Background: The cell microenvironment, especially extracellular matrix proteins, plays an important role in tumor cell response to chemotherapeutic drugs. The present study was designed to investigate whether this microenvironment can influence the antimigratory effect of an anthracycline drug, doxorubicin, when tumor cells are grown in a matrix of type I collagen, a three-dimensional (3D) context which simulates a natural microenvironment. Methods: To this purpose, we studied the migratory parameters, the integrin expression, and the activation state of focal adhesion kinase (FAK) and GTPase RhoA involved in the formation of focal adhesions and cell movement. These parameters were evaluated at non toxic concentrations which did not affect HT1080 cell proliferation. Results: We show that while doxorubicin decreased cell migration properties by 70% in conventional two-dimensional (2D) culture, this effect was completely abolished in a 3D one. Regarding the impact of doxorubicin on the focal adhesion complexes, unlike in 2D systems, the data indicated that the drug neither affected β1 integrin expression nor the state of phosphorylation of FAK and RhoA. Conclusion: This study suggests the lack of antiinvasive effect of doxorubicin in a 3D environment which is generally considered to better mimic the phenotypic behaviour of cells in vivo. Consistent with the previously shown resistance to the cytotoxic effect in a 3D context, our results highlight the importance of the matrix configuration on the tumor cell response to antiinvasive drugs.

Systemic sclerosis (SSc) is a systemic disease characterized by a great clinical and immunological heterogeneity whose pathophysiology is still being unraveled. Recently, innate immunity has been proposed to participate to the pathogenesis of SSc. In this study, we investigated the release of neutrophil extracellular traps (NETs) according to patient phenotype. Polymorphonuclear neutrophils (PMN) from 34 SSc patients and 26 healthy controls were stimulated by serum from SSc or healthy subject. NETs were visualized using epifluorescence microscope after DNA, myeloperoxidase, and Histone H3 tagging. Area of NETs were quantified using an original macro running in ImageJ^® software. PMN from SSc patients were significantly more prone to releasing NETs than control PMN after autologous stimulation. PMN from patients with severe vascular complications (pulmonary arterial hypertension, digital ulcers) produced more NETs than PMN from other SSc patients and their aberrant NET production appeared to be sustained over time. In patients with pulmonary interstitial disease or extensive cutaneous fibrosis, NET production was high at an early stage of the disease before progressively decreasing. Both serum factors and PMN activation status were involved in the enhanced production of NETs in SSc. Consequently, neutrophils and especially NETosis represent new physiopathological and therapeutic fields in SSc.

The spatial organization of proteins in a cell population or in tissues is an important parameter to study the functionality of biological specimens. In this article, we have focused on tight junctions which form network‐like features in immunofluorescence microscopy images. Usually, the organization or disorganization of tight junctions is noticed qualitatively. The aim of this article is to present a simple method to quantify the organization level of tight junction network using image analysis with a dedicated macro developed with Image J software. The method has been validated with simulated images displaying regular decrease of network organization. Then, the macro has been applied to immunofluorescence microscopy images of cells in culture and of tissue sections. © 2012 International Society for Advancement of Cytometry

Background: Many studies associated the main polyphenolic constituent of green tea, (-)-Epigallocatechin-3-gallate (EGCG), with inhibition of cancers, invasion and metastasis. To date, most of the studies have focused on the effect of EGCG on cell proliferation or death. Since cell migration is an important mechanism involved in tumor invasion, the aim of the present work was to target another approach of the therapeutic effect of EGCG, by investigating its effect on the cell migratory behavior. Methods: The effect of EGCG (at concentrations lower than 10 μg/ml) on the migration speed of invasive cells was assessed by using 2D and 3D models of cell culture. We also studied the effects of EGCG on proteinases expression by RT-PCR analysis. By immunocytochemistry, we analyzed alterations of vimentin organization in presence of different concentrations of EGCG. Results: We observed that EGCG had an inhibitory effect of cell migration in 2D and 3D cell culture models. EGCG also inhibited MMP-2 mRNA and protein expression and altered the intermediate filaments of vimentin. Conclusion: Taken together, our results demonstrate that EGCG is able to inhibit the migration of bronchial tumor cells and could therefore be an attractive candidate to treat tumor invasion and cell migration.