CXCR4/CXCL12 Participate in Extravasation of Metastasizing Breast Cancer Cells within the Liver in a Rat Model

INTRODUCTION: Organ-specific composition of extracellular matrix proteins (ECM) is a determinant of metastatic host organ involvement. The chemokine CXCL12 and its receptor CXCR4 play important roles in the colonization of human breast cancer cells to their metastatic target organs. In this study, we investigated the effects of chemokine stimulation on adhesion and migration of different human breast cancer cell lines in vivo and in vitro with particular focus on the liver as a major metastatic site in breast cancer. METHODS: Time lapse microscopy, in vitro adhesion and migration assays were performed under CXCL12 stimulation. Activation of small GTPases showed chemokine receptor signalling dependence from ECM components. The initial events of hepatic colonisation of MDA-MB-231 and MDA-MB-468 cells were investigated by intravital microscopy of the liver in a rat model and under shRNA inhibition of CXCR4. RESULTS: In vitro, stimulation with CXCL12 induced increased chemotactic cell motility (p,0.05). This effect was dependent on adhesive substrates (type I collagen, fibronectin and laminin) and induced different responses in small GTPases, such as RhoA and Rac-1 activation, and changes in cell morphology. In addition, binding to various ECM components caused redistribution of chemokine receptors at tumour cell surfaces. In vivo, blocking CXCR4 decreased extravasation of highly metastatic MDA-MB-231 cells (p < 0.05), but initial cell adhesion within the liver sinusoids was not affected. In contrast, the less metastatic MDA-MB-468 cells showed reduced cell adhesion but similar migration within the hepatic microcirculation. CONCLUSION: Chemokine-induced extravasation of breast cancer cells along specific ECM components appears to be an important regulator but not a rate-limiting factor of their metastatic organ colonization.Claudia Wendel, André Hemping-Bovenkerk, Julia Krasnyanska, Sören Torge Mees, Marina Kochetkova, Sandra Stoeppeler and Jörg Haie

Evaluation of Osseous Integration of PVD-Silver-Coated Hip Prostheses in a Canine Model

Infection associated with biomaterials used for orthopedic prostheses remains a serious complication in orthopedics, especially tumor surgery. Silver-coating of orthopedic (mega)prostheses proved its efficiency in reducing infections but has been limited to surface areas exposed to soft tissues due to concerns of silver inhibiting osseous integration of cementless stems. To close this gap in the bactericidal capacity of silver-coated orthopedic prostheses extension of the silver-coating on surface areas intended for osseous integration seems to be inevitable. Our study reports about a PVD- (physical-vapor-deposition-) silver-coated cementless stem in a canine model for the first time and showed osseous integration of a silver-coated titanium surface in vivo. Radiological, histological, and biomechanical analysis revealed a stable osseous integration of four of nine stems implanted. Silver trace elemental concentrations in serum did not exceed 1.82 parts per billion (ppb) and can be considered as nontoxic. Changes in liver and kidney functions associated with the silver-coating could be excluded by blood chemistry analysis. This was in accordance with very limited metal displacement from coated surfaces observed by laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) 12 months after implantation. In conclusion our results represent a step towards complete bactericidal silver-coating of orthopedic prostheses

Vessel Size Imaging (VSI) by Robust Magnetic Resonance (MR) Relaxometry: MR-VSI of Solid Tumors in Correlation with Immunohistology and Intravital Microscopy

The aim of this study was to evaluate a robust magnetic resonance (MR) vessel size imaging (VSI) method for the noninvasive assessment of mean vessel size in solid tumors in a clinical dose range of ultrasmall superparamagnetic particles of iron oxide (USPIO). Therefore, USPIO-enhanced MR-VSI was performed on DU-4475, MDA-MB-435, and EOMA tumor–bearing mice xenografts with known differences in angiogenic activity and vessel morphology. MR results were compared to vessel sizes determined by immunohistochemistry (anti-CD31) and by intravital microscopy (IVM). MR-VSI revealed significantly different mean vessel sizes between the xenograft models at both USPIO doses (DU-4475: 20.6 ± 4.9 mm; MDA-MB-435: 37.4 ± 8.8 μm; and EOMA: 60.3 ± 9.6 μm at 80 μmol/kg; p < .05). Immunohistochemistry revealed lower values for all tumor entities, whereas the size distribution was in line with MR-measurements. IVM corroborated the MR results for DU-4475 and MDA-MB435, but showed similar vessel sizes for MDA-MB-435 and EOMA. Our MR-VSI method allowed a noninvasive estimation of the mean vessel size in mice xenograft solid tumors with variable vascularity using a clinically relevant USPIO dose range

Flow cytometry analysis of breast cancer cells.

<p>The cell surface expression of CXCR4 (⁃) was found in different breast cancer cell lines MDA-MB-231 (a) and MDA-MB-468 (b). Downregulation of CXCR4 expression in clones MDA-MB-231 Cl 19 (c) and MDA-MB-231 Cl 27 (d). (isotype control IgG2b: ••••; negative control:▪).</p

Kinetics of CXCR4 cell surface expression.

<p>MDA-MB-231 cells were seeded at C I (left column) or FN (right column) and stimulated with CXCL12 for up to 30 min. Fixed cells without stimulation (a+b) or with CXCL12 stimulation (25 ng/ml) for 5 min (c+d), 15 min. (e+f) or 30 min (g+h) were stained for surface expression of CXCR4. The number of large CXCR4 clusters was reduced after 30 at cells in a comparable manner for both ECM components. Scale 10 µm</p

In vivo migration of breast cancer cells within the liver sinusoids.

<p>Single cell suspensions of fluorescence-labelled tumour cells were injected into Spague-Dawley rats. (a) Example of an adherent cells (white arrow) and a cell starting to migrate (black arrow) into the liver parenchyma. Magnifications show location of the cells in relation to marked sinusoid-parenchyma borders. Intravital microscopy was done to analyse adhesion and migration properties of MDA-MB-231 (b) and MDA-MB-468 cells (c). Thirty microscopic fields were analysed in 5min observation periods for semiquantitative analysis of adherent (x) and migrated cells (□). The total numbers of arrested cells (▴) were calculated. <b>CXCR4 inhibition decreased tumour cell migration in vivo.</b> The MDA-MB-231 cells were transduced with shRNA to inhibit CXCR4 expression. Two clones 19 (◊) and 27 (x) were tested for in vivo adhesion (d) and migration (e) in the rat liver and compared with untreated cells (▪). Transfected cells showed significantly (*p<0.001) decreased relative migration rates into the liver parenchyma but cell adhesion within the hepatic microcirculation was only slightly influenced by CXCR4 reduction. Relative migration rates were based on the number of arrested cells and were calculated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030046#pone.0030046-Sahai1" target="_blank">[26]</a>.</p

Cell surface expression of chemokines receptors.

<p>Cell surface expression was analysed by flow cytometry. shRNA transfection resulted in a reduction of CXCR4 expression in MDA-MB231-27 cells. Negative and IgG controls are given as examples for the controls that have been used in each measurement. IgG controls were subtype-specific.</p

Expression of integrin subunits at cell surfaces.

<p>Cell surface expression was analysed by flow cytometry. The major difference between the cells was observed for integrin ligands of LN. Negative and IgG controls are given as examples for the controls that have been used in each measurement. IgG controls were subtype-specific.</p

CXCL12 and ECM-induced activation of small Rho GTPases.

<p>Cells were treated with different concentrations of CXCL12 in single cell suspensions (a+b) or plated at various adhesive substrates (c+d). Level of activation of RhoA, Rac1 and Cdc42 assessed by pull down assays of the GTP-bound forms were determined in MDA-MB-231 (a+c) and MDA-MB-468 cells (b+d). As example for total GTPase detection independent from phosphorylation status in each experiment Total-Rho is shown for (a) and (b). These loading controls were used for standardization of optical densities (e+f). RhoA and Rac1 activation increased in a dose-dependent manner, but Cdc42 remained at baseline levels. If MDA MB 231 (c) and MDA MB 468 (d) cells were plated at C I, FN, LN, PLL and BSA differences in ECM dependent activation of GTPases were observed. Comparing relative optical densities these matrix dependent differences were confirmed (e). Synergistic activation between chemokine stimulation and ECM binding, such as demonstrated for Rho (f), were not found.</p

CXCL12 increases cell motility dependent from the ECM.

<p>(a) Cells that were attached to different ECM components were observed for one hour using time-lapse video microscopy. The length of their moving path was determined using a cell tracking software (Cell∧D®). (b) MDA-MB-231 and (c) MDA-MB-468 cells showed different patterns of chemokine stimulated motility (□ unstimulated; ▪ 25ng/ml; ///50ng/ml; ≡ 100ng/ml CXCL12). Highly metastatic MDA-MB-231 cells were more motile at CI and LN (*p<0.001) with a concentration-dependent stimulation (+p<0.05) of their motility that was not observed at FN. In contrast, low-metastatic MDA-MB-468 cells were less motile at C I and LN, but showed a concentration-dependent stimulation at FN. (d) In CXCR4-knock-down cells (MDA-MB-231-27) these CXCL12-dependent stimulating effects were not found. The results are shown as mean ± SD for three independent experiments. For statistical analysis ECM dependent migration was compared for all three ECM components and concentration dependence of CXCL12 was compared to unstimulated cells. Significances for ECM dependent cell motility are not marked for improved readability.</p