Breast Cancer Cell Models Research Articles

Trimethyl chitosan coated palladium nanoparticles as a photothermal agent and its in vitro evaluation in 2D and 3D model of breast cancer cells

The potential of palladium has been scantily explored in biomedical applications. In the present study, palladium nanoparticles (PdNPs) were synthesized and were successfully coated with trimethyl-chitosan (TMC) to improve their biocompatibility. Coating with TMC improved the nanoparticle accumulation in MDAMB231 breast cancer cells, compared to nanoparticles coated with native chitosan. The TMC coated palladium nanoparticles (TMC/PdNPs) exhibited good biocompatibility and physiological stability, as compared to the plain(uncoated) PdNPs. TMC coated PdNPs resulted in photothermal therapeutic effect, when irradiated with a near-infrared (NIR) laser having the wavelength of 808-nm. The TMC/PdNPs resulted in good cytotoxic effect upon laser treatment in both, 2D monolayers and 3D spheroids of MDAMB231 cells, the latter mimicking the tumor microenvironment. These results clearly indicated that TMC/PdNPs acted as ideal photothermal agents for anti-cancer therapy in combination with a non-invasive near-infrared laser.

Paclitaxel resistance is mediated by NF-κB on mesenchymal primary breast cancer cells.

Paclitaxel has been used widely to treat breast cancer and other types of cancer. However, resistance is a major cause of failure for treatment and results in cancer progression. The present study investigated the association between paclitaxel resistance and the mesenchymal phenotype, using a model of primary breast cancer cells and employing four different cultures, two with an epithelial phenotype (MBCDF and MBCD17) and two with a mesenchymal phenotype (MBCDF-D5 and MBCD3). Epithelial-mesenchymal markers were evaluated by western blotting; MBCDF and MBCD17 cells expressed E-cadherin, SNAIL, Slug, and Twist, low levels of N-cadherin, but not vimentin. MBCDF-D5 and MBCD3 cells expressed N-cadherin, vimentin, and higher levels of SNAIL, and low levels of E-cadherin, Slug, and Twist. Cell viability was evaluated using a crystal violet assay after paclitaxel treatment; primary breast cancer cells with mesenchymal phenotype were resistant to paclitaxel compared with the epithelial primary breast cancer cells. Furthermore, using western blotting, it was revealed that mesenchymal cells had elevated levels of nuclear factor-κΒ (NF-κB) p65 and IκB kinase (IKK). Additionally, it was demonstrated that paclitaxel-induced degradation of the inhibitor of NF-κB, activation of NF-κB in a dose-dependent manner, and Bcl-2 and Bcl-xL upregulation. Finally, employing western blotting and crystal violet assays, the effects of the proteasome inhibitor ALLN were assessed. ALLN inhibited paclitaxel-induced NF-κB activation and restored the sensitivity to paclitaxel. Together, these data suggest that targeting the NF-κB/IKK axis might be a promising strategy to overcome paclitaxel resistance.

Breast Cancer Cell Subtypes Display Different Metabolic Phenotypes That Correlate with Their Clinical Classification.

Simple SummaryRecent studies on cancer cell metabolism have achieved notable breakthroughs that have led to a new scientific paradigm. How cancer cell metabolic reprogramming is orchestrated and the decisive role of this reprogramming in the oncogenic process and tumor adaptative evolution has been characterized at the molecular level. Despite this knowledge, it is essential to understand how cancer cells can metabolically respond as a living whole to ensure their survival and adaptation potential. In this work, we investigated whether different cancers and different subtypes display different metabolic phenotypes with a focus on breast cancer cell models representative of each clinical subtype. The potential results might have significant translational implications for diagnostic, prognostic and therapeutic applications.Metabolic reprogramming of cancer cells represents an orchestrated network of evolving molecular and functional adaptations during oncogenic progression. In particular, how metabolic reprogramming is orchestrated in breast cancer and its decisive role in the oncogenic process and tumor evolving adaptations are well consolidated at the molecular level. Nevertheless, potential correlations between functional metabolic features and breast cancer clinical classification still represent issues that have not been fully studied to date. Accordingly, we aimed to investigate whether breast cancer cell models representative of each clinical subtype might display different metabolic phenotypes that correlate with current clinical classifications. In the present work, functional metabolic profiling was performed for breast cancer cell models representative of each clinical subtype based on the combination of enzyme inhibitors for key metabolic pathways, and isotope-labeled tracing dynamic analysis. The results indicated the main metabolic phenotypes, so-called ‘metabophenotypes’, in terms of their dependency on glycolytic metabolism or their reliance on mitochondrial oxidative metabolism. The results showed that breast cancer cell subtypes display different metabophenotypes. Importantly, these metabophenotypes are clearly correlated with the current clinical classifications.

Synthesis, Anticancer Activity, Structure-Activity Relationship, and Molecular Modeling Studies of α-Mangostin Derivatives as hERα Inhibitor.

α-Mangostin is one of the secondary metabolites in mangosteen pericarp, which has been reported to have anti-breast cancer activity. In our previous study, three α-mangostin derivatives were computationally designed as hERα antagonists. In this present study, the designed compounds were synthesized undergoing a benzoylation reaction between α-mangostin with three benzoyl chloride derivatives to produce three derivatives, namely, AMB-1, AMB-2, and AMB-10. The synthesized compounds were then evaluated for their antiproliferative activity against the MCF-7 breast cancer cell model with hERα as the protein target. The in vitro assay shows moderate activity (57-126 μM) for all derivatives. The dynamic behaviors of all ligands, including α-mangostin and 4-hydroxytamoxifen (4-OHT), were studied with 100 ns of MD simulation. The structure-activity relationship shows that although it does not entirely concord with the expected design, it can explain the trend of α-mangostin and its derivatives antiproliferative activities against MCF-7, which associates with hERα antagonism.

Abstract P119: Differential sensitivity to poly(ADP-ribose) polymerase inhibitors in patient-derived cell models of breast cancer

Abstract Female breast cancer (BC) became the most commonly diagnosed cancer globally in 2020. One of the hallmarks of BC is both intratumor and intertumor heterogeneity. Efforts have been made to categorize this heterogeneity based on the presence or absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2/ERBB2). Receptor expression patterns are used to stratify BC and the subsequent treatments. Much of the current knowledge of breast cancer comes from in vitro and in vivo studies using well established cell lines, some of which have been cultured since the 1950s. Given that serial passaging over many years has been reported to alter the mutational profile of cell lines and therefore their relevance to clinical decisions on the primary tumour, new near-tumor cell models need to be generated. In this study, tumour biopsies or ascitic fluids were obtained from a local cancer research biobank, as well as directly from metastatic BC patients across the globe. Using mechanical dissociation and cell separation techniques we isolated tumor cells and grew them in proprietary media. The resultant models were subjected to whole exome and RNA sequencing. In addition, the models were tested in a 3-day cell death assay against a library of more than 50 FDA-approved drugs. The models and four BC cell lines (HCC1937, ZR-75-1, MDA-MB-436 and HCC1428) were also tested in a longer term 7-day assay against a panel of four poly(ADP-ribose) polymerase (PARP) inhibitors. A total of 10 patient-derived cell (PDC) models were successfully established from biopsies or ascitic fluids taken from patients with primary (1/10) or metastatic (9/10) BC. Models were established from patients with ER+ BC, HER2+ BC and triple negative BC (TNBC) within an average of 23 days from sample arrival. All models displayed a greater sensitivity to the non-targeted drugs in the library compared with the targeted drugs. Mutations in breast cancer associated 1 (BRCA1) and BRCA2 genes that were not present in BC cell lines were identified in a subset of the PDCs. All models showed some sensitivity to PARP inhibitors, and most were exquisitely sensitive to talazoparib with IC50s of less than 100 nM. Determining cell death responses in near-tumour PDCs should thus be adopted to enable more informed clinical decisions for treatment pathways and in parallel will be promising tools for oncology drug discovery. Citation Format: Immaculate Nalubowa, Subir Singh, Yuen Ngan Fan, Rachel Howard-Jones, Albert Bezman, Dominic I. James, Geoff Muckle, Gareth J. Griffiths. Differential sensitivity to poly(ADP-ribose) polymerase inhibitors in patient-derived cell models of breast cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P119.

Zeb1-mediated autophagy enhances resistance of breast cancer cells to genotoxic drugs

Autophagy is a highly conserved process of cellular self-digestion that involves the formation of autophagosomes for the delivery of intracellular components and dysfunctional organelles to lysosomes. This process is induced by different signals including starvation, mitochondrial dysfunction, and DNA damage. The molecular link between autophagy and DNA damage is not well understood yet. Importantly, tumor cells utilize the mechanism of autophagy to cope with genotoxic anti-cancer drug therapy. Another mechanism of drug resistance is provided to cancer cells via the execution of the EMT program. One of the critical transcription factors of EMT is Zeb1. Here we demonstrate that Zeb1 is involved in the regulation of autophagy in several breast cancer cell models. On the molecular level, Zeb1 likely facilitates autophagy through the regulation of autophagic genes, resulting in increased LC3-II levels, augmented staining with Lysotracker, and increased resistance to several genotoxic drugs. The attenuation of Zeb1 expression in TNBC cells led to the opposite effect. Consequently, we propose that Zeb1 augments the resistance of breast cancer cells to genotoxic drugs, at least partially, via autophagy.Collectively, we have uncovered a novel function of Zeb1 in the regulation of autophagy in breast cancer cells.

Anticancer Activity and Mechanism of Action Evaluation of an Acylhydrazone Cu(II) Complex toward Breast Cancer Cells, Spheroids, and Mammospheres.

The purpose of this work was to screen the anticancer activity and mechanisms of action of Cu(II)-acylhydrazone complex [Cu(HL)(H2 O)](NO3 )⋅H2 O, (CuHL), to find a potential novel agent for breast chemotherapies. Cytotoxicity studies on MCF7 cells demonstrated that CuHL has stronger anticancer properties than cisplatin over breast cancer cell models. Computational simulations showed that CuHL could interact in the minor groove of the DNA dodecamer, inducing a significant genotoxic effect on both cancer cells from 0.5 to 1 μM. In this sense, molecular docking and molecular dynamics simulations showed that the compound could interact with 20S proteasome subunits. Also, cell proteasome experiments using breast cancer cells revealed that the complex can inhibit proteasomal activity. Moreover, CuHL induced apoptosis in breast cancer cells at very low micromolar concentrations (0.5-2.5 μM) and displayed relevant anticancer activity over spheroids derived from MCF7 cells. Ultimately, CuHL diminished the number of mammospheres formed, disturbing their morphology and size.

Enhanced IFNα Signaling Promotes Ligand-Independent Activation of ERα to Promote Aromatase Inhibitor Resistance in Breast Cancer.

Simple SummaryInterferon alpha (IFNα) signaling is highly upregulated in ER+ breast cancers that become resistant to estrogen deprivation therapy. This study uncovers how enhanced (IFNα)/JAK-STAT signaling directly influences estrogen receptor (ERα) activation in the absence of estrogen. We found that inhibiting IFNα signaling downregulates expression and activation of ERα. In addition, STAT1 and ERα directly interact and regulate a key interferon-stimulated gene, IFITM1, in AI-resistant breast cancer cells. We demonstrate that crosstalk occurs between IFNα and ERα pathways, which contributes to aggression and survival of AI-resistant breast cancer, thus representing a novel mechanism of acquired AI resistance.Aromatase inhibitors (AIs) reduce estrogen levels up to 98% as the standard practice to treat postmenopausal women with estrogen receptor-positive (ER+) breast cancer. However, approximately 30% of ER+ breast cancers develop resistance to treatment. Enhanced interferon-alpha (IFNα) signaling is upregulated in breast cancers resistant to AIs, which drives expression of a key regulator of survival, interferon-induced transmembrane protein 1 (IFITM1). However, how upregulated IFNα signaling mediates AI resistance is unknown. In this study, we utilized MCF-7:5C cells, a breast cancer cell model of AI resistance, and demonstrate that these cells exhibit enhanced IFNα signaling and ligand-independent activation of the estrogen receptor (ERα). Experiments demonstrated that STAT1, the mediator of intracellular signaling for IFNα, can interact directly with ERα. Notably, inhibition of IFNα signaling significantly reduced ERα protein expression and ER-regulated genes. In addition, loss of ERα suppressed IFITM1 expression, which was associated with cell death. Notably, chromatin immunoprecipitation experiments validated that both ERα and STAT1 associate with ERE sequences in the IFITM1 promoter. Overall, hyperactivation of IFNα signaling enhances ligand-independent activation of ERα, which promotes ER-regulated, and interferon stimulated gene expression to promote survival in AI-resistant breast cancer cells.

STING protects breast cancer cells from intrinsic and genotoxic-induced DNA instability via a non-canonical, cell-autonomous pathway.

STING (Stimulator of Interferon Genes) is an endoplasmic reticulum-anchored adaptor of the innate immunity best known to trigger pro-inflammatory cytokine expression in response to pathogen infection. In cancer, this canonical pathway can be activated by intrinsic or drug-induced genomic instability, potentiating antitumor immune responses. Here we report that STING downregulation decreases cell survival and increases sensitivity to genotoxic treatment in a panel of breast cancer cell lines in a cell-autonomous manner. STING silencing impaired DNA Damage Response (53BP1) foci formation and increased DNA break accumulation. These newly identified properties were found to be independent of STING partner cGAS and of its canonical pro-inflammatory pathway. STING was shown to partially localize at the inner nuclear membrane in a variety of breast cancer cell models and clinical tumor samples. Interactomics analysis of nuclear STING identified several proteins of the DNA Damage Response, including the three proteins of the DNA-PK complex, further supporting a role of STING in the regulation of genomic stability. In breast and ovarian cancer patients that received adjuvant chemotherapy, high STING expression is associated with increased risk of relapse. In summary, this study highlights an alternative, non-canonical tumor-promoting role of STING that opposes its well-documented function in tumor immunosurveillance.

Plasma membrane permeabilization to explain erythrosine B phototoxicity on in vitro breast cancer cell models.

Lipid oxidation is ubiquitous in cell life under oxygen and essential for photodynamic therapy (PDT) of carcinomas. However, the mechanisms underlying lipid oxidation in rather complex systems such as plasma membranes remain elusive. Herein, Langmuir monolayers were assembled with the lipid extract of glandular breast cancer (MCF7) cells and used to probe the molecular interactions allowing adsorption of the photosensitizer (PS) erythrosine B and subsequent photooxidation outcomes. Surface pressure (π) versus area (cm2/mL) isotherms of MCF7 lipid extract shifted to larger areas upon erythrosine incorporation, driven by secondary interactions that affected the orientation of the carbonyl groups and lipid chain organization. Light-irradiation increased the surface area of the MCF7 lipid extract monolayer containing erythrosine owing to the lipid hydroperoxidation, which may further undergo decomposition, resulting in the chain cleavage of phospholipids and membrane permeabilization. Incorporation of erythrosine by MCF7 cells induced slight toxic effects on in vitro assays, differently of the severe phototoxicity caused by light-irradiation, which significantly decreased cell viability by more than 75% at 2.5×10-6 mol/L of erythrosine incubated for 3 and 24h, reaching nearly 90% at 48h of incubation. The origin of the phototoxic effects is in the rupture of the plasma membrane shown by the frontal (FSC) and side (SSC) light scattering of flow cytometry. Consistent with hydroperoxide decomposition, membrane permeabilization was also confirmed by cleaved lipids detected in mass spectrometry and subsidizes the necrotic pathway of cell death.

Ruthenium Half-Sandwich Type Complexes with Bidentate Monosaccharide Ligands Show Antineoplastic Activity in Ovarian Cancer Cell Models through Reactive Oxygen Species Production.

Ruthenium complexes are developed as substitutes for platinum complexes to be used in the chemotherapy of hematological and gynecological malignancies, such as ovarian cancer. We synthesized and screened 14 ruthenium half-sandwich complexes with bidentate monosaccharide ligands in ovarian cancer cell models. Four complexes were cytostatic, but not cytotoxic on A2780 and ID8 cells. The IC50 values were in the low micromolar range (the best being 0.87 µM) and were similar to or lower than those of the clinically available platinum complexes. The active complexes were cytostatic in cell models of glioblastoma, breast cancer, and pancreatic adenocarcinoma, while they were not cytostatic on non-transformed human skin fibroblasts. The bioactive ruthenium complexes showed cooperative binding to yet unidentified cellular target(s), and their activity was dependent on reactive oxygen species production. Large hydrophobic protective groups on the hydroxyl groups of the sugar moiety were needed for biological activity. The cytostatic activity of the ruthenium complexes was dependent on reactive species production. Rucaparib, a PARP inhibitor, potentiated the effects of ruthenium complexes.

The FHP01 DDX3X Helicase Inhibitor Exerts Potent Anti-Tumor Activity In Vivo in Breast Cancer Pre-Clinical Models.

Simple SummaryDDX3X has been repeatedly proven to exert pro-tumorigenic effects in breast cancer. An inhibitor of DDX3X helicase function, FHP01, exhibited very effective antiproliferative and tumor killing activity in vitro and in vivo against several breast cancer cells, yet showed no systemic toxicity but good bioavailability in vivo. FHP01 may therefore represent a novel therapeutic approach with high potential as a personalized strategy for breast cancer, confirming DDX3X as a new actionable molecular target in these tumors.Inhibition of DDX3X expression or activity reduces proliferation in cells from various tumor tissues, in particular in breast cancer, and its expression often correlates to tumor aggressiveness. This makes DDX3X a prominent candidate for the design of drugs for novel personalized therapeutic strategies. Starting from an in silico drug discovery approach, a group of molecules has been selected by molecular docking at the RNA binding site of DDX3X. Here, the most promising among them, FHP01, was evaluated in breast cancer preclinical models. Specifically, FHP01 exhibited very effective antiproliferative and killing activity against different breast cancer cell types, among which those from triple-negative breast cancer (TNBC). Interestingly, FHP01 also inhibited WNT signaling, a key tumorigenic pathway already correlated to DDX3X functions in breast cancer model cell lines. Ultimately, FHP01 also caused a significant reduction, in vivo, in the growth of MDA MB 231-derived TNBC xenograft models. Importantly, FHP01 showed good bioavailability and no toxicity on normal peripheral blood mononuclear cells in vitro and on several mouse tissues in vivo. Overall, our data suggest that the use of FHP01 and its related compounds may represent a novel therapeutic approach with high potential against breast cancer, including the triple-negative subtype usually correlated to the most unfavorable outcomes because of the lack of available targeted therapies.

Gene Signatures Induced by Ionizing Radiation as Prognostic Tools in an In Vitro Experimental Breast Cancer Model.

Simple SummaryThe present work analyzed the expression of genes involved in radiation, using an in vitro experimental breast cancer model developed by the combined treatment of low doses of high linear energy transfer (LET) radiation α particle radiation and estrogen yielding different stages in a malignantly transformed breast cancer cell model called Alpha model. Results showed important findings of genes involved in cancers of the breast, lung, and nervous system, and others. Most of those genes analyzed in these studies such as ATM, selenoproteins, GABA receptor, interleukins, epsin, and cathepsin inhibitors like stefins, and metallothioneins can be used for new prognostic tools and future therapies since they affect cancer progression and metastasis. In conclusion, gene signature demonstrated to be specific to cell line types, hence cell-dependency must be considered in future radiotherapy treatment planning since molecular and clinical features affect such results. Thus, using gene technology and molecular information is possible to improve therapies and reduction of side effects.This study aimed to analyze the expression of genes involved in radiation, using an Affymetrix system with an in vitro experimental breast cancer model developed by the combined treatment of low doses of high linear energy transfer (LET) radiation α particle radiation and estrogen yielding different stages in a malignantly transformed breast cancer cell model called Alpha model. Altered expression of different molecules was detected in the non-tumorigenic Alpha3, a malignant cell line transformed only by radiation and originally derived from the parental MCF-10F human cell line; that was compared with the Alpha 5 cell line, another cell line exposed to radiation and subsequently grown in the presence 17β-estradiol. This Alpha5, a tumorigenic cell line, originated the Tumor2 cell line. It can be summarized that the Alpha 3 cell line was characterized by greater gene expression of ATM and IL7R than control, Alpha5, and Tumor2 cell lines, it presented higher selenoprotein gene expression than control and Tumor2; epsin 3 gene expression was higher than control; stefin A gene expression was higher than Alpha5; and metallothionein was higher than control and Tumor2 cell line. Therefore, radiation, independently of estrogen, induced increased ATM, IL7R, selenoprotein, GABA receptor, epsin, stefin, and metallothioneins gene expression in comparison with the control. Results showed important findings of genes involved in cancers of the breast, lung, nervous system, and others. Most genes analyzed in these studies can be used for new prognostic tools and future therapies since they affect cancer progression and metastasis. Most of all, it was revealed that in the Alpha model, a breast cancer model developed by the authors, the cell line transformed only by radiation, independently of estrogen, was characterized by greater gene expression than other cell lines. Understanding the effect of radiotherapy in different cells will help us improve the clinical outcome of radiotherapies. Thus, gene signature has been demonstrated to be specific to tumor types, hence cell-dependency must be considered in future treatment planning. Molecular and clinical features affect the results of radiotherapy. Thus, using gene technology and molecular information is possible to improve therapies and reduction of side effects while providing new insights into breast cancer-related fields.

Preparing PAMAM-NK4 nano complexes and examining their in vitro growth suppression effects in breast cancer.

This study sought to examine the suppression of the NK4 (which is a fragment that originates from the trypsin digestion of the hepatocyte growth factor) gene as mediated by new nano material polyamidoamine (PAMAM) dendrimers in the growth of breast cancer cells MDA-MB-231 and MCF-7, and the therapeutic effects in a nude mice model of transplanted tumor cell MDA-MB-231. We built PAMAM-NK4 nano particles and detected the in vitro transfection rate. Nano complexes and blank plasmid PAMAM dendrimers were transfected to MDA-MB-231 and MCF-7 cells, respectively. The western-blotting method, MTT experiment method, and bead method were used to detect the effects of the nano complexes on NK4 protein expression, cell proliferation, and cell apoptosis. The nude mice model of transplanted tumor cell MDA-MB-231 comprised 40 nude female mice who were subject to injections. The mice were randomly divided into four groups, comprising 10 mice per group. The control, blank plasmid and treatment groups were subcutaneously injected with 0.2 mL of 0.9% NaCl (Sodium chloride) solution, 0.2 mL of plasmid solution (including 100 µg PAMAM pcDNA3.1(-) blank plasmid nano complexes) and 0.2 mL of plasmid solution (including PAMAM-NK4 100 µg) beside the tumor inoculation spot, respectively. The positive control group was intraperitoneally injected with 0.2 mL of doxorubicin solution, including 100 µg doxorubicin. Western blotting was used to detect the NK4 protein expression of the transplanted tumor tissues of the various groups. NK4 protein was successfully expressed in MDA-MB-231 and MCF-7 cells transfected with PAMAM-NK4 nano particles, and cell proliferation was suppressed and cell apoptosis was induced. The tumor volumes and masses of the treatment and positive control groups were obviously smaller than those of the control group. The differences were statistically significant (P<0.05). The treatment group had an obviously higher mean value of NK4 protein expression than the control group. The differences were statistically significant (P<0.05). PAMAM-NK4 nano complexes suppress the growth of the breast cancer cells MDA-MB-231 and MCF-7, and had a treatment effect on this tumor nude mice model of breast cancer cells.

Biofunctional supramolecular hydrogels fabricated from a short self-assembling peptide modified with bioactive sequences for the 3D culture of breast cancer MCF-7 cells

Self-assembling peptides are a type of molecule with promise as scaffold materials for cancer cell engineering. We have reported a short self-assembling peptide, (FFiK)2, that had a symmetric structure connected via a urea bond. In this study, we functionalized (FFiK)2 by conjugation with various bioactive sequences for the 3D culture of cancer cells. Four sequences, RGDS and PHSRN derived from fibronectin and AG73 and C16 derived from laminin, were selected as bioactive sequences to promote cell adhesion, proliferation or migration. (FFiK)2, and its derivatives could co-assemble into supramolecular nanofibers displaying bioactive sequences and form hydrogels. MCF-7 cells were encapsulated in functionalized peptide hydrogels without significant cytotoxicity. Encapsulated MCF-7 cells proliferated under 3D culture conditions. MCF-7 cells proliferated with spheroid formation in hydrogels that displayed RGDS or PHSRN sequences, which will be able to be applied to drug screening targeting cancer stem cells. On the other hand, since MCF-7 cells migrated in a 3D hydrogel that displayed AG73, we could construct the metastatic model of breast cancer cells, which is helpful for the elucidation of breast cancer cells and drug screening against cancer cells under metastatic state. Therefore, functionalized (FFiK)2 hydrogels with various bioactive sequences can be used to regulate cancer cell function for tumor engineering and drug screening.

LightSpot®-FL-1 Fluorescent Probe: An Innovative Tool for Cancer Drug Resistance Analysis by Direct Detection and Quantification of the P-glycoprotein (P-gp) on Monolayer Culture and Spheroid Triple Negative Breast Cancer Models.

Simple SummaryTumoral drug resistance is mainly caused by multidrug resistance transporters (MDR), such as the P-gp, which presents high clinical interest. For this reason, the P-gp-mediated drug resistance diagnosis may be very relevant for optimizing anticancer treatment efficacy. However, the lack of effective analytical tools limits this clinical diagnostic approach. Therefore, our group has developed LightSpot®-FL-1, a new cell-permeant fluorescent probe able to specifically localize and quantify the P-gp inside unicellular, monolayer, and cellular mass models. The application of this innovative tool was firstly demonstrated in the preclinical field, using five triple-negative breast cancer (TNBC) cell models. The comparison between classical anti-P-gp immunostaining and LightSpot®-FL-1 P-gp staining highlighted a strong similarity with P-gp localization and expression level quantification. LightSpot®-FL-1 P-gp detection and quantification, using several fluorescence imaging methods, are easy, direct, and cost-effective and are, therefore, very promising for future clinical diagnosis development.P-gp is the most widely studied MDR protein conferring cellular resistance to many standard or targeted therapeutic agents. For this reason, P-gp chemoresistance evaluation, established before or during chemotherapy, can be very relevant in order to optimize the efficacy of treatments, particularly for aggressive tumoral subtypes such as triple-negative breast cancer (TNBC). In this context, our team developed an innovative cell-permeant fluorescent probe called the LightSpot®-FL-1, which is able to specifically localize and quantify the P-gp in cells or cell masses, as evidenced on different TNBC cell models. First, flow cytometry analysis showed LightSpot®-FL-1 cell penetration and persistence in time, in TNBC cells. Then, LightSpot®-FL-1 staining was compared to anti-P-gp immunostaining by fluorescence microscopy on five TNBC cell lines. Results showed a clear similarity of P-gp localization and expression level, confirmed by Pearson’s and Mander’s colocalization coefficients with 92.1% and 100.0%, and a strong correlation coefficient of R2 = 0.99. In addition, the LightSpot®-FL-1 staining allowed the quantification of a P-gp induction (33% expression increase) following a 6-hour spheroid model exposure to the anti-PARP Olaparib. Thus, the new LightSpot®-FL-1 cell-permeant probe, targeting P-gp, appears to be an effective tool for drug resistance evaluation in preclinical models and shows promising possibilities for future use in clinical diagnosis.

Improving Cytotoxicity against Breast Cancer Cells by Using Mixed-Ligand Ruthenium(II) Complexes of 2,2'-Bipyridine, Amino Acid, and Nitric Oxide Derivatives as Potential Anticancer Agents.

Several metal-based molecules that display cytotoxicity against multiple cell lines have been pursued in an attempt to fight against cancer and to overcome the typical side effects of drugs like cisplatin. In this scenario, ruthenium complexes have been extensively studied due to their activity in both in vitro and in vivo biological systems, including various cancer cell strains. We aimed to develop a method to synthesize novel [Ru(NO)(bpy)2L2]2+ complexes containing amino acid ligands by using an alternative Click Chemistry approach, namely the copper azide-alkyne cycloaddition reaction (CuAAC reaction), to construct nitrosyl/nitrite complexes bearing a modified lysine residue. We synthesized a new ligand by Click Chemistry approach and new compounds bearing the unprecedented ligand. Cytotoxicity was assessed by the classical MTT colorimetric assay. MCF-7 and MDAMB- 231 cells were used as breast cancer cell models. MCF-10 was used as a model of healthy cells. Amino acid ligands related to N3-Lys(Fmoc) and the new pyLys were successfully synthesized by the diazotransfer reaction and the CuAAC reaction, respectively. The latter reaction involves coupling between N3-Lys(Fmoc) and 3ethynylpyridine. Both N3-Lys(Fmoc) and the new pyLys were introduced into the ruthenium bipyridine complex I, or cis-[RuII(NO)(NO2)(bpy)2]2+, to generate the common nitro-based complex III, which was further converted to the final complex IV. Results of the MTT assay proved the cytotoxic effect of cis- [RuII(NO)(pyLysO-)(bpy)2](PF6)2 against the mammalian breast cancer cells MCF-7 and MDA-MB231. The viability assays revealed that complex IV, bearing a NO group and a modified lysine residue, was able to release NO and cross tumor cell membranes. In this work, Complex IV was observed to be the most active ruthenium bipyridine complex against the mammalian breast cancer cells MCF-7 and MDA-MB231: it was approximately twice as active as cisplatin, whilst complexes I-III proved to be less cytotoxic than complex IV. Additional tests using healthy MCF 10A cells showed that complexes II-IV were three- to sixfold less toxic than cisplatin, which suggested that complex IV was selective against cancer cells.

Abstract 2864: RET as a novel therapeutic target in breast cancer brain metastases

Abstract Brain metastases (BrM) are reported in approximately 10-30% of breast cancer patients with a poor median overall survival of 8.5 months. Current therapeutic strategies are mostly limited to surgery, chemotherapy and radiotherapy, making the study of novel druggable targets and brain-permeable small molecule inhibitors an urgent clinical need. Our lab recently reported a hybrid-capture exome RNA-sequencing study on 21 patient-matched pairs of primary breast tumors and brain metastases and identified upregulation of several receptor tyrosine kinases including RET in BrM. The ligand for RET is Glial Cell Derived Neurotrophic Factor (GDNF), which is released by astrocytes and microglia in response to neuroinflammation and brain tissue damage. Clinical BrM cell growth in ex vivo cultures was reduced by the multikinase inhibitor Cabozantinib, indicating a potential role for RET in the growth of BrM lesions. We hypothesize that the high level of brain-derived GDNF provides a permissive microenvironment for RET overexpressing breast cancer cells to form brain metastases, and will evaluate the therapeutic potential of RET targeted therapies for breast cancer BrM patients. Our preliminary data show that in RET-overexpressing (RET-OE) breast cancer cell models, 30-minute GDNF treatment induces RET phosphorylation at two distinct tyrosine residues, along with downstream signaling illustrated by ERK1/2, AKT, and STAT3 phosphorylation. This induction was effectively abrogated after shRNA-mediated RET knockdown. We employed a phospho-kinase array to identify novel downstream targets regulated by the GDNF-RET signaling axis, and found that GDNF induces phosphorylation of multiple targets (including p27 and RSK) in RET-OE cells. Interestingly, phosphorylation of multiple Src family kinases (Lck, Lyn, Fgr) was downregulated with GDNF treatment. We will further examine these downstream targets via reverse phase protein array (RPPA). GDNF triggers migration of RET-OE cell lines in both 2D wound scratch and 3D transwell migration assays. Additionally, we present results obtained through an ex vivo organotypic coculture system, in which RET-OE cells are cultured on top of 300-micron thick murine brain, kidney, or liver slices. RET overexpression increases colony formation compared with empty vector control, suggesting a role for RET signaling in the initial establishment of metastatic brain lesions. Differences in colonization between empty vector transfected and RET-OE cells remain minimal in kidney and liver sections, suggesting a role for RET specific to the brain microenvironment. We will examine RET-driven brain metastasis in vivo by employing RET-OE and RET-deficient cell lines in intracranial, intracardiac, and mammary fat pad injection. These models of BrM will be randomized to RET inhibitor or vehicle treatment to evaluate the efficacy of RET-targeted therapy in the setting of breast cancer brain metastasis. Citation Format: Simeng Liu, Geoffrey Pecar, Jagmohan Hooda, Jennifer M. Atkinson, Fangyuan Chen, Adrian V. Lee, Steffi Oesterreich. RET as a novel therapeutic target in breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2864.

Abstract 3150: A characterization of cancer vasculogenic mimicry: Extracellular matrix induced cellular signaling to lumen formation

Abstract Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can only occur in Matrigel; however, the protein component and signaling pathways involved in this process are unknown. Methods: Using an established in vitro model of VM, of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized pharmacological inhibitors, gene silencing and blocking antibodies to elucidate the signaling pathways involved in the process of VM. Immunofluorescence and sirius red staining were used to determine the glycoprotein-rich component lining the lumen of the tubular structures. Results: Differently to what observed in the presence of Matrigel, VM did not occur when cancer cells were cultivated on plastic, glass or heat denatured Matrigel (10 mins at 65°C). Using exclusively Collagen I or Laminin 111 to mimic the extracellular matrix we observed than only in the presence of Laminin 111 could VM formation occur. Laminin is secreted and deposited by HEY cells and constitutes a part of the luminal lining. Silencing of integrin β1, but not β3, by siRNA and antibody blocking prevents this process. Chemical inhibition of PI3K pathway and metalloproteases (MMP) activation demonstrate that these pathways are also essential. RNAseq analysis suggests that this process has minimal dependence on de novo transcriptional activity. Discussion and conclusion: We have shown that VM only occurs when cells are seeded on Matrigel but not on plastic, glass or heat denatured Matrigel, suggesting that this phenomenon is susceptible to substrate/matrix rigidity. Furthermore, we identified Laminin as the essential matrix protein secreted and deposited by cancer cells to allow for VM assembly. Its interaction with integrin β1, and the consequent regulation of the activity of MMP leads to the remodeling of the ECM to favor the connection of the VM channels to the microcirculation system. This pathway is not heavily dependent on transcription but requires the PI3K pathway. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets. Citation Format: Gabriel Mingo, Andres Valdivia, Varina Aldana, Javiera Pradenas, Nicole Babbitt, Pamela Gonzalez, Francisco Nualart, Jorge Díaz, Lisette Leyton, Cristina Bertocchi, Gareth Owen. A characterization of cancer vasculogenic mimicry: Extracellular matrix induced cellular signaling to lumen formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3150.

Abstract 1333: Evaluation of a novel PELP1 inhibitor for treatment of triple negative breast cancer

Abstract Background: Breast cancer (BC) is composed of distinct molecular subtypes, such as ER+ BC and triple negative BC (TNBC). Development of novel effective therapies for patients with TNBC remains the highest unmet need in patient treatment and survivorship. PELP1 plays an essential role in several pathways including hormonal signaling, cell cycle progression, ribosomal biogenesis, and DNA damage response. PELP1 expression is an independent prognostic predictor of shorter BC-specific survival and an independent prognostic factor for predicting poor survivorship in TNBC patients. Recently, we generated a small molecule inhibitor of PELP1 (SMIP34) that binds and inhibits PELP1 oncogenic signaling. The objective of this study is to test the utility of SMIP34 as a novel therapeutic for treating TNBC. Methods: We have selected seven TNBC model cell lines (BT549, MDA-MB-453, MDA-MB-231, MDA-MB-468, SUM-159, HCC1806, and HCC1937) and one human mammary epithelial (HMEC) cell line in this study. In vitro activity was assessed using Cell Titer Glo, MTT, colony formation, and matrigel invasion assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq. Xenograft tumor-derived explant (XDEX) assays and patient-derived tumor explant (PDEX) assays were used for preclinical evaluation. Results: Using a panel of BC model cell lines, we found that SMIP34 treatment reduced cell viability with an IC50 of 3-8µM with no activity in HMEC cells. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, confirming its target specificity. We confirmed the physical interaction of SMIP34 to PELP1 using biotin-SMIP34 and MST assays. SMIP34 treatment significantly reduced the invasiveness and colony formation of TNBC cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. Further, RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. Western analyses confirmed SMIP34 treatment significantly reduced known PELP1 downstream signaling. Mechanistic studies using global RNA-seq identified that SMIP34 treatment alters known PELP1 modulated pathways (ribosomal biogenesis). Using MDA-MB-231 xenograft and PDX tumor tissues in explant assays, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Accordingly, in xenograft models, SMIP34 (20mg/kg/IP) treatment resulted in a significant reduction in tumor volume compared to vehicle control. Conclusion: Our results using in vitro, ex vivo, and in vivo studies demonstrated that the PELP1 inhibitor SMIP34 has therapeutic efficacy against TNBC. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545 (R.K.V) Citation Format: Kristin A. Altwegg, Suryavathi Viswanadhapalli, Junhao Liu, Zexuan Liu, Uday P. Pratap, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Evaluation of a novel PELP1 inhibitor for treatment of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1333.