Lipid tethering of breast tumor cells enables real-time imaging of free-floating cell dynamics and drug response

Funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.Free-floating tumor cells located in the blood of cancer patients, known as circulating tumor cells (CTCs), have become key targets for studying metastasis. However, effective strategies to study the free-floating behavior of tumor cells in vitro have been a major barrier limiting the understanding of the functional properties of CTCs. Upon extracellular-matrix (ECM) detachment, breast tumor cells form tubulin-based protrusions known as microtentacles (McTNs) that play a role in the aggregation and re-attachment of tumor cells to increase their metastatic efficiency. In this study, we have designed a strategy to spatially immobilize ECM-detached tumor cells while maintaining their free-floating character. We use polyelectrolyte multilayers deposited on microfluidic substrates to prevent tumor cell adhesion and the addition of lipid moieties to tether tumor cells to these surfaces through interactions with the cell membranes. This coating remains optically clear, allowing capture of high-resolution images and videos of McTNs on viable free-floating cells. In addition, we show that tethering allows for the real-time analysis of McTN dynamics on individual tumor cells and in response to tubulin-targeting drugs. The ability to image detached tumor cells can vastly enhance our understanding of CTCs under conditions that better recapitulate the microenvironments they encounter during metastasis

Single-Cell Tracking of Breast Cancer Cells Enables Prediction of Sphere Formation from Early Cell Divisions

Summary: The mammosphere assay has become widely employed to quantify stem-like cells in a population. However, the problem is there is no standard protocol employed by the field. Cell seeding densities of 1,000 to 100,000 cells/mL have been reported. These high densities lead to cellular aggregation. To address this, we have individually tracked 1,127 single MCF-7 and 696 single T47D human breast tumor cells by eye over the course of 14 days. This tracking has given us detailed information for the commonly used endpoints of 5, 7, and 14 days that is unclouded by cellular aggregation. This includes mean sphere sizes, sphere-forming efficiencies, and a well-defined minimum size for both lines. Importantly, we have correlated early cell division with eventual sphere formation. At 24 hr post seeding, we can predict the total spheres on day 14 with 98% accuracy in both lines. This approach removes cell aggregation and potentially shortens a 5- to 14-day assay to a 24 hours. : Biology Experimental Methods; Cancer Subject Areas: Biology Experimental Methods, Cance

Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

Activation of the unfolded protein response (UPR) in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus) and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs

The Ezrin Metastatic Phenotype Is Associated with the Initiation of Protein Translation1

We previously associated the cytoskeleton linker protein, Ezrin, with the metastatic phenotype of pediatric sarcomas, including osteosarcoma and rhabdomyosarcoma. These studies have suggested that Ezrin contributes to the survival of cancer cells after their arrival at secondary metastatic locations. To better understand this role in metastasis, we undertook two noncandidate analyses of Ezrin function including a microarray subtraction of high-and low-Ezrin-expressing cells and a proteomic approach to identify proteins that bound the N-terminus of Ezrin in tumor lysates. Functional analyses of these data led to a novel and unifying hypothesis that Ezrin contributes to the efficiency of metastasis through regulation of protein translation. In support of this hypothesis, we found Ezrin to be part of the ribonucleoprotein complex to facilitate the expression of complex messenger RNA in cells and to bind with poly A binding protein 1 (PABP1; PABPC1). The relevance of these findings was supported by our identification of Ezrin and components of the translational machinery in pseudopodia of highly metastatic cells during the process of cell invasion. Finally, two small molecule inhibitors recently shown to inhibit the Ezrin metastatic phenotype disrupted the Ezrin/PABP1 association. Taken together, these results provide a novel mechanistic basis by which Ezrin may contribute to metastasis

Identification of temsirolimus exposures required to inhibit total protein synthesis.

<p>(<b>A</b>) Cells were pulse labeled with ³⁵S-methionine/cysteine and analyzed by scintillation counting for incorporated radioactivity normalized to vehicle treated control (top panel) or SDS-PAGE (lower panel). Experiments were performed in duplicate or triplicate and mean values shown +/- standard deviation. One-way ANOVA was performed for statistical analysis and asterisk denotes <i>p</i> = <0.05 for treatment condition compared to vehicle treated control. (<b>B</b>) Human rhabdomyosarcoma cells (Rh30) were treated with 2μM or 20μM temsirolimus for 1.5hrs. Cell lysates were then layered onto 15–45% linear sucrose gradients for polysome analysis. Samples were fractionated and UV absorbance was measured in real-time. The location of 40S, 60S ribosomal subunits as well as 80S ribosome monomers and larger molecular weight polysomes are indicated. (<b>C</b>) Human 143B osteosarcoma cells were treated with vehicle, 20nM or 20μM temsirolimus for the indicated times. Protein lysates were analyzed by Western blot for levels of phospho and total protein for eIF2 alpha, eEF2 and 4EBP1. (<b>D</b>) The same as in (A) using human osteosarcoma cells (HOS and 143B) as well as human rhabdomyosarcoma cells (RD) treated with vehicle, 20nM or 20μM rapamycin for 1hr. Three independent experiments were performed for each treatment condition.</p

Decreased solvent accessibility of 28S rRNA in ribosomes isolated from cells treated with temsirolimus.

<p>(<b>A</b>) Polysome profiles (upper panel) and Western blot analysis of mTOR and FKBP12 protein levels (middle panel) in fractions following sucrose density gradient fractionation. Temsirolimus levels were quantified by HPLC-MS-MS analysis from pooled fractions as indicated in the text. <b>(B</b>) Alignment of X-ray crystal structures from <i>D</i>. <i>radiodurans</i> complexed with rapamycin (PDB 1Z58) and <i>S</i>. <i>cerevisiae</i> (PDB 3U5D) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185089#pone.0185089.ref052" target="_blank">52</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185089#pone.0185089.ref054" target="_blank">54</a>]. (<b>C</b>) Secondary structure model for <i>S</i>. <i>cerevisiae</i> large subunit domains II, IV and V, adapted from the Comparative RNA Website [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185089#pone.0185089.ref055" target="_blank">55</a>]. Arrow denotes location of the entrance to the peptide exit tunnel and the location of nucleotides in (B) are labeled. (<b>D</b>) Protection of 28S rRNA from chemical modification in temsirolimus treated samples. Primer extension analysis of human 28S rRNA from ribosomes isolated from temsirolimus treated or vehicle treated human rhabdomyosarcoma cells. Purified ribosomes were treated with DMS (modifies accessible adenosine and cytosines), CMCT (modifies accessible uracil and guanines) or mock treated. RNA was then purified, quantitated and equal amounts were used as the template for fluorescent primer extension analysis. Capillary electrophoresis was used to separate and resolve products under conditions allowing for single nucleotide resolution along with a DNA size standard. Arrows and shaded peaks denote the position of a DMS or CMCT modifiable ribonucleotide which is protected from modification in temsirolimus treated samples. An asterisk (*) denotes a chemical independent stop in some samples. The human nucleotide sequence appears below the electropherogram with <i>D</i>. <i>radiodurans</i> (DR), <i>E</i>. <i>coli</i> (EC), <i>S</i>. <i>cerevisiae</i> (SC) and <i>H</i>. <i>sapiens</i> (HU) numbering to denote the position of interest. Nucleotide positions were adjusted (+1) to account for the fact that DMS and CMCT modification results in a stop one nucleotide before the modified base.</p

Pharmacologically relevant micromolar doses of temsirolimus provide maximal tumor cell growth inhibition.

<p>(<b>A</b>) Human rhabdomyosarcoma (Rh30, RD) and osteosarcoma (143B, HOS-MNNG) cells were treated for 48hrs with the indicated doses of temsirolimus followed by staining with sulforhodamine B. Growth is represented as percentage of vehicle treated control. Each cell line was assayed at least three times and mean values for each data point are shown. (<b>B</b>) Cell cycle analysis of asynchronous cells following treatment with temsirolimus at different doses. Human osteosarcoma cells were treated for the indicated times with vehicle, 20nM, 2μM or 20μM temsirolimus followed by FACS analysis of propidium iodide stained cells to determine the distribution of cells within each phase of the cell cycle. (<b>C</b>) Treatment with FK-506 rescues low dose growth inhibitory effects of temsirolimus, but not high dose effects. Human osteosarcoma cells were treated with the indicated doses of temsirolimus alone or in combination with FK-506. Both drugs bind the intracellular protein FKBP12, but only temsirolimus/FKBP12 complex can bind to mTOR. Treatment was for 48hrs. Cell growth is expressed relative to FK-506 alone and assayed by staining with sulforhodomine B. Each set of conditions were analyzed in three separate experiments and mean values are shown.</p