MCF-7 Spheroids Research Articles

Evaluation of senescence in individual MCF-7 spheroids based on electrochemical measurement of senescence-associated β-galactosidase activity

Cellular senescence is a physiological phenomenon by which normal cells irreversibly lose their proliferative potential. Understanding the mechanisms of senescence may be helpful to elucidate the causes of ageing and for the development of anti-tumor drugs. As an indicator of senescent cells, the senescence-associated β-galactosidase (SA-β-Gal) activity at pH 6.0 has been used widely for fundamental senescence research. However, the conventional SA-β-Gal assay is not optimal for evaluating complex three-dimensional multicellular samples. We established a highly sensitive method for the direct quantification of the SA-β-Gal activity of individual multicellular spheroids within 6min, based on scanning electrochemical microscopy (SECM). Using SECM, we successfully detected the differences in SA-β-Gal activity between MCF-7 spheroids undergoing all-trans retinoic acid (ATRA)-induced senescence and control spheroids. The SECM-based method demonstrated in this study may be widely applicable to characterize senescence processes involved in physiology and pathology.

Abstract 1419: Glypican-3 (GPC3) inhibits metastatic dissemination in a preclinical human breast cancer model

Abstract Although GPC3 has been linked to cancer, its role during mammary tumor progression is barely known. With the aim to develop a pre-clinical breast cancer model, we genetically modified MCF-7 (poorly-metastatic, GPC3 +) and MDA-MB231 (metastatic, GPC3 -) cell lines. GPC3 expression was blocked in MCF-7 by siRNA (generating MCF-7-shGPC3 sublines), while it was overexpressed in MDA-MB231 by viral infection (producing MDA-MB231-GPC3 sublines). First, we analyzed the in vivo tumor growth by s.c. inoculation of MCF-7 and MDA-MB231 engineered cells into nude mice. Silencing GPC3 stimulated tumorigenicity, while its overexpression inhibited it. The histological analysis of MCF-7-shGPC3 tumors revealed extensive invasion of the muscle and the dermis, while control tumors did not invade. In addition, MDA-MB231-GPC3 tumors were less invasive than MDA-MB231-vector ones. We also evaluated spontaneous metastasis capacity by lung histological analysis. While no metastases were found in MCF-7-sh scramble tumor-bearing mice, lung nodules were detected in mice inoculated with GPC3 silenced cells. On the other hand, mice bearing MDA-MB231-GPC3 tumors showed a decrease in the incidence of metastasis. Next, we did a panel of in vitro tests. Although MCF-7-shGPC3 and control cells were morphologically similar, an increase in the number of actin stress fibers was found in GPC3 silenced cells. In association with this mesenchymal characteristic, this subline exhibited a decrease in E-Cadherin expression. While MDA-MB231-vector cells presented a fibroblastic appearance, GPC3 overexpression induced a drastic change in cell morphology turning to an epithelial phenotype. In addition, although control cells showed large stress fibers, GPC3 overexpressing cells localized their actin in cortical position. Moreover, GPC3 induced a reexpression of the epithelial marker E-Cadherin. We tested anchorage-independent growth, finding that GPC3 silencing disorganized MCF-7 spheroids. MDA-MB231-GPC3 cells formed larger spheroids than those generated by controls. Blocking E-cadherin, employing a neutralizing antibody, reversed the 3-D growth ability of GPC3 overexpressing cells. The sensitivity to nutrient depletion was evaluated. We found, through propidium iodide/Höescht staining, a reduction in starved MCF-7-shGPC3 cell death. While MCF-7-sh scramble cells showed morphological alterations characteristic of apoptotic death as detected by orange acridine/ethidium bromide staining, no apoptosis was found in MCF-7-shGPC3 cells. GPC3 overexpression increased MDA-MB-231 cell susceptibility to death. Although there was no apoptosis in MDA-MB231 control cells, the overexpression of GPC3 promoted an increase in the number of cells with nuclear and cytoplasmic manifestations of apoptosis. We demonstrate a central role of GPC3 in human breast cancer biology. Our results indicate that it acts a metastasis suppressor in human mammary cancer. Citation Format: Lilian F. Castillo, Rocio Tascon, Elisa Bal de Kier Joffé, Maria G. Peters. Glypican-3 (GPC3) inhibits metastatic dissemination in a preclinical human breast cancer model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1419. doi:10.1158/1538-7445.AM2015-1419

Generation of Multicellular Tumor Spheroids with Microwell-Based Agarose Scaffolds for Drug Testing

Three dimensional multicellular aggregate, also referred to as cell spheroid or microtissue, is an indispensable tool for in vitro evaluating antitumor activity and drug efficacy. Compared with classical cellular monolayer, multicellular tumor spheroid (MCTS) offers a more rational platform to predict in vivo drug efficacy and toxicity. Nevertheless, traditional processing methods such as plastic dish culture with nonadhesive surfaces are regularly time-consuming, laborious and difficult to provide uniform-sized spheroids, thus causing poor reproducibility of experimental data and impeding high-throughput drug screening. In order to provide a robust and effective platform for in vitro drug evaluation, we present an agarose scaffold prepared with the template containing uniform-sized micro-wells in commercially available cell culture plates. The agarose scaffold allows for good adjustment of MCTS size and large-scale production of MCTS. Transparent agarose scaffold also allows for monitoring of spheroid formation under an optical microscopy. The formation of MCTS from MCF-7 cells was prepared using different-size-well templates and systematically investigated in terms of spheroid growth curve, circularity, and cell viability. The doxorubicin cytotoxicity against MCF-7 spheroid and MCF-7 monolayer cells was compared. The drug penetration behavior, cell cycle distribution, cell apoptosis, and gene expression were also evaluated in MCF-7 spheroid. The findings of this study indicate that, compared with cellular monolayer, MCTS provides a valuable platform for the assessment of therapeutic candidates in an in vivo-mimic microenvironment, and thus has great potential for use in drug discovery and tumor biology research.

A eudesmane-type sesquiterpene isolated from Pluchea odorata (L.) Cass. combats three hallmarks of cancer cells: Unrestricted proliferation, escape from apoptosis and early metastatic outgrowth in vitro

Pluchea odorata is ethno pharmaceutically used to treat inflammation-associated disorders. The dichloromethane extract (DME) was tested in the carrageenan-induced rat paw oedema assay investigating its effect on inflammation that was inhibited by 37%. Also an in vitro anti-neoplastic potential was reported. However, rather limited information about the bio-activity of purified compounds and their cellular mechanisms are available. Therefore, two of the most abundant eudesmanes in P. odorata were isolated and their anti-neoplastic and anti-intravasative activities were studied. HL-60 cells were treated with P. odorata compounds and metabolic activity, cell number reduction, cell cycle progression and apoptosis induction were correlated with relevant protein expression. Tumour cell intravasation through lymph endothelial monolayers was measured and potential causal mechanisms were analyzed by Western blotting. Compound PO-1 decreased the metabolic activity of HL-60 cells (IC50=8.9μM after 72h) and 10μM PO-1 induced apoptosis, while PO-2 showed just weak anti-neoplastic activities at concentrations beyond 100μM. PO-1 arrested the cell cycle in G1 and this correlated with induction of JunB expression. Independent of this mechanism 25μM PO-1 decreased MCF-7 spheroid intravasation through the lymph endothelial barrier. Hence, PO-1 inhibits an early step of metastasis, impairs unrestricted proliferation and induces apoptosis at low micromolar concentrations. These results warrant further testing in vivo to challenge the potential of PO-1 as novel lead compound.

Photodynamic Therapy Based on Arrabidaea chica (Crajiru) Extract Nanoemulsion: In vitro Activity against Monolayers and Spheroids of Human Mammary Adenocarcinoma MCF-7 Cells

Natural products have been sources of numerous drugs over the history. However, little is known about the therapeutic potential of Amazon forest species. This work aimed at testing the potential of the hydrophobic extract of Arrabidaea chica (ACE), an Amazon plant, as a source of photosensitizers for anticancer photodynamic therapy. ACE was tested as a nanoemulsion (ACE-NE) produced by phase inversion temperature. It was found that ACE-NE intensely absorbs red light and, under photoactivation (λ 660 nm, 4.5 J/cm2), produces reactive oxygen species. Photoactivated ACE-NE presented, in vitro, a CC50 of 1.3 μg ACE/mL against human breast adenocarcinoma MCF-7 cells, and was effective in lysing MCF-7 spheroids. In the dark, ACE was toxic neither to human mammary epithelial MCF-10A cells nor to MCF-7 cells. On that ground, this work is the first to show that A. chica is a source of photosensitizers potentially useful for anticancer photodynamic therapy.

Inhibition of 3-D tumor spheroids by timed-released hydrophilic and hydrophobic drugs from multilayered polymeric microparticles.

First-line cancer chemotherapy necessitates high parenteral dosage and repeated dosing of a combination of drugs over a prolonged period. Current commercially available chemotherapeutic agents, such as Doxil and Taxol, are only capable of delivering single drug in a bolus dose. The aim of this study is to develop dual-drug-loaded, multilayered microparticles and to investigate their antitumor efficacy compared with single-drug-loaded particles. Results show hydrophilic doxorubicin HCl (DOX) and hydrophobic paclitaxel (PTX) localized in the poly(dl-lactic-co-glycolic acid, 50:50) (PLGA) shell and in the poly(l-lactic acid) (PLLA) core, respectively. The introduction of poly[(1,6-bis-carboxyphenoxy) hexane] (PCPH) into PLGA/PLLA microparticles causes PTX to be localized in the PLLA and PCPH mid-layers, whereas DOX is found in both the PLGA shell and core. PLGA/PLLA/PCPH microparticles with denser shells allow better control of DOX release. A delayed release of PTX is observed with the addition of PCPH. Three-dimensional MCF-7 spheroid studies demonstrate that controlled co-delivery of DOX and PTX from multilayered microparticles produces a greater reduction in spheroid growth rate compared with single-drug-loaded particles. This study provides mechanistic insights into how distinctive structure of multilayered microparticles can be designed to modulate the release profiles of anticancer drugs, and how co-delivery can potentially provide better antitumor response.

Development of a magnetic 3D spheroid platform with potential application for high-throughput drug screening.

Three-dimensional (3D) cell culture has become increasingly adopted as a more accurate model of the complex in vivo microenvironment compared to conventional two-dimensional (2D) cell culture. Multicellular spheroids are important 3D cell culture models widely used in biological studies and drug screening. To facilitate simple spheroid manipulation, magnetic spheroids were generated from magnetically labeled cells using a scaffold-free approach. This method is applicable to a variety of cell types. The spheroids generated can be targeted and immobilized using magnetic field gradients, allowing media change or dilution to be performed with minimal disruption to the spheroids. Cells in magnetic spheroids showed good viability and displayed typical 3D morphology. Using this platform, a 28 day study was carried out using doxorubicin on magnetic MCF-7 spheroids. The results provided a proof-of-principle for using magnetic tumor spheroids in therapeutic studies. They can offer beneficial insights that help to bridge the gap between in vitro and in vivo models. Furthermore, this platform can be adapted for high-throughput screening in drug discovery.

Snapshot coherence-gated direct wavefront sensing for multi-photon microscopy.

Deep imaging in turbid media such as biological tissue is challenging due to scattering and optical aberrations. Adaptive optics has the potential to compensate the tissue aberrations. We present a wavefront sensing scheme for multi-photon scanning microscopes using the pulsed, near-infrared light reflected back from the sample utilising coherence gating and a confocal pinhole to isolate the light from a layer of interest. By interfering the back-reflected light with a tilted reference beam, we create a fringe pattern with a known spatial carrier frequency in an image of the back-aperture plane of the microscope objective. The wavefront aberrations distort this fringe pattern and thereby imprint themselves at the carrier frequency, which allows us to separate the aberrations in the Fourier domain from low spatial frequency noise. A Fourier analysis of the modulated fringes combined with a virtual Shack-Hartmann sensor for smoothing yields a modal representation of the wavefront suitable for correction. We show results with this method correcting both DM-induced and sample-induced aberrations in rat tail collagen fibres as well as a Hoechst-stained MCF-7 spheroid of cancer cells.

Quantified pH detection with hyperpolarized 13C-Bicarbonate

Event Abstract Back to Event Quantified pH detection with hyperpolarized 13C-Bicarbonate Johannes Scholz1, 2*, Martin A. Janich2, Angela M. Otto1, Franz Schilling1, Axel Haase1 and Marion I. Menzel2 1 Technische Universität München, Physics, Germany 2 GE Global Research Center, Germany Background and aim. pH plays a crucial role in several diseases, such as cancer, hypoxia, inflammation etc., and it is therefore desirable to detect pH in vivo. Ideally, the detection should be noninvasive and spatially localized. Despite the medical impact, a clinical tool for pH mapping is not yet available. Different approaches in pH detection were made in NMR e.g. with 31P and CEST.[1] A promising candidate to achieve pH mapping in vivo is 13C metabolic magnetic resonance (13CMMR). This technique enhances the signal of 13C-labeled bicarbonate using dissolution dynamic nuclear polarization (DNP) and then probes the pH by measuring the ratio of 13C bicarbonate and 113CO2 [2]. Methods. Hyperpolarization is achieved using dissolution DNP Hypersense polarizer. The MR acquisition is performed on pH phantoms and mammary carcinoma cell spheroids (MCF-7) using a 3T MR system (GE Signa HDx) and a 14T Bruker spectrometer. Relaxation time analysis was additionally performed with a 1T Bruker Minispec mq40 NMR analyzer. Results. In vitro tests using hyperpolarized sodium bicarbonate and phosphate buffers and pH-phantoms showed good correlation between the derived pH value and the reference pH (+/- 0.03) obtained with a pH electrode. An analysis of the signal dynamics revealed that the correct bicarbonate to CO2 signal ratio is established after 40 seconds without adding carbonic anhydrases. In tumor cell systems (MCF-7 spheroids), T1 times of bicarbonate and CO2 were determined for different field strengths (1T, 3T, 14T) and different pH values in the biological range (6-8). Comparison of the values achieved with and without tumor spheroids show the impact of the spheroids on the hyperpolarized signal. Acknowledgements This research was partly funded by BMBF grant 01EZ1114A.

Acoustical imaging of internal spheroid structures at a variety of frequencies

We have previously shown that ultrasound is capable of identifying apoptosis in individual cells and cell pellets due to changes in the ultrasound attenuation, speed of sound, and backscatter. Spheroids can be used to more accurately model non-vascularized tumors due to their three-dimensional growth pattern, cell-cell interaction, disorganized growth, and development of a necrotic core when grown to sufficiently large sizes. To examine cell death in spheroids due to necrosis or chemotherapy, quantitative ultrasound methods were used on the ultrasound backscatter power spectrum throughout the spheroid. MCF7 spheroids ranging in size from 100 to 1000 μm were probed at 25 and 55 MHz using a VEVO770 high frequency ultrasound machine, and at 80 and 200 MHz using an acoustic microscope. Changes in spheroid structure as the necrotic core develops, and after it is exposed to chemotherapeutic agents were recorded and analyzed. An increase in the ultrasound backscatter amplitude from necrotic cells within the core of the spheroid versus the viable cells around the core was observed. Changes in the ultrasound backscatter were also observed for spheroids treated with chemotherapeutics to induce apoptosis. This work furthers our understanding of non-invasively identifying the viability of cancerous tumors, and the efficacy of chemotherapeutic treatments.

Development of model systems for analysis of effects of cell‐cell and cell‐microenvironment interactions on pH regulatory proteins in breast cancer

When studying cancer development it is essential to employ models that recapitulate the tumor microenvironment (TME). We recently showed that truncated ErbB2 (ΔNErbB2) increases acid extrusion via the Na+/H+ exchanger NHE1 and the Na+,HCO3− cotransporter NBCn1 and increases NBCn1 expression in MCF‐7 breast cancer cells. Further, NHE1 inhibition strongly sensitized ΔNErbB2‐expressing MCF‐7 cells to chemotherapy‐induced death. Here, we explore the impact of specific aspects of the TME on pH‐regulatory and survival/motility signaling. Employing MCF‐7 cells +/− inducible ΔNErbB2 expression we show that: (i) TME simulation (pHe 6.5, 1% O2, 7.5 mM lactate) strongly increases Akt‐ and ERK1/2 phosphorylation/activity; TME mimicking conditions has no additional effect on NBCn1 expression but further increases ERK1/2 activity in ΔNErbB2+ cells (ii) in 2D coculture with primary human breast fibroblasts, MCF‐7 cells upregulate the lactate‐H+ cotransporter MCT1 and the epithelial‐mesenchymal transition (EMT) marker α‐smooth muscle actin; (iii) in MCF‐7 spheroids NHE1 expression and ERM‐ and Akt (S473) phosphorylation are greatly reduced compared to 2D monoculture, and pERM and apparently also NBCn1 localize to the spheroid periphery. In conclusion, the TME profoundly impacts on pH regulatory ion transporters and survival/motility signaling in MCF‐7 breast cancer cell models.Funding: Danish Cancer Society, Danish Research Council

In vitro characterisation of the anti-intravasative properties of the marine product heteronemin

Metastases destroy the function of infested organs and are the main reason of cancer-related mortality. Heteronemin, a natural product derived from a marine sponge, was tested in vitro regarding its properties to prevent tumour cell intravasation through the lymph-endothelial barrier. In three-dimensional (3D) cell cultures consisting of MCF-7 breast cancer cell spheroids that were placed on lymph-endothelial cell (LEC) monolayers, tumour cell spheroids induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer; 12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) and NF-κB activity are major factors inducing CCIDs, which are entry gates for tumour emboli intravasating the vasculature. This 3D co-culture is a validated model for the investigation of intravasation mechanisms and of drugs preventing CCID formation and hence lymph node metastasis. Furthermore, Western blot analyses, NF-κB reporter, EROD, SELE, 12(S)-HETE, and adhesion assays were performed to investigate the properties of heteronemin. Five micromolar heteronemin inhibited the directional movement of LECs and, therefore, the formation of CCIDs, which were induced by MCF-7 spheroids. Furthermore, heteronemin reduced the adhesion of MCF-7 cells to LECs and suppressed 12(S)-HETE-induced expression of the EMT marker paxillin, which is a regulator of directional cell migration. The activity of CYP1A1, which contributed to CCID formation, was also inhibited by heteronemin. Hence, heteronemin inhibits important mechanisms contributing to tumour intravasation in vitro and should be tested in vivo.

Anti-mitochondrial therapy in human breast cancer multi-cellular spheroids

During multi-cellular tumor spheroid growth, oxygen and nutrient gradients develop inducing specific genetic and metabolic changes in the proliferative and quiescent cellular layers. An integral analysis of proteomics, metabolomics, kinetomics and fluxomics revealed that both proliferative- (PRL) and quiescent-enriched (QS) cellular layers of mature breast tumor MCF-7 multi-cellular spheroids maintained similar glycolytic rates (3–5nmol/min/106 cells), correlating with similar GLUT1, GLUT3, PFK-1, and HKII contents, and HK and LDH activities. Enhanced glycolytic fluxes in both cell layer fractions also correlated with higher HIF-1α content, compared to MCF-7 monolayer cultures. On the contrary, the contents of the mitochondrial proteins GA-K, ND1, COXIV, PDH-E1α, 2-OGDH, SDH and F1-ATP synthase (20 times) and the oxidative phosphorylation (OxPhos) flux (2-times) were higher in PRL vs. QS. Enhanced mitochondrial metabolism in the PRL layers correlated with an increase in the oncogenes h-Ras and c-Myc, and transcription factors p32 and PGC-1α, which are involved in the OxPhos activation. On the other hand, the lower mitochondrial function in QS was associated with an increase in Beclin, LC3B, Bnip3 and LAMP protein levels, indicating active mitophagy and lysosome biosynthesis processes. Although a substantial increase in glycolysis was developed, OxPhos was the predominant ATP supplier in both QS and PRL layers. Therefore, targeted anti-mitochondrial therapy by using oligomycin (IC50=11nM), Casiopeina II-gly (IC50=40nM) or Mitoves (IC50=7nM) was effective to arrest MCF-7 spheroid growth without apparent effect on normal epithelial breast tissue at similar doses; canonical anti-neoplastic drugs such as cisplatin and tamoxifen were significantly less potent.

Bay11-7082 inhibits the disintegration of the lymphendothelial barrier triggered by MCF-7 breast cancer spheroids; the role of ICAM-1 and adhesion

Background:Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics.Methods:Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of ‘circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs.Results:Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively.Conclusion:Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.

Effect of homotypic and heterotypic interaction in 3D on the E-selectin mediated adhesive properties of breast cancer cell lines

Hematogenous metastasis involves a glycoprotein mediated adhesion cascade of tumor cells with E-selectin on the endothelial layer of the blood vessels. Cell–cell interactions play a major role in cancer metastasis and invasiveness. Intercellular communication between two cancer cells or between a cancer cell with a stromal cell in the microenvironment such as fibroblasts or inflammatory cells play an important role in metastatic invasion. Culturing tumor cells as 3D spheroids can recapitulate these physiologically relevant cell–cell interactions. The heterogeneity in primary tumors is attributed to cell subpopulations with varying degree of invasiveness. Co-culturing cancer cells with different phenotypes as 3D spheroids can mimic this heterogeneity. Here we report the effect of homotypic and heterotypic interactions in breast cancer cells cultured as 3D spheroids on polydimethylsiloxane (PDMS) on the adhesion phenotype to E-selectin. We show that breast cancer cell lines (BT20 and MCF7) propagating as 3D spheroids on PDMS exhibit a stronger interaction with human recombinant E-selectin when compared to their respective monolayer grown counterparts on tissue culture plate (TCP). Matrigel invasion assay also indicated that BT20 and MCF7 spheroids were more invasive than BT20 and MCF7 cells grown as monolayers. To mimic tumor heterogeneity in vitro, a co-culture model included tumorigenic cell lines BT20, MCF7 and a non-tumorigenic mammary epithelial cell line MCF10A. These cell lines were cultured together in equal seeding ratio on PDMS to generate co-culture spheroids. The heterotypic interactions in the co-culture model resulted in enhancement of the adhesion of the most invasive BT20 cell line to E-selectin. BT20 cells in co-culture bound to the greatest degree to soluble E-selectin compared to MCF7 and MCF10A cells in co-culture. Co-invasion assay with co-culture spheroids indicated that BT20 cells in co-culture were more invasive than MCF7 and MCF10A cells. The results presented here indicate that homotypic and heterotypic interaction of cancer cells favor adhesion to E-selectin thus representing a complexity beyond planar cell culture. Also, when cells of different phenotypes are mixed, the heterogeneity enhances the adhesive phenotype and invasiveness of the most invasive cell population. The results challenge the classic use of planar cell culture for evaluating the adhesion of cancer cells to E-selectin and establish our co-culture technique as a model that can help investigative studies in metastasis and invasiveness of breast and other types of cancers.

Simultaneous measurements of the TCA cycle and respiration in isolated mitochondria and intact cells with the XF24-3 Analyzer

similar glycolytic rates (3-5 nmol/min/10 cells), which correlated with similar protein contents (GLUT1, GLUT3, HKII, and LDH-A) and enzyme activities (HK and LDH). Enhanced glycolytic fluxes in both cell layer fractions also correlated with higher expression of the transcriptional factors HIF-1a and TIGAR compared to MCF-7 monolayer cultures. On the contrary, the contents of the mitochondrial proteins ND1, SDH, COXIV, PDH, 2-OGDH, glutaminase, and ATP synthase (3-20 times) as well as the respiratory chain enzyme activities (COX, SDH) and the oxidative phosphorylation (OxPhos) flux (2-times) were higher in PRL vs. QS. Enhanced mitochondrial metabolism in the PRL layers correlated with an increase in the oncogenes h-Ras and c-Myc, and transcription factors p32 and PGC1α involved in the OxPhos activation. On the other hand, the lower mitochondrial function in the QS layers was associated with an increase in Atg7, Beclin, LC3B, Bnip3 and LAMP protein levels indicating active mitophagy and lysosome biosynthesis. Although a substantial increase in glycolysis was developed, OxPhos was the main ATP supplier in both QS and PRL cell layers. Therefore, antimitochondrial therapy by using oligomycin or Casiopeina II-gly was effective to arrest MCF-7 spheroid growth (IC50≈20–25 nM) without apparent effect on normal epithelial breast tissue at similar doses; canonical anti-neoplastic drugs such as cisplatin and tamoxifen were significantly less potent.

Investigating cyclooxygenase-2 signaling in breast cancer-initiating cells.

e21107 Investigating cyclooxygenase-2 signaling in breast cancer-initiating cells Background: Only a small fraction of breast tumor cells, breast cancer-initiating cells (CSC), have the ability to initiate tumor growth and metastasis. Some characteristics of breast CSCs have been described in vitro by employing non-adherent CSC-enriching culture conditions, however, little is known regarding prostanoid signaling or prostaglandin E2 (PgE2) production in CSCs. In this study we measured cyclooxygenase-2 (COX-2) and prostaglandin E2 receptor expression in CSCs and assessed the effects of COX-2 and Ep4 inhibition on in vitro CSC spheroid formation and PgE2 production. Methods: CSCs were cultured in serum-free medium using ultra low attachment plates. Cells were treated with vehicle, a COX-2 inhibitor (Celecoxib), or an EP4 inhibitor (GW627368X) at concentrations of 0.1, 1, 5, 10, 50, or 100uM for 10 days. Spheroids were quantified using a Gelcount machine. Non-linear regression was used to calculate IC50 values. Western blots were performed using anti-COX-2, anti- EP1, 2, 3 and 4 receptor antibodies. Prostaglandin E2 production was measured using a PgE2 immunoassay kit. Results: COX-2 protein expression was observed for MDA-MB-231 and SUM149 CSCs. EP2 and EP4 protein was detected for MCF-7, MDA-MB-231, and SUM149 CSCs. The Celecoxib IC50 values for MCF-7 and SUM149 CSC spheroid formation were 0.5 and 2.2 uM, respectively; GW627386X IC50 values for MCF-7 and SUM149 CSC spheroid formation were 1.2 and 0.3uM, respectively. No significant differences in PGE2 production were observed for MCF-7 CSCs compared to adherent MCF-7 cells (11.5 ± 4.4 vs. 8.7 ± 2.1 pg/mL/106 cells; p=0.88). However, PgE2 production in MDA-MB-231 CSCs was significantly higher than adherent MDA-MB-231 cells (1507.0 ± 329.0 vs. 13.8 ± 5.7 pg/mL/106 cells; p≤0.001) and SUM149 CSC PgE2 production was significantly higher than SUM149 adherent cells (4932.9 ± 501.7 vs. 194.5 ± 31.0 pg/mL/106 cells; p≤0.001). 10uM Celecoxib treatment inhibited PGE2 production in MDA-MB-231 and MDA-MB-231 CSCs. Conclusions: MDA-MB-231 and SUM149 CSCs express COX-2, EP2 and Ep4 and produce high levels of PGE2. CSC spheroid formation is significantly decreased with COX-2 and EP4 inhibition.

P1-03-08: Adipose Tissue in Breast Cancer: Not an Idle Bystander but an Active Participant in Breast Cancer Progression.

Abstract Background: Adipose tissue is a dynamic organ that secretes a plethora of molecules called adipokines. In breast cancer we find a unique situation were genetically changed cells (the cancer cells) are in close contact with adipocytes. Moreover, obesity is a known negative prognostic marker for postmenopausal breast cancer patients. We hypothesize that adipocyte-derived factors influence breast cancer progression. Materials and methods: Adipose tissue was collected from breast cancer patients undergoing a mastectomy. After macroscopic removal of blood vessels and connective tissue, the adipose tissue was carefully cut into 2–3mm3 pieces and were incubated in specific adipose-tissue culture medium. After 24h, the medium was collected and the quality was checked by determining the concentration of total proteins, leptin, adiponectin, TNFalpha and triglycerides. This conditioned medium of adipose tissue (CM AT) was used for in vitro experimentation with MCF-7 breast cancer cells. Results: Effect of AT on morphology and aggregation: when MCF-7 cells are grown in a culture flask, they tend to form round compact islands. Under influence of CM AT, the islands form sharp edges, the cells in an island can be counted individually and they show scattering. Importantly, despite the major changes in cellular morphology, CM AT removal rescued the compact island formation of MCF-7 cells. In the slow aggregation assay, cells treated with CM AT (and a subtherapeutic concentration of a neutralizing E-cadherin antibody) lost the ability to form compact aggregates. Furthermore, MCF-7 spheroids placed inside adipose tissue showed massive reorganization into an irregularly shaped mass. Effect of AT on proliferation: starting from an equal number of cells and counting them every 2 days, it became clear that MCF-7 cells with CM AT had a higher rate of proliferation than MCF-7 cells in control medium. This stimulation of proliferation was confirmed by cell cycle analysis which revealed a doubling of cells in the G2/M phase, and western blot which showed an upregulation of cyclin A and cyclin E, both positive regulators of the cell cycle. Effect of AT on invasion: a 24h collagen type I invasion assay revealed invasive characteristics of MCF-7 cells treated with CM AT while MCF-7 cells in control conditions are round and non-invasive. In contrast, a transwell collagen test over 14 days was not able to show MCF-7 cells invading the collagen gel under influence of CM AT. However, the growth pattern of MCF-7 cells on the collagen gel was clearly disorganised when compared with the control situation. Conclusion: These findings suggest that adipose tissue-derived factors exert a dramatic selective force on patterning, invasion and growth of MCF-7 breast cancer cells. Unraveling the mechanism behind these observations may provide vital information regarding the link between obesity and poor prognosis in postmenopausal breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-08.

NF-κB mediates the 12(S)-HETE-induced endothelial to mesenchymal transition of lymphendothelial cells during the intravasation of breast carcinoma cells.

Background:The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts.Methods:To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs.Results:We show that tumour spheroids generate ‘circular chemorepellent-induced defects’ (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with ∼50% attenuation of CCID formation by treatment of cells with 10 μM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general.Interpretation:These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell–cell contacts, migration, and the generation of CCID.

The role of non-muscle myosin IIA in aggregation and invasion of human MCF-7 breast cancer cells

Human MCF-7/6 breast cancer cells differ from their MCF-7/AZ counterparts by their invasiveness in a number of assays in vitro, such as invasion of MCF-7 spheroids into embryonic chick heart fragments or type I collagen gels. Comparative proteomic analysis of these two variants revealed an identical pattern, except for a 230 kDa protein present in the invasive MCF-7/6 variant, but hardly detectable in the non-invasive MCF-7/AZ one. This protein appeared to be the non-muscle myosin IIA heavy chain (NMIIA), also coined MYH9. Experimental inhibition of NMIIA by reducing either its expression (via stable shRNA transduction) or its function (via the specific ATPase inhibitor blebbistatin) underpinned the decisive role of NMIIA in MCF-7 cell invasion. Inhibition of NMIIA indeed blocked the invasion of MCF-7/6 cells in three-dimensional invasion substrata such as embryonic chick heart fragments and type I collagen gels. Invasiveness of MCF-7/6 cells has been related to poor formation and compaction of aggregates, due to a functionally defective E-cadherin/catenin complex. Both genetic and pharmacological inhibition of NMIIA stimulated MCF-7/6 cell aggregation. Together, these data indicate that NMIIA is a decisive protein for MCF-7 cells to invade, indicating that this molecule is a candidate for targeted anti-invasive treatment.