A Heterogeneous In Vitro Three Dimensional Model of Tumour-Stroma Interactions Regulating Sprouting Angiogenesis

Angiogenesis, the formation of new blood vessels, is an essential process for tumour progression and is an area of significant therapeutic interest. Different in vitro systems and more complex in vivo systems have been described for the study of tumour angiogenesis. However, there are few human 3D in vitro systems described to date which mimic the cellular heterogeneity and complexity of angiogenesis within the tumour microenvironment. In this study we describe the Minitumour model – a 3 dimensional human spheroid-based system consisting of endothelial cells and fibroblasts in co-culture with the breast cancer cell line MDA-MB-231, for the study of tumour angiogenesis in vitro. After implantation in collagen-I gels, Minitumour spheroids form quantifiable endothelial capillary-like structures. The endothelial cell pre-capillary sprouts are supported by the fibroblasts, which act as mural cells, and their growth is increased by the presence of cancer cells. Characterisation of the Minitumour model using small molecule inhibitors and inhibitory antibodies show that endothelial sprout formation is dependent on growth factors and cytokines known to be important for tumour angiogenesis. The model also shows a response to anti-angiogenic agents similar to previously described in vivo data. We demonstrate that independent manipulation of the different cell types is possible, using common molecular techniques, before incorporation into the model. This aspect of Minitumour spheroid analysis makes this model ideal for high content studies of gene function in individual cell types, allowing for the dissection of their roles in cell-cell interactions. Finally, using this technique, we were able to show the requirement of the metalloproteinase MT1-MMP in endothelial cells and fibroblasts, but not cancer cells, for sprouting angiogenesis

Demonstration in Vivo That Stromelysin-3 Functions through Its Proteolytic Activity

Stromelysin-3 (ST3), a matrix metalloproteinase (MMP) expressed in aggressive carcinomas, has been shown to promote tumor development in different in vivo experimental models. However, the inability of its mature form to degrade extracellular matrix components casts doubt on whether ST3 functions in vivo as a protease. In this study, we evaluated whether the ST3 tumor-promoting effect could be ascribed to its proteolytic activity and whether this putative protease could be targeted with MMP inhibitors. Catalytically inactive mutant cDNA of human (h) ST3 or mouse (m) ST3 were generated and transfected into MCF7 cells. When injected into nude mice in the presence of matrigel, the mutant-bearing cells did not exhibit the enhanced tumorigenicity elicited by MCF7 cells transfected with wild-type ST3 cDNA. In a second approach, TIMP2 overproduction in MCF7 cells expressing hST3 was induced by retroviral infection. The co-expression of ST3 and TIMP2 failed to enhance the tumorigenicity of MCF7 cells. Notably, matrigel depleted of low-molecular-weight proteins and growth factors failed to promote the tumorigenicity of ST3-expressing MCF7 cells. These findings provide the first in vivo evidence that ST3 is indeed a protease that can modulate cancer progression by remodeling extracellular matrix and probably by inducing it to release the necessary microenvironmental factors. Thus, ST3 represents an interesting target for specific MMP inhibition

Fitting lactation data with two mathematical models and extension factors for milk, fat and protein of Belgian dairy goats

peer reviewedData for 18 155 test day milk yields and fat and protein percentages recorded from 15 February 1988 to 1 September 1993 were obtained from the Office de promotion des petits élevages en Wallonie. Due to irregular test intervals and a variable number of tests per lactation, production was estimated at 25 day intervals (25, 50, ..., 250 days). A total of 13 773 test day records for Anglo-Nubian, Chamoisee, Saanen and crossbreds were available for analysis. Parities were classified into 1 and ≥ 2. The inverse polynomial and the incomplete gamma functions were fitted to test day milk yields in order to define the shape of the lactation curves for the various breeds and parities. Two data subsets were created by random selection of entire lactation data from the original data set, and both functions were fitted to test day milk yields within breed-parity classes. Parameters of equations estimated on a subset of the data were validated on the other subset. Estimates of peak yields were higher and times of peak yield later by the inverse polynomial method than by the incomplete gamma but remained within ranges found in the literature. Based on the coefficient of multiple determination (R2), both equations were equally accurate in fitting lactation data of a subset. Though average residual deviations were slightly higher with the inverse polynomial than with the incomplete gamma, the crossvalidation did not reveal any particular trend of residuals for any equation. For practical reasons, extension factors for milk, fat and protein yields were derived using the inverse polynomial and are presented

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