Cultured MCF-7 Cells Research Articles

Antiproliferative Activity and Cellular Uptake of Evodiamine and Rutaecarpine Based on 3D Tumor Models.

Evodiamine (EVO) and rutaecarpine (RUT) are promising anti-tumor drug candidates. The evaluation of the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids of cancer cells would better recapitulate the native situation and thus better reflect an in vivo response to the treatment. Herein, we employed the 3D culture of MCF-7 and SMMC-7721 cells based on hanging drop method and evaluated the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids, and compared the results with those obtained from 2D monolayers. The drugs’ IC50 values were significantly increased from the range of 6.4–44.1 μM in 2D monolayers to 21.8–138.0 μM in 3D multicellular spheroids, which may be due to enhanced mass barrier and reduced drug penetration in 3D models. The fluorescence of EVO and RUT was measured via fluorescence spectroscopy and the cellular uptake of both drugs was characterized in 2D tumor models. The results showed that the cellular uptake concentrations of RUT increased with increasing drug concentrations. However, the EVO concentrations uptaken by the cells showed only a small change with increasing drug concentrations, which may be due to the different solubility of EVO and Rut in solvents. Overall, this study provided a new vision of the anti-tumor activity of EVO and RUT via 3D multicellular spheroids and cellular uptake through the fluorescence of compounds.

Abstract 44: Hexyl-benzyl-biguanide (HBB) potently and selectively inhibits CYP3A4 epoxygenase activity and inhibits EET stabilization of mitochondrial respiration in ER+HER2- breast cancer cells, inducing glycolysis and pyruvate biosynthesis

Abstract Cytochrome P450 3A4 (CYP3A4) promotes ER+HER2- breast cancer cell proliferation and survival, in part, by biosynthesis of epoxyeicosatrienoic acids (EETs). EETs are known to regulate mitochondrial function in non-transformed cells, but the roles of CYP3A4 and EETs in regulation of breast cancer bioenergetics are unknown. Hexyl-benzyl-biguanide (HBB) is useful probe of CYP3A4 epoxygenase activity and selectively inhibits EET biosynthesis (IC50 = 9 uM vs. IC50 = 50 uM for CYP2C8). HBB caused depolarization of mitochondria in MCF-7 cells, while (±)-14,15-EET provided partial protection. The soluble epoxide hydrolase (sEH) inhibitor t-AUCB ameliorated inhibition of oxygen consumption rates (OCR) by HBB (20 uM), while there was no effect on extracellular acidification rate (ECAR), indicating that the primary effect of HBB is on OCR. At 30 minutes, HBB added to MCF-7 cells transiently suppressed phosphorylation of pyruvate kinase muscle isozyme 2 (PKM2) on Tyr-105, which has been reported to favor enzymatically inactive dimer over active tetramer. Suppression of phosphorylated PKM2 correlated with subsequent PKM2 tetramer formation and increase of intracellular pyruvate and extracellular lactate at 1 hour. The (±)-14,15-EET regioisomer reduced the pro-glycolytic PKM2 tetramer at 1 hour, suggesting that HBB may promote PKM2 tetramer, in part, through reduction of EET. Prolonged exposure to HBB (20 uM) in cultured cells activated phosphorylation of PKM2 on Tyr-105, but there was increased cellular necrosis correlating with reduced mitochondrial respiration and reduction of ATP stores, indicating that loss of respiration was the dominant effect. HBB inhibited the ER+HER2- MCF-7 xenograft, similar to CYP3A4 silencing. HBB promoted phosphorylation of intratumoral PKM2 on Tyr-105, consistent with long-term exposure to HBB in cultured MCF-7 cells. Notably, MCF-7 tumor response to HBB did not correlate with phosphorylation of AMPK-alpha on Thr-172, a marker of AMPK activation. Metformin (5 mM) exhibited no effect on PKM2 or its phosphorylation in cultured MCF-7 cells. Together, these results indicate that part of the inhibitory effect of HBB on ER+HER2- breast cancer is mediated through inhibition of respiration. Significance: These results establish HBB as a useful chemical probe of respiration, with indirect effects on PKM2 regulation. HBB may also be useful as a potential therapeutic candidate for ER+HER2- breast cancer. Citation Format: Zhijun Guo, Irina Sevrioukova, Eric Hanse, Xia Zhang, Ilia Denisov, Ting-Lan Chiu, Rebecca Cuellar, Christian Torres, Julia Wulfkuhle, Emanuel Petricoin, Qing Cao, Haitao Chu, Beverly Norris, Robert Schumacher, Ameeta Kelekar, Ian Blair, Jorge Capdevila, John Falck, Thomas Poulos, Steven Sligar, Gunda Georg, Elizabeth Amin, David A. Potter. Hexyl-benzyl-biguanide (HBB) potently and selectively inhibits CYP3A4 epoxygenase activity and inhibits EET stabilization of mitochondrial respiration in ER+HER2- breast cancer cells, inducing glycolysis and pyruvate biosynthesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 44.

Abstract 980: The long non-coding RNA CAI2 is a tumor suppressor gene

Abstract We have recently reported on the discovery of a long non-coding RNA imbedded in intron 2 of the p16/ARF (CDKN2A) locus at 9p21. CAI2 is a non-conserved, RNA pol II regulated gene with whose expression is highly correlated with parent genes p16 and ARF. However, CAI2 is expressed in cell lines deleted for p16, ARF and even p15, and is expressed cell lines epigenetically silenced for p16 and/or ARF. Furthermore, treatment of p16 and/or ARF epigenetically silenced cell lines with 5-aza-2’-deoxycytidin allows p16 and/or ARF re-expression while having no influence on CAI2, confirming independent regulation. Speculating, therefore, that the regulatory region of CAI2 lies imbedded in the non-transcribed portion of intron 2, we created expression constructs of the immediate 5’ region of CAI2. However, consistent with a bioinformatical analysis, no regulatory activity was detected. This suggests that this non-coding RNA is non-traditionally controlled by remote but yet to be identified regulatory element(s) and/or mechanisms. By colony formation assay, CAI2 overexpression can inhibit cell growth in the p16 intact HEK293T and SK-BR-3 breast cancer cell lines. RNA Immunoprecipitation has revealed that CAI2 interacts with H3K27me3, a recruiter of Polycomb Repressive Complex 1 (PRC1) that modulates chromatin structure and gene expression, and BMI1, a PRC1 complex protein that is a negative regulator of p16. Consistent with this effect, CAI2 can induce p16 expression in HEK293T cells, and this was hypothesized to be the mechanism by which CAI2 inhibits cell growth. However, we now show that CAI2 can also inhibit colony formation in the p16/ARF deleted MCF7 and MDA-MB-231 breast cancer cell lines. Interestingly however, CAI2 overexpression had little to no effect on 2D cultures of HEK293T or MCF7 cells including DNA synthesis, XTT metabolic activity or cell count. As CAI2 is imbedded in the p16/ARF gene, standard siRNAs may inadvertently influence p16 and/or ARF expression due to actions on their pre-mRNAs. Therefore, we created a series of partial CAI2 knockouts using CRISPR technology and compared their growth by colony formation assay. Notably, clones containing a partial knockout of the 5’ region of CAI2 proliferated faster than mock transfected clones, consistent with the growth suppressive properties observed with CAI2 overexpression. In 2D cultures, a small increase in DNA synthesis was also observed in the partial deletions. The HEK293T clones containing partial deletions of CAI2 also exhibited small increases in expression of the remaining intact portions of the gene with a concomitant decrease in p16 expression, all consistent with an influence of CAI2 on p16 expression and cell growth; no influence on ARF expression was observed. Taken together, the data demonstrate that CAI2 is capable of influencing p16 expression, but this influence is insufficient to fully account for the ability of CAI2 to regulate growth, and we conclude that CAI2 is a bona fide tumor suppressor gene in its own right. Citation Format: Olga Cohen, Ruey-Jen Lin, Alice L. Yu, Mitchell B. Diccianni. The long non-coding RNA CAI2 is a tumor suppressor gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 980.

Abstract 4105: Dissecting the mechanisms of hepatocyte replacement in breast cancer liver metastases

Abstract The liver is a common site of metastasis in metastatic breast cancer and is associated with significant mortality. Histopathological examination of human breast cancer liver metastases (BCLMs) reveals that the majority of these tumours present with a ‘replacement growth pattern.’ In this growth pattern, the breast cancer cells freely invade the liver parenchyma and replace the resident hepatocytes. In advanced BCLMs, breast cancer cells can eventually replace a significant volume of the liver parenchyma which, ultimately, leads to organ failure and death. However, the mechanism through which breast cancer cells replace the resident hepatocytes is unknown. Here we set out to establish how this replacement of hepatocytes by breast cancer cells occurs in BCLMs. To do so, we established two in vivo models of advanced BCLM: (a) an ER+PR+HER2- model using the MCF7 cell line, and (b) a triple negative patient-derived xenograft model using breast cancer cells isolated from a pleural effusion. We have also established an in vitro co-culture system, where MCF7 cells are co-cultured with HepG2 cells, which is designed to mimic the breast cancer cell-hepatocyte interaction observed in vivo. Both in vivo models exhibit a histology that closely mimics the replacement growth pattern of human BCLMs, where breast cancer cells replace hepatocytes at the tumour-liver interface. To address whether hepatocytes are killed by adjacent breast cancer cells in vivo, we evaluated hepatocyte death by apoptosis using appropriate markers (cleaved-PARP and cleaved-caspase-3). Surprisingly, although extensive apoptotic death of breast cancer cells in the tumour mass could be observed, no evidence for apoptotic death of hepatocytes could be found. However, we observed extensive cell-in-cell invasion at the tumour-liver interface, where live breast cancer cells enter the cytoplasm of adjacent hepatocytes. We also observe a similar process of cell-in-cell invasion within in vitro cultures of MCF-7 cells with HepG2 cells. Although cell-in-cell invasion has been described as a mechanism of non-apoptotic cell death in breast cancer cells, the role of cell-in-cell invasion between breast cancer cells and hepatocytes in BCLMs has not been investigated. Our current studies are focused in two areas: (a) we are using intra-vital microscopy and time-lapse microscopy to investigate whether cell-in-cell invasion between breast cancer cells and hepatocytes mediates non-apoptotic death of hepatocytes both in vivo and in vitro, and (b) we are investigating the molecular mechanisms that mediate cell-in-cell invasion between breast cancer cells and hepatocytes. In conclusion, we present preliminary evidence for a novel mechanism via which breast cancer cells could replace hepatocytes in BCLMs. Further studies aimed at elucidating the molecular basis of this mechanism may reveal novel targets for preventing the replacement of the liver parenchyma by metastatic breast cancer cells. Citation Format: Mark R. Nathan, Lefteris Kostaras, Victoria Bridgeman, Shane Foo, Peter Vermeulen, Andrew Reynolds. Dissecting the mechanisms of hepatocyte replacement in breast cancer liver metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4105.

Tunicamycin-induced endoplasmic reticulum stress reduces in vitro subpopulation and invasion of CD44+/CD24- phenotype breast cancer stem cells

Tunicamycin is an inhibitor of glycosylation that disturbs protein folding machinery in eukaryotic cells. Tunicamycin causes accumulation of unfolded proteins in cell endoplasmic reticulum (ER) and induces ER stress. ER stress is an essential mechanism for cellular homeostasis has role in cell death via reprogramming of protein processing, regulation of autophagy and apoptosis. In this study we show effect of tunicamycin on subpopulation and invasion of CD44+/CD24- MCF7 breast cancer stem cells. CD44+/CD24- cells were isolated from MCF7 cell line by fluorescence activated cell sorting (FACS) and treated with tunicamycin. ER stress was monitored by evaluation of X-box binding protein 1(XBP-1) mRNA splicing, cleaved activating transcription factor 6 (ATF6) nuclear translocation and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. CD44+/CD24- subpopulation was analyzed using flow cytometry. Invasion was investigated by scratch assay, trypan blue staining, 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation and in vitro migration assays. Increased level of spliced XBP-1, ATF6 nuclear translocation and CHOP protein expression were detected in CD44+/CD24- and original MCF7 cells treated with tunicamycin. Also, a significant decline in CD44+/CD24- cell subpopulation was determined in the cells treated with tunicamycin. The results also showed inhibited invasion, increased cell death, suppressed proliferation and reduced migration in the CD44+/CD24- and CD44+/CD24- rich MCF7 cell culture, under effect of tunicamycin. Our results indicate that CD44+/CD24- phenotype MCF7 cells are susceptible to tunicamycin. The results showed that tunicamycin-induced ER stress suppresses CD44+/CD24- phenotype cell subpopulation and in vitro invasion and accelerates tumorosphore formation. These results suggest that tunicamycin-induced ER stress inhibits CD44+/CD24- phenotype MCF7 breast cancer stem cells. We conclude that using ER-targeting chemicals like tunicamycin is an interesting approach to target breast cancer stem cells inside tumor.

The estrogenic properties of Danggui Buxue Tang, a Chinese herbal decoction, are triggered predominantly by calycosin in MCF-7 cells

BackgroundDanggui Buxue Tang (DBT), a Chinese herbal decoction containing Astragali Radix (AR; roots of Astragalus memebranaceus (Fisch.) Bunge var. mongholicus (Bunge) Hsiao) and Angelicae Sinensis Radix (ASR; roots of Angelica sinensis Oliv.) at a weight ratio of 5:1, is used to improve menopausal syndromes in women. Several lines of evidence indicate that DBT has strong estrogenic property; however, the action mechanism of this herbal decoction is not known. Calycosin, a major flavonoid in AR, shares similar structure with β-estradiol, and thus which is hypothesized to be the key compound of DBT in responsible for such estrogenic properties. AimsWe aimed to determine the role of calycosin in DBT in terms of its estrogenic functions by the creation of calycosin-depleted DBT (DBTΔcal) and calycosin-added DBT (DBT+cal) herbal extracts. MethodsThe signalings triggered by DBT∆cal, DBT+cal, and parental DBT were compared in cultured MCF-7 cells by determining: (i) the activation of estrogen responsive element; (ii) the phosphorylation of estrogen receptor α (ERα); and (iii) the phosphorylation of Erk1/2. The DBT-induced responses were in dose- and/or time-dependent manners. ResultsThe estrogenic signals triggered by DBT were markedly reduced in DBTΔcal, and in contrast the addition of calycosin in DBT, i.e. DBT+cal, enhanced the responses by 2–5 folds; however, calycosin alone did not show such properties. In parallel, the DBT-induced responses could be significantly blocked by inhibitors for estrogen receptor and mitogen activated protein kinases. ConclusionThus, we hypothesize that calycosin is an indispensable chemical in DBT, and which plays a linker in orchestrating multi-components of DBT as to achieve the maximal estrogenic functions. These discoveries should be invaluable in drug development and in investigating the modernization of traditional Chinese medicine from a new perspective.

Real-time imaging of non-adherent cells maintained in contact with adherent cell layers

Abstract Cell-cell interactions are crucial to the survival and function of cells. Imaging these interactions in real-time is a challenging task with non-adherent cells due to their mobility in suspension, not to mention their communication with other heterotypic cells. Here we present a novel method to trap non-adherent cells in precise locations with a layer of adherent cells in order to study and image their interactions while in contact over time. This platform uses a well plate format to integrate 8 individual microfluidic units. Each unit has a 3 mm × 3 mm × 40 μm (L × W × H) chamber for cell culture, and an array of one hundred 72 × 72 × 15 μm (L × W × H) trap areas within each chamber to immobilize non-adherent cells underneath. The chamber in each unit can be addressed with programmable and on-demand perturbation of up to 2 different reagents to perform live cell imaging assays without disrupting the cell cultures. We have successfully cultured MCF-7 cells using this design, with Jurkat cells subsequently loaded and trapped above the layer of MCF-7. Upon reagent introduction, time-lapse images confirmed that the Jurkat cells remained in place. In conclusion, the microfluidic design allows investigation of cell-cell interactions in a wide range of applications requiring the confinement of non-adherent cells in contact with a different adherent cell layer. The on-demand perturbation feature further increases the capability to control the microenvironment exposed to the cell culture.

A Multi-Functional Tumor Theranostic Nanoplatform for MRI Guided Photothermal-Chemotherapy.

To develop a multi-functional theranostic nanoplatform with increased tumor retention, improving antitumor efficacy and decreased side effects of chemotherapy drugs. GO@Gd nanocomposites was synthesized via decorating gadolinium (Gd) nanoparticles (GdNP) onto graphene oxide (GO), and then functionalized by polyethylene glycol (PEG2000), folic acid (FA), a widely used tumor targeting molecule, was linked to GO@Gd-PEG, finally, doxorubicin (DOX) was loaded onto GO@Gd-PEG-FA and obtained a tumor-targeting drug delivery system (GO@Gd-PEG-FA/DOX). GO@Gd-PEG-FA/DOX was characterized and explored its theranostic applications both in a cultured MCF-7 cells and tumor-bearing mice. GO@Gd-PEG-FA/DOX could efficiently cross the cell membranes, lead to more apoptosis and afford higher antitumor efficacy without obvious toxic effects to normal organs owing to its prolonged blood circulation and 7.6-fold higher DOX uptake of tumor than DOX. Besides, GO@Gd-PEG-FA/DOX also served as a powerful photothermal therapy (PTT) agent for thermal ablation of tumor and a strong T1-weighted contrast agent for tumor MRI diagnosis. The multi-functional nanoplatform also could selectively kill cancer cells in highly localized regions via the excellent tumor-targeting and MRI guided PTT abilities. GO@Gd-PEG-FA/DOX exhibited excellent photothermal-chemotherapeutic efficacy, tumor-targeting property and tumor diagnostic ability.

Protein Profiles Associated with Anoikis Resistance of Metastatic MDA-MB-231 Breast Cancer Cells.

Resistance to anoikis, a cell-detachment induced apoptosis, is one of the malignant phenotypes which support tumor metastasis. Molecular mechanisms underlying the establishment of this phenotype require further investigation. This study aimed at exploring protein expression profiles associated with anoikis resistance of a metastatic breast cancer cell. Cell survival of suspension cultures of non-metastatic MCF-7 and metastatic MDA-MB-231 cells were compared with their adherent cultures. Trypan blue exclusion assays demonstrated a significantly higher percentage of viable cells in MDA-MB-231 than MCF-7 cell cultures, consistent with analysis of annexin V-7-AAD stained cells indicating that MDA-MB-231 possess anti-apoptotic ability 1.7 fold higher than MCF-7 cells. GeLC-MS/MS analysis of protein lysates of MDA-MB-231 and MCF-7 cells grown under both culture conditions identified 925 proteins which are differentially expressed, 54 of which were expressed only in suspended and adherent MDA-MB-231 but not in MCF-7 cells. These proteins have been implicated in various cellular processes, including DNA replication and repair, transcription, translation, protein modification, cytoskeleton, transport and cell signaling. Analysis based on the STITCH database predicted the interaction of phospholipases, PLC and PLD, and 14-3-3 beta/alpha, YWHAB, with the intrinsic and extrinsic apoptotic signaling network, suggesting putative roles in controlling anti-anoikis ability. MDA-MB-231 cells grown in the presence of inhibitors of phospholipase C, U73122, and phospholipase D, FIPI, demonstrated reduced ability to survive in suspension culture, indicating functional roles of PLC and PLD in the process of anti-anoikis. Our study identified intracellular mediators potentially associated with establishment of anoikis resistance of metastatic cells. These proteins require further clarification as prognostic and therapeutic targets for advanced breast cancer.

ELF5 and DOK7 regulation in anti-estrogen treated cells and tumors.

Background Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops. This study characterizes the expression of hormone receptors, and the mRNA and DNA methylation levels of docking protein 7 (DOK7), and E74-like factor 5 (ELF5), in 21 novel tamoxifen-resistant cell lines and extends the findings to primary and recurrent human breast tumors. MethodsTwenty-one tamoxifen-selected cell lines were developed through cloning by limiting dilution of an MCF-7 cell culture treated with 1 μM tamoxifen for 6 months. The parent (MCF-7) and tamoxifen-selected cell lines were characterized for protein expression of ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) using immunohistochemistry (IHC). The mRNA levels of ER, DOK7, and ELF5 were assessed using quantitative RT-PCR. Promoter methylation levels of DOK7 and ELF5 were determined by pyrosequencing of bisulfite-modified DNA. The relationship between hormone receptor status and promoter methylation of DOK7 and ELF5 was further examined using available methylation array data (Illumina HM450) from a set of paired primary and second breast tumors from 24 women.ResultsAll 21 of the novel tamoxifen-selected cell lines are ER-positive, and HER2-negative, and 18 of the cell lines are PR-negative while the MCF-7 cells were scored as ER-positive, modestly PR-positive and HER2 negative. Expression of DOK7 and ELF5 is significantly up-regulated in half of the tamoxifen-selected cell lines as compared to the parental MCF-7. In contrast, the previously established ER-negative TMX2-28 cell line has decreased expression of both DOK7 and ELF5 and increased DNA methylation in the transcriptional start site region of these genes. ELF5 methylation was lower in second versus primary tumors in women who received anti-estrogen treatment, in PR-negative versus PR-positive tumors, and in the subset of PR-positive first tumors from the group of women who had second PR-negative tumors as compared to those who had second PR-positive tumors.ConclusionsThe distinct ELF5 methylation of PR-positive primary tumors from women who had a PR-negative recurrence indicates the possibility of stratification of women for tailored treatment in the early stages of disease.

Abstract P1-16-08: Inhibition of β-catenin pathway to overcome endocrine resistance in breast cancer

Abstract Background: It is well known that Wnt/β-catenin signaling pathway is involved in hormone receptor negative breast cancer, especially triple-negative breast cancer. In the case of hormone resistant breast cancer cells, they rarely showed hormone receptor expression. So we aimed to investigate whether the β-catenin pathway becomes activated in endocrine-resistant breast cancer cells and inhibition of β-catenin pathway can overcome endocrine resistance. Methods: We have established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with gradually increasing 4-hydroxytamoxifen concentration till 3μM. The levels of protein expression and mRNA transcripts were determined using western blot analysis and real-time quantitative PCR. The transcriptional activity of β-catenin was measured using luciferase activity assay. We used ICG-001 as inhibitor of β-catenin transcription activity. Results: The expression of estrogen receptor was significantly decreased in TamR cells. On the other hand, the expressions of HER2 and EGFR were increased in TamR cells than in control cells. The active (uncomplexed form) β-catenin level was increased in TamR cells, and also showed a significantly increased β-catenin transcriptional activity. The ICG-001, small-molecular inhibitor of Wnt/β-catenin pathway, treatment significantly reduced β-catenin transcriptional activity in TamR cells. The ICG-001 reduced cell viability fo TamR cells which showed resistance to tamoxifen by 65.3%, and also inhibited target gene cyclin D1 expression. The combination of ICG-001 and mTOR inhibitor, rapamycin reduced cell viability of TamR cells by 81.7% and there was an additive effect of two drugs as a combination index of 1.022. Conclusions: The β-catenin pathway is activated in endocrine-resistant breast cancer cells and inhibition of that pathway would be a new therapeutic strategy which overcomes endocrine resistance in breast cancer. Citation Format: Won HS, Lee KE, Lee KM, Nam EM, Mun Y-C, Seong C-M, Lee SN. Inhibition of β-catenin pathway to overcome endocrine resistance in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-16-08.

A double-network poly(Nɛ-acryloyl l-lysine)/hyaluronic acid hydrogel as a mimic of the breast tumor microenvironment

To mimic the structure of breast tumor microenvironment, novel double-network poly(Nɛ-acryloyl l-lysine)/hyaluronic acid (pLysAAm/HA) hydrogels were fabricated by a two-step photo-polymerization process for in vitro three-dimensional (3D) cell culture. The morphology, mechanical properties, swelling and degradation behaviors of pLysAAm/HA hydrogels were investigated. The growth behavior and function of MCF-7 cells cultured on the hydrogels and standard 2D culture plates were compared. The results showed that pLysAAm/HA hydrogels had a highly porous microstructure with a double network and that their mechanical properties, swelling ratio and degradation rate depended on the degree of methacrylation of HA. The results of in vitro studies revealed that the pLysAAm/HA hydrogels could support MCF-7 cell adhesion, promote cell proliferation, and induce the diversification of cell morphologies and overexpression of VEGF, IL-8 and bFGF. The MCF-7 cells cultured on 3D hydrogels showed significantly increased migration and invasion abilities as compared to 2D-cultured cells. Preliminary in vivo results confirmed that the 3D culture of MCF-7 cells resulted in greater tumorigenesis than their 2D culture. These results indicate that the pLysAAm/HA hydrogels can provide a 3D microenvironment for MCF-7 cells that is more representative of the in vivo breast cancer. Statement of SignificanceTraditional 2D cell cultures cannot ideally represent their in vivo physiological conditions. In this work, we reported a method for preparing double-network poly(Nɛ-acryloyl l-lysine)/hyaluronic acid hydrogel, and demonstrated its suitability for use in mimicing breast tumor microenvironment. Results showed the prepared hydrogels had controllable mechanical properties, swelling ratio and degradation rate. The MCF-7 cells cultured in hydrogels expressed much higher levels of pro-angiogenic growth factors and displayed significantly enhanced migration and invasion abilities. The tumorigenic capability of MCF-7 cells pre-cultured in 3D hydrogels was enhanced significantly. Therefore, the novel hydrogel may provide a more physiologically relevant 3D in vitro model for breast cancer research. To our knowledge, this is the first report assessing a HA-based double-network hydrogel used as a tumor model.

Detection of apoptosis in cancer cell lines using Surface Plasmon Resonance imaging

Induction of apoptosis in cancer cells by therapeutic agents is an important event to detect the potential effectiveness of therapies. Here we explore the potential of Surface Plasmon Resonance imaging (SPRi) to assess apoptosis in cancer cells exposed to therapeutic agents by measuring the cytochrome C release of apoptotic cells. Spots on the SPR sensor were coated with anti-cytochrome C, anti-EpCAM, anti-CD49e monoclonal antibodies and combinations thereof. Cells from the breast cancer cell line MCF7 were introduced into a flow cell, captured on a sensor surface and exposed to culture medium with and without paclitaxel. The cells were followed for 72h. Clear SPRi responses were observed on the anti-EpCAM coated spots, indicating binding of the MCF7 cells with strong time and drug presence dependent increases in SPRi responses on the spots coated with both anti-EpCAM as well as anti-cytochrome C. This suggests a release of cytochrome C by the MCF7 cells in these specific locations. In addition offline experiments were performed where cultured MCF7 cells were exposed to complete culture medium with paclitaxel, Trastuzumab antibody and Trastuzumab T-DM1 (an antibody drug conjugate). The supernatant of these cells was analyzed and also their drug concentration dependent cytochrome C presence was detected. These preliminary results suggest SPRi to be a unique tool to measure real time response of cancer cells exposed to drugs or drug combinations.

Electrochemical Imaging for Single-cell Analysis of Cell Adhesion Using a Collagen-coated Large-scale Integration (LSI)-based Amperometric Device

We here report the electrochemical imaging of cell adhesion using a large-scale integration (LSI)-based amperometric device, called a Bio-LSI device. The device consists of 400 sensor electrodes arranged with a pitch of 250 µm. The device surface was modified with collagen to assist in the culture of MCF-7 cells and promote their adhesion. The cells disturb the electrochemical reaction of redox mediators, allowing the electrochemical signal to be used to evaluate cell adhesion at the single-cell level. This approach was applied to a cell detachment test. The results show that the Bio-LSI device is a promising tool for single-cell analysis.

A photo-polymerized poly(Nε-acryloyl l-lysine) hydrogel for 3D culture of MCF-7 breast cancer cells.

The most common in vitro cell culture platform, standard two-dimensional (2D) monolayer cell culture, often fails to mimic the tumor microenvironment, while animal models complicate research on the effect of individual factors on cell behaviors. Both are unsatisfactory in the research of molecular mechanisms of tumor development and progression and the discovery and development of anticancer drugs. In vitro three-dimensional (3D) cell culture can partially simulate in vivo conditions and 3D-cultured cancer cells can recapture many essential features of native tumor tissues. In this study, to mimic the in vivo breast tumor microenvironment, novel reduction-responsive poly(Nε-acryloyl l-lysine) (pLysAAm) hydrogels were synthesized by rapid photo-polymerization of Nε-acryloyl l-lysine and using N,N'-bis(acryloyl)-(l)-cystine as a crosslinker, and their physicochemical properties were characterized systemically. The results showed that the pLysAAm hydrogels were formed within 93 s under UV irradiation and exhibited almost total elastic recovery from compressions as high as 75%. The lyophilized hydrogel samples displayed a highly porous structure with interconnected pores, had an equilibrium swelling ratio of about 20, and were degraded faster in a glutathione-containing solution than in PBS solution. The biological versatility of the pLysAAm hydrogels was demonstrated by both in vitro MCF-7 cell culture and in vivo tumor formation. Compared to cells cultured as 2D monolayers, the 3D-cultured cells presented 3D cell morphology, exhibited better cell viability, expressed higher levels of pro-angiogenic factors, and showed significantly greater migration and invasion abilities. The results from assay of tumorigenicity in nude mice and histologic analysis demonstrated the enhanced tumorigenic and angiogenic capabilities of the MCF-7 cells pre-cultured in pLysAAm hydrogels. These findings suggest that pLysAAm hydrogels may be used to bridge the gap between standard in vitro cell cultures and living tissues, aid breast cancer research, and help researchers to develop novel anticancer therapeutics.

Human IGF1 pro-forms induce breast cancer cell proliferation via the IGF1 receptor.

IGF1 is a key regulator of tissue growth and development and has been implicated in the initiation and progression of various cancers, including breast cancer. Through IGF1 mRNA splicing different precursor pro-peptides, i.e., the IGF1Ea, IGF1Eb and IGF1Ec pro-forms, are formed whose biological roles in the pathogenesis of breast cancer have not been established yet. The objective of this study was to assess the biological activity of the IGF1 pro-forms in human breast cancer-derived cells. The different IGF1 pro-forms were generated through transient transfection of HEK293 cells with the respective vector constructs. The resulting conditioned media were applied in vitro to MCF7, T47D and ZR751 breast cancer-derived cell cultures. The recombinant human IGF1 pro-forms were also tested for their binding affinity to an anti-IGF1 specific antibody by immunoprecipitation. To determine whether the IGF1 pro-forms induce cell proliferation, mature IGF1 was neutralised in HEK293-derived conditioned media. We found that the IGF1 pro-forms were the only forms that were produced intra-cellularly, whereas both mature IGF1 and the IGF1 pro-forms were detected extra-cellularly. We also found that E peptides can impair the IGF1 pro-form binding affinity for the anti-IGF1 antibody and, thus, hamper an accurate measurement of the IGF1 pro-forms. Additionally, we found that the IGF1 antibody can completely inhibit IGF1-induced breast cancer cell proliferation and IGF1 receptor (IGF1R) phosphorylation, wheras the same antibody was found to only partially inhibit the biological activity of the pro-forms. Moreover, we found that the IGF1 pro-form activities can completely be inhibited by neutralising the IGF1R. Finally, we compared the bioactivity of the IGF1 pro-forms to that of mature IGF1, and found that the IGF1 pro-forms were less capable of phosphorylating the IGF1R in the breast cancer-derived cells tested. Our data indicate that IGF1 pro-forms can induce breast cancer cell proliferation via the IGF1R, independent from the mature IGF1 form. These results underline the importance of an accurate assessment of the presence of IGF1 pro-forms within the breast cancer microenvironment.

Abstract B49: Inhibition of β-catenin pathway to overcome endocrine resistance in tamoxifen-resistant breast cancer cell line

Abstract Background: It is well known that Wnt/β-catenin signaling pathway is involved in hormone receptor negative breast cancer, especially triple-negative breast cancer. In the case of hormone resistant breast cancer cells, they rarely showed hormone receptor expression. So we aimed to investigate whether the β-catenin pathway becomes activated in endocrine-resistant breast cancer cells and inhibition of β-catenin pathway can overcome endocrine resistance. Methods: We have established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with gradually increasing 4-hydroxytamoxifen concentration till 3μM. The levels of protein expression and mRNA transcripts were determined using western blot analysis and real-time quantitative PCR. The transcriptional activity of β-catenin was measured using luciferase activity assay. We used ICG-001 as inhibitor of β-catenin transcription activity. Results: The expression of estrogen receptor was significantly decreased in TamR cells. On the other hand, the expressions of HER2 and EGFR were increased in TamR cells than in control cells. The active (uncomplexed form) β-catenin level was increased in TamR cells, and also showed a significantly increased β-catenin transcriptional activity. The ICG-001, small-molecular inhibitor of Wnt/β-catenin pathway, treatment significantly reduced β-catenin transcriptional activity in TamR cells. The ICG-001 reduced cell viability fo TamR cells which showed resistance to tamoxifen by 65.3%, and also inhibited target gene cyclin D1 expression. The combination of ICG-001 and mTOR inhibitor, rapamycin reduced cell viability of TamR cells by 81.7% and there was an additive effect of two drugs as a combination index of 1.022. Conclusions: The β-catenin pathway is activated in endocrine-resistant breast cancer cells and inhibition of that pathway would be a new therapeutic strategy which overcomes endocrine resistance in breast cancer. Citation Format: Hye Sung Won, Kyoung Eun Lee, Eun Mi Nam, Yeung-Chul Mun, Chu-Myong Seong, Soon Nam Lee. Inhibition of β-catenin pathway to overcome endocrine resistance in tamoxifen-resistant breast cancer cell line. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B49.

MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels.

Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17β-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models.

Trastuzumab and docetaxel in a preclinical organotypic breast cancer model using tissue slices from mammary fat pad: Translational relevance.

With the ever-increasing number of drugs approved to treat cancers, selection of the optimal treatment regimen for an individual patient is challenging. Breast cancer complexity requires novel predictive methods and tools. In the present study, we set up experimental conditions to obtain an 'ex vivo' organotypic culture from xenotransplanted mice aiming at recapitulating the human clinical condition. The effect of trastuzumab (large biological molecule) and docetaxel (small chemical entity) was subsequently investigated on this organotypic model and compared with in vivo and in vitro activity on tumor cells. Tissue slices of 200 µm were obtained from mammary fat pad of SCID mice xenotransplanted with human MCF-7 breast cancer cells. Viability and proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and Ki-67 immunohistochemistry,and apoptosis by cleaved caspase-3 immunohistochemistry. In vivo antitumor activity of trastuzumab and docetaxel was determined by caliper measurement of tumor volume and Ki-67 expression on explanted masses by immunohistochemistry. A Teflon support and normoxia were necessary experimental conditions to obtain high viability of excised breast cancer infiltrated mammary fat pad slices upon 48 h cultivation, as shown by MTT proliferation assay, and Ki-67 expression. Breast cancer tissue slices treated for 48 h with trastuzumab or docetaxel showed a significant dose-dependent reduction of viability by MTT assay. Consistently, both drugs down-modulated Ki-67 and increased cleaved caspase-3. Tumor masses collected from docetaxel-or trastuzumab-treated mice showed a similar reduction of proliferation markers. By contrast, MCF-7 cell cultures were significantly inhibited by docetaxel but not by trastuzumab. Tumor tissue slices represent a more predictive experimental cancer model compared to cell cultures for both small and large molecule antitumor efficacy. This observation supports the relevance of microenvironment in the overall tumor biology and response to therapeutics.

Rhamnolipids functionalized AgNPs-induced oxidative stress and modulation of toxicity pathway genes in cultured MCF-7 cells

Rhamnolipids extracted from Pseudomonas aeruginosa strain JS-11 were utilized for synthesis of stable silver nanoparticles (Rh-AgNPs). The Rh-AgNPs (23nm) were characterized by Fourier transform infra-red (FTIR) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). The cytotoxicity assays suggested significant decrease in viability of Rh-AgNPs treated human breast adenocarcinoma (MCF-7) cells, compared with normal human peripheral blood mononuclear (PBMN) cells. Flow cytometry data revealed 1.25-fold (p<0.05) increase in the fluorescence of 2′,7′-dichlorofluorescein (DCF) at 0.25μg/mL. However, at Rh-AgNPs concentrations of 0.5 and 1.0μg/mL, much lesser fluorescence was noticed, which is attributed to cell death. Results with the fluorescent probe Rh123 demonstrated change in inner mitochondrial membrane and dissipation of membrane potential. The cell cycle analysis suggested 19.9% (p<0.05) increase in sub-G1 peak with concomitant reduction in G1 phase at 1μg/mL of Rh-AgNPs, compared to 2.7% in untreated control. The real-time RT2 Profiler™ PCR array data elucidated the overexpression of seven oxidative stress and DNA damage pathways genes viz. BAX, BCl2, Cyclin D1, DNAJA1, E2F transcription factor 1, GPX1 and HSPA4, associated with apoptosis signaling, proliferation and carcinogenesis, pro inflammatory and heat shock responses in Rh-AgNPs treated cells. Thus, the increased ROS production, mitochondrial damage and appearance of sub-G1 (apoptotic) population suggested the anti-proliferative activity, and role of oxidative stress pathway genes in Rh-AgNPs induced death of MCF-7 cancer cells.