Calcium measurements.

<p>The diagram shows the mean calcium influx in CCL21 stimulated cells. An increase in cytosolic calcium levels was only observed in M13HS-2 and M13HS-8 hybrid cells. To prove whether the CCL21 induced calcium influx in these cell lines depended on PLC-β/γ activity, appropriate calcium measurements within the presence of 5 µM U73122 were performed. Cells were preincubated for 5 minutes with U73122 prior to analysis. Shown are the mean±SD of n = 3 independent experiments. Statistical significance was calculated using Student's <i>t</i>-test: n.s.  =  not significant; **  =  <i>p</i><0.01; ***  =  <i>p</i><0.001.</p

CCR7 expression of parental cell lines and hybrid cell lines.

<p>CCR7 expression was detected by Western Blot analysis, whereby elf4E expression was used as an internal control. Shown are representative Western Blot data of n = 3 independent experiments.</p

Knockdown of CCR7 expression abolish the CCL21 induced migration of M13HS hybrid cell lines.

<p>The migratory activity was analyzed using the 3D collagen matrix migration assay combined with time-lapse video-microscopy. For a better comparison the locomotory activities of CCL21 stimulated cells were calculated in relation to untreated control cells, which were set to 100%. Cells were stimulated with 500 ng/ml CCL21. Shown are the mean±SD of n = 3 independent experiments. <b>(A)</b> HS578T-Hyg breast cancer cells, <b>(B)</b> M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics, <b>(C)</b> M13HS-2 hybrid cells, <b>(D)</b> M13HS-8 hybrid cells. Statistical significance was calculated using Student's <i>t</i>-test: ***  =  <i>p</i><0.001.</p

Fusion of CCL21 Non-Migratory Active Breast Epithelial and Breast Cancer Cells Give Rise to CCL21 Migratory Active Tumor Hybrid Cell Lines

<div><p>The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.</p></div

Analysis of signal transduction cascades initiated by CCL21/CCR7 signaling.

<p>Cells were treated with 500 ng/ml CCL21 for 0, 1, 2, 5, and 10 minutes. For inhibitor studies cells were preincubated for 60 minutes with 500 nM Ly29004 or 30 minutes with 500 nM PD98059, respectively. Cells were analyzed for AKT phosphorylation and MAPK^p42/44 (MAPK) phosphorylation, whereby elf4E was used as a housekeeping gene. Values indicate the relative intensities of proteins in relation to elf4E, which was set to 100%. Shown are representative Western Blot data of n = 3 independent experiments. <b>(A)</b> HS578T-Hyg breast cancer cells, <b>(B)</b> M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics, <b>(C)</b> M13HS-2 hybrid cells, <b>(D)</b> M13HS-8 hybrid cells.</p