Ridha Limame

Comparative Analysis of Dynamic Cell Viability, Migration and Invasion Assessments by Novel Real-Time Technology and Classic Endpoint Assays

Background Cell viability and motility comprise ubiquitous mechanisms involved in a variety of (patho)biological processes including cancer. We report a technical comparative analysis of the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform, with conventional label-based endpoint methods, hereby indicating performance characteristics and correlating dynamic observations of cell proliferation, cytotoxicity, migration and invasion on cancer cells in highly standardized experimental conditions. Methodology/Principal Findings Dynamic high-resolution assessments of proliferation, cytotoxicity and migration were performed using xCELLigence technology on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines. Proliferation kinetics were compared with the Sulforhodamine B (SRB) assay in a series of four cell concentrations, yielding fair to good correlations (Spearmans Rho 0.688 to 0.964). Cytotoxic action by paclitaxel (0–100 nM) correlated well with SRB (Rho>0.95) with similar IC50 values. Reference cell migration experiments were performed using Transwell plates and correlated by pixel area calculation of crystal violet-stained membranes (Rho 0.90) and optical density (OD) measurement of extracted dye (Rho>0.95). Invasion was observed on MDA-MB-231 cells alone using Matrigel-coated Transwells as standard reference method and correlated by OD reading for two Matrigel densities (Rho>0.95). Variance component analysis revealed increased variances associated with impedance-based detection of migration and invasion, potentially caused by the sensitive nature of this method. Conclusions/Significance The xCELLigence RTCA technology provides an accurate platform for non-invasive detection of cell viability and motility. The strong correlations with conventional methods imply a similar observation of cell behavior and interchangeability with other systems, illustrated by the highly correlating kinetic invasion profiles on different platforms applying only adapted matrix surface densities. The increased sensitivity however implies standardized experimental conditions to minimize technical-induced variance.

Integrated miRNA and mRNA expression profiling of the inflammatory breast cancer subtype

Background:MicroRNAs (miRNAs) are key regulators of gene expression. In this study, we explored whether altered miRNA expression has a prominent role in defining the inflammatory breast cancer (IBC) phenotype.Methods:We used quantitative PCR technology to evaluate the expression of 384 miRNAs in 20 IBC and 50 non-IBC samples. To gain understanding on the biological functions deregulated by aberrant miRNA expression, we looked for direct miRNA targets by performing pair-wise correlation coefficient analysis on expression levels of 10 962 messenger RNAs (mRNAs) and by comparing these results with predicted miRNA targets from TargetScan5.1.Results:We identified 13 miRNAs for which expression levels were able to correctly predict the nature of the sample analysed (IBC vs non-IBC). For these miRNAs, we detected a total of 17 295 correlated miRNA–mRNA pairs, of which 7012 and 10 283 pairs showed negative and positive correlations, respectively. For four miRNAs (miR-29a, miR-30b, miR-342-3p and miR-520a-5p), correlated genes were concordant with predicted targets. A gene set enrichment analysis on these genes demonstrated significant enrichment in biological processes related to cell proliferation and signal transduction.Conclusions:This study represents, to the best of our knowledge, the first integrated analysis of miRNA and mRNA expression in IBC. We identified a set of 13 miRNAs of which expression differed between IBC and non-IBC, making these miRNAs candidate markers for the IBC subtype.

Quantitative methylation profiling in tumor and matched morphologically normal tissues from breast cancer patients.

BackgroundIn the present study, we determined the gene hypermethylation profiles of normal tissues adjacent to invasive breast carcinomas and investigated whether these are associated with the gene hypermethylation profiles of the corresponding primary breast tumors.MethodsA quantitative methylation-specific PCR assay was used to analyze the DNA methylation status of 6 genes (DAPK, TWIST, HIN-1, RASSF1A, RARβ2 and APC) in 9 normal breast tissue samples from unaffected women and in 56 paired cancerous and normal tissue samples from breast cancer patients.ResultsNormal tissue adjacent to breast cancer displayed statistically significant differences to unrelated normal breast tissues regarding the aberrant methylation of the RASSF1A (P = 0.03), RARβ2 (P = 0.04) and APC (P = 0.04) genes. Although methylation ratios for all genes in normal tissues from cancer patients were significantly lower than in the cancerous tissue from the same patient (P ≤ 0.01), in general, a clear correlation was observed between methylation ratios measured in both tissue types for all genes tested (P < 0.01). When analyzed as a categorical variable, there was a significant concordance between methylation changes in normal tissues and in the corresponding tumor for all genes tested but RASSF1A. Notably, in 73% of patients, at least one gene with an identical methylation change in cancerous and normal breast tissues was observed.ConclusionsHistologically normal breast tissues adjacent to breast tumors frequently exhibit methylation changes in multiple genes. These methylation changes may play a role in the earliest stages of the development of breast neoplasia.

Cancer-associated adipose tissue promotes breast cancer progression by paracrine Oncostatin M and Jak/STAT3 signaling.

Increasing evidence supports the critical roles played by adipose tissue in breast cancer progression. Yet, the mediators and mechanisms are poorly understood. Here, we show that breast cancer-associated adipose tissue from freshly isolated tumors promotes F-actin remodeling, cellular scattering, invasiveness, and spheroid reorganization of cultured breast cancer cells. A combination of techniques, including transcriptomics, proteomics, and kinomics enabled us to identify paracrine secretion of oncostatin M (OSM) by cancer-associated adipose tissue. Specifically, OSM, expressed by CD45(+) leucocytes in the stromal vascular fraction, induced phosphorylation of STAT3 (pSTAT3-) Y705 and S727 in breast cancer cells and transcription of several STAT3-dependent genes, including S100 family members S100A7, S100A8, and S100A9. Autocrine activation of STAT3 in MCF-7 cells ectopically expressing OSM-induced cellular scattering and peritumoral neovascularization of orthotopic xenografts. Conversely, selective inhibition of OSM by neutralizing antibody and Jak family kinases by tofacitinib inhibited STAT3 signaling, peritumoral angiogenesis, and cellular scattering. Importantly, nuclear staining of pSTAT3-Y705 identified at the tumor invasion front in ductal breast carcinomas correlates with increased lymphovascular invasion. Our work reveals the potential of novel therapeutic strategies targeting the OSM and STAT3 axis in patients with breast cancer harboring nuclear pSTAT3-Y705.

Is there a role for mammary stem cells in inflammatory breast carcinoma?: a review of evidence from cell line, animal model, and human tissue sample experiments.

Stem cells are pluripotent cells, with a large replicative potential, which perform normal physiological functions such as tissue renewal and damage repair. However, because of their long lifespan and high replicative potential, stem cells are ideal targets to accumulate multiple mutations. Therefore, they can be regarded as being responsible for the initiation of tumor formation. In the past, numerous studies have shown that the presence of an elaborate stem cell compartment within a tumor is associated with aggressive tumor cell behavior, frequent formation of metastases, resistance to therapy, and poor patient survival. From this perspective, tumors from patients with inflammatory breast cancer (IBC), an aggressive breast cancer subtype with a dismal clinical course, are most likely to be associated with stem cell biology. To date, this hypothesis is corroborated by evidence resulting from in vitro and in vivo experiments. Both gene and microRNA expression profiles highlighted several stem cell‐specific signal transduction pathways that are hyperactivated in IBC. Also, these stem cell‐specific signal transduction pathways seem to converge in the activation of nuclear factor‐kappa B, a molecular hallmark of IBC, and induction of epithelial‐to‐mesenchymal transition. Recently, the latter mechanism was identified as a prerequisite for the induction of stem cell characteristics in breast cancer cells. Cancer 2010;116(11 suppl):2794–805.

Quantitative assessment of DNA hypermethylation in the inflammatory and non-inflammatory breast cancer phenotypes

In this study, a comparative quantitative methylation profiling of inflammatory breast cancer (IBC) and non-IBC was set up for the identification of tumor-specific methylation patterns. Methylation ratios of six genes measured in benign breast tissues (n=9) and in tumor samples from non-IBC (n=81) and IBC (n=19) patients using quantitative methylation-specific PCR. Median methylation ratios observed in breast cancer (n=100) were significantly higher than those observed in benign breast tissues for 5 of 6 genes (TWIST, HIN-1, RASSF1A, RARβ2 and APC). Only one of the individual genes studied, RARβ2, showed differential methylation ratios in IBC and non-IBC (P=0.016). Using the maximal methylation ratio observed in benign breast tissue as a threshold, the methylation frequency of two genes, RARβ2 and APC, was significantly increased in IBC (n=19) when compared to non-IBC (n=81): 53% vs. 23% for RARβ2 (P=0.012) and 84% vs. 54% for APC (P=0.017). Using hierarchical clustering, methylation patterns could not classify breast cancers according to their phenotype. The finding of differential frequencies of methylation in IBC and non-IBC for 2 out of 6 genes suggests that gene-specific patterns of methylation could provide a basis for molecular classification of IBC Testing for additional genes could help to define the IBC phenotype based on patterns of aberrant gene promoter methylation.

Abstract 2633: Gene signatures derived from in vitro isolated migratory and invasive breast cancer cells indicate early metastatic relapse.

Background Cancer cell motility and tissue invasion are cornerstones of metastatic dissemination. Adequate molecular profiling of chemotaxis-driven tumor cells is impeded by the transient and stochastic nature of motility occurring in only a small subset of tumor cells. To unveil molecular pathways implicated in breast cancer cell invasion and migration, an in vitro set-up was designed to extract total RNA for microarray hybridisation from isolated migratory and invasive breast cancer cells at an early and a late time point. These target populations were compared to corresponding non-migratory and non-invasive cells for migration- and invasion-related expression profiling to assess potential prognostic relevance. Methods and results A 24-well Transwell set-up was used to perform both migration and invasion experiments on MDA-MB-231 breast cancer cells after 24h serum starvation (400000 cells/insert). A Matrigel coating (20%/15μL) was applied to the bottom-side of insert membranes in both settings. For invasion experiments, a layer of Matrigel (20%/20μL) was added on top of the membranes. Total RNA was isolated at early and late timepoints from both migratory / invasive and reference cells and hybridized onto Illumina HumanHT-12 v4 BeadChips in biological triplicates. Normalization and differential gene expression analysis were performed using the Limma-package in BioConductor. Lists of differentially expressed genes were analyzed using Ingenuity software. Using the nearest shrunken centroid algorithm gene signatures characteristic for early or late invasive or migratory cells were generated. The resulting models were applied onto six distinct gene expression data sets of pre-treatment samples from patients with mostly lymph node negative breast cancer. Survival analysis was performed using distant metastasis-free or disease-free survival as endpoint. Although all signatures were associated with survival in at least one data set, the gene signature associated with late invasion was associated with survival (range HR: 1.403 - 1.929; all P Conclusions Gene expression characteristics of invasive and migratory breast cancer cells indicate poor prognosis on several publicly available breast cancer expression data sets. For both migration and invasion, the strongest prognostic relevance was associated with the late time points, possibly by extended differences caused by early events. The role of identified target genes is being functionally validated and additionally, these data can be subjected to multivariate regression analysis to study association with the molecular subtypes. Citation Format: Ridha Limame, Ken Op de Beeck, Steven Van Laere, Guy Van Camp, Marc Peeters, Luc Dirix, Filip Lardon, Olivier De Wever, Patrick Pauwels. Gene signatures derived from in vitro isolated migratory and invasive breast cancer cells indicate early metastatic relapse. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2633. doi:10.1158/1538-7445.AM2013-2633

Abstract 1898: Potential molecular mechanisms of intrinsic resistance to EGFR-targeting monoclonal antibodies in HNSCC

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Head and Neck Squamous Cell Carcinoma (HNSCC) comprises 90% of all head and neck cancers and is still a major cause of morbidity and mortality worldwide. The epidermal growth factor receptor (EGFR) is overexpressed in 80-95% of all HNSCC tumors. Since EGFR initiates important signal transduction pathways in HNSCC carcinogenesis, anti-EGFR therapeutics were developed in order to prevent its activation. However, many tumors remain non-responsive, since the single-agent response rate of the monoclonal antibody cetuximab is less than 15%. Therefore, biomarkers predicting sensitivity to EGFR-targeted therapy are needed. Methods: First, the IC50 (drug dose reducing cell growth to 50%) of cetuximab and panitumumab was determined on seven HNSCC cell lines by the sulforhodamine B assay. In addition, these cell lines were screened for mutations in K-Ras by High Resolution Melting Analysis, EGFRvIII mutation and HPV infection by PCR. Cell growth was monitored in real-time using an xCELLigence system (Roche Applied Science) in order to determine the optimal incubation period at which a clear effect of cetuximab treatment on cell growth could be detected. Next, “sensitive” and “resistant” cell lines were treated with 15nM cetuximab for the predefined incubation period and RNA was isolated using the Trizol method. A genome-wide gene expression analysis was performed by comparison of expression profiles of “sensitive” and “resistant” HNSCC cells. Results: Neither K-Ras nor EGFRvIII mutation and no HPV DNA could be detected. Based on the IC50 values for cetuximab and panitumumab, cell lines were either classified as “sensitive” (n=3), “resistant” (n=2) or “intermediate” (n=2). The IC50 values of the “sensitive” cell lines ranged between 0.05 - 0.43nM for cetuximab and 0.01 - 0.45nM for panitumumab. Discussion: The IC50 values of cetuximab and panitumumab were very similar, as expected since both drugs belong to the same family of EGFR-targeting therapeutics. The cell lines used in our study did not show K-Ras or EGFRvIII mutations and were HPV negative. The xCELLigence results indicate that cetuximab (15nM) starts showing a clear effect on cell growth after 13h, implying that the signalling pathways elicited by cetuximab have been activated at this time point. Preliminary results from microarray analysis indicate that intrinsic cetuximab-resistance in HNSCC can be mediated cell line-dependently by different signaling pathways. In addition, putative characteristics of epithelial-to-mesenchymal transition were seen in “resistant” compared to “sensitive” cell lines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1898. doi:1538-7445.AM2012-1898

Abstract 4300: Comparative assessment of cell viability and motility kinetics by novel real-time technology and classic endpoint assays

Background Elucidation of biological mechanisms underlying deregulated proliferation and motility of cancer cells and identification of potential therapeutic targets require accurate cell-based monitoring. The xCELLigence Real Time Cell Analysis (RTCA) technology (Roche Applied Science) constitutes a non-invasive and label-free approach to assess cell viability and motility in real time, hereby countering key features of classic label-based endpoint detection methods. Here we show results of in vitro detection of cell viability and migration on 2 cancer cell lines using xCELLigence RTCA DP and correlate these with data obtained from parallel experiments carried out using established assessment methods for each process. Methods and results Kinetic viability and migration measurements were carried out on the MDA-MB-231 (breast cancer) and A549 (lung cancer) cell lines using the xCELLigence RTCA DP instrument. Cell viability was assessed during 10 days of incubation using modified 16-well plates (E-plate) containing microelectrodes at the well bottoms for impedance-based detection of attachment, spreading and proliferation, expressed as a Cell Index (CI) value. Cell migration was measured during 38 hours using 16-well plates (CIM16-plate) consisting of an upper and a lower chamber separated by a microporous membrane equipped with a similar detection system at the bottom side. The Sulforhodamine B (SRB) assay and a 24-well Transwell system served as reference tools to assess viability and migratory kinetics. Viability was estimated by optical density (OD) reading (540 nm) of solubilized cells that were fixed and stained with SRB at a rate of 1 plate per day. Good correlations were observed between SRB and RTCA CI for 5x103 and 104 MDA-MB-231 cells/mL (Spearman9s ≤ = 0.79 and 0.84 resp) and similar doubling times (p = 0.459). The experimental Transwell design allowed dynamic quantitation of cancer cell migration by fixing and staining of the insert membranes in methanol and crystal violet in duplicates at 10 time points during a 24 hour-incubation. Pixel area quantitation showed strong correlation with xCELLigence CI (Spearman9s ≤ = 0.90 for both cell lines). However, OD measurements (590 nm) correlated even stronger with CI (Spearman9s ≤ = 0.96 and 1.00 for MDA-MB-231 and A549). Moreover, analysis of random migration indicated a significant difference between RTCA CI and area / OD (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4300. doi:1538-7445.AM2012-4300

Abstract 4319: Gene expression profiling of isolated small migratory and invasive breast cancer cells at different times during chemotaxis

Background Chemotactic cell motility and tissue invasion are key processes initiating cancer cell dissemination and metastasis. Adequate molecular profiling of chemotaxis-driven tumor cells is impeded by the transient and stochastic nature of motility occurring in only a small subset of tumor cells. To unveil molecular pathways implicated in breast cancer cell invasion and migration, an in vitro set-up was designed to isolate total RNA for microarray hybridisation from sequestered migratory and invasive breast cancer cell subpopulations at two predefined incubation stages. These target populations were compared to non-migratory and non-invasive cells to obtain a molecular picture of migratory and invasive breast cancer cell behavior. Methods and results A 24-well Transwell set-up was used to perform both migration and Matrigel invasion experiments on MDA-MB-231 breast cancer cells after 24h serum starvation (4x105 cells/insert). A Matrigel coating (20%/15μL) was applied to the bottom-side of insert membranes in both settings. For invasion experiments, a layer of Matrigel (20%/20μL) was added to the top side of the membranes. Initially, an “early” and a “late” timepoint for RNA-extraction have been selected by monitoring migration and invasion using the xCELLigence system (Roche Applied Science). High-quality total RNA was isolated at these predefined timepoints from both migratory / invasive and reference cells and hybridized onto Illumina HumanHT-12 v4 BeadChips in biological triplicates. Differential gene expression analysis and pathway analysis were done using BioConductor in R and Ingenuity Pathway Analysis respectively, considering only FDR-corrected p-values Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4319. doi:1538-7445.AM2012-4319