new perspectives to study this disease and to test new therapeutical agents.
Angiogenesis has become a major target in cancer therapy. However, current therapeutic strategies have their limitations and raise several problems. In most tumours, anti-angiogenesis treatment ...targeting VEGF (vascular endothelial growth factor) has only limited overall survival benefit compared with conventional chemotherapy alone, and reveals several specific forms of resistance to anti-VEGF treatment. There is growing evidence that anti-VEGF treatment may induce tumour cell invasion by selecting highly invasive tumour cells or hypoxia-resistant cells, or by up-regulating angiogenic alternative pathways such as FGFs (fibroblast growth factors) or genes triggering new invasive programmes. We have identified new genes up-regulated during glioma growth on the chick CAM (chorioallantoic membrane). Our results indicate that anti-angiogenesis treatment in the experimental glioma model drives expression of critical genes which relate to disease aggressiveness in glioblastoma patients. We have identified a molecular mechanism in tumour cells that allows the switch from an angiogenic to invasive programme. Furthermore, we are focusing our research on alternative inhibitors that act, in part, independently of VEGF. These are endogenous molecules that play a role in the control of tumour growth and may constitute a starting point for further development of novel therapeutic or diagnostic tools.
Experimental in vivo tumor models are essential for comprehending the dynamic process of human cancer progression, identifying therapeutic targets, and evaluating antitumor drugs. However, current ...rodent models are limited by high costs, long experimental duration, variability, restricted accessibility to the tumor, and major ethical concerns. To avoid these shortcomings, we investigated whether tumor growth on the chick chorio-allantoic membrane after human glioblastoma cell grafting would replicate characteristics of the human disease. Avascular tumors consistently formed within 2 days, then progressed through vascular endothelial growth factor receptor 2-dependent angiogenesis, associated with hemorrhage, necrosis, and peritumoral edema. Blocking of vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor signaling pathways by using small-molecule receptor tyrosine kinase inhibitors abrogated tumor development. Gene regulation during the angiogenic switch was analyzed by oligonucleotide microarrays. Defined sample selection for gene profiling permitted identification of regulated genes whose functions are associated mainly with tumor vascularization and growth. Furthermore, expression of known tumor progression genes identified in the screen (IL-6 and cysteine-rich angiogenic inducer 61) as well as potential regulators (lumican and F-box-only 6) follow similar patterns in patient glioma. The model reliably simulates key features of human glioma growth in a few days and thus could considerably increase the speed and efficacy of research on human tumor progression and preclinical drug screening.
Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is ...crucial for the identification of new therapeutic targets against pathological angiogenesis.
corrected We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21-66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process.
Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis.
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