carbocyanines and Breast-Neoplasms

This review covers the application of heptamethine cyanine dye (HMCD) mediated drug delivery. A relatively small number of HMCDs possess tumor targeting abilities, and this has spurred interest from research groups to explore them as drug delivery systems. Their tumor selectivity is primarily attributed to their uptake by certain isoforms of organic anion transporting polypeptides (OATPs) which are overexpressed in cancer tissues, although there are other possible mechanisms for the observed selectivity still under investigation. This specificity is confirmed using various cancer cell lines and is accompanied by moderate cytotoxicity. Their retention in tumor tissue is facilitated by the formation of albumin adducts as revealed by published mechanistic studies. HMCDs are also organelle selective dyes with specificity toward mitochondria and lysosomes, and with absorption and emission in the near-infrared region. This makes them valuable tools for biomedical imaging, especially in the field of fluorescence-guided tumor surgery. Furthermore, conjugating antitumor agents to HMCDs is providing novel drugs that await clinical testing. HMCD development as theranostic agents with dual tumor targeting and treatment capability signals a new approach to overcome drug resistance (mediated through evasion of efflux pumps) and systemic toxicity, the two parameters which have long plagued drug discovery.

Topics: Antineoplastic Agents; Brain Neoplasms; Breast Neoplasms; Burkitt Lymphoma; Carbocyanines; Coloring Agents; Drug Delivery Systems; Drug Discovery; Drug Resistance, Neoplasm; Female; Humans; Kidney Neoplasms; Male; Precision Medicine; Prostatic Neoplasms

To evaluate the efficacy of the near-infrared (NIR) dye Omocianine in a placebo-controlled, dose-escalating multicenter trial for the detection of malignant breast lesions by using a NIR imaging system.. The study was approved by the ethical review board of Berlin and Münster,, and all participants provided written informed consent. Fifty-two consecutive patients were examined with NIR imaging before, during, and after intravenous injection of Omocianine. Three-dimensional absorption and fluorescence diffuse optical tomography scans were recorded simultaneously on a prototype NIR imaging unit (Computed Tomography Laser Mammography, Imaging Diagnostic Systems, Inc., Ft. Lauderdale, FL). Two readers assessed the images in consensus and assigned visibility scores to lesions seen on the absorption and absorption-corrected fluorescence diffuse optical tomography mammograms. Imaging results were compared with histopathologic findings. To analyze whether lesion detection rate for malignant lesions depended on the size of the lesion, lesions were dichotomized into those measuring less than 20 mm and those measuring 20 mm or more. Moreover, the shortest diameter between the center of the target lesions and the skin was measured on axial optical mammography data.. There were a total of 53 target lesions. Histopathologically, 22 target lesions were diagnosed as benign and 31 target lesions as malignant. In the absorption mode, a detection rate of 11.8% for benign and 44.4% for malignant lesions across all dose groups was found. In the fluorescence mode, a detection rate of 17.6% was revealed for benign and 55.6% for malignant lesions across all dose groups. For dose group 0.1 mg/kg, a detection rate of 100% was found for malignant lesions in the fluorescence mode and 71.4% in the absorption mode. Across all dose groups in the fluorescence mode, detection rate for malignant target lesions in breasts smaller than the median axial breast diameter of 12.8 cm was higher with 69.2% than in median diameters ≥ 12.8 cm with 46.2%. Omocianine-enhanced fluorescence optical mammography allowed a better detection of more superficially located lesions, with detection rates for a lesion-skin distance <20 mm of 63.6%, for <30 mm of 47.4% and for ≥ 30 mm of 25%. Malignant target lesions with a diameter ≥ 20 mm were slightly better detected with 61.5% in contrast to suspicious lesions <20 mm with 53.8%. Optimal imaging time points varied strongly among the different target lesions and Omocianine dose groups, with a mean optimal time point for malignant lesions at 188 ± 385 minutes.. Preliminary data suggest that fluorescence imaging after Omocianine administration has the potential to detect malignant breast lesions. As our study showed considerable variations in the detection of breast cancer at different fluorophore concentrations ranging from 20% to 100%, future work needs to be done to assess the suitable dose for NIR imaging.

Topics: Aged; Aged, 80 and over; Breast Neoplasms; Carbocyanines; Female; Fluorescent Dyes; Germany; Humans; Image Enhancement; Image Interpretation, Computer-Assisted; Indoles; Infrared Rays; Lasers; Magnetic Resonance Imaging; Mammography; Middle Aged; Pattern Recognition, Automated; Placebos; Tomography, Optical

The CDK4/6 inhibitor palbociclib, combined with endocrine therapy, has been shown to be effective in postmenopausal women with estrogen receptor-positive, HER2-negative advanced or metastatic breast cancer. However, palbociclib is not as effective in the highly aggressive, triple-negative breast cancer that lacks sensitivity to chemotherapy or endocrine therapy. We hypothesized that conjugation of the near-infrared dye MHI-148 with palbociclib can produce a potential theranostic in triple-negative, as well as estrogen receptor-positive, breast cancer cells. In our study, the conjugate was found to have enhanced activity in all mammalian cell lines tested in vitro. However, the conjugate was cytotoxic and did not induce G1 cell cycle arrest in breast cancer cells, suggesting its mechanism of action differs from the parent compound palbociclib. The study highlights the importance of investigating the mechanism of conjugates of near-infrared dyes to therapeutic compounds, as conjugation can potentially result in a change of mechanism or target, with an enhanced cytotoxic effect in this case.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carbocyanines; CHO Cells; Cricetulus; Cytotoxins; Female; HEK293 Cells; Humans; Indoles; Piperazines; Pyridines

Topics: Animals; Breast Neoplasms; Carbocyanines; Cytokines; Female; Fibronectins; Humans; Mice; Multimodal Imaging; Peptides; Tissue Distribution; Tumor Microenvironment

Due to the hydrophobicity of the cyanine dye and the huge conjugated plane, the cyanine dye is prone to H-aggregation in aqueous solution, and the ultraviolet absorption is blue-shifted. Here, a hydrophilic quaternary stereo-specific cyanine (HQS-Cy) dye has been synthesized and polypeptide based nanoparticles have been prepared, which improve the water solubility of the cyanine in two aspects. First, at the molecular level, the sulfonic acid group increases the water solubility of the dye molecule while the dimethyl-ammonium functional group repels the molecule through the charge-charge interaction, destroying the planar characteristics of the cyanine structure, increasing the molecular distance between the dye molecules, and preventing the accumulation of cyanine. Secondly, at the nano-micelle level, the use of amphiphilic polypeptide blocks to encapsulate the dye increases the water solubility of the dye while also increasing its biocompatibility. The HQS-Cy@P NPs prepared by the above methods exhibit the maximum absorption at 985 nm and maximum fluorescence emission at 1050 nm in aqueous solution. HQS-Cy@P exhibits good photothermal stability and significant photothermal conversion efficiency of about 35.5%, and both in vitro and in vivo studies revealed that it is an efficient system for NIR-II imaging-guided photothermal therapy of cancer.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cell Line; Cell Proliferation; Cell Survival; Coloring Agents; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Humans; Infrared Rays; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Molecular Structure; Optical Imaging; Photothermal Therapy; Solubility; Uterine Cervical Neoplasms

Nitroimidazoles are one of the most common radiosensitizers investigated to combat hypoxia-induced resistance to cancer radiotherapy. However, due to poor selectivity distinguishing cancer cells from normal cells, effective doses of radiosensitization are much closer to the doses of toxicity induced by nitroimidazoles, limiting their clinical application. In this work, a tumor-targeting near-infrared (NIR) cyanine dye (IR-808) was utilized as a targeting ligand and an NIR fluorophore tracer to chemically conjugate with different structures of hypoxia-affinic nitroimidazoles. One of the NIR fluorophore-conjugated nitroimidazoles (808-NM2) was identified to preferentially accumulate in hypoxic tumor cells, sensitively outline the tumor contour, and effectively inhibit tumor growth synergistically by chemotherapy and radiotherapy. More importantly, nitroimidazoles were successfully taken into cancer cell mitochondria via 808-NM2 conjugate to exert the synergistic effect of chemoradiotherapy. Regarding the important roles of mitochondria on cancer cell survival and metastasis under hypoxia, 808-NM2 may be hopeful to fight against hypoxic tumors.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Chemoradiotherapy; Coloring Agents; Female; Humans; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Nitroimidazoles; Tumor Hypoxia

Discriminative identification of homologous miRNAs in miRNA family with high specificity and sensitivity is crucial for accurate classification, diagnosis and prognosis of breast cancer. Herein, we report a reliable, sensitive, and selective assay by coupling fluorescence resonance energy transfer (FRET) with cascade signal amplification. The strategy is developed by designing two programmable DNA probes that can be triggered to shift from "off" to "on" state in a cascade hybridization reaction in the presence of target miRNA let-7a, leading to the generation of an amplified signal. The assay can detect concentrations as low as ∼3.0 pM let-7a and discriminate let-7a from other highly homologous members in the let-7 miRNA family. Moreover, it can also be used to determine let-7a levels at single-cell resolution and evaluate the drug efficacy of let-7a expression among various molecular types of breast cancer cell lines. The advantage of this assay is a combined result of signal generation and amplification triggered by target miRNA, which can satisfy an assay of analogous miRNA in a downregulated manner with high specificity. It has promising potential as a selective assay for homologous miRNAs in precision medicine.

Topics: Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; DNA Probes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Inverted Repeat Sequences; MicroRNAs; Microscopy, Confocal; Microscopy, Fluorescence; Nucleic Acid Hybridization; Paclitaxel; Proof of Concept Study

Exosomal microRNAs (miRNAs) are reliable and noninvasive biomarkers for the early diagnosis of cancer. Yet, accurate and feasible detection of exosomal miRNAs is often hampered by the low abundance of miRNAs in exosomes and the requirement for RNA extraction in large sample volumes. Here we show a thermophoretic sensor implemented with nanoflares for in situ detection of exosomal miRNAs, without resorting to either RNA extraction or target amplification. Thermophoretic accumulation of nanoflare-treated exosomes leads to an amplified fluorescence signal upon the binding of exosomal miRNAs to nanoflares, allowing for direct and quantitative measurement of exosomal miRNAs down to 0.36 fM in 0.5 μL serum samples. One of the best markers, exosomal miR-375, showed an accuracy of 85% for detection of estrogen receptor-positive breast cancer at early stages (stages I, II). This work provides a feasible tool to improve the diagnosis of cancer.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; DNA; Exosomes; Fluorescent Dyes; Gold; Humans; Infrared Rays; Metal Nanoparticles; MicroRNAs; Middle Aged; Nucleic Acid Hybridization; Spectrometry, Fluorescence; Temperature

Mitochondria plays pivotal roles in energy production and apoptotic pathways. Mitochondria-targeting strategy has been recognized as a promising way for cancer theranostics. Thus, spatiotemporally manipulating the prolonged retention of theranostic agents within mitochondria is considerably significant in cancer diagnosis and therapy. Herein, as a proof-of concept, we for the first time report a sulfenic acid-responsive platform on controlled immobilization of probes within mitochondria for prolonged tumor imaging. A novel near-infrared (NIR) probe DATC constructed with a NIR dye (Cy5) as signal unit, a cationic triphenylphosphonium (TPP) for mitochondria targeting, and a sulfenic acid-reactive group (1,3-cyclohexanedione) for mitochondrial fixation was rationally designed and synthesized. This probe displayed good target ability to mitochondria and could act as a promising fluorescent probe for specific visualization of endogenous protein sulfenic acids expressed in the mitochondria. Moreover, the probe could be spontaneously fixed on site through the specific reaction and covalent binding to the sulfenic acids of oxidized proteins under oxidative stress, resulting in enhanced intracellular uptake and prolonged retention. We thus believe that this mitochondria-targeted and locational immobilization strategy may offer a new insight for long-term tumor imaging and effective therapy.

Topics: 3T3 Cells; Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Fluorescent Dyes; Infrared Rays; Mice; Mitochondria; Molecular Structure; Optical Imaging; Sulfenic Acids

Photodynamic therapy (PDT) is currently one of the cancer treatment options. PDT requires the application of a photosensitizer (such as: porphyrins, chlorines, and phthalocyanines) that selectively targets malignant cells. It is a dilemma to find a proper photosensitizer. In our study, we have tested a new in-vitro group of cyanine dyes. These dyes are widely applied in biotechnology as fluorescent markers. Two malignant adenocarcinoma cell lines (MCF-7/WT and MCF-7/DOX) were investigated using photodynamic reaction (PDR) with four cyanine dyes (KF-570, HM-118, FBF-749, and ER-139). KF-570 and HM-118 were irradiated with red light (630 nm), whereas FBF-749 and ER-139 with green light (435 nm). To evaluate PDR efficiency, a clonogenic test was conducted. Apoptosis was investigated by TUNEL and NCA (neutral comet) assays. Proteins selected as indicators of the apoptotic pathway (AIF, sPLA2, Smac/Diablo) and intracellular response markers (SOD-1 and GST-pi) were detected using western blot. The highest number of apoptotic cells (ca. 100%) was observed after PDR with HM-118 and KF-570 in both conducted tests, in both cell lines. The results showed that HM-118 and KF-570 cyanine dyes demonstrated a major phototoxic effect causing apoptosis in doxorubicin-resistant and sensitive cell lines.

Topics: Apoptosis; Breast Neoplasms; Carbocyanines; Caspase 3; Cell Line, Tumor; Humans; MCF-7 Cells; Photochemotherapy; Photosensitizing Agents

MicroRNAs (miRNAs) are involved in the essential progresses of many diseases and have emerged as therapeutic and diagnostic biomarkers. The combination of miRNA aberrant expression and tumor microenvironment (TME) features holds great potential for precise tumor imaging diagnosis but has been minimally explored. Herein, we rationally design a DNA@Cu-MOF nanosystem containing copper metal-organic frameworks (Cu-MOF) and a DNAzyme-assisted signal amplification procedure for deregulated miRNA-related hypoxic tumor diagnosis. The nanoprobes comprising a signal strand block Cu-specific DNAzyme precursor and a substrate strand are assembled on the surface of the hypoxia-responsive Cu-MOF. Under TME characterized by hypoxia, the DNA@Cu-MOF nanosystem disassociates and accomplishes the release of abundant Cu

Topics: Animals; Biosensing Techniques; Breast Neoplasms; Carbocyanines; Cell Survival; Copper; DNA, Catalytic; Female; Fluorescent Dyes; Humans; Mammary Neoplasms, Experimental; MCF-7 Cells; Metal-Organic Frameworks; Mice; Mice, Inbred BALB C; MicroRNAs; Optical Imaging

Topics: Animals; Biocompatible Materials; Breast Neoplasms; Carbocyanines; Cell Proliferation; Female; Iodine Radioisotopes; Mammary Neoplasms, Experimental; Mice; Molecular Structure; Optical Imaging; Peptides; Ribosomal Proteins; Salmonella typhimurium

Organic small-molecule-based photothermal agents such as cyanine dyes have received increasing attention in developing novel cancer therapies with potential clinical utility but suffer from poor stability, low photothermal efficiency, and limited accumulation at tumor sites in molecular forms. Self-assembly of small-molecule dyes into supramolecular assemblies may address these concerns by controlling the molecular organization of dye monomers to form structures of a higher order. Among them, H-aggregates of dyes favor face-to-face contacts with strongly overlapping areas, which always have a negative connotation to exhibit low or no fluorescence in most cases but may emanate energy in nonradiative forms such as heat for photothermal cancer therapy applications. Here, the synergistic self-assembly of cyanine dyes into H-aggregates is developed as a new supramolecular strategy to fabricate small-molecule-based photothermal nanomaterials. Compared to the free cyanine dyes, the H-aggregates assembled from pyrene or tetraphenylethene (TPE) conjugating cyanine exhibit the expected absorption spectral blue shift and fluorescence self-quenching but unique photothermal properties. Remarkably, the obtained H-aggregates are saucer-shaped nanoparticles that exhibit passive tumor-targeting properties to induce imaging-guided photothermal tumor ablation under irradiation. This supramolecular strategy presented herein may open up new opportunities for constructing next-generation small-molecule-based self-assembly nanomaterials for PTT cancer therapy in clinics.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Fluorescent Dyes; Mice; Mice, Nude; Particle Size; Photothermal Therapy; Pyrenes; Small Molecule Libraries; Stilbenes; Surface Properties

This report presents the synthesis and folate receptor target-specificity of amino-functionalized polyacrylamide nanoparticles (AFPAA NPs) for near-infrared (NIR) fluorescence imaging of cancer. For the synthesis of desired nano-constructs, the AFPAA NPs (hereafter referred to as NPs) were reacted with a NIR cyanine dye (CD) bearing carboxylic acid functionality by following our previously reported approach, and the resulting conjugate (NP-CD) on further reaction with folic acid (FA) resulted in a new nano-construct, FA-NP-CD, which demonstrated significantly higher uptake in folate receptor-positive breast cancer cells (KB+) and in folate receptor over-expressed tumors in vivo. The target-specificity of these nanoparticles was further confirmed by inhibition assay in folate receptor-positive (KB+) and -negative (HT-1080) cell lines. To show the advantages of polyacrylamide (PAA)-based NPs in folate receptor target-specificity, the CD used in preparing the FA-NP-CD construct was also reacted with folic acid alone and the synthetic conjugate (CD-FA) was also investigated for its target-specificity. Interestingly, in contrast to NPs (FA-NP-CD), the CD-FA conjugate did not show any significant in vitro or in vivo specificity toward folate receptors, showing the advantages of PAA-based nanotechnology in delivering the desired agent to tumor cells.

Topics: Acrylic Resins; Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Fibroblasts; Fibrosarcoma; Fluorescent Dyes; Folate Receptors, GPI-Anchored; Folic Acid; Glycoconjugates; Heterografts; Humans; Infrared Rays; KB Cells; Mice; Mice, Nude; Nanoparticles; Optical Imaging

A cyanine-derivative photosensitizer, CYBF2, was synthesized for photodynamic therapy of cancer. The photosensitizer can be activated by red-light with enhanced photostability compared to traditional cyanine photosensitizers. CYBF2 was able to generate singlet oxygen inside the cell mitochondria, which efficiently resulted in apoptosis of cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carbocyanines; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Humans; MCF-7 Cells; Microscopy, Confocal; Mitochondria; Molecular Structure; Optical Imaging; Photochemical Processes; Photochemotherapy; Photosensitizing Agents; Structure-Activity Relationship

The development of tumor targeted probes with strong signal and high contrast is always challenging in cancer imaging. Here, a unique multilayered activatable nanoprobe (MAN) is prepared to fulfill this long-standing goal. MAN adopts a versatile layer-by-layer fabrication technique that sequentially assembles multifunctional polyelectrolytes onto nanoparticles via charge-charge interaction. Unlike the common one-probe-one-fluorochrome construct, MAN offers a dramatic fluorescence enhancement by transporting a large quantity of quenched fluorochromes for maximal signal and contrast. Excellent signal amplification and retention with negligible cytotoxicity is observed in cell study. Upon systemic injection into mice, MAN quickly accumulates in tumor and its fluorescent signal is turned on by proteases overexpressed in tumors, resulting in >700% tumor-to-normal-tissue contrast. This multilayered fabrication provides a simple and powerful universal platform to design sensitive tumor imaging probes.

Topics: Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Survival; Female; Heterografts; Humans; Injections, Intravenous; Light; Mice; Mice, Nude; Molecular Imaging; Molecular Probes; Nanoparticles; Neoplasm Proteins; Peptide Hydrolases; Polyelectrolytes; Polylysine; Signal-To-Noise Ratio; Static Electricity

Because the degree of labeling (DOL) of cell-bound antibodies, often required in quantitative fluorescence measurements, is largely unknown, we investigated the effect of labeling with two different fluorophores (AlexaFluor546, AlexaFluor647) in a systematic way using antibody stock solutions with different DOLs. Here, we show that the mean DOL of the cell-bound antibody fraction is lower than that of the stock using single molecule fluorescence measurements. The effect is so pronounced that the mean DOL levels off at approximately two fluorophores/IgG for some antibodies. We developed a method for comparing the average DOL of antibody stocks to that of the isolated, cell-bound fraction based on fluorescence anisotropy measurements confirming the aforementioned conclusions. We created a model in which individual antibody species with different DOLs, present in an antibody stock solution, were assumed to have distinct affinities and quantum yields. The model calculations confirmed that a calibration curve constructed from the anisotropy of antibody stocks can be used for determining the DOL of the bound fraction. The fluorescence intensity of the cell-bound antibody fractions and of the antibody stocks exhibited distinctly different dependence on the DOL. The behavior of the two dyes was systematically different in this respect. Fitting of the model to these data revealed that labeling with each dye affects quantum yield and antibody affinity differentially. These measurements also implied that fluorophores in multiply labeled antibodies exhibit self-quenching and lead to decreased antibody affinity, conclusions directly confirmed by steady-state intensity measurements and competitive binding assays. Although the fluorescence lifetime of antibodies labeled with multiple fluorophores decreased, the magnitude of this change was not sufficient to account for self-quenching indicating that both dynamic and static quenching processes occur involving H-aggregate formation. Our results reveal multiple effects of fluorophore conjugation, which must not be overlooked in quantitative cell biological measurements.

Topics: Antibodies, Monoclonal; Antibody Affinity; Binding, Competitive; Breast Neoplasms; Carbocyanines; Female; Fluorescence; Fluorescence Polarization; Humans; Lung Neoplasms; Quinolinium Compounds; Receptor, ErbB-2; Spectrometry, Fluorescence; Tumor Cells, Cultured

Electrochemotherapy became one of the therapeutic protocols successfully used in oncology. However, biological effects occurring in cells, especially those which are drug resistant, have not been studied thoroughly. This study presents response of wild and drug resistant breast cancer cells to classical photodynamic therapy with Photofrin or experimental photodynamic therapy with cyanine IR-775, combined with electroporation. Photodynamic reaction or electroporation alone had no cytotoxic effect, but their combination significantly disturbed cellular functions. Applying electroporation allowed the drugs to increase its accumulation, especially for a poorly permeant cyanine in drug resistant cells. FACS analysis showed that even at relatively mild electric field, ca. 90% of cells were permeabilized. High intracellular concentration of drugs triggered the cellular defense system through increased expression of glutathione S-transferase and multidrug resistance proteins (MDR1 and MRP7), particularly in drug resistant cells. Finally, expressively decreased cell metabolism and proliferation, as well as formation of apoptotic bodies and fragmentation of cells were observed after the combined treatment. The results show that electroporation can be used for effective delivery of photosensitizers, even to drug resistant breast cancer cells, which was not tested before. This shows that electro-photodynamic treatment could be a promising approach to overcome a problem of drug resistance in cancer cells.

Topics: Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Membrane Permeability; Cell Survival; Dihematoporphyrin Ether; Drug Resistance, Neoplasm; Electrochemotherapy; Female; Humans; MCF-7 Cells; Photosensitizing Agents

Nanogels that are amenable to facile multi-functionalization with imaging, therapeutic, and targeting agents are attractive theranostic platforms for addressing challenges in conventional diagnostics and therapy. In this work, reactive copolymers containing poly(ethylene glycol), maleimide, and pendant hydroxyl groups as side chains are used to construct nanogels by employing their thermoresponsive self-assembly in aqueous media. Subsequent cross-linking of these nanosized aggregates with dithiols using thiol-maleimide chemistry yields nanogels containing maleimide, thiol, and hydroxyl groups. The hydroxyl groups are readily activated to N-hydroxysuccinimide based carbonates that undergo conjugation with amine-containing molecules through carbamate linkage under mild conditions. As a demonstration of multi-functionalization, the maleimide, thiol, and activated carbonate groups were functionalized with a thiol-containing cancer cell targeting peptide, a maleimide-containing fluorescent indocyanine Cy5 dye, and an anticancer drug doxorubicin, respectively. It was observed that enhanced drug release from nanogels occurs under acidic conditions. While the parent nanogel vehicles did not possess any toxicity, drug conjugated constructs with and without targeting group were cytotoxic against MDA-MB-231 breast cancer cells. The cyclic peptide containing targeted nanogel exhibited slightly higher cytotoxicity than its counterpart devoid of any targeting group. Furthermore, higher level of drug internalization into MDA-MB-231 cells was observed for the targeting group containing construct. It can be envisioned that facile fabrication and multi-functionalization of these reactive nanogels simultaneously through nonreversible and reversible linkages offers a modular platform that can be configured as a theranostic agent for addressing challenges in conventional therapy of various diseases.

Topics: Antineoplastic Agents; Boron Compounds; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Doxorubicin; Drug Carriers; Drug Delivery Systems; Female; Fluorescent Dyes; Gels; Humans; Nanostructures; Optical Imaging; Polyethylene Glycols; Polymethacrylic Acids; Theranostic Nanomedicine

Silver nanoparticles (AgNPs) have a high affinity for sulfhydryl (thiol) groups, which can be exploited for functionalization with various tracking and targeting moieties. Here, we describe how to reliably and reproducibly functionalize AgNPs with the fluorescent moiety cyanine3-polyethelyne glycol (5000 molecular weight)-thiol (Cy3-PEG

Topics: Breast Neoplasms; Carbocyanines; Cell Culture Techniques; Female; Fluorescent Dyes; Humans; Metal Nanoparticles; Molecular Imaging; Silver; Spectrophotometry, Ultraviolet; Tumor Cells, Cultured

IR-783 is a kind of heptamethine cyanine dye that exhibits imaging, cancer targeting and anticancer properties. A previous study reported that its imaging and targeting properties were related to mitochondria. However, the molecular mechanism behind the anticancer activity of IR-783 has not been well demonstrated. In this study, we showed that IR-783 inhibits cell viability and induces mitochondrial apoptosis in human breast cancer cells. Exposure of MDA-MB-231 cells to IR-783 resulted in the loss of mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) depletion, mitochondrial permeability transition pore (mPTP) opening and cytochrome c (Cyto C) release. Furthermore, we found that IR-783 induced dynamin-related protein 1 (Drp1) translocation from the cytosol to the mitochondria, increased the expression of mitochondrial fission proteins mitochondrial fission factor (MFF) and fission-1 (Fis1), and decreased the expression of mitochondrial fusion proteins mitofusin1 (Mfn1) and optic atrophy 1 (OPA1). Moreover, knockdown of Drp1 markedly blocked IR-783-mediated mitochondrial fission, loss of MMP, ATP depletion, mPTP opening and apoptosis. Our in vivo study confirmed that IR-783 markedly inhibited tumour growth and induced apoptosis in an MDA-MB-231 xenograft model in association with the mitochondrial translocation of Drp1. Taken together, these findings suggest that IR-783 induces apoptosis in human breast cancer cells by increasing Drp1-mediated mitochondrial fission. Our study uncovered the molecular mechanism of the anti-breast cancer effects of IR-783 and provided novel perspectives for the application of IR-783 in the treatment of breast cancer.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Survival; Cytochromes c; Dynamins; Female; Gene Expression Regulation, Neoplastic; GTP Phosphohydrolases; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Proteins; RNA, Small Interfering; Signal Transduction; Xenograft Model Antitumor Assays

Starting with a previously reported linear breast cancer targeting decapeptide WxEAAYQkFL, here we report the synthesis of a novel cyclic peptide analogue cyclic WXEAAYQkFL. The N- to C-terminus amide cyclized peptide with one d-amino acid (k) displayed higher uptake by breast cancer cells, with minimal uptake by the noncancerous cells compared to the linear peptide with two d-amino acids (x and k), and was stable toward proteolytic degradation. When immobilized on gold microcantilever surface, the cyclic peptide was able to capture breast cancer cells specifically and sense samples with ≥25 cancer cells/mL. Animal studies using mice carrying orthotopic breast MDA-MB-231 tumors showed that the cyclic peptide preferentially accumulates in tumor (2 h after injection) and is rapidly cleared from all other organs except kidneys and liver. The study highlights the discovery of a novel proteolytically stable cyclic peptide that can be used for targeted drug delivery or for enumerating circulating breast tumor cells.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Gold; Humans; Immobilized Proteins; Mice, Nude; Peptides, Cyclic; Proteolysis; Xenograft Model Antitumor Assays

Traceable poly(ethylene oxide)-poly(ester) micelles were developed through chemical conjugation of a near-infrared (NIR) dye to the poly(ester) end by click chemistry. This strategy was tried for micelles with poly(ε-caprolactone) (PCL) or poly(α-benzyl carboxylate-ε-caprolactone) (PBCL) cores. The surface of both micelles was also modified with the breast cancer targeting peptide, P18-4. The results showed the positive contribution of PBCL over PCL core on micellar thermodynamic and kinetic stability as well as accumulation in primary orthotopic MDA-MB-231 tumors within 4-96 h following intravenous administration in mice. This was in contrast to in vitro studies where better uptake of PEO-PCL versus PEO-PBCL micelles by MDA-MB-231 cells was observed. The presence of P18-4 enhanced the in vitro cell uptake and homing of both polymeric micelles in breast tumors, but only at early time points. In conclusion, the use of developed NIR labeling technique provided means for following the fate of PEO-poly(ester) based nano-carriers in live animals. Our results showed micellar stabilization through the use of PBCL over PCL cores, to have a more significant effect in enhancing the level and duration of nano-carrier accumulation in primary breast tumors than the modification of polymeric micellar surface with breast tumor targeting peptide, P18-4.

Topics: Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Drug Delivery Systems; Female; Fluorescent Dyes; Mice, Nude; Micelles; Peptides; Polyesters; Polyethylene Glycols

A variety of biomarkers are present on cells in peripheral blood of patients with a variety of disorders, including solid tumor malignancies. While rare, characterization of these cells for specific protein levels with the advanced technology proposed, will lead to future validation studies of blood samples as "liquid biopsies" for the evaluation of disease status and therapeutic response. While circulating tumor cells (CTCs) have been isolated in the blood samples of patients with solid tumors, the exact role of CTCs as clinically useful predictive markers is still debated. Current commercial technology has significant bias in that a positive selection technology is used that preassumes specific cell surface markers (such as EpCAM) are present on CTCs. However, CTCs with low EpCAM expression have been experimentally demonstrated to be more likely to be missed by this method. In contrast, this application uses a previously developed, technology that performs a purely negative enrichment methodology on peripheral blood, yielding highly enriched blood samples that contain CTCs as well as other, undefined cell types. The focus of this contribution is the use of multispectral imaging of epifluorescent, microscopic images of these enriched cells in order to help develop clinically relevant liquid biopsies from peripheral blood samples.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carbocyanines; Cell Count; Cell Line, Tumor; Epithelial Cell Adhesion Molecule; Female; Fluorescent Dyes; Humans; Hydrazines; Image Processing, Computer-Assisted; Indoles; Keratins; Leukocyte Common Antigens; Leukocytes, Mononuclear; Liquid Crystals; Neoplastic Cells, Circulating; Optical Imaging; Software; Succinimides

The RAS and mTOR inhibitor S-trans-trans-farnesylthiosalicylic acid (FTS) is a promising anticancer agent with moderate potency, currently undergoing clinical trials as a chemotherapeutic agent. FTS has displayed its potential against a variety of cancers including endocrine resistant breast cancer. However, the poor pharmacokinetics profile attributed to its high hydrophobicity is a major hindrance for its continued advancement in clinic. One of the ways to improve its therapeutic potential would be to enhance its bioavailability to cancer tissue by developing a method for targeted delivery. In the current study, FTS was conjugated with the cancer-targeting heptamethine cyanine dye 5 to form the FTS-dye conjugate 11. The efficiency of tumor targeting properties of conjugate 11 against cancer cell growth and mTOR inhibition was evaluated in vitro in comparison with parent FTS. Cancer targeting of 11 in a live mouse model of MCF7 xenografts was demonstrated with noninvasive, near-infrared fluorescence (NIRF) imaging. The results from our studies clearly suggest that the bioavailability of FTS is indeed improved as indicated by log P values and cancer cell uptake. The FTS-dye conjugate 11 displayed higher potency (IC

Topics: Animals; Antineoplastic Agents; Biological Availability; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Proliferation; Drug Delivery Systems; Farnesol; Female; Humans; MCF-7 Cells; Mice; Mice, Nude; ras Proteins; Salicylates; Tissue Distribution; TOR Serine-Threonine Kinases

Slow drug release at the tumor tissue and poor tumor penetration are two big challenges for the successful application of nanosystems in tumor therapy. Here, we report that a high concentration of the natural reducing agent vitamin C (VC) triggers rapid extracellular PTX release from PTX-loaded shell-sheddable PEG-SS-PCL micelles (SSM) in tumors in vivo. An in vivo tolerance study showed that VC at a blood concentration of 40mM had little toxicity to nude mice. Notably, SSM rapidly disassembled and released the payloads (Cy5 or PTX) in response to 40mM VC. In vivo near-infrared imaging of tumor-bearing mice showed that with post-injection of VC to establish a blood concentration of 40mM, Cy5 was quickly released from the micelles and diffused deep into the tumor tissue. Biodistribution studies revealed that 6h after the injection of PTX-loaded micelles the highest tumor accumulation was reached, which was set as the injection time for VC. The antitumor efficacy of a combination therapy of PTX-loaded micelles and VC was evaluated in both MCF-7 and U87MG tumor models. In both tumor models, single injections of VC didn't show any antitumor effect, while sequential administration of PTX-loaded SSM and VC exhibited significantly higher tumor inhibition effects and better survival rates as compared to single treatment with PTX-loaded micelles, demonstrating that exogenous administration of VC effectively triggered the release of PTX from SSM in vivo. The combination of reduction-sensitive nanomedicines with exogenous VC appears a promising approach to achieve potent treatment of malignant tumors.

Topics: Animals; Antineoplastic Agents; Ascorbic Acid; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Delayed-Action Preparations; Drug Carriers; Drug Liberation; Drug Synergism; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Micelles; Neoplasm Transplantation; Oxidation-Reduction; Paclitaxel; Polyesters; Polyethylene Glycols; Succinimides; Surface Properties

Perfluorocarbon (PFC) droplets were studied as new generation ultrasound contrast agents via acoustic or optical droplet vaporization (ADV or ODV). Little is known about the ODV irradiated vaporization mechanisms of PFC-microparticle complexs and the stability of the new bubbles produced. In this study, fluorescent perfluorohexane (PFH) poly(lactic-co-glycolic acid) (PLGA) particles were used as a model to study the process of particle vaporization and bubble stability following excitation in two-dimensional (2-D) and three-dimensional (3-D) cell models. We observed localization of the fluorescent agent on the microparticle coating material initially and after vaporization under fluorescence microscopy. Furthermore, the stability and growth dynamics of the newly created bubbles were observed for 11 min following vaporization. The particles were co-cultured with 2-D cells to form 3-D spheroids and could be vaporized even when encapsulated within the spheroids via laser irradiation, which provides an effective basis for further work.

Topics: Acoustics; Breast Neoplasms; Carbocyanines; Contrast Media; Female; Fluorescent Dyes; Fluorocarbons; Humans; Lactic Acid; Lasers; MCF-7 Cells; Microbubbles; Microscopy, Fluorescence; Models, Biological; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Spheroids, Cellular; Ultrasonography, Mammary; Volatilization

In this study, poly(d,l-lactide-co-glycolide) nanoparticles (PLGA NPs) with biotinylated chitosan (Bio-CS)-surface modification were prepared to be usded as a tumor-targeted and prolonged delivery system for anticancer drugs. Epirubicin (EPB), as a model drug, was encapsulated into Bio-CS surface modified PLGA (Bio-CS-PLGA) NPs with a drug encapsulation efficiency of 84.1 ± 3.4%. EPB-loaded Bio-CS-PLGA NPs were spherical shaped, and had a larger size and higher positive zeta potential compared to the unmodfied EPB-loaded PLGA NPs. The in vitro drug releases showed that EPB-loaded Bio-CS-PLGA NPs exhibited relatively constant drug release kinetics during the first 48 h and the drug burst release significantly decreased in comparison to the unmodified PLGA NPs. The results of MTS assays showed that Bio-CS-PLGA NPs markedly increased the cytotoxicity of EPB, compared to both the unmodified PLGA NPs and the CS-PLGA NPs. The uptakes of NPs in human breast cancer MCF-7 cells were evaluated by the flow cytometry and the confocal microscope. The results revealed that Bio-CS-PLGA NPs exhibited a greater extent of cellular uptake than the unmodified PLGA NPs and CS-PLGA NPs. Moreover, the cellular uptake of Bio-CS-PLGA NPs was evidently inhibited by the endocytic inhibitors and the receptor ligand, indicating that biotin receptor-mediated endocytosis was perhaps involved in the cell entry of Bio-CS-PLGA NPs. In MCF-7 tumor-bearing nude mice, EPB-loaded Bio-CS-PLGA NPs were efficiently accumulated in the tumors. In summary, Bio-CS-PLGA NPs displayed great potential for application as the carriers of anticancer drugs.

Topics: Animals; Antibiotics, Antineoplastic; Biotinylation; Breast Neoplasms; Carbocyanines; Cell Survival; Chitosan; Endocytosis; Epirubicin; Humans; Kinetics; Lactic Acid; MCF-7 Cells; Mice, Nude; Microscopy, Confocal; Microscopy, Electron, Scanning; Nanoparticles; Particle Size; Photoelectron Spectroscopy; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Surface Properties; Xenograft Model Antitumor Assays

Nanoparticle delivery is becoming an increasingly more valuable technique in cancer drug treatments. The use of fluorescent probes, in particular, can provide noninvasive strategies to interrogate the internalization mechanisms of cancer cells and aid in drug design. Here we describe the delivery of fluorescently labeled polysaccharide-based nanoparticles to breast cancer cells in vitro and their subsequent immunofluorescence microscopy examination. The description of the synthesis, preparation, and delivery of the nanoparticles can be widely applicable to other in vitro drug delivery studies.

Topics: Animals; Biological Transport; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cryopreservation; Fluorescent Dyes; Intracellular Space; Mice; Microscopy, Fluorescence; Nanoparticles; Particle Size; Polysaccharides; Sterilization

Metabolic sugar labeling followed by the use of reagent-free click chemistry is an established technique for in vitro cell targeting. However, selective metabolic labeling of the target tissues in vivo remains a challenge to overcome, which has prohibited the use of this technique for targeted in vivo applications. Herein, we report the use of targeted ultrasound pulses to induce the release of tetraacetyl N-azidoacetylmannosamine (Ac4 ManAz) from microbubbles (MBs) and its metabolic expression in the cancer area. Ac4 ManAz-loaded MBs showed great stability under physiological conditions, but rapidly collapsed in the presence of tumor-localized ultrasound pulses. The released Ac4 ManAz from MBs was able to label 4T1 tumor cells with azido groups and significantly improved the tumor accumulation of dibenzocyclooctyne (DBCO)-Cy5 by subsequent click chemistry. We demonstrated for the first time that Ac4 ManAz-loaded MBs coupled with the use of targeted ultrasound could be a simple but powerful tool for in vivo cancer-selective labeling and targeted cancer therapies.

Topics: Animals; Azides; Breast; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Click Chemistry; Drug Delivery Systems; Female; Fluorescent Dyes; Hexosamines; Mice, Inbred BALB C; Microbubbles; Optical Imaging; Ultrasonography, Mammary

The high rate of recurrence in patients with pancreatic ductal adenocarcinoma (PDAC) could be reduced by supporting the surgeons in discriminating healthy from diseased tissues with intraoperative fluorescence-guidance. Here, we studied the suitability of Cetuximab, a therapeutic monoclonal antibody targeting the human epidermal growth factor receptor (EGFR), near-infrared (NIR) fluorescently labeled as a new tool for fluorescence-guided surgery. Distribution and binding of systemically injected Cetuximab Alexa Fluor 647 conjugate (Cetux-Alexa-647) and the co-injected control human IgG Alexa Fluor 750 conjugate (hIgG-Alexa-750) was studied over 48 h by NIR fluorescence imaging in mice bearing human orthotopic AsPC-1 and MIA PaCa-2 PDAC tumors. Cetux-Alexa-647, but not the control hIgG-Alexa-750 fluorescence, was specifically detected in vivo in both primary pancreatic tumors with maximum fluorescence intensities at 24 h, and in metastases of AsPC-1 tumors as small as 1 mm. Lifetime analysis and NIR fluorescence microscopy of tumor sections confirmed the binding specificity of Cetux-Alexa-647 to PDAC cells. Comparable results were obtained with Cetuximab conjugated to Alexa Fluor 750 dye (Cetux-Alexa-750). Fluorescence-guided dissection, performed 24 h after injection of Cetuximab conjugated to IRDye 800CW (Cetux-800CW), enabled a real-time delineation of AsPC-1 tumor margins, and small metastases. Odyssey scans revealed that only the vital part of the tumor, but not the necrotic part was stained with Cetux-800CW. NIR fluorescently labeled Cetuximab may be a promising tool that can be applied for fluorescence-guided surgery to visualize tumor margins and metastatic sites in order to allow a precise surgical resection.

Topics: Animals; Breast Neoplasms; Carbocyanines; Carcinoma, Pancreatic Ductal; Cetuximab; ErbB Receptors; Female; Fluorescent Dyes; Heterografts; Humans; Male; MCF-7 Cells; Mice; Mice, Nude; Microscopy, Fluorescence; Pancreatic Neoplasms; Spectroscopy, Near-Infrared; Succinimides

Recently, photothermal therapy (PTT) has become viewed as an ideal auxiliary therapeutic treatment for cancers. However, the development of safe, convenient, and highly effective photothermal agents remains a great challenge. In this study, we prepared single-walled carbon nanotubes (SWNTs) for PTT against breast tumors under the guidance of infrared fluorescent cyanines. Tumors were accurately located using near-infrared imaging (NIR) and then exposed to laser irradiation. Both the in vivo and in vitro results showed that the SWNTs have high stability and low cytotoxicity. Introducing polyethylene glycol into our nanoparticles increased the blood-circulation time. Our in vivo results further showed that Cy5.5-conjugated SWNTs mediated PTT, resulting in efficient tumor suppression in mice under the guidance of near-infrared imaging. Due to the small amount of absorption at 808-nm, Cy5.5 increased the efficiency of PTT. Breast tumors significantly shrunk after irradiation under the 808-nm near-infrared laser. The treated mice developed scabs, but otherwise recovered after 15 days, and their physical conditions restored gradually. These data indicate that our unique photothermal-responsive SWNT-Cy5.5-based theranostic agent can serve as a promising candidate for PTT.

Topics: Animals; Breast Neoplasms; Carbocyanines; Contrast Media; Female; Fluorescent Dyes; Humans; Infrared Rays; Low-Level Light Therapy; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Nanomedicine; Nanotubes, Carbon; Optical Imaging; Time Factors; Tissue Distribution; Tumor Burden

Recent clinical trials have shown that bisphosphonate drugs improve breast cancer patient survival independent of their antiresorptive effects on the skeleton. However, because bisphosphonates bind rapidly to bone mineral, the exact mechanisms of their antitumor action, particularly on cells outside of bone, remain unknown. Here, we used real-time intravital two-photon microscopy to show extensive leakage of fluorescent bisphosphonate from the vasculature in 4T1 mouse mammary tumors, where it initially binds to areas of small, granular microcalcifications that are engulfed by tumor-associated macrophages (TAM), but not tumor cells. Importantly, we also observed uptake of radiolabeled bisphosphonate in the primary breast tumor of a patient and showed the resected tumor to be infiltrated with TAMs and to contain similar granular microcalcifications. These data represent the first compelling in vivo evidence that bisphosphonates can target cells in tumors outside the skeleton and that their antitumor activity is likely to be mediated via TAMs.. Bisphosphonates are assumed to act solely in bone. However, mouse models and clinical trials show that they have surprising antitumor effects outside bone. We provide unequivocal evidence that bisphosphonates target TAMs, but not tumor cells, to exert their extraskeletal effects, offering a rationale for use in patients with early disease.

Topics: Animals; Bone Density Conservation Agents; Breast Neoplasms; Calcinosis; Carbocyanines; Diphosphonates; Disease Models, Animal; Female; Humans; Macrophages; Mice; Middle Aged; Neoplasm Grading; Neoplasm Invasiveness; Neoplasms; Phagocytosis; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; Xenograft Model Antitumor Assays

In this work, we studied indolium and benzothiazolium pentamethine salts 1-3 as novel type of receptors for the recognition of sulphated signalling molecules (sulphated steroids: oestrone, pregnenolone and cholesterol sulphate). A recognition study was performed in an aqueous medium (1mM phosphate buffer (H2O:MeOH; 99:1 (v/v))) at pH 7.34. The tested salts displayed a high affinity for these sulphated analytes, mainly for cholesterol sulphate. However, no interaction between the salts and control, non-sulphated sterol analytes (cholesterol and bile acid) was observed. The highest affinity for the sulphated steroids was observed for benzothiazole salt 1. This salt also displayed different spectral behaviour from that observed for carbocyanine salts 2 and 3. In this presence of cholesterol sulphate, benzothiazole salt 1 displayed significant spectral changes depending on the medium used: a blue shift in the aqueous medium and a red shift in the methanolic one (H2O:MeOH; 2:1 (v/v)). Subsequently preliminary in vivo study showed that, salt 1 significantly inhibits a growth of breast carcinoma on Nu/nu mice model.

Topics: Animals; Antineoplastic Agents; Benzothiazoles; Breast Neoplasms; Carbocyanines; Cholesterol Esters; Estrone; Female; Heterocyclic Compounds; Mice, Nude; Pregnenolone; Xenograft Model Antitumor Assays

To synthesize a fluorescent Her2-NLP peptide conjugate consisting of Her2/neu targeting peptide and nuclear localization sequence peptide (NLP) and assess its cellular uptake and intracellular localization for radionuclide cancer therapy targeting Her2/neu-positive circulating breast cancer cells (CBCC). Fluorescent Cy5.5 Her2-NLP peptide conjugate was synthesized by coupling a bivalent peptide sequence, which consisted of a Her2-binding peptide (NH2-GSGKCCYSL) and an NLP peptide (CGYGPKKKRKVGG) linked by a polyethylene glycol (PEG) chain with 6 repeating units, with an activated Cy5.5 ester. The conjugate was separated and purified by HPLC and then characterized by Maldi-MS. The intracellular localization of fluorescent Cy5.5 Her2-NLP peptide conjugate was assessed by fluorescent microscopic imaging using a confocal microscope after incubation of Cy5.5-Her2-NLP with Her2/neu positive breast cancer cells and Her2/neu negative control breast cancer cells, respectively. Fluorescent signals were detected in cytoplasm of Her2/neu positive breast cancer cells (SKBR-3 and BT474 cell lines), but not or little in cytoplasm of Her2/neu negative breast cancer cells (MDA-MB-231), after incubation of the breast cancer cells with Cy5.5-Her2-NLP conjugates in vitro. No fluorescent signals were detected within the nuclei of Her2/neu positive SKBR-3 and BT474 breast cancer cells, neither Her2/neu negative MDA-MB-231 cells, incubated with the Cy5.5-Her2-NLP peptide conjugates, suggesting poor nuclear localization of the Cy5.5-Her2-NLP conjugates localized within the cytoplasm after their cellular uptake and internalization by the Her2/neu positive breast cancer cells. Her2-binding peptide (KCCYSL) is a promising agent for radionuclide therapy of Her2/neu positive breast cancer using a β(-) or α emitting radionuclide, but poor nuclear localization of the Her2-NLP peptide conjugates may limit its use for eradication of Her2/neu-positive CBCC using I-125 or other Auger electron emitting radionuclide.

Topics: Active Transport, Cell Nucleus; Amino Acid Sequence; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Nucleus; Chemistry Techniques, Synthetic; Fluorescent Dyes; Humans; Ligands; Microscopy, Fluorescence; Molecular Sequence Data; Neoplastic Cells, Circulating; Nuclear Localization Signals; Receptor, ErbB-2

Near-infrared (NIR) fluorophores have several advantages over visible-light fluorophores, including superior tissue penetration and lower autofluorescence. We recently accessed a new class of readily synthesized NIR cyanines containing a novel C4'-O-alkyl linker, which provides both high chemical stability and excellent optical properties. In this study, we provide the first in vivo analysis of this new class of compounds, represented by the tetrasulfonate FNIR-774 (Frederick NIR 774). Monoclonal antibody (mAb) conjugates of FNIR-774 were compared to conjugates of the commercially available dye (IRDye800CW (IR800)), one of the most widely used NIR fluorophores for clinical translation. Both dyes were conjugated to panitumumab (pan) or cetuximab (cet) with ratios of 1:2 or 1:5. Conjugates of both dyes demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. In contrast, in vivo imaging in mice showed different pharmacokinetics between pan-FNIR-774 (1:5) and pan-IR800 (1:5), or cet-FNIR-774 (1:5) and cet-IR800 (1:5). Particularly at the higher labeling density, mAb-FNIR-774 conjugates showed superior specific accumulation in tumors compared with mAb-IR800 conjugates. Thus, FNIR-774 conjugates showed superior in vivo pharmacokinetics compared with IR800 conjugates, independent of the mAb. These results suggest that FNIR-774 is a promising fluorescent probe for NIR optical imaging.

Topics: Alkylation; Animals; Antibodies, Monoclonal; Antineoplastic Agents; BALB 3T3 Cells; Breast Neoplasms; Carbocyanines; Cells, Cultured; Cetuximab; Female; Flow Cytometry; Fluorescent Dyes; Mice; Mice, Nude; Microscopy, Fluorescence; Panitumumab; Spectroscopy, Near-Infrared; Tissue Distribution

Demarking lymph node networks is important for cancer staging in clinical practice. Here, we demonstrate in vivo dual-color photoacoustic lymphangiography using all-organic nanoformulated naphthalocyanines (referred to as nanonaps). Nanonap frozen micelles were self-assembled from two different naphthalocyanine dyes with near-infrared absorption at 707 nm or 860 nm. These allowed for noninvasive, nonionizing, high resolution photoacoustic identification of separate lymphatic drainage systems in vivo. With both types of nanonaps, rat lymph nodes buried deeply below an exogenously-placed 10 mm thick layer of chicken breast were clearly visualized in vivo. These results show the potential of multispectral photoacoustic imaging with nanonaps for detailed mapping of lymphatic drainage systems.

Topics: Acoustics; Animals; Breast Neoplasms; Carbocyanines; Cell Movement; Chickens; Color; Female; Humans; Lymph Nodes; Mice; Mice, Inbred BALB C; Micelles; Nanoparticles; Nanotechnology; Neoplasm Metastasis; Neoplasm Staging; Optics and Photonics; Rats; Rats, Sprague-Dawley; Sentinel Lymph Node Biopsy; Spectrophotometry

Fragments of the extracellular matrix component hyaluronan (HA) promote tissue inflammation, fibrosis and tumor progression. HA fragments act through HA receptors including CD44, LYVE1, TLR2, 4 and the receptor for hyaluronan mediated motility (RHAMM/HMMR). RHAMM is a multifunctional protein with both intracellular and extracellular roles in cell motility and proliferation. Extracellular RHAMM binds directly to HA fragments while intracellular RHAMM binds directly to ERK1 and tubulin. Both HA and regions of tubulin (s-tubulin) are anionic and bind to basic amino acid-rich regions in partner proteins, such as in HA and tubulin binding regions of RHAMM. We used this as a rationale for developing bioinformatics and SPR (surface plasmon resonance) based screening to identify high affinity anionic RHAMM peptide ligands. A library of 12-mer peptides was prepared based on the carboxyl terminal tail sequence of s-tubulin isoforms and assayed for their ability to bind to the HA/tubulin binding region of recombinant RHAMM using SPR. This approach resulted in the isolation of three 12-mer peptides with nanomolar affinity for RHAMM. These peptides bound selectively to RHAMM but not to CD44 or TLR2,4 and blocked RHAMM:HA interactions. Furthermore, fluorescein-peptide uptake by PC3MLN4 prostate cancer cells was blocked by RHAMM mAb but not by CD44 mAb. These peptides also reduced the ability of prostate cancer cells to degrade collagen type I. The selectivity of these novel HA peptide mimics for RHAMM suggest their potential for development as HA mimetic imaging and therapeutic agents for HA-promoted disease.

Topics: Amino Acid Sequence; Biomimetic Materials; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Movement; Drug Evaluation, Preclinical; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Dyes; Humans; Hyaluronan Receptors; Hyaluronic Acid; Ligands; Male; Molecular Sequence Data; Neoplasm Invasiveness; Peptide Library; Prostatic Neoplasms; Surface Plasmon Resonance; Tubulin

Dual-modality imaging combines the complementary advantages of different modalities, and offers the prospect of improved preclinical research. The combination of fluorescence imaging and magnetic resonance imaging (MRI) provides cross-validated information and direct comparison between these modalities. Here, we report on the application of a novel tumor-targeted, dual-labeled nanoparticle (NP), utilizing iron oxide as the MRI contrast agent and near infrared (NIR) dye Cy5.5 as the fluorescent agent. Results of in vitro experiments verified the specificity of the NP to tumor cells. In vivo tumor targeting and uptake of the NPs in a mouse model were visualized by fluorescence and MR imaging collected at different time points. Quantitative analysis was carried out to evaluate the efficacy of MRI contrast enhancement. Furthermore, tomographic images were also acquired using both imaging modalities and cross-validated information of tumor location and size between these two modalities was revealed. The results demonstrate that the use of dual-labeled NPs can facilitate the dual-modal detection of tumors, information cross-validation, and direct comparison by combing fluorescence molecular tomography (FMT) and MRI.

Topics: Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Contrast Media; Ferric Compounds; Fluorescent Dyes; Humans; Image Enhancement; Magnetic Resonance Imaging; Microscopy, Fluorescence; Multimodal Imaging; Nanoparticles; Particle Size; Reproducibility of Results; Sensitivity and Specificity; Staining and Labeling; Tomography

We present label-free, in situ monitoring of individual DNA hybridization in microfluidics. By immobilizing molecular sentinel probes on nanoporous gold disks, we demonstrate sensitivity approaching the single-molecule limit via surface-enhanced Raman scattering which provides robust signals without photobleaching for more than an hour. We further demonstrate that a target concentration as low as 20 pM can be detected within 10 min under diffusion-limited transport.

Topics: Breast Neoplasms; Carbocyanines; Diffusion; DNA; DNA, Single-Stranded; Female; Gold; Humans; Light; Metal Nanoparticles; Microfluidic Analytical Techniques; Microfluidics; Nanostructures; Nanotechnology; Nucleic Acid Hybridization; Porosity; Receptor, ErbB-2; Spectrum Analysis, Raman; Surface Plasmon Resonance

In this proof-of-concept study, a new activatable imaging agent based on two luminophores and two different quenching mechanisms is reported. Both partial and total activation of the luminescence signal can be achieved, either in solution or in vitro. Bond cleavage makes the compound suitable for luminescence lifetime imaging.

Topics: Animals; Breast Neoplasms; Carbocyanines; Cell Line; Cell Line, Tumor; Coordination Complexes; Female; Iridium; Luminescence; Luminescent Agents; Luminescent Measurements; Mice; Microscopy, Confocal; Neoplasms; Optical Imaging

Choline kinase alpha (ChoK) expression is increasingly being recognized as an important indicator of breast cancer prognosis; however, previous efforts to noninvasively measure ChoK status have been complicated by the spectral limitations of in vivo magnetic resonance spectroscopy (MRS) and the complex network of enzymes involved in choline metabolism. The most effective ChoK inhibitors are symmetric and contain quaternary ammonium groups within heterocyclic head groups connected by an aliphatic spacer. Characterization of these bis-pyridinium and bis-quinolinium compounds has led to phase I clinical trials to assess small-molecule inhibitors of ChoK for solid tumor treatment. We report the development of a novel carbocyanine dye, JAS239, whose bis-indolium structure conforms to the parameters established for ChoK specificity and whose spacer length confers fluorescence in the near-infrared (NIR) window. Fluorimetry and confocal microscopy were used to demonstrate that JAS239 rapidly enters breast cancer cells independent of the choline transporters, with accumulation in the cytosolic space where ChoK is active. Radio-tracing and (1)H MRS techniques were used to determine that JAS239 binds and competitively inhibits ChoK intracellularly, preventing choline phosphorylation while inducing cell death in breast cancer cell lines with similar efficacy to known ChoK inhibitors. Fluorescent molecules that report on ChoK status have potential use as companion diagnostics for noninvasive breast tumor staging, because NIR fluorescence allows for detection of real-time probe accumulation in vivo. Furthermore, their ability as novel ChoK inhibitors may prove effective against aggressive, therapy-resistant tumors.

Topics: Adenosine Triphosphate; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Survival; Choline Kinase; Cytosol; Enzyme Inhibitors; Female; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Humans; Magnetic Resonance Spectroscopy; Microscopy, Confocal; Prognosis; Spectroscopy, Near-Infrared

The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo.

Topics: Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Membrane; Cell Survival; Cell Tracking; Disease Models, Animal; Female; Immunohistochemistry; Immunotherapy, Adoptive; Interferon-gamma; Interleukin-2; Ionomycin; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Spectroscopy, Near-Infrared; T-Lymphocytes; Transplantation, Homologous

Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood—so-called circulating tumor cells (CTCs)—may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.

Topics: Base Sequence; Biomarkers, Tumor; Breast Neoplasms; Cadherins; Carbocyanines; Cell Line, Tumor; DNA, Antisense; Female; Fibronectins; Gold; Green Fluorescent Proteins; Humans; Luminescent Proteins; Metal Nanoparticles; Microscopy, Fluorescence; Nanotechnology; Neoplastic Cells, Circulating; Red Fluorescent Protein; RNA, Messenger; Transplantation, Heterologous; Vimentin

Ineffective drug delivery is a vast problem of anticancer therapies. The aim of this study was to investigate the possibility of enhancement of cyanines transport through the cell membrane by electroporation and to evaluate a photodynamic activity of these compounds.. We evaluated in vitro the effectiveness of photodynamic reaction with cyanines on breast adenocarcinoma cells (MCF-7/WT) and normal Chinese hamster ovary cells (CHO) lacking voltage-dependent ion channels, alone and combined with electropermeabilization. Among six cyanines tested, two compounds could be indicated as good therapeutic candidates: IR-775 and IR-786. Cellular effects were assessed with MTT assay reporting cell mitochondrial activity and with SRB assay based on the measurement of cellular protein content. Cyanines localization was observed with confocal microscope.. Photodynamic reaction of MCF-7/WT cells with IR-775 and IR-786 did not result in cellular dysfunction. Electric field intensities and pulse duration, non-toxic for cells, significantly increased photocytotoxicity of the cyanines after electropermeabilization with IR-775 and IR-786. Much shorter exposure times were efficient for cyanines in photodynamic reaction assisted by electroporation (10 min instead of 24h).. Our results indicate that electroporation of cancerous cells in the presence of cyanine dyes could increase the uptake of the photosensitizer, which correlates with a higher cytotoxicity in the breast adenocarcinoma cell line. Electroporation may be an attractive delivery system for photosensitizers in photodynamic therapy, enabling application of new compounds and reduction of drug dose and exposure time.

Topics: Animals; Apoptosis; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Electroporation; Female; Humans; Photosensitizing Agents

Development of a heptamethine cyanine based tumor-targeting PET imaging probe for noninvasive detection and diagnosis of breast cancer.. Tumor-specific heptamethine-cyanine DOTA conjugate complexed with Cu-64 (PC-1001) was synthesized for breast cancer imaging. In vitro cellular uptake studies were performed in the breast cancer MCF-7 and noncancerous breast epithelial MCF-10A cell lines to establish tumor specificity. In vivo time-dependent fluorescence and PET imaging of breast tumor xenografts in mice were performed. Blood clearance, biodistribution, and tumor-specific uptake and plasma binding of PC-1001 were quantified. Tumor histology (H&E staining) and fluorescence imaging were examined.. PC-1001 displayed similar fluorescence properties (ε=82,880cm(-1)M(-1), Ex/Em=750/820nm) to the parental dye. Time-dependent cellular accumulation indicated significantly higher probe uptake (>2-fold, 30min) in MCF-7 than MCF-10A cells and the uptake was observed to be mediated by organic anion transport peptides (OATPs) system. In vivo studies revealed that PC-1001 has desirable accumulation profile in tumor tissues, with tumor versus muscle uptake of about 4.3 fold at 24h and 5.8 fold at 48h post probe injections. Blood half-life of PC-1001 was observed to be 4.3±0.2h. Microscopic fluorescence imaging of harvested tumor indicated that the uptake of PC-1001 was restricted to viable rather than necrotic tumor cells.. A highly efficient tumor-targeting PET/fluorescence imaging probe PC-1001 is synthesized and validated in vitro in MCF-7 breast cancer cells and in vivo in mice breast cancer xenograft model.

Topics: Animals; Biological Transport; Breast Neoplasms; Carbocyanines; Cell Transformation, Neoplastic; Chemistry Techniques, Synthetic; Copper Radioisotopes; Female; Heterocyclic Compounds, 1-Ring; Humans; MCF-7 Cells; Mice; Positron-Emission Tomography; Spectrometry, Fluorescence; Tumor Burden

A DNA tetrahedron is employed for efficient delivery of doxorubicin into drug-resistant breast cancer cells. The drug delivered with the DNA nanoconstruct is considerably cytotoxic, whereas free doxorubicin is virtually non-cytotoxic for the drug-resistant cells. Thus, the DNA tetrahedron, made of the inherently natural and biocompatible material, can be a good candidate for the drug carrier to overcome MDR in cancer cells.

Topics: Breast Neoplasms; Carbocyanines; Cell Survival; DNA; Doxorubicin; Drug Carriers; Drug Resistance, Neoplasm; Female; Humans; MCF-7 Cells; Nanostructures

Previous studies have demonstrated the feasibility of translocator protein (TSPO) imaging to visualize and quantify human breast adenocarcinoma (MDA-MB-231) cells in vivo using a TSPO-targeted near-infrared (NIR) probe (NIR-conPK11195). This study aimed to extend the use of the TSPO-targeted probe to a more biologically relevant and clinically important tumor microenvironment as well as to assess our ability to longitudinally detect the presence and progression of breast cancer cells in the brain. The in vivo biodistribution and accumulation of NIR-conPK11195 and free (unconjugated) NIR dye were quantitatively evaluated in intracranial MDA-MB-231-bearing mice and non-tumor-bearing control mice longitudinally once a week from two to five weeks post-inoculation. The in vivo time-activity curves illustrate distinct clearance profiles for NIR-conPK11195 and free NIR dye, resulting in preferential accumulation of the TSPO-targeted probe in the intracranial tumor bearing hemisphere (TBH) with significant tumor contrast over normal muscle tissue (p < 0.005 at five weeks; p < 0.01 at four weeks). In addition, the TSPO-labeled TBHs demonstrated significant contrast over the TBHs of mice injected with free NIR dye (p < 0.001 at four and five weeks) as well as over the TSPO-labeled non-tumor-bearing hemispheres (NTBHs) of control mice (p < 0.005 at four and five weeks). Overall, TSPO-targeted molecular imaging appears useful for visualizing and quantifying breast cancer xenografts propagated in the murine brain and may assist in preclinical detection, diagnosis and monitoring of metastatic disease as well as drug discovery. Furthermore, these results indicate it should be possible to perform TSPO-imaging of breast cancer cells in the brain using radiolabeled TSPO-targeted agents, particularly in light of the fact that [11C]-labeled TSPO probes such as [11C]-PK 11195 have been successfully used to image gliomas in the clinic.

Topics: Adenocarcinoma; Animals; Brain; Brain Neoplasms; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Humans; Isoquinolines; Mice; Mice, Nude; Molecular Imaging; Molecular Probes; Neoplasm Transplantation; Receptors, GABA; Tissue Distribution; Transplantation, Heterologous; Whole Body Imaging

There is great clinical interest in developing novel nanocarriers for hydrophobic cyanine dyes used as photosensitizing agents in photodynamic therapy (PDT). In the present study we have employed nanoemulsion-templated oil-core multilayer nanocapsules as robust nanocarriers for a cyanine-type photosensitizer IR-786. These nanoproducts were fabricated via layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes (PEs), i.e., anionic PSS and cationic PDADMAC on nanoemulsion liquid cores created by dicephalic or bulky saccharide-derived cationic surfactants. All nanocapsules, with different thicknesses of the PE shell and average size <200 nm (measured by DLS) demonstrated good capacity for IR-786 encapsulation. The nanocarriers were visualized by SEM and AFM and their photo-induced anticancer effect and cellular internalization in human breast carcinoma MCF-7/WT cells were determined. Biological response of the cell culture, expressed as dark and photocytotoxicity as well as fluorescence of drug molecules loaded in the multilayer vehicles, analyzed by the FACS and CLSM techniques, have indicated that the delivered IR-786 did not aggregate inside the cells and could, therefore, act as an effective third-generation photosensitizing agent. In vitro biological experiments demonstrated that the properties of studied nanostructures depended upon the PE type and the envelope thickness as well as on the surfactant architecture in the nanoemulsion-based templates employed for the nanocapsule fabrication. Similarity of results obtained for stored (three weeks in the dark at room temperature) and freshly-prepared nanocapsules, attests to viability of this stable, promising drug delivery system for poorly water-soluble cyanines useful in PDT.

Topics: Breast Neoplasms; Carbocyanines; Coloring Agents; Emulsions; Humans; Indoles; MCF-7 Cells; Nanocapsules; Photochemotherapy; Photosensitizing Agents; Polyethylenes; Polystyrenes; Quaternary Ammonium Compounds; Surface-Active Agents

We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.

Topics: Animals; Antibodies, Monoclonal, Humanized; Biomarkers, Tumor; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Fluorescent Dyes; Hydrogen-Ion Concentration; Mice; Mice, Nude; Microscopy, Fluorescence; Sensitivity and Specificity; Spectroscopy, Near-Infrared; Trastuzumab

We describe a novel multicomponent graphene nanostructured system that is biocompatible, and has strong NIR optical absorbance and superparamagnetic properties. The fabrication of the multicomponent nanostructure system involves the covalent attachment of 3 components; Fe(3)O(4)(Fe) nanoparticles, PAMAM-G4-NH(2) (G4) dendrimer and Cy5 (Cy) on a graphene oxide (GO) surface to synthesize a biologically relevant multifunctional system. The resultant GO-G4-Fe-Cy nanosystem exhibits high dispersion in an aqueous medium, and is magnetically responsive and fluorescent. In vitro experiments provide a clear indication of successful uptake of the GO-G4-Fe-Cy nanosystem by MCF-7 breast cancer cells, and it is seen to behave as a bright and stable fluorescent marker. The study also reveals varied cellular distribution kinetics profile for the GO nanostructured system compared to free Cy. Furthermore, the newly developed GO nanostructured system is observed to be non-toxic to MDA-MB-231 cell growth, in striking contrast to free G4 dendrimer and GO-G4 conjugate. The GO-G4-Fe-Cy nanostructured system characterized by multifunctionality suggests the merits of graphene for cellular bioimaging and the delivery of bioactives.

Topics: Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Survival; Dendrimers; Female; Fluorescent Dyes; Graphite; Humans; Magnetite Nanoparticles; Microscopy, Confocal; Models, Molecular; Nylons; Optical Imaging

The aim of this research is to study the dynamics and efficiency of liposome accumulation in sensitive and resistant human breast cancer cells.. Methods of fluorescence microscopy, fluorescence microspectroscopy and MTT-test have been used.. The liposome-to-cell interaction and dye cellular uptake in sensitive, cisplatin-resistant and doxorubicin-resistant MCF-7 human breast cancer cells have been analyzed using time changes in both fluorescence resonance energy transfer signal from the donor probe DiO to the acceptor one DiI preloaded in liposomes and cell image brightness.. Obtained results show that resistant cells accumulate dye-loaded liposomes more effectively and reveal more effective dye molecule cellular uptake.

Topics: Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cisplatin; Doxorubicin; Drug Carriers; Drug Resistance, Neoplasm; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Lipid Bilayers; Liposomes; MCF-7 Cells; Microscopy, Fluorescence; Molecular Structure; Phosphatidylcholines; Spectrometry, Fluorescence; Tissue Distribution

Estrogen induced proliferation of existing mutant cells is widely understood to be the major risk determining factor in the development of breast cancer. Hence determination of the Estrogen Receptor[ER] status is of paramount importance. We have carried out the synthesis and characterization of a novel NIR fluorescent dye conjugate aimed at measuring ER+ve status in-vivo. The conjugate was synthesized by ester formation between 17-β estradiol and a cyanine dye namely: bis-1, 1-(4-sulfobutyl) indotricarbocyanine-5-carboxylic acid, sodium salt. The replacement of the sodium ion in the ester by a larger glucosammonium ion was found to enhance the hydrophilicity and reduce the toxic effect on cell lines. The excitation and emission peaks for the dye were recorded as 750 and 788 nm respectively; ideal for non-invasive optical imaging owing to minimal tissue attenuation and auto-fluorescence at these wavelengths. The dye (NIRDC1) has a significant drop in plasma-protein binding therefore leading to marked improvement in pharmacokinetic profile such as dye evacuation in comparison to ICG. In addition the dye showed enhanced fluorescence quantum yield, molar extinction coefficient and linearity in fluorescence relative to ICG. This dye can be potentially used as a target specific exogenous contrast agent in molecular optical imaging for early detection of breast cancer.

Topics: Breast Neoplasms; Carbocyanines; Early Diagnosis; Esterification; Estradiol; Female; Humans; Molecular Imaging; Spectroscopy, Near-Infrared

DNA damage responses (DDR) occur during oncogenesis and therapeutic responses to DNA damaging cytotoxic drugs. Thus, a real-time method to image DNA damage in vivo would be useful to diagnose cancer and monitor its treatment. Toward this end, we have developed fluorophore- and radioisotope-labeled immunoconjugates to target a DDR signaling protein, phosphorylated histone H2A variant H2AX (γH2AX), which forms foci at sites of DNA double-strand breaks. Anti-γH2AX antibodies were modified by the addition of diethylenetriaminepentaacetic acid (DTPA) to allow (111)In labeling or the fluorophore Cy3. The cell-penetrating peptide Tat (GRKKRRQRRRPPQGYG) was also added to the immunoconjugate to aid nuclear translocation. In irradiated breast cancer cells, confocal microscopy confirmed the expected colocalization of anti-γH2AX-Tat with γH2AX foci. In comparison with nonspecific antibody conjugates, (111)In-anti-γH2AX-Tat was retained longer in cells. Anti-γH2AX-Tat probes were also used to track in vivo DNA damage, using a mouse xenograft model of human breast cancer. After local X-ray irradiation or bleomycin treatment, the anti-γH2AX-Tat probes produced fluorescent and single photon emission computed tomography signals in the tumors that were proportionate to the delivered radiation dose and the amount of γH2AX present. Taken together, our findings establish the use of radioimmunoconjugates that target γH2AX as a noninvasive imaging method to monitor DNA damage, with many potential applications in preclinical and clinical settings.

Topics: Amino Acid Sequence; Animals; Antibodies; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; DNA Damage; DNA, Neoplasm; Female; Gene Products, tat; Histones; Humans; Immunoconjugates; Indium Radioisotopes; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Sequence Data; Pentetic Acid; Transplantation, Heterologous

Breast cancer presents greatest challenge in health care in today's world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube's D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 min of photothermal therapy treatment by 1.5 W/cm(2) power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly.

Topics: Aptamers, Nucleotide; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Gold; Humans; Metal Nanoparticles; Nanostructures; Nanotubes, Carbon; Phototherapy; Spectrum Analysis, Raman

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from CLADONIA ARBUSCULA and (-)-usnic acid from ALECTORIA OCHROLEUCA were shown to be equally effective inhibitors of DNA synthesis, with IC (50) 4.2 microg/mL and 4.0 microg/mL for (+) and (-)-usnic acid against T-47D, and 5.3 microg/mL and 5.0 microg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

Topics: Antineoplastic Agents, Phytogenic; Benzimidazoles; Benzofurans; Breast Neoplasms; Carbocyanines; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA; Dose-Response Relationship, Drug; Female; Flow Cytometry; Humans; Inhibitory Concentration 50; Lichens; Membrane Potential, Mitochondrial; Necrosis; Pancreatic Neoplasms; Phytotherapy; Plant Extracts

Fluorophore mediated bio-signal retrieval has been extensively used in molecular imaging. However, only a limited number of fluorophores can be used for humans and their quantum yield is usually low. Another important issue is emitting fluorescence at the disease site, with a minimal non-specific emission at any other sites. Artificial quenching and enhancing of fluorescence was found to be possible by manipulating the distance between a fluorophore and a certain type of nanometal particle. Utilizing this unique property, we have designed a novel, FRET-like, fluorophore-nanoparticle complex. The complex emits fluorescence conditionally only at a disease site at an enhanced level. As a model system, our complex is designed to target breast cancer. As an initial step for developing this cancer locator, fluorescence alteration was studied when a spacer at various lengths is placed between a nanogold particle and a safe fluorophore.

Topics: Breast Neoplasms; Carbocyanines; Colloids; Female; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Gold; Humans; Metal Nanoparticles; Molecular Imaging

We present a new application of multispectral analysis for subcellular measurement of multiple proteins in formalin-fixed paraffin embedded tissue and cells. Typically, the targets of interest are present in the same or spatially overlapping cellular compartments. Such co-localization can complicate analysis and interpretation of the images obtained using traditional fluorescence, especially when spectrally overlapping labels are present. The spectral properties of currently available fluorescent dyes set an upper limit to the number of molecules that can be detected simultaneously with traditional fluorescence. By exciting a set of fluorophores at the same wavelength and unmixing their emission signals from background autofluorescence, we were able to image three targets in a single channel. This parallel imaging approach provides significant advantages for multiplexed analysis of tissues and cells.

Topics: Algorithms; Animals; Biomarkers; Breast; Breast Neoplasms; Carbocyanines; Cyclic AMP Response Element-Binding Protein; Female; Humans; Image Processing, Computer-Assisted; Lung; Male; Mice; Microscopy, Fluorescence; Microscopy, Fluorescence, Multiphoton; Prostate; Proteins; Signal Processing, Computer-Assisted; Tissue Array Analysis

Iron oxide nanoparticles offer a feasible tool for combined imaging and delivery of small interfering RNA (siRNA) to tumors, stimulating active interest in exploring different imaging and delivery platforms suitable for detection by a variety of modalities. In this study, we describe the synthesis and testing of a tumor-targeted nanodrug (MN-EPPT-siBIRC5) that is designed to specifically shuttle siRNA to human breast tumors. The nanodrug binds the tumor-specific antigen uMUC-1, which is found in >90% of human breast adenocarcinomas. MN-EPPT-siBIRC5 consists of superparamagnetic iron oxide nanoparticles [for magnetic resonance imaging (MRI)], the dye Cy 5.5 (for near-IR optical imaging), peptides (EPPT) that specifically target uMUC-1, and a synthetic siRNA that targets the tumor-specific antiapoptotic gene BIRC5. Nanodrug uptake by human breast adenocarcinoma cells resulted in a significant downregulation of BIRC5. Following i.v. delivery into subcutaneous mouse models of breast cancer, the nanodrug showed a preferential tumor uptake, which could be visualized by MRI and near-IR optical imaging. Furthermore, MRI could be used to quantitatively monitor nanodrug bioavailability in the tumor tissue throughout the course of treatment. Intravenous injection of the agent once a week over 2 weeks resulted in the induction of considerable levels of necrosis and apoptosis in the tumors, translating into a significant decrease in tumor growth rate. Our strategy permits the simultaneous tumor-specific delivery of siRNA to tumors and the imaging of the delivery process. More generally, it illustrates the potential to apply this approach to many human cancer studies, including for basic tumor biology and therapy.

Topics: Amino Acid Sequence; Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Ferric Compounds; Flow Cytometry; Gene Knockdown Techniques; Humans; Inhibitor of Apoptosis Proteins; Mice; Mice, Nude; Microtubule-Associated Proteins; Molecular Sequence Data; Nanoparticles; Peptides; RNA, Small Interfering; Survivin; Xenograft Model Antitumor Assays

Monitoring and targeting protein kinases is widely accepted as a promising approach for disease diagnosis and drug discovery. For this purpose, the authors have developed an original type of peptide array as a high-throughput screening assay for quantitatively evaluating kinase activity. A volume of 2 nL of peptide solution was spotted onto a formyl group-modified glass slide by using an arrayer, which was designed for use with protein chip technology. The phosphorylation was recognized by fluorescence-label antibody and detected with an automatic microarray scanner widely used in DNA chip technology. The system needs low sample volume, provides a high-density peptide array, and supplies high reproducibility. It provided enough sensitivity for inhibitor screening, even though a relatively low concentration of purified kinase was employed. The assay also proved useful for the detection of intracellular kinase activity as well as for the measurement of the fluctuations of intracellular protein kinase activity with drug stimulation. Thus, this peptide array would be applicable for kinase-targeted diagnosis, cell-based drug screening, and signal pathway investigation.

Topics: Animals; Antibodies; Breast Neoplasms; Carbocyanines; Cell Extracts; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Female; Fluorescent Dyes; Humans; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Peptides; Phosphorylation; Protein Array Analysis; Protein Kinases; Reproducibility of Results; Sensitivity and Specificity

Epidermal growth factor receptor (EGFR) overexpression is associated with several key features of cancer development and growth. Therefore, EGFR is a very promising biological target for tumor diagnosis and anticancer therapy. Characterization of EGFR expression is important for clinicians to select patients for EGFR-targeted therapy and evaluate therapeutic effects.. To investigate whether near-infrared (NIR) fluorescent dye Cy5.5-labeled anti-EGFR monoclonal antibody Erbitux can characterize EGFR expression level in MDA-MB-231 and MCF-7 breast cancer xenografts using an in vivo NIR imaging method.. A fluorochrome probe was designed by coupling Cy5.5 to Erbitux through acidylation, and the fluorescence property of the Erbitux-Cy5.5 conjugate was characterized by fluorospectroscopy. Flow cytometry and laser confocal microscopy were used to test the EGFR specificity of the antibody probe in vitro. Erbitux-Cy5.5 was also injected intravenously into immune-deficient mice bearing MDA-MB-231 or MCF-7 tumors. Whole-body and region-of-interest fluorescence images were acquired and analyzed. The EGFR expression was also analyzed and confirmed by immunohistochemical assay.. The maximum excitation/emission wavelength for the Erbitux-Cy5.5 probe was 674/697 nm, similar to that of free Cy5.5 (674/712 nm). Confocal microscopy confirmed receptor-specific uptake in MDA-MB-231 and MCF-7 cells. In flow cytometry probe specificity assay, Erbitux-Cy5.5 showed a 9.32-fold higher affinity for MDA-MB-231 than MCF-7 cells. In vivo NIR imaging also indicated specific uptake in EGFR-positive tumors. Probe uptake rate and maximum intake dose in MDA-MB-231 were significantly higher than those in MCF-7 xenografts (P < 0.001). Immunohistochemical staining confirmed the in vivo imaging results, showing differentiated EGFR expression in MDA-MB-231 (+ + +) and MCF-7 (+) tumor tissues.. Erbitux-Cy5.5 may be used as a specific NIR contrast agent for the noninvasive characterization of EGFR expression level in breast cancer xenografts.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cetuximab; ErbB Receptors; Female; Flow Cytometry; Humans; Immunoenzyme Techniques; Mice; Mice, Nude; Microscopy, Confocal; Neoplasm Transplantation; Sensitivity and Specificity; Spectroscopy, Near-Infrared; Statistics, Nonparametric

Non-biological signal (or noise) has been the bane of microarray analysis. Hybridization effects related to probe-sequence composition and DNA dye-probe interactions have been observed in differential methylation hybridization (DMH) microarray experiments as well as other effects inherent to the DMH protocol.. We suggest two models to correct for non-biologically relevant probe signal with an overarching focus on probe-sequence composition. The estimated effects are evaluated and the strengths of the models are considered in the context of DMH analyses.. The majority of estimated parameters were statistically significant in all considered models. Model selection for signal correction is based on interpretation of the estimated values and their biological significance.

Topics: Base Sequence; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; DNA Methylation; DNA Probes; Female; Fluorescent Dyes; Gene Expression Profiling; Humans; Methylation; Models, Genetic; Models, Statistical; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Sequence Analysis, DNA; Signal Transduction

Use of fluorescence imaging in oncology is evolving rapidly, and nontargeted fluorochromes are currently being investigated for clinical application. Here, we investigated whether the degree of tumour angiogenesis can be assessed in vivo by planar and tomographic methods using the perfusion-type cyanine dye SIDAG (1,1'-bis- [4-sulfobutyl]indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium).. Mice were xenografted with moderately (MCF7, DU4475) or highly vascularized (HT1080, MDA-MB435) tumours and scanned up to 24 hours after intravenous SIDAG injection using fluorescence reflectance imaging. Contrast-to-noise ratio was calculated for all tumours, and fluorochrome accumulation was quantified using fluorescence-mediated tomography. The vascular volume fraction of the xenografts, serving as a surrogate marker for angiogenesis, was measured using magnetic resonance imaging, and blood vessel profile (BVP) density and vascular endothelial growth factor expression were determined.. SIDAG accumulation correlated well with angiogenic burden, with maximum contrast to noise ratio for MDA-MB435 (P < 0.0001), followed by HT1080, MCF7 and DU4475 tumours. Fluorescence-mediated tomography revealed 4.6-fold higher fluorochrome concentrations in MDA-MB435 than in DU4475 tumours (229 +/- 90 nmol/l versus 49 +/- 22 nmol/l; P < 0.05). The vascular volume fraction was 4.5-fold (3.58 +/- 0.9% versus 0.8 +/- 0.53%; P < 0.01), blood vessel profile density 5-fold (399 +/- 36 BVPs/mm2 versus 78 +/- 16 BVPs/mm2) and vascular endothelial growth factor expression 4-fold higher for MDA-MB435 than for DU4475 tumours.. Our data suggest that perfusion-type cyanine dyes allow assessment of angiogenesis in vivo using planar or tomographic imaging technology. They may thus facilitate characterization of solid tumours.

Topics: Adenocarcinoma; Animals; Blotting, Western; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Contrast Media; Fibrosarcoma; Fluorescence; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Magnetic Resonance Imaging; Melanoma; Mice; Neoplasms; Neovascularization, Pathologic; Optics and Photonics; Tomography; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Lovastatin, an inhibitor of cellular cholesterol synthesis, has an apparent anti-cancer property, but the detailed mechanisms of its anti-cancer effects remain poorly understood. We investigated the molecular mechanism of Lovastatin anti-tumor function through the study of its effect on membrane ion flow, gap junctional intercellular communication (GJIC), and the pathways of related signals in MCF-7 mammary cancer cells. After treatment for 24-72 h with 4, 8 or 16 micromol/L Lovastatin, cellular proliferation was examined via the MTT assay, and changes in membrane potential and cellular [Ca(2+)](i) were monitored using confocal laser microscopy. In addition, the expression of plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA was analyzed via RT-PCR, the GJIC function was examined using the scrape-loading dye transfer (SLDT) technique, and MAPK phosphorylation levels were tested with the kinase activity assay. The results showed that Lovastatin treatment significantly inhibited the growth of MCF-7 breast cancer cells. It also increased the negative value of the membrane potential, leading to the hyperpolarization of cells. Moreover, Lovastatin treatment continuously enhanced [Ca(2+)](i), although the levels of PMCA1 mRNA were unchanged. GJIC was also upregulated in MCF-7 cells, with transfer of LY Fluorescence reaching 4 to 5 rows of cells from the scraped line after treatment with 16 micromol/L Lovastatin for 72 h. Finally, downregulation of ERK1 and p38(MAPK) phosphorylation were found in Lovastatin-treated MCF-7 cells. It could be deduced that Lovastatin can induce changes in cellular hyperpolarization and intracellular Ca(2+) distributions, and increase GJIC function. These effects may result in changes in the downstream signal cascade, inhibiting the growth of MCF-7 cells.

Topics: Antineoplastic Agents; Benzothiazoles; Breast Neoplasms; Calcium; Carbocyanines; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Female; Fluorescence; Gap Junctions; Gene Expression Regulation, Neoplastic; Humans; Ion Transport; Lovastatin; Membrane Potentials; Microscopy, Confocal; Mitogen-Activated Protein Kinase 3; p38 Mitogen-Activated Protein Kinases; Plasma Membrane Calcium-Transporting ATPases; RNA, Messenger; Signal Transduction

Dielectrophoresis (DEP) was used to examine a panel of MCF-7 cell lines comprising parental MCF-7 cells and MDR derivatives: MCF-7TaxR (paclitaxel-resistant, P-glycoprotein (P-gp) positive), MCF-7DoxR (doxorubicin-resistant MRP2 positive) plus MCF-7MDR1 (MDR1 transfected, P-gp positive). MCF-7DoxR and MCF-7MDR1 were broadly cross-resistant to natural product anticancer agents, whereas MCF-7TaxR cells were not, contrary to P-gp expression. Whilst DEP revealed modest membrane changes in MDR sub-lines, we saw significant changes in their cytoplasmic conductivity: MCF-7TaxR

Topics: Anthracyclines; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cell Survival; Cisplatin; Colchicine; Cytoplasm; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Electrophoresis; Etoposide; Female; Fluorescent Dyes; Humans; Inhibitory Concentration 50; Membrane Potentials; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Paclitaxel; Phenotype

Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin-alpha or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT.

Topics: Benzimidazoles; Breast Neoplasms; Carbocyanines; Cations, Divalent; Cell Line, Tumor; Copper; DNA-Binding Proteins; Female; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases; Membrane Potentials; Metallothionein; Metals, Heavy; Mitochondria; Plasmids; Response Elements; Time Factors; Transcription Factor MTF-1; Transcription Factors; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53; Zinc

Differential in-gel electrophoresis (DIGE) experiments allow three protein samples to be run per gel. The three samples are labeled with the spectrally resolvable fluorescent dyes, Cy2, Cy3, and Cy5, respectively. Here, we show that protein-specific dye effects exist, and we present a linear mixed model for analysis of DIGE data which takes dye effects into account. A Java implementation of the model, called DIGEanalyzer, is freely available at http://bioinfo.thep.lu.se/digeanalyzer.html. Three DIGE experiments from our laboratory, with 173, 64, and 24 gels, respectively, were used to quantify and verify the dye effects. DeCyder 5.0 and 6.5 were used for spot detection and matching. The fractions of proteins with a statistically significant (0.001 level) dye effect were 19, 34, and 23%, respectively. The fractions of proteins with a dye effect above 1.4-fold change were 1, 4, and 6%, respectively. The median magnitude of the dye effect was 1.07-fold change for Cy5 versus Cy3 and 1.16-fold change for Cy3 versus Cy2. The maximal dye effect was a seven-fold change. The dye effects of spots corresponding to the same protein tend to be similar within each of the three experiments, and to a smaller degree across experiments.

Topics: Algorithms; Animals; Brain Chemistry; Breast Neoplasms; Carbocyanines; Computational Biology; Electrophoresis, Gel, Two-Dimensional; Female; Fluorescent Dyes; Humans; Image Processing, Computer-Assisted; Internet; Linear Models; Ovarian Neoplasms; Proteins; Proteomics; Rats; Software; Tandem Mass Spectrometry

Spotted cDNA microarrays generally employ co-hybridization of fluorescently-labeled RNA targets to produce gene expression ratios for subsequent analysis. Direct comparison of two RNA samples in the same microarray provides the highest level of accuracy; however, due to the number of combinatorial pair-wise comparisons, the direct method is impractical for studies including large number of individual samples (e.g., tumor classification studies). For such studies, indirect comparisons using a common reference standard have been the preferred method. Here we evaluated the precision and accuracy of reconstructed ratios from three indirect methods relative to ratios obtained from direct hybridizations, herein considered as the gold-standard.. We performed hybridizations using a fixed amount of Cy3-labeled reference oligonucleotide (RefOligo) against distinct Cy5-labeled targets from prostate, breast and kidney tumor samples. Reconstructed ratios between all tissue pairs were derived from ratios between each tissue sample and RefOligo. Reconstructed ratios were compared to (i) ratios obtained in parallel from direct pair-wise hybridizations of tissue samples, and to (ii) reconstructed ratios derived from hybridization of each tissue against a reference RNA pool (RefPool). To evaluate the effect of the external references, reconstructed ratios were also calculated directly from intensity values of single-channel (One-Color) measurements derived from tissue sample data collected in the RefOligo experiments. We show that the average coefficient of variation of ratios between intra- and inter-slide replicates derived from RefOligo, RefPool and One-Color were similar and 2 to 4-fold higher than ratios obtained in direct hybridizations. Correlation coefficients calculated for all three tissue comparisons were also similar. In addition, the performance of all indirect methods in terms of their robustness to identify genes deemed as differentially expressed based on direct hybridizations, as well as false-positive and false-negative rates, were found to be comparable.. RefOligo produces ratios as precise and accurate as ratios reconstructed from a RNA pool, thus representing a reliable alternative in reference-based hybridization experiments. In addition, One-Color measurements alone can reconstruct expression ratios without loss in precision or accuracy. We conclude that both methods are adequate options in large-scale projects where the amount of a common reference RNA pool is usually restrictive.

Topics: Adenocarcinoma; Breast Neoplasms; Carbocyanines; Carcinoma, Renal Cell; DNA, Complementary; DNA, Neoplasm; Female; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms

Various thermotherapies are based on the induction of lethal heat in target tissues. Spatial and temporal instabilities of elevated temperatures induced in therapy targets require optimized treatment protocols and reliable temperature control methods during thermotherapies. Heat-stress induced effects on mitochondrial transmembrane potentials were analyzed in breast cancer cells, species MX1, using the potential sensor JC-1 (Molecular Probes, Invitrogen, Germany). Potential dependant labeling of heat-stressed cells was imaged and evaluated by fluorescence microscopy and compared with control cells. JC-1 stains mitochondria in cells with high mitochondrial potentials by forming orange-red fluorescent J-aggregates while in cells with depolarized or damaged mitochondria the sensor dye exists as green fluorescent monomers. In MX1 cells orange-red and green fluorescence intensities were correlated with each other after various heat-stress treatments and states of mitochondrial membrane potentials were deduced from the image data. With increasing stress temperatures the intensity of red fluorescent J-aggregates decreased while the green fluorescence intensity of JC-1 monomers increased. This heat-stress response happened in a nonlinear manner with increasing temperatures resulting in a nonlinear increase of red/green fluorescence ratios. These data indicated that mitochondria in MX1 cells were increasingly depolarized in response to increasing ambient temperatures.

Topics: Benzimidazoles; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Female; Fluorescent Dyes; Hot Temperature; Humans; Hyperthermia, Induced; Membrane Potential, Mitochondrial; Microscopy, Fluorescence

The objective of this study was to assess the effect of hydrophilic/hydrophobic block chain lengths on the internalization of poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles by cancer cells. PEO-b-PCL block copolymers with varied PEO and PCL chain lengths were synthesized, assembled to polymeric micelles and loaded with a hydrophobic fluorescent probe (DiI) through a co-solvent evaporation method of physical encapsulation. The slow release of the fluorescent probe from the micellar structure was evidenced following DiI transfer to lipid vesicles. The extent of micellar uptake by cancer cells was investigated through their incubation with MCF-7 cells followed by measurement of the fluorescent emission intensity of DiI (lambda=550 nm) in separated lysed cells. Cellular internalization of polymeric micelles was confirmed by laser scanning microscopy. The mechanism of micellar uptake was investigated by pretreatment of MCF-7 cells with chlorpromazine and cytochalasin B. Encapsulation of DiI in PEO-b-PCL micelles lowered the extent and rate of hydrophobic probe internalization by cancer cells. For polymeric micelles with 5000 gmol(-1) of PCL and varied PEO molecular weights of 2000, 5000 and 13,000 gmol(-1), maximum uptake was observed at a PEO molecular weight of 5000 gmol(-1). For polymeric micelles with 5000 gmol(-1) of PEO and varied PCL molecular weights of 5000, 13,000 and 24,000 gmol(-1), maximum uptake was observed at 13,000 gmol(-1) of PCL. Chlorpromazine reduced the cellular uptake of PEO-b-PCL micelles independent from the block copolymer structure, pointing to the involvement of clathrin mediated endocytosis mechanisms in the uptake of polymeric micelles by cancer cells. Inhibition of cellular uptake of PEO-b-PCL micelles by cytochalasin B, on the other hand, was found to be dependent on the chemical structure of the core/shell forming blocks.

Topics: Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Cold Temperature; Drug Delivery Systems; Female; Fluorescent Dyes; Humans; Magnetic Resonance Spectroscopy; Micelles; Microscopy, Confocal; Polyesters; Staining and Labeling

The most widely used microarray experiment design includes the use of a reference standard. Comparisons of gene expression between samples are facilitated because each sample is directly measured against the reference standard, using two fluorescent dyes. Numerous reports indicate that some genes incorporate the two commonly used dyes with different efficiencies, contributing to inaccurate data. However, it is widely assumed that these effects will not corrupt results if the reference standard is labeled with the same dye on each microarray. We demonstrate that this assumption is not reliable and that dye orientation can significantly influence measured changes in gene expression.

Topics: Animals; Bias; Breast; Breast Neoplasms; Carbocyanines; Carcinoma; Cell Line; DNA, Complementary; Equipment Design; Equipment Failure Analysis; Fluorescent Dyes; Gene Expression Profiling; Humans; Mice; Mice, Nude; Mutagenesis, Insertional; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Precancerous Conditions; Quality Control; Reference Standards; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Transplantation, Heterologous

The estrogenic flavanone rac-8-prenylnaringenin (8-PN) and 3 derivatives (rac-7-(O-prenyl)naringenin-4'-acetate (7-O-PN), rac-5-(O-prenyl)naringenin-4',7-diacetate (5-O-PN), and rac-6-(1,1-dimethylallyl)naringenin (6-DMAN) were prepared by chemical synthesis and analyzed with respect to their toxicity and possible cell cycle effects in human acute myeloid leukemia (HL-60) cells. With the exception of 5-O-PN, all the other naringenins showed only weak toxic effects at concentrations below 50 micromol/l. A cell cycle analysis over several cell generations up to 4 days was carried out using the fluorescent dye carboxyfluorescein diacetate N-succinimidyl ester (CFSE) followed by propidium iodide (PI) staining at the end of the experiment. The well-studied flavonol quercetin was included in the analysis as a reference substance. All flavonoids affected cell proliferation, but the extent and the resulting changes in the proliferation pattern were specific for each substance. In contrast to the radical scavenging activity of quercetin, the tested flavanones showed no anti-oxidative properties using several different test systems. Similarly, the mitochondrial membrane potential (DeltaPsim) was hardly effected by these compounds, while both menadione and quercetin strongly reduced the potential after 1 h of treatment. The reported chemical modification of interesting lead substances (like the strongly estrogenic 8-PN) presents a promising approach to modulate the properties of a relevant substance in a pharmacologically desirable way. The low toxicity and weak cytostatic properties of the tested naringenin derivatives is encouraging for further studies on known naringenin target molecules.

Topics: Benzimidazoles; Breast Neoplasms; Carbocyanines; Cell Division; Cell Line, Tumor; Cell Membrane; Dose-Response Relationship, Drug; Flavanones; Fluorescent Dyes; Free Radical Scavengers; Germany, East; HL-60 Cells; Humans; Membrane Potentials; Mitochondria; Quercetin; Reactive Oxygen Species; Time Factors; Vitamin K 3

The specificity of a novel epidermal growth factor (EGF)-Cy5.5 fluorescent optical probe in the detection of EGF receptor (EGFr) was assessed using continuous-wave fluorescence imaging accomplished via an intensified charge-coupled device (CCD) camera. Human mammary MDA-MB-468 (EGFr+) and MDA-MB-435 (EGFr-) cancer cells were incubated with Cy5.5, EGF-Cy5.5, or the anti-EGFr monoclonal antibody C225 or EGF followed by EGF-Cy5.5 and examined under a fluorescence microscope. In vivo imaging was performed on mice with s.c. MDA-MB-468 and MDA-MB-435 tumors. Images were obtained every 6 s for 20 min after i.v. injection of each agent and every 24 h after injection for up to 192 h. Additionally, mice with MDA-MB-468 tumors were injected i.v. with C225 24 h before injection of EGF-Cy5.5. EGF-Cy5.5, but not Cy5.5 or indocyanine green dye (ICG), bound to MDA-MB-468 cells. Binding of EGF-Cy5.5 was blocked by C225 and by EGF. In contrast, binding of EGF-Cy5.5 to MDA-MB-435 cells was not observed. Monitoring of the time-fluorescence intensity in mice confirmed that ICG and Cy5.5 had no favorable binding to tumor regardless of EGFr expression level. In contrast, EGF-Cy5.5 accumulated only in MDA-MB-468 tumors. Moreover, tumor uptake of EGF-Cy5.5 was blocked by C225. ICG and Cy5.5 fluorescence was completely absent from the tumor site, regardless of EGFr expression level, 24 h after injection. Little EGF-Cy5.5 fluorescence was detected in MDA-MB-435 tumors 24 h after injection. In MDA-MB-468 tumors, our data suggest that EGF-Cy5.5 may be used as a specific NIR contrast agent for noninvasive imaging of EGFr expression and monitoring of responses to molecularly targeted therapy.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Carbocyanines; Cell Line, Tumor; Epidermal Growth Factor; ErbB Receptors; Female; Fluorescent Dyes; Humans; Mice; Mice, Nude; Neoplasm Transplantation; Spectroscopy, Near-Infrared; Substrate Specificity; Transplantation, Heterologous

Topics: Breast Neoplasms; Carbocyanines; Fluorescent Dyes; Humans; Lasers

Optical mammography with near-infrared (NIR) light using time-domain, frequency-domain, or continuous-wave techniques is a novel imaging modality to locate human breast tumors. By investigating excised specimens of normal and diseased mamma tissue we were able to demonstrate that differences in their scattering properties are a poor predictive parameter for normal and diseased mamma tissue. This paper describes the application of a NIR dye to improve the differentiation between breast tumors and normal tissue in a rat model. The NIR dye furnished a high tumor-to-tissue contrast ratio (6:1) in fluorescence images. Furthermore, this dye was used to develop liquid scattering phantoms with absorbing and fluorescent inhomogeneities. Using frequency-domain and time-domain instrumentation these inhomogeneities were localized at sufficient contrast by their increased absorption and fluorescence. Contrast between inhomogeneities and surrounding medium could be improved by combining fluorescence and transmittance images.

Topics: Breast Neoplasms; Carbocyanines; Contrast Media; Female; Fluorescent Dyes; Humans; Infrared Rays; Mammography; Optics and Photonics; Phantoms, Imaging; Reference Values; Scattering, Radiation

Confocal laser scanning microscopy (CLSM) is an extensive but reliable tool for assessing the hybridisation signals in fluorescence in situ hybridisation (FISH). Most CLSMs are equipped with an argon-laser and a helium/neon-laser illumination system with excitation wavelengths of 488, 543 and 633 nm. A protocol for an optimal nuclear counterstaining in combination with dual-colour FISH for these laser illumination systems has not been established so far. Here, we determined the suitability of eleven dimeric and monomeric cyanine nucleic acid stains on paraffin sections of breast carcinoma specimens in combination with dual-colour FISH (Her-2/neu and centromere 17) for CLSM application. Strong staining of cell nuclei was observed for TO-PRO-3 and YO-PRO-3, YOYO-1 and propidium iodide (PI), but only TO-PRO-3 showed specific staining of nuclei without any staining of the cytoplasm. A specific emission in exclusively one distinct fluorescence channel was shown for TO-PRO-3 (633 nm excitation) as well as YOYO-1, BO-PRO-1 and Sytox Green (488 nm excitation), evaluated by a CLSM and confirmed by 3-D fluorescence spectra. High stability of fluorescence intensity was shown for the far-red dyes TO-PRO-3, YO-PRO-3, YOYO-3 and Syto-59 as well as YOYO-1 and PI. Only TO-PRO-3 was due to its high specificity and stability suitable for detection of an amplification of the Her-2/neu gene by dual-colour FISH and CLSM evaluation.

Topics: Breast Neoplasms; Carbocyanines; Carcinoma, Ductal, Breast; Cell Nucleus; Drug Stability; Fluorescent Dyes; Genes, erbB-2; Humans; In Situ Hybridization, Fluorescence; Microscopy, Confocal; Microtomy; Nucleic Acids; Paraffin Embedding; Sensitivity and Specificity; Staining and Labeling

Laser scanning cytometry (LSC) is a relatively new slide-based technology developed for commercial use by CompuCyte (Cambridge, MA) for performing multiple fluorescence measurements on individual cells. Because techniques developed for performing four or more measurements on individual lymphoid cells based on light scatter as a triggering parameter for cell identification are not suitable for the identification of fixed epithelial tumor cells, an alternative approach is required for the analysis of such cells by LSC. Methods for sample preparation, event triggering, and the performance of multiple LSC measurements on disaggregated fixed human cells were developed using normal lymphocytes and two human breast cancer cell lines, JC-1939 and MCF-7, as test populations. Optimal conditions for individual cell identification by LSC were found to depend on several factors, including deposited cell density (cells per unit area), the dynamic range of probe fluorescence intensities, and intracellular distribution of the fluorescent probe. Sparsely deposited cells exhibited the least cell overlap and the brightest immunofluorescent staining. Major advantages of using DNA probes over a cytoplasmic immunofluorescent protein marker such as tubulin for event triggering are that the former exhibit greater fluorescence intensity within a relatively sharply demarcated nuclear region. The DNA-binding dye LDS-751 was found to be suboptimal for quantitative DNA measurements but useful as a triggering measurement that permits the performance of simultaneous fluorescein isothiocyanate-, phycoerythrin-, and indodicarbocyanine-based measurements on each cell. A major potential advantage of LSC over flow cytometry is the high yields of analyzable cells by LSC, permitting the performance of multiple panels of multicolor measurements on each tumor. In conclusion, we have developed and optimized a technique for performing multiple fluorescence measurements on fixed epithelial cells by LSC based on event triggering using the DNA-binding dye LDS 751. Although not ideal for quantitative measurements of cell DNA content, the large Stokes shift of this dye permits the performance of three or more additional fluorescence measurements on each cell.

Topics: Breast Neoplasms; Carbocyanines; Carcinoma; Cell Nucleus; DNA, Neoplasm; Female; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fluoroimmunoassay; Humans; Microscopy, Confocal; Organic Chemicals; Phycoerythrin; Sensitivity and Specificity; Tissue Fixation; Tumor Cells, Cultured

Topics: Breast; Breast Neoplasms; Carbocyanines; Female; Glycosaminoglycans; Histocytochemistry; Humans; Hyaluronic Acid; Hydrogen-Ion Concentration; Neoplasm Proteins; Nucleic Acids; Phosphoproteins; Pregnancy; Quinolines; Sialoglycoproteins; Staining and Labeling