Influence of (A) material properties (Young’s modulus for cytoplasm and nucleus are as first and second value in parenthesis), (B) cell dimensions, (C) thickness of the actin cortex in percent of the cell radius, and (D) cell embedding (Young’s modulus for agar in parenthesis) on natural frequencies of MCF10A cells (A) or MCF7 cells (A-D).

<p>Influence of (A) material properties (Young’s modulus for cytoplasm and nucleus are as first and second value in parenthesis), (B) cell dimensions, (C) thickness of the actin cortex in percent of the cell radius, and (D) cell embedding (Young’s modulus for agar in parenthesis) on natural frequencies of MCF10A cells (A) or MCF7 cells (A-D).</p

Increased death of MCF7 and MDA-MB-231 cells after irradiation with an ultrasonic frequency of 24.5 kHz.

<p>(A) Cells either cultivated in 2D culture or (B) growing in 3D culture on alginate beads (gems) were treated with 24.5 kHz and four different intensities for 4 min; 1 h later the proportion of dead cells (propidium iodide (PI) positive cells) was determined by FACS analysis. Results represent the means of data from eight (A) or three (B) independent experiments; the error bars represent the standard errors; p-values were calculated by the two-sided, paired Student’s t-test with * p<0.05, ** p<0.01.</p

Characterization of Dynamic Behaviour of MCF7 and MCF10A Cells in Ultrasonic Field Using Modal and Harmonic Analyses

<div><p>Treatment options specifically targeting tumour cells are urgently needed in order to reduce the side effects accompanied by chemo- or radiotherapy. Differences in subcellular structure between tumour and normal cells determine their specific elasticity. These structural differences can be utilised by low-frequency ultrasound in order to specifically induce cytotoxicity of tumour cells. For further evaluation, we combined <i>in silico</i> FEM (finite element method) analyses and <i>in vitro</i> assays to bolster the significance of low-frequency ultrasound for tumour treatment. FEM simulations were able to calculate the first resonance frequency of MCF7 breast tumour cells at 21 kHz in contrast to 34 kHz for the MCF10A normal breast cells, which was due to the higher elasticity and larger size of MCF7 cells. For experimental validation of the <i>in silico</i>-determined resonance frequencies, equipment for ultrasonic irradiation with distinct frequencies was constructed. Differences for both cell lines in their response to low-frequent ultrasonic treatment were corroborated in 2D and in 3D cell culture assays. Treatment with ~ 24.5 kHz induced the death of MCF7 cells and MDA-MB-231 metastases cells possessing a similar elasticity; frequencies of > 29 kHz resulted in cytotoxicity of MCF10A. Fractionated treatments by ultrasonic irradiation of suspension myeloid HL60 cells resulted in a significant decrease of viable cells, mostly significant after threefold irradiation in intervals of 3 h. Most importantly in regard to a clinical application, combined ultrasonic treatment and chemotherapy with paclitaxel showed a significantly increased killing of MCF7 cells compared to both monotherapies. In summary, we were able to determine for the first time for different tumour cell lines a specific frequency of low-intensity ultrasound for induction of cell ablation. The cytotoxic effect of ultrasonic irradiation could be increased by either fractionated treatment or in combination with chemotherapy. Thus, our results will open new perspectives in tumour treatment.</p></div

Decreased survival of MCF7 cells after irradiation with an ultrasonic frequency of 24.5 kHz as determined by XCelligence (Roche).

<p>Decreased survival of MCF7 cells after irradiation with an ultrasonic frequency of 24.5 kHz as determined by XCelligence (Roche).</p

Decreased survival of HL60 cells after fractionated irradiation.

<p>HL60 suspension cells were treated by ultrasonic irradiation once, twice or three times at intervals of 3h (2x 3 h, 3x 3 h) or 6 h (2x 6 h). The number of vital cells was determined by FACS 1 h after each irradiation. (The number of vital cells of the untreated control was set as 100%.) Results represent the means of data from three independent experiments; the error bars represent the standard errors; p-values were calculated by the two-sided, paired Student’s t-test with * p<0.05, ** p<0.01, *** p<0.001.</p

Harmonic vibration analysis of (A) MCF7 and (B) MCF10A cells (minimal sizes for both cell types) with external hydrostatic pressure in a frequency range of 20 kHz up to 60 kHz showing the displacement amplitudes.

<p>The red horizontal lines depict the maximum cell size which allows the amplitudes with the cell dimension to be compared.</p

Treatment of MCF7 cells with either (A) ultrasonic irradiation with 23.22 kHz and two different intensities (0.3 W/cm² or 1 W/cm², dark grey bars), (B) paclitaxel with 100 nM or 200 nM (light grey bars) or (C) combinations of both treatments (ultrasonic irradiation followed by paclitaxel treatment; white bars) with a) constant concentration of paclitaxel and different intensities of ultrasonic irradiation, and b) constant intensity and different concentrations of paclitaxel.

<p>Results represent the means of data from eight independent experiments; the error bars represent the standard errors; p-values were calculated by the two-sided, paired Student’s t- test with * p<0.05, ** p<0.01, *** p<0.001.</p

Resolving Tumor Heterogeneity: Genes Involved in Chordoma Cell Development Identified by Low-Template Analysis of Morphologically Distinct Cells

<div><p>The classical sacrococcygeal chordoma tumor presents with a typical morphology of lobulated myxoid tumor tissue with cords, strands and nests of tumor cells. The population of cells consists of small non-vacuolated cells, intermediate cells with a wide range of vacuolization and large heavily vacuolated (physaliferous) cells. To date analysis was only performed on bulk tumor mass because of its rare incidence, lack of suited model systems and technical limitations thereby neglecting its heterogeneous composition. We intended to clarify whether the observed cell types are derived from genetically distinct clones or represent different phenotypes. Furthermore, we aimed at elucidating the differences between small non-vacuolated and large physaliferous cells on the genomic and transcriptomic level. Phenotype-specific analyses of small non-vacuolated and large physaliferous cells in two independent chordoma cell lines yielded four candidate genes involved in chordoma cell development. <i>UCHL3</i>, coding for an ubiquitin hydrolase, was found to be over-expressed in the large physaliferous cell phenotype of MUG-Chor1 (18.7-fold) and U-CH1 (3.7-fold) cells. The mannosyltransferase <i>ALG11</i> (695-fold) and the phosphatase subunit <i>PPP2CB</i> (18.6-fold) were found to be up-regulated in large physaliferous MUG-Chor1 cells showing a similar trend in U-CH1 cells. <i>TMEM144</i>, an orphan 10-transmembrane family receptor, yielded contradictory data as cDNA microarray analysis showed up- but RT-qPCR data down-regulation in large physaliferous MUG-Chor1 cells. Isolation of few but morphologically identical cells allowed us to overcome the limitations of bulk analysis in chordoma research. We identified the different chordoma cell phenotypes to be part of a developmental process and discovered new genes linked to chordoma cell development representing potential targets for further research in chordoma tumor biology.</p></div

Expression analyses of MUG-Chor1 candidate genes in U-CH1 cells.

<p>RT-qPCR was done on AB7900 TaqMan (Applied Biosystems; Foster City, CA). Normalization (<i>GAPDH</i> and <i>ACTB</i>) and statistical analysis was done with GenEx Professional (MultiD Analysis; Version 5.3.5.6; see also 2.7). Cut-off for multiple testing (<i>ALG11</i>, <i>UCHL3</i>, <i>TMEM144</i> and <i>PPP2CB</i>) was p = 0.01274.</p>a<p>calculated as mean values from quadruplicate or triplicate (in case the Cq value could not be defined) biological samples.</p>b<p>Cq values were normalized to <i>GAPDH</i> and <i>ACTB</i> (ΔCq). Differential expression (ΔΔCq) is given as positive (up-regulated in large cells) or negative (down-regulated in large cells) fold change ( = 2^ΔΔCq).</p

Morphological and immunohistochemical characterization of the chordoma tumor giving rise to MUG-Chor1 cell line

<p>. A) Hematoxylin/eosin stained section show lobulated myxoid tumor tissue with cords, strands and nests of tumor cells with pale/eosinophilic to vacuolated cytoplasm. B, C) In detail, the tumor is composed of small cells with eosinophilic cytoplasm and partly spindle cell morphology and large vacuolated/physaliferous tumor cells including “signet ring” shaped cells. D) All cell phenotypes yield the chordoma-specific nuclear staining for brachyury. Arrowheads: small cells; asterisks: large vacuolated/physaliferous cells; arrows: “signet ring” cells. Scale bars: 500 µm (A), 50 µm (B, C); D: original magnification X20.</p