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Tasdemir et. al.
1 Resource Report 2 3 Comprehensive 2D and 3D phenotypic characterization of human 4 invasive lobular carcinoma cell lines 5 1,2 1,2 1,2 3 1,2 6 Nilgun Tasdemir , Emily Bossart , Zheqi Li , Zhu Li , Kevin M. Levine , Britta M. 4 3,5 6,7 1,2,* 7 Jacobsen , George C. Tseng , Nancy E. Davidson , Steffi Oesterreich
8 9 1Womens Cancer Research Center, University of Pittsburgh Medical Center (UPMC) Hillman Cancer 10 Center (HCC), Magee Womens Research Institute, Pittsburgh, PA 15213, USA 11 2Department of Pharmacology & Chemical Biology, University of Pittsburgh, Pittsburgh, PA 15213, 12 USA 13 3Department of Biostatistics, University of Pittsburgh, Pittsburgh, PA 15213, USA 14 4Dept. of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO 15 5Department of Computational & Systems Biology, Pittsburgh, PA 15213, USA 16 6Fred Hutchinson Cancer Center, Seattle, WA 98109, USA 17 7University of Washington, Seattle WA 98195, USA 18 19 Running Title: Characterizing human invasive lobular carcinoma cell lines 20 21 Keywords: invasive lobular carcinoma, ILC, 3D modeling, extracellular matrix, ECM 22 23 Financial Support: The work is in part funded by a Department of Defense Breakthrough 24 Fellowship Award to NT (BC160764), Shear Family Foundation grant and Susan G. Komen 25 Leadership grant to SO (SAC160073), Breast Cancer Research Foundation grants to NED and 26 SO, and a grant (#4100070287) with Pennsylvania Department of Health. The Pennsylvania 27 Department of Health specifically disclaims responsibility for any analyses, interpretations or 28 conclusions. EB is supported by a National Institutes of Health (NIH) Ruth L. Kirschstein Award 29 (1F31CA203055-01). ZL is supported by the University of Pittsburgh John S. Lazo Cancer 30 Pharmacology Fellowship. KML is supported by an individual fellowship from the NIH/NCI 31 (5F30CA203095) The project used the UPMC Hillman Cancer Center Biostatistics and Tissue 32 and Research Pathology Services (TARPS) Cores, in part supported by P30CA047904. 33 34 *Corresponding Author: 35 Steffi Oesterreich, PhD 36 [email protected] 37 Phone: 4126418555 38 Women’s Cancer Research Center 39 UPMC Hillman Cancer Center 40 204 Craft Avenue 41 Pittsburgh, PA 15213, USA
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42 Conflict of interest statement: None to report 43 44 45 46 47 Authors’ Contributions 48 49 Conception and design: N. Tasdemir, NE. Davidson, S. Oesterreich 50 Development of methodology: N. Tasdemir, L. Zhu, GC. Tseng, S. Oesterreich 51 Acquisition of data (performed experiments, processed data, etc.): N. Tasdemir, E. Bossart, Z. 52 Li, Z. Li 53 Analysis and interpretation of data (e.g. biological interpretation, statistical analysis, 54 computational analysis): N. Tasdemir, Z. Li, KM. Levine, NE. Davidson, S. Oesterreich 55 Writing, review and/or revision of the manuscript: N. Tasdemir, Z. Li, KM. Levine, BM. 56 Jacobson, GC. Tseng, NE. Davidson, S. Oesterreich 57 Study supervision: NE. Davidson and S. Oesterreich 58 59 60 61 62 63 Word count: 5,000 (main text) 64 Total number of Figures: 7 main figures, 6 supplementary figures 65 Total Number of Tables: 3 supplementary tables 66 Total Number of References: 50 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82
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83 Abstract (250 words) 84 85 Invasive lobular carcinoma (ILC) is the second most common subtype of breast cancer following 86 invasive ductal carcinoma (IDC) and characterized by the loss of E-cadherin-mediated adherens 87 junctions. Despite displaying unique histological and clinical features, ILC still remains a 88 chronically understudied disease with limited knowledge on the available laboratory research 89 models. To this end, herein we report a comprehensive 2D and 3D phenotypic characterization of 90 four Estrogen Receptor-positive human ILC cell lines - MDA-MB-134, SUM44, MDA-MB-330 91 and BCK4. Compared to the IDC cell lines MCF7, T47D and MDA-MB-231, ultra-low 92 attachment culture conditions revealed a remarkable anchorage-independence ability that was 93 unique to the ILC cells, a feature not evident in soft agar gels. 3D Collagen I and Matrigel 94 culture indicated a generally loose morphology for the ILC cell lines, which exhibited differing 95 preferences for adhesion to ECM proteins in 2D. Furthermore, ILC cells had limited migration 96 and invasion ability in wound-scratch and transwell assays with the exception of haptotaxis to 97 Collagen I. Transcriptional comparison of the cell lines confirmed the decreased cell 98 proliferation and E-cadherin-mediated intercellular junctions in ILC, while uncovering the 99 induction of novel pathways related to cyclic nucleotide phosphodiesterase activity, ion 100 channels, drug metabolism and alternative cell adhesion molecules such as N-cadherin, some of 101 which were also differentially regulated in ILC versus IDC tumors. Altogether, these studies will 102 serve as an invaluable resource for the breast cancer research community and facilitate further 103 functional discoveries towards understanding ILC, identifying novel drug targets and ultimately 104 improving the outcome of patients with ILC. 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123
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124 Introduction 125 126 Invasive lobular carcinoma (ILC) is the second most common type of breast cancer following 127 invasive ductal carcinoma (IDC), accounting for 10-15% of all cases (1). At an annual number of th 128 ~25-38,000, which is higher than ovarian cancer or melanoma, ILC is the 6 most common 129 cancer among women in US (2). Histologically IDC tumors form palpable masses or lumps, 130 while ILCs grow as small, dyscohesive cells in a single-file pattern (1,3). This unique growth 131 pattern makes mammographic detection and surgical removal of ILC difficult, complicating 132 breast conservation (3). In addition, compared to IDCs, ILCs present more frequently as multi- 133 centric and bilateral and with metastases to ovaries, peritoneum and gastrointestinal tract (1,4). 134 Paradoxically, while patients with ILC display favorable prognostic and predictive factors 135 (Estrogen Receptor [ER]-positive, Progesterone Receptor-[PR] positive, HER2-negative, low 136 Ki67 index) and are mostly treated with endocrine therapy, they exhibit more long-term
137 recurrences compared to patients with IDC, indicative of endocrine resistance (4,5). 138 139 Despite its distinctive histological and clinical features, ILC has remained a gravely understudied 140 subtype of breast cancer. The most characteristic feature of ILC is the lack of E-cadherin- 141 mediated adherens junctions, thought to be largely responsible for its single-file growth pattern 142 (6). This hallmark E-cadherin loss, found in 95% of all ILC tumors versus in only 7% of IDCs, 143 occurs through truncating mutations and loss-of-heterozygosity (6-8). Our knowledge of ILC as 144 a unique subtype of breast cancer is only recently emerging with comprehensive reports from big 145 consortia such as The Cancer Genome Atlas (TCGA) (7) and Rational Therapy for Breast Cancer 146 (RATHER) (9). Multi-omics profiling of human tumors has begun to reveal candidate disease 147 drivers such as HER2, HER3, FOXA1 and PIK3CA mutations, PTEN loss and ESR1 148 amplifications, events more frequently observed in ILC compared to IDC (7,9,10). However, the 149 functional validation of these potential drivers is hindered by the availability of few ER-positive 150 human ILC cell lines for use in the laboratory and limited knowledge on their biological 151 phenotypes. Thus there is urgent need to develop additional cell line models, as well as 152 thoroughly characterizing the cellular behaviors of the existing ones. 153 154 Our laboratory has recently reported the first profiling of ER function and endocrine response in 155 ER-positive human ILC cell lines (11). Here we go one step beyond and characterize their 156 growth and morphologies in 3D environments such as in ultra-low attachment (ULA) culture 157 (12), soft agar (13), and within/on top of ECM proteins (14,15), as well as their adhesion 158 properties in 2D (16). Using IDC cell lines for comparison, we probe their migration potential in 159 response to both soluble attractants in chemotaxis assays (17) and to substrate bound ECM 160 proteins in haptotaxis assays (18). In addition, we report on their abilities to invade Collagen I 161 and Matrigel, as well as assessing their use of amoeboid invasion in non-cross-linked Collagen I 162 gels (19,20). Comparison of transcriptional profiling data of ER-positive human ILC and IDC 163 cell lines identified a number of clinically relevant genes and pathways that provide important
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164 insights into the sub-type specific gene expression programs likely responsible for their divergent 165 biological phenotypes. Combined, our studies serve as invaluable resource for modeling ILC in 166 the laboratory and pave the way for a promising direction of research for ILC biology towards 167 new discoveries. 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203
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204 Materials and Methods 205 206 Cell culture 207 208 MDA-MB-134-VI (MDA-MB-134), MDA-MB-330, MCF-7, T47D and MDA-MB-231 were 209 obtained from the American Type Culture Collection. SUM44PE (SUM44) was purchased from 210 Asterand and BCK4 was kindly provided by Britta Jacobsen, University of Colorado Anschutz, 211 CO. Cell lines were maintained in the following media (Life Technologies) with 10% FBS: 212 MDA-MB-134 and MDA-MB-330 in 1:1 DMEM:L-15, MCF7 and MDA-MB-231 in DMEM, 213 T47D in RPMI, BCK4 in MEM with non-essential aminoacids (Life Technologies) and insulin 214 (Sigma-Aldrich). SUM44 was maintained as described (11) in DMEM-F12 with 2% charcoal 215 stripped serum and supplements. Cell lines were routinely tested to be mycoplasma free, 216 authenticated by the University of Arizona Genetics Core by Short Tandem Repeat DNA 217 profiling and kept in continuous culture for <6 months. PIK3CA plasmids in pBABE (Addgene) 218 and PTEN shRNAs in pMLPE (a kind gift from Scott W. Lowe, Memorial Sloan Kettering 219 Cancer Center, NY) were packaged as previously described (21) and cells were selected with 220 1µg/ml puromycin (Life Technologies). shRNA sequences are provided in Supplementary Table 221 1. 222 223 Immunoassays and trichrome staining 224 225 Western blots were performed as previously described (22) using 5% milk powder for blocking 226 and developed using ECL (Sigma-Aldrich). For immunofluorescence, cells grown on coverslips 227 were fixed with 4% paraformaldehyde, permeabilized and blocked with 5% Normal Goat Serum. 228 Coverslips were incubated with antibodies, washed and mounted using DAPI- containing media 229 (Thermo Fisher Scientific). Slides were imaged using a Nikon A1 advanced confocal system. 230 Details of the antibodies used are included in Supplementary Table 1. Masson’s Trichrome 231 (Sigma-Aldrich) staining was performed on a tissue microarray using manufacturer’s protocol 232 without the acetic acid step after the aniline blue. 233 234 Anchorage-independence and stemness assays 235 236 For 2D and ULA growth assays, ILC (15,000/96-well; 300,000/6-well) and IDC (5,000/96-well; 237 100,000/6-well) cells were seeded in regular (Thermo Fisher Scientific) or ULA (Corning Life 238 Sciences) 96-well plates and assayed using CellTiter-Glo (Promega) on a Promega GloMax plate 239 reader. Soft agar assays with ILC (50,000/plate) and IDC (10,000/plate) cells were performed in 240 35-mm plates (Thermo Fisher Scientific) as previously described (13,23). For mammosphere 241 assays, ILC (60,000/well) and IDC (20,000/well) cells were seeded in 6-well ULA plates 242 (Corning Life Sciences) as previously described (24) in 1:1 DMEM/Ham’s F-12 media with 20 243 ng/mL bFGF (BD Biosciences), 20 ng/mL EGF (BD Biosciences), B27 (Gibco), 2.5 mL
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244 Penicillin/Streptomycin, and 4 µg/mL Heparin (Sigma-Aldrich). All images were taken on an 245 Olympus IX83 inverted microscope. For stem cell expression experiments, cells were stained 246 with the indicated antibodies and analyzed on an LSRII flow cytometer (BD Biosciences). 247 Gating was performed using the BD FACS Diva Software and isotype antibody stainings. Details 248 of the antibodies used are included in Supplementary Table 1. 249 250 3D ECM assays 251 252 ILC (15,000/well) and IDC (5,000) cells were embedded in rat-tail Collagen I (Corning Life 253 Sciences) at 4mg/ml in 24-well plates following manufacturer’s recommendations. For Matrigel 254 (BD Biosciences) assays, ILC (5,000/well) and IDC (4,000/well) cells were seeded into single
255 wells of 8-well LabTek Chamber Slides (Thermo Fisher Scientific) as previously described (14). 256 Colonies were imaged on an Olympus IX83 inverted microscope. 257 258 ECM adhesion assays 259 260 ILC (100,000-200,000/well) and IDC (50,000-10,000/well) cells were seeded into 96-well plates 261 with the indicated coatings (Corning Life Sciences) following detachment with PBS containing 262 2mM EDTA. After incubation, plates were imaged on an Olympus IX83 inverted microscope, 263 washed twice with PBS and quantified using FluoReporter dsDNA kit (Life Technologies) 264 following manufacturer’s protocol. 265 266 Migration and invasion assays 267 268 Wound-scratch assays were performed as previously described (25,26) using the IncuCyte Zoom 269 Live Cell Imaging System (Essen Bioscience). PMA (Sigma-Aldrich) was used at 100 nM. For 270 transwell experiments, cells were serum starved overnight, plated into 8 µm inserts (Thermo 271 Fisher Scientific and Cell Biolabs) and quantified using Crystal violet following manufacturer’s 272 protocol. Images of inserts were taken on an Olympus SZX16 dissecting microscope. 273 274 Differential gene expression, pathway and survival analyses 275 276 RNA-Sequencing data of the cell lines was obtained from Marcotte et al. (27). The R package 277 DESeq2 was used for differential expression analysis and pathway enrichment analysis was 278 implemented using KEGG, BIOCARTA, REACTOME and KEGG databases as previously 279 described (22). Survival analysis was performed using the METABRIC dataset (28) as 280 previously described (22). 281 282 283
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284 Statistical analysis 285 286 Data analysis was performed using GraphPad Prism. Data is presented as mean +/- standard 287 deviation. Statistical tests used for each figure are indicated in the respective figure legends. 288 289 Supplementary methods 290 291 Detailed methods are described in Supplementary Text. 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323
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324 Results 325 326 Hormone receptor and adherens junction status 327 328 In this study, we focused on four ER-positive human ILC cell lines - MDA-MB-134, SUM44, 329 MDA-MB-330 and BCK4 and utilized the IDC cell lines MCF7, T47D and MDA-MB-231 for 330 comparative studies. As seen in Fig. 1A, Western blotting confirmed ER expression in all cell 331 lines, except for the ER-negative MDA-MB-231 cells (29). Of note, although there are 332 conflicting reports on the ER status of MDA-MB-330 cells (29,30), they displayed abundant ER 333 levels in our hands. Only BCK4 and T47D had detectable PR expression at these exposure 334 conditions. As expected, E-cadherin was absent from MDA-MB-134, SUM44, which harbor 335 CDH1 truncating mutations (30), from BCK4 cells, as well as from MDA-MB-231 cells, in 336 which the CDH1 promoter is hypermethylated (31). 337 338 Consistent with the down-regulation and/or mislocalization of other junction components in the 339 absence of E-cadherin (32,33), β-catenin expression was absent from MDA-MB-134, SUM44 340 and BCK4 cells – a result different from that in MDA-MB-231 cells, which retained β-catenin 341 expression without E-cadherin. Interestingly, MDA-MB-330 cells, which harbor a bi-allelic, 342 truncating CTNNA1 mutation (30), still expressed E-cadherin and β-catenin in the absence of α- 343 catenin. In contrast, p120-catenin (p120) was detected in all cell lines with the weakest 344 expression in BCK4 cells, which also exhibited lower α-catenin levels. Co-immunofluorescence 345 staining confirmed the absence of functional E-cadherin in ILC cell lines, which was 346 mislocalized to the cytoplasm in MDA-MB-330 cells (Fig. 1B; top). Similarly, p120 was also 347 largely cytoplasmic in ILC cell lines and in MDA-MB-231 cells, unlike its normal membranous 348 co-localization with E-cadherin in IDC cells (Fig. 1B; bottom). Collectively, these data confirm 349 the absence of functional adherens junctions in ILC cells. 350 351 352 Anchorage-independence ability 353 354 Next we assessed the anchorage-independence ability of ILC cell lines by growing them in ULA 355 conditions, which forces them into a suspension culture (12). ILC cell lines exhibited a 356 dyscohesive, scattered morphology in 2D plates consistent with their lack of adherens junctions, 357 while growing as large floating clusters in ULA plates (Fig. 2A; top). In contrast, MCF7 and 358 T47D cells were more cohesive in 2D and formed tight spheres in ULA (Fig. 2A; bottom). 359 Despite having overall slower proliferation rates compared to the IDC cells, the ILC cell lines 360 had a remarkable ability to grow equally well in 2D and ULA plates, with BCK4 showing the 361 least robust ULA phenotype (Fig. 2B). Importantly, this anchorage-independence was unique to 362 the ILC cells, as the IDC cell lines had much poorer growth in ULA versus 2D culture. 363 Interestingly, while MDA-MB-231 cells with no adherens junctions displayed a loose ULA
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364 morphology more similar to ILC than IDC cells, they had the poorest ULA growth of all the IDC 365 cell lines. 366 367 Given the superior anchorage-independent growth of ILC cell lines compared to IDC, we also 368 assessed their ability to form mammospheres, which are similarly grown in ULA plates but with 369 a selective media that enriches for stem-like cells (24). Assessment of stemness in the ILC cell 370 lines was also of interest to us given the higher expression of stem cell markers such as 371 ALDH1A1 in ILC versus IDC tumors (34,35). Despite their robust growth in ULA conditions, 372 ILC cell lines formed poorly defined, loose mammospheres that were difficult to quantify, unlike 373 the tighter MCF7 and T47D spheres (Supplementary Fig. S1A). Flow cytometric analysis of low high high low 374 stem cell markers similarly did not identify a putative CD24 /CD44 or CD49f /EPCAM 375 stem cell population in the ILC cell lines (Supplementary Fig. S1B-C). Although such a 376 population was present in MDA-MB-231 cells, this cell line did not form mammospheres as 377 robustly as MCF7 and T47D cells. Consistent with previous literature, these results indicate poor 378 mammosphere formation in cells with disrupted adherens junctions and a discordance between 379 stem cell expression and mammosphere formation ability (36). 380 381 Another form of anchorage-independence is the ability to grow in suspension in soft agar gels 382 (13). In general, ILC cell lines exhibited limited, dyscohesive growth in this semi-solid medium, 383 with BCK4 cells forming the smallest colonies (Fig. 3A; top). The ILC growth was similar to the 384 growth of MCF7 and T47D cells, the latter displaying a tighter morphology. As expected, MDA- 385 MB-231 cells formed the most robust soft agar colonies, serving as a positive control. 386 Altogether, these assays indicate that ILC cells exhibit a unique anchorage-independence ability 387 in ULA conditions, a phenotype not replicated in soft agar or mammosphere culture. 388 389 390 3D ECM growth and cell-matrix interactions 391 392 In tissues, ILC tumors grow as a single-file of cells within a dense layer of stroma rich in ECM 393 (1,6). This phenomenon can be visualized by staining human tumors with Masson’s Trichrome, 394 which clearly demonstrates higher levels of collagen fibers in ILC compared to IDC 395 (Supplementary Fig. S2). However, the 3D ECM growth of human ILC cell lines has not 396 previously been systematically analyzed. Therefore, we first embedded ILC cell lines in thick 397 Collagen I gels, where MDA-MB-134 and SUM44 cells exhibited the most robust growth, while 398 MDA-MB-330 cells displayed a looser morphology and BCK4 formed the smallest colonies 399 (Fig. 3A). Similar results were obtained when the cells were either embedded within or cultured 400 on top of Matrigel, displaying a “grape-like” morphology previously described for cells with 401 poor cell-cell adhesion (37). In contrast, MCF7 and T47D cells formed very tight colonies in all 402 ECM environments (Fig. 3B). Interestingly, MDA-MB-330 cells exhibited protrusive structures
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403 in Matrigel culture (Fig. 3A), which are more characteristic of MDA-MB-231 cells with a 404 “stellate” morphology and known invasive potential (Fig. 3B) (37). 405 406 In addition to growing cells within 3D gels, we also assayed the adhesion of cells to ECM 407 proteins in 2D to gain a deeper understanding of their cell-matrix interactions (16). To this end, 408 we seeded ILC and IDC cell lines onto plates coated with Collagen I, Collagen IV, Fibronectin, 409 Laminin or Matrigel in serum free media. We also utilized uncoated plates for comparison and 410 bovine serum albumin (BSA) coated plates as negative control for background adhesion levels. 411 While a 2-hour incubation indicated a low level of overall binding in ILC cell lines, especially in 412 MDA-MB-134 and SUM44 cells (Supplementary Fig S3A), a 16 hour-incubation resulted in 413 more efficient binding and varying cell morphologies on different matrices (Fig. 4A and 414 Supplementary Fig. S3B). The ECM protein most preferred for binding in general was 415 Collagen I (Fig. 4B), on which most cells displayed prominent adhesive protrusions (Fig. 4A), 416 followed by Collagen IV and Matrigel. 417 418 Of the ILC cell lines analyzed, MDA-MB-134 cells displayed a unique matrix interaction profile, 419 with a less overall adhesion to ECM proteins than to uncoated plates and no visible adhesive 420 protrusions on any matrix. Interestingly, unsupervised clustering of the ILC and IDC cell lines 421 using publicly available transcript profiling data (27) showed that MDA-MB-134 cells clustered 422 separately from the other ILC cell lines, displaying a unique expression pattern of genes 423 encoding both integrins (Supplementary Fig. S3C) and matrix metalloproteinases (MMPs) 424 (Supplementary Fig. S3D), which are well known mediators of cell-matrix adhesion (16). 425 Combined, these data indicate that ILC cell lines have differing morphologies in 3D ECM gels 426 and divergent adhesive properties on matrix proteins. 427 428 429 Migration and invasion potential 430 431 Next we assessed cell migration employing the commonly used wound-scratch assay, in which a 432 gap (“a wound”) is introduced into the middle of a monolayer to induce directional movement of 433 cells from the wound edges (17,25). Using the IncuCyte live-cell imaging system and capturing 434 images of cells every 4 hours, we observed very limited basal migration in the ILC cell lines 435 (Fig. 5A; left). This was in stark contrast to the IDC cells (Fig. 5A; right), which completely 436 closed the wound in as early as 24 hours (MDA-MB-231). To exogenously induce cell 437 migration, we treated the cells with phorbol myristate acetate (PMA), which activates the PKC 438 pathway and downstream actin cytoskeleton reorganization (38). PMA treatment clearly 439 triggered migratory protrusions at the edges of the wound in MCF7 cells (Fig. 5B; right) and 440 substantially increased their migration rate (Fig. 5C; right). In contrast, however, despite 441 inducing protrusions in the otherwise-round ILC cell lines (Fig. 5B; left), PMA had limited
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442 effect on their movement (Fig. 5C; left). While the strongest PMA effect was in BCK4, this cell 443 line still failed to close the wound after 72 hours. 444 445 As an alternative to the wound-scratch assay, we next utilized transwell Boyden chambers to 446 assess migration and invasion (17). As expected, the highly migratory MDA-MB-231 cells 447 exhibited substantial chemotaxis to FBS, while MCF7 and T47D cell were weakly migratory 448 (Fig. 6A). However, the ILC cell lines exhibited very limited migration to FBS in this assay. 449 Given the ECM-rich stroma of ILC tumors (1,6) (see Supplementary Fig. S2) , we also assayed 450 migration to substrate bound ECM in haptotaxis experiments (18), in which the undersides of the 451 inserts were coated with a thin layer of Collagen I. Interestingly, SUM44 and MDA-MB-330 452 cells displayed abundant haptotaxis to Collagen I over BSA in this assay (Fig. 6B), despite no 453 chemotaxis to FBS (see Fig. 6A). This result was different from MCF7 and MDA-MB-231 cells, 454 which exhibited Collagen I haptotaxis (Fig. 6B) but also chemotaxis to FBS (see Fig. 6A), 455 highlighting the unique requirement of matrix only by ILC cells for migration. However, this 456 finding did not extend to BCK4 or MDA-MB-134 cells, which did not migrate substantially in 457 either assay (Fig. 6A-B), with the phenotype of the latter being consistent with its weak ECM 458 adhesion (see Fig. 4). 459 460 Almost half of all human ILC tumors harbor activating, hotspot mutations in PIK3CA and 13% 461 have PTEN loss due to inactivating mutations or deletions (7). These alterations are known to 462 activate downstream Akt signaling, which can induce cell migration and invasion (39). While 463 MCF7 and T47D both harbor PIK3CA mutations, human ILC cell lines are wild type for 464 PIK3CA and PTEN (27). We therefore overexpressed PIK3CA mutants and knocked down 465 PTEN in MDA-MB-134 cells to potentially augment their migration ability. Interestingly, 466 compared to the respective controls, only the H1047R but not the E545K mutation activated 467 downstream Akt signaling, along with all four PTEN shRNAs in MDA-MB-134 cells, which 468 was different from the highly active MCF7 cells that harbor the endogenous E545K mutation 469 (Supplementary Fig. 4A). However, when assayed in either transwell Boyden chambers 470 (Supplementary Fig. 4B-C) or wound-scratch assays (Supplementary Fig. 4D), none of the 471 tested alterations alone were sufficient to induce cell migration in MDA-MB-134 cells. 472 473 Using the transwell Boyden chambers, we also assayed the invasion capacity of human ILC cell 474 lines. When plated in top chambers coated on the inside with either acid-extracted cross-linked 475 Collagen I (Fig. 6C) or Matrigel (Fig. 6D), the highly invasive MDA-MB-231 cells were the 476 only cell line that exhibited robust invasion; while MCF7 and T47D were weakly invasive. The 477 ILC cell lines, however, had limited invasion of either Collagen I or Matrigel in response to FBS 478 in bottom chambers. In addition to mesencyhmal invasion of cross-linked ECM proteins, cells 479 can also exhibit amoeboid invasion by squeezing through the pores in non-cross-linked ECM 480 (19,20). Given their morphological similarity to the small, round cells of melanoma and non- 481 small cell lung cancer that utilize amoeboid invasion, we also assayed this type of invasion in
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482 ILC cell lines using transwell chambers coated with pepsin-extracted Collagen I. In contrast to 483 the invasive MDA-MB-231 cells, however, ILC and IDC cell lines exhibited limited ameoboid 484 invasion in this assay (Supplementary Figure 5). Altogether, these results suggest that ILC cell 485 lines exhibit limited migration and invasion in traditional laboratory assays with the exception of 486 haptotaxis to Collagen I. 487 488 489 Transcriptional comparison of ILC and IDC cell lines and tumors 490 491 Our comprehensive analysis of the 2D and 3D phenotypes of human ILC cell lines clearly 492 demonstrated unique biological properties. In order to delineate the gene expression programs 493 that may underlie the divergent cellular phenotypes of the ILC and IDC cell lines, we performed 494 transcriptional comparison analyses using publicly available data sets (27), which covered all of 495 the cell lines used in this study except for BCK4. Importantly, of the ILC cell lines with available 496 data, we only focused on MDA-MB-134 and SUM44 cells in order to capture the differential 497 expression of E-cadherin in ILC versus IDC and therefore excluded MDA-MB-330 cells that do 498 not harbor the hallmark CDH1 mutation. In addition, ER-negative MDA-MB-231 cells were also 499 excluded from the analyses to ensure comparison between cell lines belonging to the same 500 molecular subtype (i.e. luminal), leaving MCF7 and T47D. 501 502 Despite the small number of cell lines analyzed, unsupervised hierarchical clustering of the ILC 503 and IDC cells clustered MDA-MB-134 and SUM44 closer to each other and away from MCF7 504 and T47D (Supplementary Fig. S6A). Differential expression analysis, based on a fold-change 505 cut-off value of 1.5 and false discovery rate (FDR) value of 0.05, identified 320 genes that were 506 expressed higher in ILC versus IDC cell lines and 387 that were expressed lower (Fig. 7A and 507 Supplementary Table 2). Pathway enrichment analysis on the differentially expressed genes 508 (Fig. 7B and Supplementary Table 3) indicated upregulated transmembrane protein tyrosine 509 kinase pathway in ILC cell lines, consisting of genes such as FGFR1, a known amplified 510 oncogene in ILC (40). Additional pathways revealed from this analysis included ion channel 511 activity, tyrosine metabolism, biological oxidation, cyclic nucleotide phosphodiesterase activity, 512 drug metabolism cytochrome P450 and alternative cell adhesion, with genes such as CDH2. 513 Conversely, the downregulated pathways confirmed the decreased intercellular junctions and 514 proliferation in ILC versus IDC cell lines (6,30), as well as extending to further categories such 515 as interferon signaling, amyloids, RNA Pol I transcription, extracellular structure and 516 organization, and focal adhesions, with the last category mediating cell-matrix interactions (Fig. 517 7C and Supplementary Table 3). 518 519 We recently reported a transcriptomic comparison of ILC and IDC tumors from the TCGA (7) 520 and METABRIC (28) cohorts (22). We therefore wished to determine to what extent the in vitro 521 transcriptional differences from the cell lines correlated with their in vivo counterpart from the
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522 tumors. To this end, we initially performed a principal component (PCA) analysis of all the 523 TCGA ER-positive, luminal A ILC (n=174) and IDC (n=774) tumors used in our original study 524 (22) and the four cell lines used herein (MDA-MB-134, SUM44, MCF7, T47D), which 525 somewhat separated the ILC and IDC tumors but clustered all the cell lines separately and away 526 from the tumors (Supplementary Fig. 6B). Nevertheless, overlap of the differentially expressed 527 genes between ILC and IDC tumors (22) and those between ILC and IDC cell lines 528 (Supplementary Table 2) identified 14 upregulated and 17 downregulated genes, the latter 529 including CDH1 (Fig. 7D-E). The query of these genes in the METABRIC dataset (28) revealed 530 that higher expression of PPFIBP2, as seen in cell lines and tumors from ILC versus IDC, was 531 significantly associated with worse disease-specific survival in ILC but not in IDC (Fig. 7F; 532 top). Similarly, lower expression of PLOD2, as seen in ILC versus IDC tumors and cell lines, 533 exhibited a significant association with worse disease-specific survival in ILC but not in IDC 534 (Fig. 7F; bottom). Collectively, these data highlight the sub-type specific, clinically relevant 535 gene expression programs that may account for the divergent biological phenotypes between ILC 536 and IDC cell lines and should be more deeply explored. 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564
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565 Discussion 566 567 ILC is a special subtype of breast cancer with distinct histological and clinical features from the 568 more common subtype IDC (1). Despite the clear need to expand our understanding of the 569 unique biology of ILC, there are currently few laboratory models available for research and a 570 huge gap in our knowledge on their biological properties beyond endocrine response (11). Our 571 study is the first comprehensive report on the 2D and 3D phenotypic characterization of four ER- 572 positive human ILC cell lines. Using a number of IDC cell lines for comparison, herein we 573 profiled their 2D and 3D growth, matrix interactions, migratory and invasive properties and sub- 574 type specific gene expression programs. Although a number of ER-negative ILC cell lines are 575 available (30), we limited our focus to the ER-positive models given that approximately 90% of 576 ILC tumors are of this molecular subtype (1,6). Interestingly, despite the controversial ER status 577 of MDA-MB-330 cells (29,30), we included them in our studies since in our hands they 578 exhibited abundant expression of ER, the functionality of which needs further investigation. 579 580 The unique anchorage-independence ability we observed in the human ILC cell lines suggests 581 resistance to anoikis - detachment induced apoptosis - , and is in agreement with findings in 582 mouse cell lines from a transgenic model of ILC (41). Given the well-accepted role of 583 anchorage-independence in metastasis (42), our interesting result may have important clinical 584 implications. While ILC and IDC tumors both progress to the stage of disseminated disease, 585 patients with ILC present more often with long-term endocrine-resistant recurrences (1,4,5). The 586 ability to survive in the absence of attachment to matrix may allow ILC cells to stay dormant in 587 foreign ECM environments for extended periods of time prior to re-growth and colonization. 588 Interestingly, our results indicated that this anchorage-independence ability was unique to the 589 ILC cells in the ULA settings and not evident in soft agar or mammosphere culture, suggesting a 590 context-dependent phenotype. Importantly, based on our data, testing of therapeutic agents for 591 ILC in future studies in both 2D and ULA conditions might allow uncoupling of potential effects 592 on cell proliferation versus metastasis. 593 594 Our 3D ECM experiments revealed generally loose, poorly defined colonies for the ILC cell 595 lines, which is not surprising given their defect in adherens junctions (6). Nevertheless, different 596 cell lines still displayed varying morphologies and abilities to grow in these settings. Such a 597 divergent pattern was especially evident in the ECM adhesion experiments, where MDA-MB- 598 134 generally exhibited a less preference for interacting with matrix proteins. Our in silico 599 analysis revealed a putative list of integrins and MMP proteins that may account for this 600 phenotype. Interestingly, none of the ILC cell lines analyzed assumed their native, in vivo single- 601 file morphology within or on top of 3D ECM gels on 2D matrix coatings. Since the single-file 602 pattern of the cancer cells in ILC tumors may provide important spatial and polarity cues, forcing 603 ILC cell lines to grow in linear patterns using platforms such as micro-patterned ECM surfaces 604 (43) may allow better modeling of ILC in the laboratory. Based on our data, the poor ECM
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605 adhesion of MDA-MB-134 cells makes them a less suitable choice for such future studies 606 compared to the remaining ILC cell lines. 607 608 Given their dyscohesive morphology, we expected that ILC cell lines may exhibit single cell 609 migration as opposed to the collective migration of cell lines such as MCF7. To our surprise, 610 however, in traditional wound-scratch assays, ILC cells exhibited a remarkable inability to 611 robustly migrate even with PKC or Akt activation. This result may be due to their lack of 612 adherens junctions, which makes it difficult to grow them into a complete monolayer and likely 613 prevents them from experiencing the same loss of cell polarity at the wound edge as IDC cells. In 614 transwell Boyden chambers, ILC cell lines did not exhibit substantial migration or invasion 615 except for haptotaxis of SUM44 and MDA-MB-330 cells to Collagen I, which was consistent 616 with their matrix adhesion properties and highlights the need for incorporating ECM proteins 617 into such assays. While our attempts at studying amoeboid invasion of ILC cells in non- 618 crosslinked Collagen I did not reveal much movement towards FBS, there is a clear need for 619 more sophisticated, alternative assays using micro-patterned surfaces and stromal cell types such 620 as fibroblasts to generate physiologically-relevant confined spaces and ECM tracks (43,44). 621 622 Our transcriptional comparison of ILC and IDC cell lines confirmed previously known 623 differences in E-cadherin-mediated cell-cell junctions, cell proliferation and expression of 624 transmembrane receptor tyrosine kinases such as FGFR1 (6,30,40). In addition, this analysis 625 revealed a number of novel differences in pathways such as ion channel activity, drug 626 metabolism cytochrome P450 and extracellular structure and organization. Interestingly, one 627 pathway upregulated in ILC versus IDC cell lines was related to cell-cell adhesion and consisted 628 of genes such as CDH2, which encodes N-cadherin. This pathway may provide alternative cell- 629 cell communication in the absence of E-cadherin and may be indicative of a partial epithelial-to- 630 mesenchymal transition in ILC, a result consistent with the recent RATHER report on ILC 631 tumors (9). Collectively, the genes and pathways we identified may account for the divergent 632 biological phenotypes of the ILC and IDC cell lines we observed throughout our studies. 633 634 Overlaying the gene expression data from ILC and IDC cell lines with that from tumors, we 635 observed a completely separate clustering and very little overlap. This phenomenon has 636 previously been reported for ovarian cancer (45) and is not surprising given the much higher 637 complexity of in vivo settings compared to in vitro (15,46). Future gene expression profiling 638 studies of the cell lines cultured on ECM proteins analyzed in our study and/or in the presence of 639 stromal cell types such as fibroblasts should yield a better recapitulation of tumor transcriptional 640 programs. Nevertheless, our analysis identified a number of differentially regulated genes that 641 were common between the cell lines and tumors, some of which exhibited a significant 642 correlation with disease-free survival of ILC but not IDC tumors. This approach helped generate 643 a short list of clinically relevant genes that may be pertinent to the unique ILC biology. 644
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645 PPFIBP2, also known as Liprin Beta 2, encodes a protein involved in the plasma membrane 646 recruitment of leukocyte common antigen-related receptor (LAR) protein-tyrosine phosphatases, 647 which regulate focal adhesions and mammary gland development (47). It is commonly fused to 648 oncogenes such as RET in thyroid cancer and the hypo-methylation of its enhancer is associated 649 with increased breast cancer risk (48). Since our data links PPFIB2 to poor survival specifically 650 in the ILC cohort, further functional interrogation of this understudied, candidate oncogenic 651 driver may implicate it as a novel therapeutic target in ILC. PLOD2 encodes an enzyme involved 652 in the hydroxylation of lysyl residues in collagen-like peptides and ECM remodeling (49). In 653 contrast to its promotion of metastasis in lung adenocarcinoma (50), the association of PLOD2 654 with better survival specifically in the ILC cohort suggests that decreased collagen crosslinking 655 may create a microenvironment more permissive to growth and dissemination in ILC, 656 underlining the importance of studying amoeboid migration and invasion. 657 658 In conclusion, our comprehensive characterization of the 2D and 3D phenotypes of ER-positive 659 human ILC lines revealed important insights into the unique biology of ILC. With increasing 660 interest in ILC in the laboratory and a growing list of candidate disease drivers from next- 661 generation sequencing efforts, our study will serve as an invaluable resource for the breast cancer 662 research community and as a platform to facilitate functional validation of potential therapeutic 663 targets towards improving the clinical outcome of patients with ILC. 664 665 666 667 668 669 670 Acknowledgments 671 The authors thank Dr. Jennifer Xavier for critical reading of the manuscript. 672 673 674 675 676 677 678 679 680 681 682 683 684
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685 References 686 687 1. Rakha EA, Ellis IO. Lobular breast carcinoma and its variants. Semin Diagn Pathol 688 2010;27:49-61 689 2. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin 2017;67:7-30 690 3. Evans WP, Warren Burhenne LJ, Laurie L, O'Shaughnessy KF, Castellino RA. Invasive 691 lobular carcinoma of the breast: mammographic characteristics and computer-aided 692 detection. Radiology 2002;225:182-9 693 4. Pestalozzi BC, Zahrieh D, Mallon E, Gusterson BA, Price KN, Gelber RD, et al. Distinct 694 clinical and prognostic features of infiltrating lobular carcinoma of the breast: combined 695 results of 15 International Breast Cancer Study Group clinical trials. J Clin Oncol 696 2008;26:3006-14 697 5. Metzger Filho O, Giobbie-Hurder A, Mallon E, Gusterson B, Viale G, Winer EP, et al. 698 Relative Effectiveness of Letrozole Compared With Tamoxifen for Patients With Lobular 699 Carcinoma in the BIG 1-98 Trial. J Clin Oncol 2015;33:2772-9 700 6. McCart Reed AE, Kutasovic JR, Lakhani SR, Simpson PT. Invasive lobular carcinoma of 701 the breast: morphology, biomarkers and 'omics. Breast Cancer Res 2015;17:12 702 7. Ciriello G, Gatza ML, Beck AH, Wilkerson MD, Rhie SK, Pastore A, et al. 703 Comprehensive Molecular Portraits of Invasive Lobular Breast Cancer. Cell 704 2015;163:506-19 705 8. Rakha EA, Abd El Rehim D, Pinder SE, Lewis SA, Ellis IO. E-cadherin expression in 706 invasive non-lobular carcinoma of the breast and its prognostic significance. 707 Histopathology 2005;46:685-93 708 9. Michaut M, Chin SF, Majewski I, Severson TM, Bismeijer T, de Koning L, et al. 709 Integration of genomic, transcriptomic and proteomic data identifies two biologically 710 distinct subtypes of invasive lobular breast cancer. Sci Rep 2016;6:18517 711 10. Desmedt C, Zoppoli G, Gundem G, Pruneri G, Larsimont D, Fornili M, et al. Genomic 712 Characterization of Primary Invasive Lobular Breast Cancer. J Clin Oncol 2016;34:1872- 713 81 714 11. Sikora MJ, Cooper KL, Bahreini A, Luthra S, Wang G, Chandran UR, et al. Invasive 715 lobular carcinoma cell lines are characterized by unique estrogen-mediated gene 716 expression patterns and altered tamoxifen response. Cancer Res 2014;74:1463-74 717 12. Rotem A, Janzer A, Izar B, Ji Z, Doench JG, Garraway LA, et al. Alternative to the soft- 718 agar assay that permits high-throughput drug and genetic screens for cellular 719 transformation. Proc Natl Acad Sci U S A 2015;112:5708-13 720 13. Macpherson I, Montagnier L. Agar Suspension Culture for the Selective Assay of Cells 721 Transformed by Polyoma Virus. Virology 1964;23:291-4 722 14. Lee GY, Kenny PA, Lee EH, Bissell MJ. Three-dimensional culture models of normal 723 and malignant breast epithelial cells. Nat Methods 2007;4:359-65
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724 15. Stock K, Estrada MF, Vidic S, Gjerde K, Rudisch A, Santo VE, et al. Capturing tumor 725 complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug 726 discovery. Sci Rep 2016;6:28951 727 16. Yue J, Zhang K, Chen J. Role of integrins in regulating proteases to mediate extracellular 728 matrix remodeling. Cancer Microenviron 2012;5:275-83 729 17. Justus CR, Leffler N, Ruiz-Echevarria M, Yang LV. In vitro cell migration and invasion 730 assays. J Vis Exp 2014 731 18. Carter SB. Haptotaxis and the mechanism of cell motility. Nature 1967;213:256-60 732 19. Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. 733 Nat Rev Cancer 2003;3:362-74 734 20. Sahai E, Marshall CJ. Differing modes of tumour cell invasion have distinct requirements 735 for Rho/ROCK signalling and extracellular proteolysis. Nat Cell Biol 2003;5:711-9 736 21. Fellmann C, Hoffmann T, Sridhar V, Hopfgartner B, Muhar M, Roth M, et al. An 737 optimized microRNA backbone for effective single-copy RNAi. Cell Rep 2013;5:1704- 738 13 739 22. Du T ZL, Levine KM, Tasdemir N, Lee AV, Houten BV, Tseng GC, Oesterreich S. 740 Invasive lobular and ductal breast carcinoma differ in immune response, protein 741 translation efficiency and metabolism. Sci Rep 2018;Accepted 742 23. Manni A, Wright C, Buck H. Growth factor involvement in the multihormonal regulation 743 of MCF-7 breast cancer cell growth in soft agar. Breast Cancer Res Treat 1991;20:43-52 744 24. Lombardo Y, de Giorgio A, Coombes CR, Stebbing J, Castellano L. Mammosphere 745 formation assay from human breast cancer tissues and cell lines. J Vis Exp 2015 746 25. Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive 747 method for analysis of cell migration in vitro. Nat Protoc 2007;2:329-33 748 26. Kramer N, Walzl A, Unger C, Rosner M, Krupitza G, Hengstschlager M, et al. In vitro 749 cell migration and invasion assays. Mutat Res 2013;752:10-24 750 27. Marcotte R, Sayad A, Brown KR, Sanchez-Garcia F, Reimand J, Haider M, et al. 751 Functional Genomic Landscape of Human Breast Cancer Drivers, Vulnerabilities, and 752 Resistance. Cell 2016;164:293-309 753 28. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The genomic 754 and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature 755 2012;486:346-52 756 29. Dai X, Cheng H, Bai Z, Li J. Breast Cancer Cell Line Classification and Its Relevance 757 with Breast Tumor Subtyping. J Cancer 2017;8:3131-41 758 30. Christgen M, Derksen P. Lobular breast cancer: molecular basis, mouse and cellular 759 models. Breast Cancer Res 2015;17:16 760 31. Hollestelle A, Peeters JK, Smid M, Timmermans M, Verhoog LC, Westenend PJ, et al. 761 Loss of E-cadherin is not a necessity for epithelial to mesenchymal transition in human 762 breast cancer. Breast Cancer Res Treat 2013;138:47-57
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763 32. Shibata T, Kokubu A, Sekine S, Kanai Y, Hirohashi S. Cytoplasmic p120ctn regulates 764 the invasive phenotypes of E-cadherin-deficient breast cancer. Am J Pathol 765 2004;164:2269-78 766 33. Schackmann RC, van Amersfoort M, Haarhuis JH, Vlug EJ, Halim VA, Roodhart JM, et 767 al. Cytosolic p120-catenin regulates growth of metastatic lobular carcinoma through 768 Rock1-mediated anoikis resistance. J Clin Invest 2011;121:3176-88 769 34. Zhao H, Langerod A, Ji Y, Nowels KW, Nesland JM, Tibshirani R, et al. Different gene 770 expression patterns in invasive lobular and ductal carcinomas of the breast. Mol Biol Cell 771 2004;15:2523-36 772 35. Bertucci F, Orsetti B, Negre V, Finetti P, Rouge C, Ahomadegbe JC, et al. Lobular and 773 ductal carcinomas of the breast have distinct genomic and expression profiles. Oncogene 774 2008;27:5359-72 775 36. Manuel Iglesias J, Beloqui I, Garcia-Garcia F, Leis O, Vazquez-Martin A, Eguiara A, et 776 al. Mammosphere formation in breast carcinoma cell lines depends upon expression of E- 777 cadherin. PLoS One 2013;8:e77281 778 37. Kenny PA, Lee GY, Myers CA, Neve RM, Semeiks JR, Spellman PT, et al. The 779 morphologies of breast cancer cell lines in three-dimensional assays correlate with their 780 profiles of gene expression. Mol Oncol 2007;1:84-96 781 38. Sumagin R, Robin AZ, Nusrat A, Parkos CA. Activation of PKCbetaII by PMA 782 facilitates enhanced epithelial wound repair through increased cell spreading and 783 migration. PLoS One 2013;8:e55775 784 39. Samuels Y, Diaz LA, Jr., Schmidt-Kittler O, Cummins JM, Delong L, Cheong I, et al. 785 Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 786 2005;7:561-73 787 40. Reis-Filho JS, Simpson PT, Turner NC, Lambros MB, Jones C, Mackay A, et al. FGFR1 788 emerges as a potential therapeutic target for lobular breast carcinomas. Clin Cancer Res 789 2006;12:6652-62 790 41. Derksen PW, Liu X, Saridin F, van der Gulden H, Zevenhoven J, Evers B, et al. Somatic 791 inactivation of E-cadherin and p53 in mice leads to metastatic lobular mammary 792 carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 793 2006;10:437-49 794 42. Mori S, Chang JT, Andrechek ER, Matsumura N, Baba T, Yao G, et al. Anchorage- 795 independent cell growth signature identifies tumors with metastatic potential. Oncogene 796 2009;28:2796-805 797 43. Paul CD, Mistriotis P, Konstantopoulos K. Cancer cell motility: lessons from migration 798 in confined spaces. Nat Rev Cancer 2017;17:131-40 799 44. Gaggioli C, Hooper S, Hidalgo-Carcedo C, Grosse R, Marshall JF, Harrington K, et al. 800 Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases 801 in leading and following cells. Nat Cell Biol 2007;9:1392-400
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802 45. Domcke S, Sinha R, Levine DA, Sander C, Schultz N. Evaluating cell lines as tumour 803 models by comparison of genomic profiles. Nat Commun 2013;4:2126 804 46. Abbott A. Cell culture: biology's new dimension. Nature 2003;424:870-2 805 47. Sarhan AR, Patel TR, Cowell AR, Tomlinson MG, Hellberg C, Heath JK, et al. LAR 806 protein tyrosine phosphatase regulates focal adhesions through CDK1. J Cell Sci 807 2016;129:2962-71 808 48. Wang Y, Hao DP, Li JJ, Wang L, Di LJ. Genome-wide methylome and chromatin 809 interactome identify abnormal enhancer to be risk factor of breast cancer. Oncotarget 810 2017;8:44705-19 811 49. Rautavuoma K, Takaluoma K, Passoja K, Pirskanen A, Kvist AP, Kivirikko KI, et al. 812 Characterization of three fragments that constitute the monomers of the human lysyl 813 hydroxylase isoenzymes 1-3. The 30-kDa N-terminal fragment is not required for lysyl 814 hydroxylase activity. J Biol Chem 2002;277:23084-91 815 50. Du H, Chen Y, Hou X, Huang Y, Wei X, Yu X, et al. PLOD2 regulated by transcription 816 factor FOXA1 promotes metastasis in NSCLC. Cell Death Dis 2017;8:e3143 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 841
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842 Figure Legends 843 844 Figure 1. Characteristics of the human ILC and IDC cell lines used in the study. 845 846 A. Western blotting using the indicated antibodies on whole cell lysates from ILC (left; red) and 847 IDC (right; blue) cell lines. β-Actin was used as a loading control. B. Merged images of co- 848 immunofluorescence staining for E-cadherin (red) and p120 (green) in ILC (top) and IDC 849 (bottom) cell lines. DAPI (blue) was used for counterstaining to mark nuclei. Arrows indicate 850 cytoplasmic co-localization of E-cadherin and p120 in MDA-MB-330 cells. Scale bar: 10 µm. 851 852 853 Figure 2. Human ILC cell lines exhibit superior growth in ULA culture than human IDC 854 cell lines. 855 856 A. Phase contrast light microscopy images of ILC (red; top) and IDC (blue; bottom) cell lines in 857 6-well 2D and ULA plates 4 days post plating. Scale bar: 100 µm. B. Relative growth curves 858 showing fold growth normalized to day 0 at each time point over 6 days for ILC (red; left) and 859 IDC (blue; right). Graphs show representative data from three experiments (n=6). p-values are 860 two-way ANOVA comparison of 2D and ULA. * p ≤ 0.05; **** p ≤ 0.0001. 861 862 863 Figure 3. ILC and IDC cell lines exhibit varying morphologies in 3D culture. 864 865 Phase contrast light microscopy images of (A) ILC (red) and (B) IDC (blue) cell lines in soft 866 agar (4 weeks post plating), Collagen I, Matrigel embedded and Matrigel on-top culture (ILC: 3 867 weeks post plating; IDC: 1 week post plating). Scale bar: 100 µm. 868 869 870 Figure 4. Human ILC cell lines have differing preferences for adhesion to ECM proteins. 871 872 A. Phase contrast light microscopy images of ILC (red; left) and IDC (blue; right) cell lines in 873 uncoated, Collagen I or BSA-coated plates 16 hours post-plating. Scale bar: 100 µm. B. Fold 874 adhesion (normalized to BSA) of ILC (red) and IDC (blue) cell lines 16 hours post-plating. 875 Graphs show representative data from two experiments (n=4). p-values are from ordinary one- 876 way ANOVA with Dunnett’s multiple comparison test to uncoated conditions for each cell line. 877 * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001. 878 879 880 881
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882 Figure 5. Human ILC cell exhibit limited migration ability in wound-scratch assays. 883 884 A. Snapshots of the scratch wounds in ILC (red; left) and IDC (blue; right) cell lines at the 885 indicated time points. Scale bar: 100 µm. B. Snapshots of the scratch wounds in BCK4 (red) and 886 MCF7 (blue) cell lines treated with 100 nM PMA. Arrows indicate migratory protrusions at the 887 wound edge. Scale bar: 300 µm. C. Relative wound densities over hour 0 in ILC (red; left) and 888 IDC (blue; right) cell lines over time with and without PMA treatment. Graphs shows 889 representative data from two-three experiments (n=6-8). p-values are from two-way ANOVA 890 comparison of -PMA vs +PMA. * p ≤ 0.05; **** p ≤ 0.0001. 891 892 893 Figure 6. Human ILC cell lines exhibit limited migration and invasion towards FBS but 894 SUM44 and MDA-MB-330 exhibit haptotaxis to Collagen I in transwell Boyden chamber 895 assays. 896 897 A-D. Images (top) and quantification (bottom) of crystal violet-stained inserts with ILC (red; 898 left) and IDC (blue; right) cell lines from (A) Chemotaxis (B) Haptotaxis (C) Collagen I invasion 899 and (D) Matrigel invasion assays towards the indicated attractants after 72 hours. Graphs show 900 representative data from two independent experiments (n=3 biological replicates). p-values are 901 from unpaired t-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. 902 903 904 Figure 7. Transcriptional differences between human ILC and IDC cell lines and tumors. 905 906 A. Supervised clustering heat map of ILC (red) and IDC (blue) cell lines using differentially 907 expressed genes. B-C. Lists of representative (B) upregulated and (C) downregulated pathways 908 and genes in ILC versus IDC cell lines. q-values are from Fisher’s exact test with FDR<0.05. D- 909 E. Venn diagrams (D) and lists (E) of commonly upregulated and downregulated genes between 910 ILC and IDC cell lines and tumors. F. Disease-specific survival curves for PPFIBP2 (top) and 911 PLOD2 (bottom) in ER+, Luminal A ILC (red; left) and IDC (blue; right) patients in the 912 METABRIC dataset. Patients were divided into two groups by PPFIBP2 (q3: third quadrant) and 913 PLOD2 (q1: first quadrant) expression. p-values are from log-rank test. q-values were calculated 914 using the Benjamini-Hochberg method to correct p-values for multiple comparisons testing 915 within histology. The small ILC sample size (n=60) allows for limited statistical power in 916 detecting survival differences after multiple testing correction. 917
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A B
MDA-MB-134SUM44MDA-MB-330BCK4 MCF7T47DMDA-MB-231 MDA-MB-134 MDA-MB-330 BCK4 ERα SUM44
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α-Catenin MCF7 T47D MDA-MB-231 β-Catenin p120
p120 /
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Figure 1. Characteristics of the human ILC and IDC cell lines used in the study. A. Western blotting using the indicated antibodies on whole cell lysates from ILC (left; red) and IDC (right; blue) cell lines. β-Actin was used as a loading control. B. Merged images of co-immunofluorescence staining for E-cadherin (red) and p120 (green) in ILC (top) and IDC (bottom) cell lines. DAPI (blue) was used for counterstaining to mark nuclei. Arrows indicate cytoplasmic co-localization of E-cadherin and p120 in MDA-MB-330 cells. Scale bar: 10 μm. bioRxiv preprint doi: https://doi.org/10.1101/341313; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A MDA-MB-134 SUM44 MDA-MB-330 BCK4 2D ULA
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Figure 2. Human ILC cell lines exhibit superior growth in ULA culture than human IDC cell lines. A. Phase contrast light microscopy images of ILC (red; top) and IDC (blue; bottom) cell lines in 6-well 2D and ULA plates 4 days post plating. Scale bar: 100 μm. B. Relative growth curves showing fold growth normalized to day 0 at each time point over 6 days for ILC (red; left) and IDC (blue; right). Graphs show representative data from three experiments (n=6). p-values are from two-way ANOVA comparison of 2D and ULA. ** p ≤ 0.01; **** p ≤ 0.0001. bioRxiv preprint doi: https://doi.org/10.1101/341313; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A MDA-MB-134 SUM44 MDA-MB-330 BCK4 Soft agar Collagen I Matrigel embed Matrigel on-top
MCF7 T47D MDA-MB-231 B Soft agar Collagen I Matrigel embed Matrigel on-top
Figure 3. ILC and IDC cell lines exhibit varying morphologies in 3D culture. Phase contrast light microscopy images of (A) ILC (red) and (B) IDC (blue) cell lines in soft agar (4 weeks post plating), Collagen I, Matrigel embedded and Matrigel on-top culture (ILC: 3 weeks post plating; IDC: 1 week post plating). Scale bar: 100 μm. bioRxiv preprint doi: https://doi.org/10.1101/341313; this version posted June 7, 2018. The copyright holder for this preprint (which was not A certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. uncoated Collagen I BSA uncoated Collagen I BSA MCF7 MDA-MB-134 T47D SUM44 MDA-MB-231 MDA-MB-330 BCK4
B 16 hours uncoated
10 **** Col I **** Col IV Fibronectin ****
BSA) ver Laminin (o **** Matrigel 5 *** **** **** **** ** BSA ** * **** **** **** **** * ****
dh es ion **** ****
a ** **** ** ** **** ******** **** **** Fold 0
47D BCK4 MCF7 T SUM44
MDA-MB-134 MDA-MB-330 MDA-MB-231
Figure 4. Human ILC cell lines have differing preferences for adhesion to ECM proteins. A. Phase contrast light microscopy images of ILC (red; left) and IDC (blue; right) cell lines in uncoated, Collagen I or BSA-coated plates 16 hours post-plating. Scale bar: 100 μm. B. Fold adhesion (normalized to BSA) of ILC (red) and IDC (blue) cell lines 16 hours post-plating. Graph shows representative data from two experiments (n=4). p-values are from ordinary one-way ANOVA with Dunnett’s multiple comparison test to uncoated conditions for each cell line. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001. bioRxiv preprint doi: https://doi.org/10.1101/341313; this version posted June 7, 2018. The copyright holder for this preprint (which was not A 0 hrs certified 24by peer hrs review) is the author/funder.48 hrs All rights reserved. No 0reuse hrs allowed without permission.24 hrs 48 hrs MCF7 MDA-MB-134 T47D SUM44 MDA-MB-231 MDA-MB-330
B BCK4 MCF7 BCK4 + PMA
C MDA-MB-134 SUM44 D MCF7
100 100 100 **** ce nt) ce nt) -PMA ce nt) -PMA 80 +PMA 80 +PMA 80 * 60 60 60 -PMA 40 40 40 +PMA
20 20 20 ensity (per ve wound d ensity (per ve wound d ensity ensity (per ve wound d ensity 0 0 0 0 12 24 36 48 60 72 0 12 24 36 48 60 72 0 12 24 36 48 60 72 Relati Relati Relati Time (hours) Time (hours) Time (hours)
MDA-MB-330 BCK4 T47D and MDA-MB-231 100 100
ce nt) 100 ce nt) -PMA -PMA ce nt) +PMA 80 +PMA 80 80
60 60 60
40 40 **** 40 MDA-MB-231 20 20 20
ensity (per ve wound d ensity T47D ensity (per ve wound d ensity ensity (per ve wound d ensity 0 0 0 0 12 24 36 48 60 72 0 12 24 36 48 60 72 Relati Relati 0 12 24 36 48 60 72 Relati Time (hours) Time (hours) Time (hours) Figure 5. Human ILC cell lines exhibit limited migration ability in wound-scratch assays. A. Snapshots of the scratch wounds in ILC (red; left) and IDC (blue; right) cell lines at the indicated time points. Scale bar: 100 μm. B. Snapshots of the scratch wounds in BCK4 (red) and MCF7 (blue) cell lines treated with 100 nM PMA. Arrows indicate migratory protrusions at the wound edge. Scale bar: 300 μm. C-D. Relative wound densities over hour 0 in (C) ILC (red) and (D) IDC (blue) cell lines over time with and without PMA treatment. Graphs show representative data from two-three experiments (n=6-8). p-values are from two-way ANOVA comparison of -PMA vs +PMA. * p ≤ 0.05; **** p ≤ 0.0001. bioRxiv preprint doi: https://doi.org/10.1101/341313; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A - FBS + FBS - FBS + FBS B BSA COL I BSA COL I MCF7 MCF7 MDA-MB-134 MDA-MB-134 T47D T47D SUM44 SUM44 MDA-MB-231 MDA-MB-330 MDA-MB-231 MDA-MB-330
Chemotaxis COL I Haptotaxis 2.0 *** 2.5 **** BSA -FBS BCK4
BCK4 2.0 COL I 1.5 +FBS 1.0 540 nm) 540 nm) ** ( ce ( ce 1.0 ** 0.5 *** 0.5 **
*** Absorban Absorban *** ** 0.0 0.0
47D 47D BCK4 MCF7 T BCK4 MCF7 T SUM44 - FBS + FBS SUM44
MDA-MB-134 MDA-MB-330 MDA-MB-231 MDA-MB-134 MDA-MB-330 MDA-MB-231
C - FBS + FBS - FBS + FBS D - FBS + FBS - FBS + FBS MCF7 MCF7 MDA-MB-134 MDA-MB-134 T47D T47D SUM44 SUM44 MDA-MB-231 MDA-MB-231 MDA-MB-330 MDA-MB-330
Collagen I invasion Matrigel invasion 0.6 1.5 -FBS * BCK4 BCK4 -FBS +FBS +FBS **** 0.4
540 nm) 1.0 * 540 nm) ( ce ( ce
0.2 ** *** 0.5 *** * ** * Absorban Absorban 0.0 0.0
47D BCK4 MCF7 T 47D SUM44 BCK4 MCF7 T SUM44
MDA-MB-134 MDA-MB-330 MDA-MB-231 MDA-MB-134 MDA-MB-330 MDA-MB-231
Figure 6. Human ILC cell lines exhibit limited migration and invasion towards FBS but SUM44 and MDA-MB-330 exhibit haptotaxis to Collagen I in transwell Boyden chamber assays. Images (top) and quantification (bottom) of crystal violet-stained inserts with ILC (red; left) and IDC (blue; right) cell lines from (A) Chemotaxis (B) Haptotaxis (C) Collagen I invasion and (D) Matrigel invasion assays towards the indicated attractants after 72 hours. Graphs shows representative data from two independent experiments (n=3 biological replicates). p-values are from unpaired t-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. bioRxiv preprint doi: https://doi.org/10.1101/341313; this version posted June 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. A standardized log2tpm D up in ILC vs IDC up in ILC vs IDC tumors cell lines −1.5 0 1
840 14 306 IDC cell lines ILC cell lines
down in ILC vs IDC down in ILC vs IDC tumors cell lines
590 17 370
SUM44 MCF7 T47D AMB134 D M B Pathways upregulated in ILC vs IDC cells E Genes commonly regulated in both cell lines and tumors Pathway Genes q-value up down CACNA1C, CLCA2, Ion channel activity 0.001 KCNC1, KCND3 CDH1 HIST1H3H TFAP2B CCR2 TGFBI DDC, HGD, CPXM2 WDR72 ISG15 Tyrosine metabolism BBOX1 PDE4B C16orf45 MAOB, TAT 0.001 CX3CR1 KCNF1 GJB2 TSPAN7 CPEB1 PXDN PDE7B EEF1A2 CYP27B1 CYP4F3, GCLM, CLEC3B RASSF6 DPP3 Biological oxidations 0.001 NR3C2 SULF1 PGR GSTA4, PTGIS PTGIS PPFIBP2 APRT PLOD2 PCDHGB4 Cyclic nucleotide PDE10A, PDE4B, 0.007 phosphodiesterase activity PDE7B Drug metabolism ALDH3B2, GSTM1, F PPFIBP2 LumA ILC PPFIBP2 LumA IDC 0.007 Cytochrome P450 UGT1A4 Strata + PPFIBP2
0.00 q = 0.4805 0.00 q = 0.898 Disease−specific su Disease−specific su 0 5 10 15 0 5 10 15 C Years Years Pathways downregulated in ILC vs IDC cells Number at risk Number at risk 45 35 16 4 391 309 168 52
ata ata 1 r 15 11 4 3 r 0 131 90 50 16 Pathway Genes q-value St − St − − 0 5 10 15 − 0 5 10 15 Years Years HERC5, HLA-DRB1, Interferon signaling 1.88E-07 ICAM1, ISG15 GSN, HIST1H2BC, PLOD2 LumA ILC PLOD2 LumA IDC Amyloids 3.46E-06 HIST2H4A, TGFBI Strata + PLOD2
HIST1H2BK, v v 1.00 ++ 1.00 +++++ 7.30E-05 vi + vi ++++++++++++++++ RNA Pol I transcription r +++++++ + r ++++++++++++++ HIST1H3H, TAF1C ++ +++++++++++++++ +++++++++ ++++ ++++++++++++++++ 0.75 + 0.75 +++++++++++++ ++++++++++++++++ Extracellular structure AGRN, PCDHB11, ++++ + + ++++++++++++++++++++++ 0.0005 + + + + + ++ + and organization PCDHB9, SPINK5 0.50 0.50 EGFR, FLT4, LIF, 0.25 0.25 Regulation of 0.002 p = 0.0093 p = 0.57 TBX2, TIMP1 0.00 q = 0.2883 0.00 q = 0.768 cell proliferation Disease−specific su Disease−specific su CDH1, CLDN1, 0 5 10 15 0 5 10 15 Intercellular junction 0.008 Years Years AMOTL1, GJB2 Number at risk Number at risk 15 10 4 0 131 99 58 24
ata ata 0 CAV1, CAV2, r 45 36 16 7 r 1 391 300 160 44 St − St − Focal adhesion 0.008 − 0 5 10 15 − 0 5 10 15 COL5A1, MYL9 Years Years Figure 7. Transcriptional differences between human ILC and IDC cell lines and tumors. A. Supervised clustering heat map of ILC (red) and IDC (blue) cell lines using differentially expressed genes. B-C. Lists of representative (B) upregulated and (C) downregulated pathways and genes in ILC vs IDC cell lines. q-values are from Fisher’s exact test with FDR<0.05. D-E. Venn diagrams (D) and lists (E) of commonly upregulated and downregulated genes between ILC and IDC cell lines and tumors. F. Disease-specific survival curves for PPFIBP2 (top) and PLOD2 (bottom) in ER+, Luminal A ILC (red; left) and IDC (blue; right) patients in the METABRIC dataset. Patients were divided into two groups by PPFIBP2 (q3: third quadrant) and PLOD2 (q1: first quadrant) expression. p-values are from log-rank test. q-values were calculated using the Benjamini- Hochberg method to correct p-values for multiple comparisons testing within histology. The small ILC sample size (n=60) allows for limited statistical power in detecting survival differences after multiple testing correction.