Molecular Predictors of 3 D Morphogenesis by Breast Cancer Cell Lines in 3 D Culture

Correlative analysis of molecular markers with phenotypic signatures is the simplest model for hypothesis generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology was imaged through phase contrast microscopy, and computational methods were developed to segment and represent each colony at multiple dimensions. Subsequently, subpopulations from these morphological responses were identified through consensus clustering to reveal three clusters of round, grape-like, and stellate phenotypes. In some cases, cell lines with particular pathobiological phenotypes clustered together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grape-like phenotype). Next, associations with molecular features were realized through (i) differential analysis within each morphological cluster, and (ii) regression analysis across the entire panel of cell lines. In both cases, the dominant genes that are predictive of the morphological signatures were identified. Specifically, PPARc has been associated with the invasive stellate morphological phenotype, which corresponds to triple-negative pathobiology. PPARc has been validated through two supporting biological assays. Citation: Han J, Chang H, Giricz O, Lee GY, Baehner FL, et al. (2010) Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture. PLoS Comput Biol 6(2): e1000684. doi:10.1371/journal.pcbi.1000684 Editor: Uwe Ohler, Duke University, United States of America Received January 27, 2009; Accepted January 25, 2010; Published February 26, 2010 Copyright: 2010 Han et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research (contract DE-AC0205CH11231); the National Institutes of Health under grants U54 CA112970, CA58207, and CA090788; Susan G. Komen for the Cure (KG091136); and the Avon Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: B_Parvin@lbl.gov ¤ Current address: Genentech, South San Francisco, California, United States of America

[1]  O. Petersen,et al.  A new diploid nontumorigenic human breast epithelial cell line isolated and propagated in chemically defined medium , 1987, In Vitro Cellular & Developmental Biology.

[2]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[3]  N. Copeland,et al.  The paralemmin protein family: identification of paralemmin-2, an isoform differentially spliced to AKAP2/AKAP-KL, and of palmdelphin, a more distant cytosolic relative. , 2001, Biochemical and biophysical research communications.

[4]  C. Larabell,et al.  Reversion of the Malignant Phenotype of Human Breast Cells in Three-Dimensional Culture and In Vivo by Integrin Blocking Antibodies , 1997, The Journal of cell biology.

[5]  J. Zha,et al.  Axl as a potential therapeutic target in cancer: role of Axl in tumor growth, metastasis and angiogenesis , 2009, Oncogene.

[6]  J. Daugman.  Uncertainty relation for resolution in space, spatial frequency, and orientation optimized by two-dimensional visual cortical filters. , 1985, Journal of the Optical Society of America. A, Optics and image science.

[7]  O. Petersen,et al.  Trisomy 7p and malignant transformation of human breast epithelial cells following epidermal growth factor withdrawal. , 1996, Cancer research.

[8]  Qing Nie,et al.  Ovol2 Suppresses Cell Cycling and Terminal Differentiation of Keratinocytes by Directly Repressing c-Myc and Notch1* , 2009, The Journal of Biological Chemistry.

[9]  John A. Hartigan,et al.  Clustering Algorithms , 1975 .

[10]  Bahram Parvin,et al.  Graphical methods for quantifying macromolecules through bright field imaging , 2009, Bioinform..

[11]  Majid Ahmadi,et al.  Pattern recognition with moment invariants: A comparative study and new results , 1991, Pattern Recognit..

[12]  Mina J. Bissell,et al.  3D culture models of normal and malignant breast epithelial cells , 2006 .

[13]  Guoxiong Xu,et al.  Role of prostaglandin E2 receptors in migration of murine and human breast cancer cells. , 2003, Experimental cell research.

[14]  Yusuke Nakamura,et al.  Involvement of TMEM22 overexpression in the growth of renal cell carcinoma cells. , 2009, Oncology reports.

[15]  Ian O Ellis,et al.  Basal-like breast cancer: a critical review. , 2008, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[16]  M. Bissell,et al.  Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[17]  Kai-Kuang Ma,et al.  Rotation-invariant and scale-invariant Gabor features for texture image retrieval , 2007, Image Vis. Comput..

[18]  W. May,et al.  Tnk1/Kos1 knockout mice develop spontaneous tumors. , 2008, Cancer research.

[19]  Yoshihiro Yamanishi,et al.  Extraction of correlated gene clusters from multiple genomic data by generalized kernel canonical correlation analysis , 2003, ISMB.

[20]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[21]  M. Teague.  Image analysis via the general theory of moments , 1980 .

[22]  Z. Herceg,et al.  Depletion of the 110-Kilodalton Isoform of Poly(ADP-Ribose) Glycohydrolase Increases Sensitivity to Genotoxic and Endotoxic Stress in Mice , 2004, Molecular and Cellular Biology.

[23]  Mina J Bissell,et al.  Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. , 2010, Journal of cell science.

[24]  Shaker A Mousa,et al.  The role of tissue factor pathway inhibitor in tumor growth and metastasis. , 2007, Seminars in thrombosis and hemostasis.

[25]  Jill M Seargent,et al.  GW9662, a potent antagonist of PPARγ, inhibits growth of breast tumour cells and promotes the anticancer effects of the PPARγ agonist rosiglitazone, independently of PPARγ activation , 2004, British journal of pharmacology.

[26]  Xinrong Ma,et al.  Prostaglandin E receptor EP4 antagonism inhibits breast cancer metastasis. , 2006, Cancer research.

[27]  Taesung Park,et al.  Response projected clustering for direct association with physiological and clinical response data , 2008, BMC Bioinformatics.

[28]  R. Sharan,et al.  Cluster analysis and its applications to gene expression data. , 2002, Ernst Schering Research Foundation workshop.

[29]  J. Emerman,et al.  Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes , 1977, In Vitro.

[30]  B. S. Manjunath,et al.  Texture Features for Browsing and Retrieval of Image Data , 1996, IEEE Trans. Pattern Anal. Mach. Intell..

[31]  Heidi R. Kast-Woelbern,et al.  Rosiglitazone Induction of Insig-1 in White Adipose Tissue Reveals a Novel Interplay of Peroxisome Proliferator-activated Receptor γ and Sterol Regulatory Element-binding Protein in the Regulation of Adipogenesis* , 2004, Journal of Biological Chemistry.

[32]  P. Howe,et al.  Disabled-2 (Dab2) Is Required for Transforming Growth Factor β-induced Epithelial to Mesenchymal Transition (EMT)* , 2005, Journal of Biological Chemistry.

[33]  George M. Church,et al.  Biclustering of Expression Data , 2000, ISMB.

[34]  Manabu T. Nakamura,et al.  Gene regulation of mammalian desaturases. , 2002, Biochemical Society transactions.

[35]  T Suzuki,et al.  Peroxisome proliferator-activated receptor gamma in human breast carcinoma: a modulator of estrogenic actions. , 2006, Endocrine-related cancer.

[36]  Geoffrey J McLachlan,et al.  Selection bias in gene extraction on the basis of microarray gene-expression data , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Timothy H Murphy,et al.  Paralemmin-1, a modulator of filopodia induction is required for spine maturation. , 2008, Molecular biology of the cell.

[38]  Gordon K. Smyth,et al.  limma: Linear Models for Microarray Data , 2005 .

[39]  King-Jen Chang,et al.  Over-expression of EphB3 enhances cell-cell contacts and suppresses tumor growth in HT-29 human colon cancer cells. , 2009, Carcinogenesis.

[40]  M. Duncan,et al.  Attenuation of junctional adhesion molecule-A is a contributing factor for breast cancer cell invasion. , 2008, Cancer research.

[41]  Mitsuko Masutani,et al.  PolyADP‐ribosylation and cancer , 2007, Cancer science.

[42]  M J Bissell,et al.  Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. , 1989, Development.

[43]  M. Carey,et al.  The Enhanceosome and Transcriptional Synergy , 1998, Cell.

[44]  Jill P. Mesirov,et al.  Consensus Clustering: A Resampling-Based Method for Class Discovery and Visualization of Gene Expression Microarray Data , 2003, Machine Learning.

[45]  Renchi Yang,et al.  Peroxisome proliferator-activated receptor gamma in malignant diseases. , 2006, Critical reviews in oncology/hematology.

[46]  King-Jen Chang,et al.  Over-expression of EphB 3 enhances cell – cell contacts and suppresses tumor growth in HT-29 human colon cancer cells , 2009 .

[47]  Roded Sharan,et al.  Discovering statistically significant biclusters in gene expression data , 2002, ISMB.

[48]  Gordon K Smyth,et al.  Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2004, Statistical applications in genetics and molecular biology.

[49]  E. Aubrey Thompson,et al.  Peroxisome Proliferator-activated Receptor γ Promotes Epithelial to Mesenchymal Transformation by Rho GTPase-dependent Activation of ERK1/2* , 2006, Journal of Biological Chemistry.

[50]  Akira Masuda,et al.  Significant up-regulation of a novel gene, CLCP1, in a highly metastatic lung cancer subline as well as in lung cancers in vivo , 2002, Oncogene.

[51]  Genee Y. Lee,et al.  The morphologies of breast cancer cell lines in three‐dimensional assays correlate with their profiles of gene expression , 2007, Molecular oncology.