Live Breast Cancer Cells Research Articles

Anion selective chromogenic and fluorogenic chemosensor and its application in breast cancer live cell imaging

A novel anion selective chromogenic and fluorogenic chemosensor (L) bearing catechol and phenol groups was developed. The sensorLwas tested for live breast cancer cell imaging and can be applied to the breast tumour diagnosis.

High-speed transport-of-intensity phase microscopy with an electrically tunable lens

We present a high-speed transport-of-intensity equation (TIE) quantitative phase microscopy technique, named TL-TIE, by combining an electrically tunable lens with a conventional transmission microscope. This permits the specimen at different focus position to be imaged in rapid succession, with constant magnification and no physically moving parts. The simplified image stack collection significantly reduces the acquisition time, allows for the diffraction-limited through-focus intensity stack collection at 15 frames per second, making dynamic TIE phase imaging possible. The technique is demonstrated by profiling of microlens array using optimal frequency selection scheme, and time-lapse imaging of live breast cancer cells by inversion the defocused phase optical transfer function to correct the phase blurring in traditional TIE. Experimental results illustrate its outstanding capability of the technique for quantitative phase imaging, through a simple, non-interferometric, high-speed, high-resolution, and unwrapping-free approach with prosperous applications in micro-optics, life sciences and bio-photonics.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Abstract LB-240: A novel approach to detecting RNA expression in living breast cancer cells.

Abstract When studying the complexities of cancer biomarkers, the ability to monitor gene expression within live cells that have become cancerous provides better insight than examining fixed or lysed tissues. However, monitoring RNA within intact cells can prove to be challenging with current techniques, which involve complex RNA isolation procedures that compromise cell viability. Further, amplification methods can create false positives or erroneously inflate differences. Here we describe a probe-based detection method that allows for the detection of ErbB-2 and EGFR mRNA levels across live intact SK-BR-3 breast cancer cells without the need for transfection reagents. The detection methodology is based upon an oligonucleotide gold nanoparticle conjugate capable of detecting intracellular levels of mRNA and miRNA in living cells. The probes enter the cell by means of the cell's native endocytosis machinery. Upon binding to the complementary target RNA sequence, a fluorescent signal (flare) is released, which allows the cells to be detected on any fluorescence analysis platform. In contrast to traditional RNA detection methods, since our technique allows for detection within live cells without altering their gene expression, the same cells that were profiled for EGFR and ErbB-2 expression could be sorted based on their specific mRNA content and subsequently further studied. Stimulation or treatments could then be performed on the previously profiled cells and assessed using probes for additional RNA markers or more traditional techniques such as antibody staining, qRT-PCR, or Array profiling. For this purpose, we combined immunostaining for cell surface EGFR with fluorogenic nanoparticle-based detection of EGFR mRNA in 4 breast cancer cell lines known to range in EGFR expression levels, and observed significant correlation for the signals in two-color flow cytometry. Thus, detecting RNA expression levels in live cancer cells with the ability to utilize the same cells in downstream testing gives researchers the ability to perform experiments which were previously thought to be impossible. Citation Format: Don Weldon, Kevin Su, Alex Ko, Grace Johnston, Matthew Hsu, Lucas Armstrong. A novel approach to detecting RNA expression in living breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-240. doi:10.1158/1538-7445.AM2013-LB-240

Quantitative FRET Imaging to Visualize the Invasiveness of Live Breast Cancer Cells

Matrix metalloproteinases (MMPs) remodel tumor microenvironment and promote cancer metastasis. Among the MMP family proteases, the proteolytic activity of the pro-tumorigenic and pro-metastatic membrane-type 1 (MT1)-MMP constitutes a promising and targetable biomarker of aggressive cancer tumors. In this study, we systematically developed and characterized several highly sensitive and specific biosensors based on fluorescence resonant energy transfer (FRET), for visualizing MT1-MMP activity in live cells. The sensitivity of the AHLR-MT1-MMP biosensor was the highest and five times that of a reported version. Hence, the AHLR biosensor was employed to quantitatively profile the MT1-MMP activity in multiple breast cancer cell lines, and to visualize the spatiotemporal MT1-MMP activity simultaneously with the underlying collagen matrix at the single cell level. We detected a significantly higher level of MT1-MMP activity in invasive cancer cells than those in benign or non-invasive cells. Our results further show that the high MT1-MMP activity was stimulated by the adhesion of invasive cancer cells onto the extracellular matrix, which is precisely correlated with the cell’s ability to degrade the collagen matrix. Thus, we systematically optimized a FRET-based biosensor, which provides a powerful tool to detect the pro-invasive MT1-MMP activity at single cell levels. This readout can be applied to profile the invasiveness of single cells from clinical samples, and to serve as an indicator for screening anti-cancer inhibitors.

Accurate Sensitivity of Quantum Dots for Detection of HER2 Expression in Breast Cancer Cells and Tissues

Here we introduce novel optical properties and accurate sensitivity of Quantum dot (QD)-based detection system for tracking the breast cancer marker, HER2. QD525 was used to detect HER2 using home-made HER2-specific monoclonal antibodies in fixed and living HER2(+) SKBR-3 cell line and breast cancer tissues. Additionally, we compared fluorescence intensity (FI), photostability and staining index (SI) of QD525 signals at different exposure times and two excitation wavelengths with those of the conventional organic dye, FITC. Labeling signals of QD525 in both fixed and living breast cancer cells and tissue preparations were found to be significantly higher than those of FITC at 460-495nm excitation wavelengths. Interestingly, when excited at 330-385nm, the superiority of QD525 was more highlighted with at least 4-5 fold higher FI and SI compared to FITC. Moreover, QDs exhibited exceptional photostability during continuous illumination of cancerous cells and tissues, while FITC signal faded very quickly. QDs can be used as sensitive reporters for in situ detection of tumor markers which in turn could be viewed as a novel approach for early detection of cancers. To take comprehensive advantage of QDs, it is necessary that their optimal excitation wavelength is employed.

Abstract 5183: Raster-image-correlation spectroscopy of paxillin-GFP-expressing breast cancer cell in vitro and in vivo

Abstract Raster-image-correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in the living cell, including concentrations of molecules and their diffusion coefficients. Any cell containing a fluorophore that can be imaged with a laser scanning microscope can be analyzed with RICS. We obtained RICS images with an Olympus FluoView FV1000 confocal microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Paxillin is involved in the assembly of focal adhesions, which was linked to green fluorescent protein (GFP) for the current study. In this study, we describe RICS of paxillin-GFP expression in breast cancer cells (MDA-MB-231) in vitro and in vivo. Slow-moving membrane-bound paxillin proteins were measured in live breast cancer cells in vitro. Paxillin-GFP-expressing breast cancer cells (1×106) were injected in the epigastric cranials vein of the nude mouse. Paxillin-GFP-expressing breast cancer cells became attached to the inner vessel wall within 3 hours after injection. Rapidly-moving cytosolic paxillin-GFP molecules were imaged with RICS. With the ability to measure the molecular dynamics of paxillin in cancer cells in vitro and in vivo by RICS, we are now capable of studying the role of both slow-moving paxillin in the cell membrane and rapidly-moving cytosolic paxillin in cancer-cell behavior. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5183. doi:1538-7445.AM2012-5183

Raman and Fluorescence Spectral Imaging of Live Breast Cancer Cells Incubated with PEGylated Gold Nanorods

The optical properties of PEGylated gold nanorods (PEG-GNR) in interaction with cells have been investigated with Raman and fluorescence microspectroscopic imaging. The emission spectra were compared with those from dispersions of GNR, which can be characterized by a broad emission bandwidth of approximately 60 nm with a band maximum around 675 nm. These properties are in good agreement with observations from various other gold substrates and (nano)particles. The emission spectra from cells incubated with PEG-GNR were dominated by Raman scattering from locations where no GNR were present. Intense fluorescence spectral lines, with peak amplitude comparable with the Raman scattering from cells, were observed from locations containing GNR. The frequency range of the fluorescence emission spectra coincided mainly with the Raman fingerprint region from 500 cm−1 to 1800 cm−1, excited by the laser emission line at 647.1 nm. No surface-enhanced Raman spectra were observed. It was furthermore observed from cluster analysis of the Raman and fluorescence hyperspectral datasets that the GNR-related integrated fluorescence emission band from an individual cell could be sub-divided in multiple bands with slightly varying band maxima. Raman difference spectra of cells with GNR minus control cells showed that the amplitude of lipid signal in cells incubated with PEG-GNR was increased. An excellent correlation was found between the increased lipid signals and locations of the nanorods. This positive correlation between Raman signals from lipids and fluorescence signals from gold nanorods supports that gold nanorods are locally accumulating in lipid vesicles within the cells.

Facile Synthesis of Hollow Mesoporous Hydroxyapatite Nanoparticles for Intracellular Bio-imaging

The effects of several critical factors including the amount of EG, the reaction time, the concentrations of Ca(CH3COO)2 and NaHCO3, the concentration of H3PO4, and the amount of acetic acid were investigated for synthesizing hollow mesoporous hydroxyapatite nanoparticles (hm-HANPs). We optimized the reaction conditions as follows: the concentration of Ca(CH3COO)2 and NaHCO3 was 0.3 M, the ratio of Ca(CH3COO)2 and NaHCO3 to ethylene glycol (EG) was 1:5, the reaction time for the formation of hydroxyapatite was 3 hr, the concentration of H3PO4 was 0.01 M, and the ratio of hm-HANP solution to acetic acid was 1:3.125. Monodispersed hm- HANPs with a defined particle size could be obtained. A fluorescent dye named fluorescein isothiocyanate (FITC) was successfully linked to the surface of hm-HANPs for the bioimaging of hm-HANPs in living breast cancer cells (BT-20). The FITC-hm-HANPs were taken up by the cells and stayed in the cytoplasm with high biocompatibility. The information obtained in this study will benefit the synthesis of hydroxyapatite nanostructures for many biomedical-related applications.

Distinguishing breast cancer cells using surface-enhanced Raman scattering

The detection and identification of epidermal growth factor receptor 2 (HER2)-positive breast cancer cells is crucial for the clinic therapy of breast cancer. For the aim of the detection, a novel surface-enhanced Raman scattering (SERS) probe for distinguishing breast cancers at different HER2 statuses is reported in this paper. In such a probe, anti-HER2 antibody-conjugated silver nanoparticles have been synthesized for specific targeting of HER2-positive breast cancer cells. More importantly, different from the previously reported SERS probe for targeting cancer cells, p-mercaptobenzoic acid is utilized as both the Raman reporter and the conjugation agent for attaching antibody molecules, which leads to a much simplified structure. For investigating the ability of such a probe to distinguish breast cancer cells, SKBR3 and MCF7 cells were chosen as two model systems, which are HER2-positive- and HER2-negative-expressing cells, respectively. The experimental results reveal that SKBR3 cells exhibit much stronger SERS signals than MCF7 cells, indicating that the probe could be utilized to distinguish breast cancer cells at different HER2 statuses. This kind of SERS probe holds a potential for a direct detection of living breast cancer cells with the advantages of easy fabrication, high SERS sensitivity, and biocompatibility.

Abstract 1769: Phage display selection of cancer cell-binding and -internalizing ligands using random peptide libraries constructed at N-terminus of P99 cephalosporinase

Abstract The cancer researchers today are focused in developing new types of cancer treatments that use drugs or other substances to identify and kill cancer cells while doing very little or no damage to normal healthy cells. It is believed that an ability to home in on cancer cells makes chemotherapy both more effective and less likely to cause side effects. One of the strategies to achieve this goal depends on developing ligands that specifically bind/internalize to cancer cells and exert therapeutic effects of their own or guide a targeted delivery of a therapeutic agent (cytotoxic drug, gene, radionuclide, or enzyme) to the tumor cells. Phage display is one example of high throughput technologies that has been successfully utilized for the generation of massive ligand libraries and their selection against specific targets. We constructed novel phage-displayed random linear dodecapeptide (X12) and cysteine-constrained random decapeptide (CX10C) libraries at N-terminal end of the Enterobacter cloacae P99 cephalosporinase molecule (P99 β-lactamase: β-lactam hydrolase, E.C. # 3.5.2.6). The peptide-β-lactamase fusions were displayed at the amino terminus of the minor coat pIII protein of M13 filamentous phage. Several cancer cell-specific binding β-lactamase-peptide fusion ligands were isolated by selecting these libraries on live BT-474 human breast cancer cells. The identified ligands shared several significant motifs, which showed their selectivity and possible binding to some common cancer cell targets. β-Lactamase is a high turnover enzyme with several available chromogenic and fluorogenic substrates. The peptide fusion to this enzyme made the post-panning steps very fast and simple. The concentrations of β-lactamase-peptide fusions in phage samples, lysates and purified fusion proteins were normalized based on the β-lactamase activity. The clone screening was also based on assaying the residual cell-bound β-lactamase activity. Some of the cancer cell binding β-lactamase-peptide fusion proteins showed cell internalization by both immunofluorescence and β-lactamase activity-based procedures. The staining with FRET-based substrate CCF4 confirmed the functional integrity of the internalized β-lactamase, and demonstrated that at least a part of the internalized enzyme conjugate could escape the endosomal degradation pathway in intact form and reacted with the substrate present in the cytoplasm. The specificity of the selected ligands was assessed by studying their binding and internalization in 11 other cancer and non-cancer cell lines. The β-lactamase fusion to peptides not only accelerates the clone screening process but can also facilitate the tracking of ligand molecules in various biological studies. The cancer-specific affinity reagents selected from these libraries have a potential for their use in targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1769. doi:10.1158/1538-7445.AM2011-1769

Abstract P6-04-06: Stratification of Breast Cancers Based on Functional Phosphoprotein Signaling Profiles Elicited from Live Tumor Cells

Abstract Background: Abnormal signal transduction networks are frequent targets of existing and emerging molecularly targeted agents (MTAs). Unfortunately, most predictive biomarkers to guide therapeutic selection are based on indirect assessment of signal transduction through DNA mutations or transcriptional profiles rather than dynamic assessment of signal transduction proteins themselves. Classification of breast cancer based on functional signaling profiles derived from a set of signaling phosphoproteins induced upon growth factor stimulation of live breast cancer cells is likely to provide a more accurate system for MTA selection than indirect methods utilizing fixed or frozen tissue. Objective: This study aimed to demonstrate stratification of multiple breast cancer model systems based on functional signaling profiles elicited from live tumor cells in response to ex vivo stimuli. Methods: Breast cancer cell lines (MCF-7, HCC-1937, MDA-MB-231, BT474, and SKBR3) were exposed to either vehicle (control) or stimulated with 200 ng/ml epidermal growth factor (EGF) for 5 minutes then lysed and proteins extracted. Mean Fluorescence Intensity (MFI) levels of six phosphoproteins (pEGFR, pErk, pAkt, pP70S6K, pGSK3β, and pSTAT3) were determined in sextuplet using a multiplexed bead-immunoassay (BioPlex, BioRad) and a modulation score (MS), defined as the log 2 (MFI stimulated /MFI contrd), calculated for each. Scores were ranked by percentile relative to the median (0.66) and inter-quartile range (IQR) (154). Moderate responders were classified as those with MS between the 75th percentile (2.20) and the 75th percentile plus the IQR (3.74). High responders were those MS > 3.74. Low responders were those MS falling between the IQR and 75th percentile (1.54-2.20) whereas non responders were classified as MS < 1.54 Results: EGF stimulation resulted in high levels of EGFR-phosphorylation in all cells except BT474, which responded moderately (2.57). MS for pErk were high in MCF-7 cells (3.92), moderate in HCC -1937 (2.89) and none for the other lines tested. Moderate STAT-3 phosphorylation was observed in only MCF-7cells (2.34) whereas low pAkt MS were observed in only SKBR3 (1.78). All other markers across the five cell lines tested were non responders (< 1.54), with pGSK3β and pP70S6K yielding MS < 1.0 for all five cell lines. Interestingly, the relative MS rank order of all six proteins differed across each cell line suggesting further opportunity for stratification. Conclusions: Our data demonstrate that different breast cancer cell lines display unique functional phosphoprotein signaling profiles, thereby providing a mechanism for stratifying tumors based on individual signal transduction pathway activation. Further studies are underway to correlate the functional signaling profiles identified here to sensitivity and resistance to specific MTA treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-04-06.

Abstract 1977: BAL27862: A unique microtubule-targeted drug that suppresses microtubule dynamics, severs microtubules, and overcomes Bcl-2- and tubulin subtype-related drug resistance

Abstract Microtubule-targeted drugs (MTDs) are widely used to treat human malignancies such as ovarian and breast cancers. However, a major limitation of their efficacy is the development of resistance modulated by P-gp over-expression or modification of the microtubular target and/or the expression/function of apoptotic pathway proteins such as Bcl-2 [1]. BAL27862 is a novel, orally bioavailable synthetic MTD that triggers apoptosis in cancer cell lines. BAL27862 induces a G2/M arrest, destabilizing microtubules (MT) to produce a unique MT phenotype distinct from that observed with vinca alkaloids (e.g. vinblastine), colchicine and taxanes. Moreover, BAL27862 retains its anti-proliferative activity against multi-drug-resistant, P-gp over-expressing tumor cells [2]. To investigate the mechanism of action of BAL27862, we analyzed its effects on MT dynamics in living breast cancer cells (SKBr3 cells). As measurements were not possible using equipotent cytotoxic concentrations of the MT destabilizer vinblastine, it was compared to the MT stabilizer paclitaxel. BAL27862 suppressed MT dynamic instability, supressing MT catastrophes and growth rate and increasing the mean duration of pauses. In contrast, paclitaxel suppressed the MT shortening rate and did not affect the MT growth rate. At equipotent cytotoxic concentrations, the suppression of MT dynamics by BAL27862 was 2-fold lower than paclitaxel. Strikingly, BAL27862 displayed a unique MT severing activity. We then determined the anti-proliferative potency of BAL27862 in paclitaxel- and Vinca-alkaloid-resistant ovarian cancer cells (A2780-TC1 cells) in comparison to the chemosensitive parental line (A2780-wt cells). A2780-TC1 cell resistance is mediated by P-gp over-expression, Bcl-2 down-regulation and modifications in tubulin subtype composition [1]. Importantly, BAL27862 remained fully active against A2780-TC1 cells. Indeed, when comparing EC50, the resistance factors observed were 1.14 (NS) and 147 (p<0.01) for BAL27862 and paclitaxel, respectively. Furthermore, in the presence of verapamil (P-gp inhibitor), resistance factors were 1.17 (NS) and 11.43 (p<0.01) for BAL27862 and paclitaxel, respectively. These results confirm that BAL27862 overcomes P-gp overexpression-mediated resistance, and demonstrate that Bcl-2 status and modifications in tubulin composition do not necessarily affect its cytotoxicity. In conclusion, BAL27862 shares properties with other MTDs such as suppression of MT dynamics, but displays unique features such as MT severing. In ovarian tumor cells, activity appears independent of Bcl-2 and tubulin modifications. These features make BAL27862 a promising compound for clinical development. 1. Estève MA et al, Mol Cancer Ther, 2006 2. Lane H. et al, EORTC-NCI-AACR 2008, Abstract 444 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1977.

Abstract A170: High-content screening for inhibitors of oncogenic transcription by c-Myc

Abstract There is evidence that Myc partners with another molecule called TRRAP to regulate genes required for transformation. Our hypothesis is that blocking the interaction of Myc with TRRAP will selectively kill breast cancer cells. Traditional drug screening approaches to identify compounds that will break apart two interacting proteins have rarely been successful. However, our approach is to screen compounds in live breast cancer cells for those that prevent Myc and TRRAP from binding to each other. Since the cell is constantly replacing Myc, a compound that prevents it from binding TRRAP should kill even established tumors. Importantly, we previously mapped the places on Myc and TRRAP that bind them together and showed they don't involve the parts of the protein involved in other important functions. Therefore, compounds we find that prevent Myc-TRRAP binding should not be toxic to normal cells. Methods: We have established a new assay in which Myc and TRRAP are expressed as fusion proteins to Cerulean and Citrine fluorescence proteins, respectively. We are developing two different but related assays for the interaction between the two proteins. In one we rely on proximity resulting in complementation between non-fluorescent fragments of the Cerulean fluorescence protein. In this assay heterodimerization results in increased fluorescence. In the other assay proximity results in fluorescence resonance energy transfer (FRET) between the Cerulean and Citrine fluorescence proteins. We detect FRET by fluorescence lifetime imaging (FLIM). Results: A novel robotic microscope that can automatically perform high speed FLIM and thereby detect Myc-TRRAP binding quickly and accurately has been assembled and tested. Our automated microscope has had an environmental stage for live cell imaging fitted and tested. Both FRET standards and constructs based on the Cerulean-Myc / TRRAP-Citrine pair have been measured to validate the assay. Cell lines are being optimized for use for screening. Conclusions: High speed FLIM can be used to detect FRET between Cerulean-Myc and TRRAP-Citrine in live cells. Using our automated microscope we can test individually the effect of large numbers of small molecules in live breast cancer cells. This will let us identify compounds that are not only effective but that get into breast cancer cells and are not toxic to normal cells. Relevance/Impact: To stimulate the development of new drugs effective against a wide spectrum of cancers, we are identifying small drug like molecules that disrupt the function of a particularly potent cell growth gene called Myc, which is often misregulated in breast cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A170.

Endoplasmic reticulum-associated calcium regulates taxol-induced apoptosis in breast cancer cells.

Abstract Abstract #4163 Background: The body eliminates unhealthy cells through programmed cell-suicide called apoptosis. Calcium (Ca2+), one of the key regulators of cell survival, is also important in regulating apoptosis. Although the chemotherapeutic agent Taxol employs apoptosis to induce cell death in breast cancer treatment, the exact mechanism of how it induces apoptosis and the role of Ca2+ in this process remain unclear. The endoplasmic reticulum (ER), the main intracellular Ca2+ store, is newly-recognized as an important gateway in apoptosis, and possibly provides a target for Taxol.
 Objective/Hypothesis: Putting these facts together, our hypothesis is that ER Ca2+ changes induced by Taxol determine breast cancer cell susceptibility to apoptosis and thus play a key role in ER-associated apoptosis. Therefore, this study investigated whether Ca2+ changes, especially associated with the ER, were generated and related to this apoptotic event.
 Study Design and Methods: For this study, we used the MDA-MB-468 breast cancer cell line. The dynamic Ca2+ changes induced by Taxol were determined in living breast cancer cells by two methods: free cytosolic Ca2+ changes were measured using Fluo4-AM Ca2+ dye; and ER Ca2+ changes were measured by a novel Ca2+ cameleon, D1ER. After inducing and evaluating Taxol-induced apoptosis in this breast cancer cell line, the effects of different Ca2+ interfering agents on the apoptotic event were tested to determine whether Taxol-induced apoptosis is Ca2+ dependent.
 Results: Taxol has a direct effect on Ca2+ homeostasis. Taxol induces a rapid ER Ca2+ release and results in a transient increase in cytosolic Ca2+ levels. A gradual ER Ca2+ depletion developed and contributed to the final sustained cytosolic Ca2+ increase. Interfering with these Ca2+ changes inhibited the Taxol-induced apoptosis, indicating that ER Ca2+ promotes Taxol-induced apoptosis.
 Relevance: It is critical to understand the process through which the widely used anticancer agent Taxol induces apoptosis. This research addressed the question of whether intracellular Ca2+ changes play a critical or marginal role in mediating Taxol-induced apoptosis. Elucidating the role of calcium in this process will not only help to clarify the mechanism of Taxol, but also aid in more effective application of Taxol in breast cancer treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4163.

Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted goldnanoshells and two-photon excitation microscopy

We demonstrate the capability of using immunotargeted gold nanoshells as contrast agentsfor in vitro two-photon microscopy. The two-photon luminescence properties ofdifferent-sized gold nanoshells are first validated using near-infrared excitation at 780 nm.The utility of two-photon microscopy as a tool for imaging live HER2-overexpressing breastcancer cells labeled with anti-HER2-conjugated nanoshells is then explored and imagingresults are compared to normal breast cells. Five different imaging channels aresimultaneously examined within the emission wavelength range of 451–644 nm. Our resultsindicate that under near-infrared excitation, superior contrast of SK-BR-3 cancer cellslabeled with immunotargeted nanoshells occurs at an emission wavelength ranging from590 to 644 nm. Luminescence from labeled normal breast cells and autofluorescencefrom unlabeled cancer and normal cells remain imperceptible under the sameconditions.

Cell tracking and detection of molecular expression in live cells using lipid-enclosed CdSe quantum dots as contrast agents for epi-third harmonic generation microscopy

We demonstrated that lipid-enclosed CdSe quantum dots (LEQDs) can function as versatile contrast agents in epi-detection third harmonic generation (THG) microscopy for biological applications in vivo. With epi-THG intensities 20 times stronger than corresponding fluorescence intensities from the same LEQDs under the same conditions of energy absorption, such high brightness LEQDs were proved for the abilities of cell tracking and detection of specific molecular expression in live cancer cells. Using nude mice as an animal model, the distribution of LEQD-loaded tumor cells deep in subcutaneous tissues were imaged with high THG contrast. This is the first demonstration that THG contrast can be manipulated in vivo with nanoparticles. By linking LEQDs with anti-Her2 antibodies, the expression of Her2/neu receptors in live breast cancer cells could also be easily detected through THG. Compared with fluorescence modalities, the THG modality also provides the advantage of no photobleaching and photoblinkin g effects. Combined with a high penetration 1230 nm laser, these novel features make LEQDs excellent THG contrast agents for in vivo deep-tissue imaging in the future.

Dynamics of the CapG actin-binding protein in the cell nucleus studied by FRAP and FCS

FRAP (fluorescence recovery after photobleaching) and FCS (fluorescence correlation spectroscopy) are spectroscopic methods for monitoring the dynamic distribution of proteins inside the nucleus of living cells. As an example we report our studies on the intracellular mobility of the actin-binding protein CapG in live breast cancer cells. This Gelsolin-related protein is a putative oncogene. It appears to be overexpressed especially in metastasizing breast cancer. Furthermore, the CapG protein is known to be involved in the motility control of non-muscle benign cells. Its increased expression triggers an increase in cell motility of benign cells. Thus it can be expected that in cancer cells overexpressing the CapG protein, motility, invasiveness and metastasis might be particularly promoted. Since the nuclear CapG fraction seems to be pivotal to the increase in cell motility, we focused our studies on the CapG mobility in cell nuclei of live breast cancer cells. Using FCS and FRAP we showed that the eGFP-tagged CapG is monomeric and characterized its diffusional properties on the microsecond to minute timescale. This information about the mobility and compartmentalization of CapG might help to provide insight into its function within the cell nucleus and give clues about its altered cellular function in malignant dedifferentiation.

P35 Phage display-derived human scFv antibodies isolated by binding to live primary breast cancer cells recognize GRP78

P35 Phage display-derived human scFv antibodies isolated by binding to live primary breast cancer cells recognize GRP78

Analysis of Estrogen Receptor α-Sp1 Interactions in Breast Cancer Cells by Fluorescence Resonance Energy Transfer

Estrogen-dependent regulation of several genes associated with cell cycle progression, proliferation, and nucleotide metabolism in breast cancer cells is associated with interactions of estrogen receptor (ER)alpha/Sp1 with GC-rich promoter elements. This study investigates ligand-dependent interactions of ERalpha and Sp1 in MCF-7 breast cancer cells using fluorescence resonance energy transfer (FRET). Chimeric ERalpha and Sp1 proteins fused to cyan fluorescent protein or yellow fluorescent protein were transfected into MCF-7 cells, and a FRET signal was induced after treatment with 17beta-estradiol, 4'-hydroxytamoxifen, or ICI 182,780. Induction of FRET by these ERalpha agonists/antagonists was paralleled by their activation of gene expression in cells transfected with a construct (pSp1(3)) containing three tandem Sp1 binding sites linked to a luciferase reporter gene. In contrast, interactions between ERalpha and Sp1DeltaDBD [a DNA binding domain (DBD) deletion mutant of Sp1] are not observed, and this is consistent with the critical role of the C-terminal DBD of Sp1 for interaction with ERalpha. Results of the FRET assay are consistent with in vitro studies on ERalpha/Sp1 interactions and transactivation, and confirm that ERalpha and Sp1 interact in living breast cancer cells.