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Concept: MCF-7

170

Multidrug resistance driven by ABC membrane transporters is one of the major reasons for treatment failure in human malignancy. Some limited evidence has previously been reported on the cell cycle dependence of ABC transporter expression. However, it has never been demonstrated that the functional activity of these transporters correlates with the cell cycle position. Here, we studied the rate of intrinsic ABC transport in different phases of the cell cycle in cultured MCF-7 breast cancer cells. The rate was characterized in terms of the efflux kinetics from cells loaded with an ABC transporter substrate. As averaging the kinetics over a cell population could lead to errors, we studied kinetics of ABC transport at the single-cell level. We found that the rate of ABC transport in MCF-7 cells could be described by Michaelis-Menten kinetics with two classical parameters, V(max) and K(M). Each of these parameters showed similar unimodal distributions with different positions of maxima for cell subpopulations in the 2c and 4c states. Compared to the 2c cells, the 4c cells exhibited greater V(max) values, indicating a higher activity of transport. They also exhibited a greater V(max)/K(M) ratio, indicating a higher efficiency of transport. Our findings suggest that cell cycle-related modulation of MDR may need to be taken into account when designing chemotherapy regimens which include cytostatic agents.

Concepts: Gene expression, Cancer, Breast cancer, Cell division, Chemotherapy, Chemotherapy regimens, Transport, MCF-7

169

PURPOSE: Development of a heptamethine cyanine based tumor-targeting PET imaging probe for noninvasive detection and diagnosis of breast cancer. METHODS: 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. RESULTS: PC-1001 displayed similar fluorescence properties (ε=82,880cm(-1)M(-1), E(x)/E(m)=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. CONCLUSIONS: 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.

Concepts: Cancer, Breast cancer, Oncology, In vivo, Benign tumor, Tumor, Neoplasm, MCF-7

28

In this study, a novel amphiphilic copolymer designed as N-octyl-N-phthalyl-3,6-O-(2-hydroxypropyl) chitosan (OPHPC) were synthesized and then conjugated with folic acid (FA-OPHPC) to produce a targeted drug carrier for tumor-specific drug delivery. OPHPC and FA-OPHPC were characterized by FT-IR, (1)H NMR, (13)C NMR and elemental analysis. Paclitaxel (PTX) loaded OPHPC micelles (PTX-OPHPC) with well-defined spherical shape and homogeneous distribution exhibited drug-loading rate ranging from 33.6% to 45.3% and entrapment efficiency from 50.5% to 82.8%. In the cellular uptake studies, PTX-OPHPC brought about a significantly higher amount of PTX accumulated in human breast adenocarcinoma cell line (MCF-7 cells) compared with Taxol. Moreover, the cellular uptake of PTX in PTX loaded FA-OPHPC micelles (PTX-FA-OPHPC) was 3.2-fold improved in comparison with that of PTX-OPHPC. The results revealed that OPHPC micelle might be a promising drug carrier for promoting PTX cellular uptake and FA-OPHPC micelle could be used as a potential tumor-targeted drug vector.

Concepts: Breast cancer, Lung cancer, Chemotherapy, Vesicle, Taxane, Paclitaxel, Docetaxel, MCF-7

28

Molecular beacons (MBs) have shown fascinating applications in many biological fields. However, exploration of cost-effective, sensitive, stable and efficient MB for in situ live cell- based assay has still room for improvement. In this regards, we have developed a novel MB which bears a spherical graphite nanoparticle (GN) as a fluorescent quencher. The GN resulted in the high quenching efficiency, and the presence of GN enhanced the biological stability and transfection of the MB into the cells, thereby enabling the real-time survivin mRNA detection and quantification in the MCF-7 breast cancer cells. These results demonstrated that the advancement of the proposed MB containing a GN nanoquencher can be used as a robust molecular probe for genetic analysis in the cells.

Concepts: DNA, Genetics, Gene expression, Cancer, Breast cancer, Molecular biology, Carcinoma in situ, MCF-7

27

A star-shaped biodegradable polymer, mannitol-core poly(D,L-lactide-co-glycolide)-D-α-tocopheryl polyethylene glycol 1000 succinate (M-PLGA-TPGS), was synthesized with the intent to provide novel nanoformulation for breast cancer chemotherapy in this research. The novel copolymer was prepared by a core-first approach through three parts of chemical reaction, and characterized by NMR, GPC and TGA. The docetaxel-loaded M-PLGA-TPGS nanoparticles (NPs), prepared by a modified nano-precipitation method, were observed to be near-spherical shape with narrow size distribution. The CLSM results showed the uptake level of M-PLGA-TPGS NPs was higher than PLGA NPs and PLGA-TPGS NPs in MCF-7 cells. A significantly higher level of cytotoxicity was achieved by docetaxel-loaded M-PLGA-TPGS NPs than that of commercial Taxotere(®), docetaxel-loaded PLGA-TPGS and PLGA NPs. The study on drug loading and encapsulation efficiency proved that star-shaped M-PLGA-TPGS could carry higher level of drug than linear polymer. The in vivo experiment showed docetaxel-loaded M-PLGA-TPGS NPs have the highest anti-tumor efficacy. In conclusion, the star-like M-PLGA-TPGS copolymer could be used as a potential and promising drug-loaded biomaterial applied in developing novel nanoformulation for breast cancer therapy.

Concepts: Cancer, Breast cancer, Metastasis, Oncology, Chemotherapy, Copolymer, Chemotherapy regimens, MCF-7

26

The trapping or immobilization of individual cells at specific locations in microfluidic platforms is essential for single cell studies, especially those requiring cell stimulation and downstream analysis of cellular content. Selectivity for individual cell types is required when mixtures of cells are analyzed in heterogeneous and complex matrices, such as the selection of metastatic cells within blood samples. Here, we demonstrate a microfluidic device based on direct current (DC) insulator-based dielectrophoresis (iDEP) for selective trapping of single MCF-7 breast cancer cells from mixtures with both mammalian peripheral blood mononuclear cells (PBMC) as well MDA-MB-231 as a second breast cancer cell type. The microfluidic device has a teardrop iDEP design optimized for the selective capture of single cells based on their differential DEP behavior under DC conditions. Numerical simulations adapted to experimental device geometries and buffer conditions predicted the trapping condition in which the dielectrophoretic force overcomes electrokinetic forces for MCF-7 cells, whereas PBMCs were not trapped. Experimentally, selective trapping of viable MCF-7 cells in mixtures with PBMCs was demonstrated in good agreement with simulations. A similar approach was also executed to demonstrate the selective trapping of MCF-7 cells in a mixture with MDA-MB-231 cells, indicating the selectivity of the device for weakly invasive and highly invasive breast cancer cells. The DEP studies were complemented with cell viability tests indicating acceptable cell viability over the course of an iDEP trapping experiment.

Concepts: Cancer, Breast cancer, Metastasis, Lung cancer, Cell division, Chemotherapy, PBMC, MCF-7

24

A diiodo distyryl boron dipyrromethene (BODIPY) core was conjugated to two ferrocenyl quenchers through acid-labile ketal and/or thiol-cleavable disulfide linkers, of which the fluorescence and photosensitizing properties were significantly quenched through a photoinduced electron-transfer process. The two symmetrical analogues that contained either the ketal or disulfide linkers could only be activated by a single stimulus, whereas the unsymmetrical analogue was responsive to dual stimuli. Upon interaction with acid and/or dithiothreitol (DTT), these linkers were cleaved selectively. The separation of the BODIPY core and the ferrocenyl moieties restored the photoactivities of the former in phosphate buffered saline and inside the MCF-7 breast cancer cells, rendering these compounds as potential activable photosensitizers for targeted photodynamic therapy. The dual activable analogue exhibited the greatest enhancement in intracellular fluorescence intensity in both an acidic environment (pH 5) and the presence of DTT (4 mm). Its photocytotoxicity against MCF-7 cells also increased by about twofold upon preincubation with 4 mm of DTT. The activation of this compound was also demonstrated in nude mice bearing a HT29 human colorectal carcinoma. A significant increase in fluorescence intensity in the tumor was observed over 9 h after intratumoral injection.

Concepts: Cancer, Breast cancer, Metastasis, Acid, Cancer staging, Colorectal cancer, Buffer solution, MCF-7

5

Strong epidemiologic evidence documents the protective effect of physical activity on breast cancer risk, recurrence, and mortality, but the underlying mechanisms remain to be identified. Using human exercise-conditioned serum for breast cancer cell incubation studies and murine exercise interventions, we aimed to identify exercise factors and signaling pathways involved in the exercise-dependent suppression of breast cancer. Exercise-conditioned serum from both women with breast cancer (n = 20) and healthy women (n = 7) decreased MCF-7 (hormone-sensitive) and MDA-MB-231 (hormone-insensitive) breast cancer cell viability in vitro by 11% to 19% and reduced tumorigenesis by 50% when preincubated MCF-7 breast cancer cells were inoculated into NMRI-Foxn1(nu) mice. This exercise-mediated suppression of cell viability and tumor formation was completely blunted by blockade of β-adrenergic signaling in MCF-7 cells, indicating that catecholamines were the responsible exercise factors. Both epinephrine (EPI) and norepinephrine (NE) could directly inhibit breast cancer cell viability, as well as tumor growth in vivo EPI and NE activate the tumor suppressor Hippo signaling pathway, and the suppressive effect of exercise-conditioned serum was found to be mediated through phosphorylation and cytoplasmic retention of YAP and reduced expression of downstream target genes, for example, ANKRD1 and CTGF. In parallel, tumor-bearing mice with access to running wheels showed reduced growth of MCF-7 (-36%, P < 0.05) and MDA-MB-231 (-66%, P < 0.01) tumors and, for the MCF-7 tumor, increased regulation of the Hippo signaling pathway. Taken together, our findings offer a mechanistic explanation for exercise-dependent suppression of breast cancer cell growth. Cancer Res; 77(18); 1-11. ©2017 AACR.

Concepts: Cancer, Breast cancer, Metastasis, Oncology, Cell division, Tumor, Neoplasm, MCF-7

3

Finding advanced anti-cancer agents with selective toxicity in tumor tissues is the goal of anticancer delivery systems. This study investigated potential application of nanostructured lipid carriers (NLCs) in increasing melatonin induced cytotoxicity and apoptosis in MCF-7 breast cancer cells.

Concepts: Cancer, Breast cancer, Metastasis, Oncology, Cancer staging, Chemotherapy, Pleural effusion, MCF-7

2

An engineered supercharged coiled-coil protein (CSP) and the cationic transfection reagent Lipofectamine 2000 are combined to form a lipoproteoplex for the purpose of dual delivery of siRNA and doxorubicin. CSP, bearing an external positive charge and axial hydrophobic pore, demonstrates the ability to condense siRNA and encapsulate the small molecule chemotherapeutic, doxorubicin. The lipoproteoplex demonstrates improved doxorubicin loading relative to Lipofectamine 2000. Furthermore, it induces effective transfection of GAPDH (60% knockdown) in MCF-7 breast cancer cells with efficiencies comparing favorably to Lipofectamine 2000. When the lipoproteoplex is loaded with doxorubicin, the improved doxorubicin loading (~40 µg Dox/mg CSP) results in a substantial decrease in MCF-7 cell viability.

Concepts: DNA, Cancer, Breast cancer, Metastasis, Electric charge, Molecule, Chemotherapy, MCF-7