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ECM Microenvironment Regulates Collective Migration PNAS PLUS and Local Dissemination in Normal and Malignant Mammary Epithelium

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ECM Microenvironment Regulates Collective Migration PNAS PLUS and Local Dissemination in Normal and Malignant Mammary Epithelium ECM microenvironment regulates collective migration PNAS PLUS and local dissemination in normal and malignant mammary epithelium Kim-Vy Nguyen-Ngoca,b,1, Kevin J. Cheunga,b,1, Audrey Brenotc, Eliah R. Shamira,b, Ryan S. Graya,b, William C. Hinesd, Paul Yaswend, Zena Werbc,2, and Andrew J. Ewalda,b,c,2 Departments of aCell Biology and bOncology, Center for Cell Dynamics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205; cDepartment of Anatomy, University of California, San Francisco, CA 94143; and dLife Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 Contributed by Zena Werb, July 27, 2012 (sent for review March 10, 2012) Breast cancer progression involves genetic changes and changes in experimental cancer models (15). Additionally, basement mem- the extracellular matrix (ECM). To test the importance of the ECM in brane proteins and their integrin receptors have been shown to tumor cell dissemination, we cultured epithelium from primary regulate carcinoma cell behavior (16–18). A major challenge today human breast carcinomas in different ECM gels. We used basement is to distinguish the relative contributions of specific genetic and membrane gels to model the normal microenvironment and colla- microenvironmental changes to the migration and local dissemi- gen I to model the stromal ECM. In basement membrane gels, nation of carcinoma cells. malignant epithelium either was indolent or grew collectively, In vivo, there are vast differences in the soluble signals, the without protrusions. In collagen I, epithelium from the same tumor stromal cells, and the ECM microenvironments surrounding car- invaded with protrusions and disseminated cells. Importantly, cinomas and normal ducts (9). It is difficult to manipulate these collagen I induced a similar initial response of protrusions and signals independently in an intact tumor and even more challenging dissemination in both normal and malignant mammary epithelium. to assess the acute cell behavioral consequences of experimental However, dissemination of normal cells into collagen I was transient manipulations. The relative optical inaccessibility of mammalian and ceased as laminin 111 localized to the basal surface, whereas tissues led our laboratory and others to establish 3D ex vivo models dissemination of carcinoma cells was sustained throughout culture, of both normal and malignant mammary epithelial growth (5, 19– and laminin 111 was not detected. Despite the large impact of ECM 24). We have applied these techniques to test the relative impor- on migration strategy, transcriptome analysis of our 3D cultures tance of genetic and microenvironmental changes in regulating the revealed few ECM-dependent changes in RNA expression. However, pattern of collective cell migration and the likelihood of local we observed many differences between normal and malignant dissemination. epithelium, including reduced expression of cell-adhesion genes in tumors. Therefore, we tested whether deletion of an adhesion gene Results could induce sustained dissemination of nontransformed cells into An epithelial cell in a mammary duct exists in a highly structured CELL BIOLOGY collagen I. We found that deletion of P-cadherin was sufficient for 3D environment and receives extensive inputs from cell–cell, cell– sustained dissemination, but exclusively into collagen I. Our data matrix, and soluble signals. We previously identified the critical reveal that metastatic tumors preferentially disseminate in specific conditions that enable primary mammary epithelium to undergo an ECM microenvironments. Furthermore, these data suggest that organotypic program of branching morphogenesis (2). We found breaks in the basement membrane could induce invasion and that, despite extensive cell migration, normal mammary morpho- dissemination via the resulting direct contact between cancer cells genesis in 3D Matrigel cultures and in vivo occurs without ECM- and collagen I. directed protrusions (2, 3). In contrast, carcinomas in vivo can migrate with protrusions and can disseminate cells locally and to ollective cell migration is an important mechanism for both distant sites (6, 25). Because the tumor microenvironment changes Cnormal epithelial development and cancer invasion (1). Dur- in parallel with genetic changes in the cancer cells (10), it is unclear ing collective cell migration, cells move in coordinated groups and whether the protrusive migration and dissemination of carcinoma maintain cell–cell adhesion. In the normal mammary gland, ducts cells are the result of cell-intrinsic motility differences or of inter- transition from a polarized bilayer into a proliferative, motile, actions of the cancer cells with their microenvironment. Therefore, multilayered epithelium and then migrate collectively through the we exploited organotypic culture techniques to isolate and culture stromal tissue (2, 3). Mammary carcinomas also originate from fragments from individual primary human mammary carcinomas a polarized adult epithelium, transition from a simple to multi- layered organization, and migrate collectively (4, 5). Despite these similarities, normal ductal morphogenesis in vivo does not involve Author contributions: K.-V.N.-N., K.J.C., A.B., E.R.S., R.S.G., Z.W., and A.J.E. designed re- local dissemination of cells and eventually results in restoration of search; K.-V.N.-N., K.J.C., A.B., E.R.S., R.S.G., Z.W., and A.J.E. performed research; W.C.H. polarized simple epithelial architecture. In contrast, breast carci- and P.Y. contributed new reagents/analytic tools; K.-V.N.-N., K.J.C., A.B., E.R.S., R.S.G., Z.W., nomas continue to grow, disseminate cells locally, and frequently and A.J.E. analyzed data; and K.-V.N.-N., K.J.C., E.R.S., Z.W., and A.J.E. wrote the paper. metastasize to distant sites (6). These observations raise the fun- The authors declare no conflict of interest. damental question: What features of tumor progression can reg- Data deposition: The microarray data reported in this paper have been deposited in the ulate the transition from a collective to a disseminative phenotype? National Center for Biotechnology Information Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE39173). Movies are available via the Ameri- Cancer is a genetic disease, and sequencing has revealed that can Society for Cell Biology (ASCB) Cell: An Image Library, http://www.cellimagelibrary.org genes encoding cell–cell and cell–matrix adhesion proteins fre- (accession nos. 42151–42168). quently are mutated (7, 8). However, breast cancer also involves 1K.-V.N.-N. and K.J.C. contributed equally to this work. characteristic changes in the ECM and the tumor microenviron- 2To whom correspondence may be addressed. E-mail: [email protected] or aewald2@ ment (9–12). For example, collagen I is enriched and aligned at the jhmi.edu. stromal border in breast tumors (10, 13), changes in collagen I See Author Summary on page 15543 (volume 109, number 39). organization are independent negative prognostic indicators (14), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and increased collagen I crosslinking accelerates progression in 1073/pnas.1212834109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1212834109 PNAS | Published online August 23, 2012 | E2595–E2604 in different ECM microenvironments (Fig. 1A and SI Materials and thousand cells. We allocated fragments of the same tumor to dif- Methods). We first optimized the medium conditions to yield ferent 3D ECM microenvironments and observed ECM-de- consistent branching morphogenesis in samples of normal human pendent carcinoma migration strategies (Fig. 1 B–C′′). In 3D breast epithelium (Fig. S1 A–D). We then focused on two ECM Matrigel, we observed both indolent behavior and collective epi- environments: a gel composed of basement membrane proteins thelial migration (Fig. 1 B–B′′). We observed single-cell protrusions (Matrigel) to model the normal breast epithelial microenvironment from the epithelium in Matrigel only rarely and did not observe and 3 mg/mL collagen I to model the stromal matrix encountered robust collective protrusive migration. In contrast, fragments from by invading mammary carcinomas (10). Although fibrillar collagen the same primary human mammary carcinoma exhibited pro- I is present near normal mammary ducts, it remains outside an trusive migration and disseminated cells extensively into 3D gels intact basement membrane even during branching morphogenesis, of 3 mg/mL collagen I (Fig. 1 C–C′′, H, and I). Although the limiting contact with normal epithelial cells (26). extent of invasion and dissemination varied among tumor frag- ments (Fig. S2 A–C), the borders of carcinoma fragments cultured Human Mammary Carcinomas Invade and Disseminate Preferentially in Matrigel maintained an epithelial appearance without pro- into Collagen I. We explanted fragments from primary human trusions (150/155 fragments from five human tumors) (Fig. 1H), mammary carcinomas (n = 7 tumors) (Fig. S1E and SI Materials whereas the borders of carcinoma fragments in collagen I were and Methods) into 3D ECM cultures (Fig. 1A). The starting point protrusive (90/109 fragments from five human tumors) and exhibited for culture was epithelial fragments of a few hundred to a few extensive local dissemination (89/109 fragments) (Fig. 1 H and I). Free epithelium Re-embed Matrigel to Matrigel (M-M) A from 3D gels in 3D gels Matrigel to Collagen I (M-C) Matrigel Collagen I to Matrigel (C-M) Primary human breast carcinoma Collagen I Collagen I to Collagen I (C-C) 1st Round of Culture 2nd Round of Culture B Matrigel B’ B’’ 50 µm µ 20µm 0h 15h 30h 50 m F-Actin DAPI C Collagen I C’ C’’ First Round Culture µ 50 m µ 20µm 0h 15h 30h 50 m F-Actin DAPI D M-M H Protrusive Migration 100 90/109 75 40/62 50 µm 0h 45h 50 10/22 E M-C 25 % of Fragments 5/155 1/42 0/22 0 50 µm 0h 45h M M-M C-M C M-C C-C F C-M I Local Dissemination 100 89/109 50 µm 75 1h 45h 37/62 50 Second Round Culture (Matrix Switching) G C-C 5/22 25 25/155 % of Fragments 2/42 0/22 50 µm 0 1h 45h M M-M C-M C M-C C-C Fig.
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