Tumor initiating but differentiated luminal-like breast cancer cells are highly invasive in the absence of basal-like activity Jiyoung Kima, René Villadsena, Therese Sørlieb,c, Louise Fogha, Signe Z. Grønlunda,d, Agla J. Fridriksdottira, Irene Kuhne, Fritz Rankf,1, Vera Timmermans Wielengaf, Hiroko Solvangb,g, Paul A. W. Edwardsh, Anne-Lise Børresen-Daleb,i, Lone Rønnov-Jessend, Mina J. Bisselle,2, and Ole William Petersena,2 aDepartment of Cellular and Molecular Medicine, Centre for Biological Disease Analysis and Danish Stem Cell Centre, Faculty of Health Sciences, University of Copenhagen, DK-2200 Copenhagen N, Denmark; bDepartment of Genetics, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Montebello 0310, Oslo, Norway; cCancer Stem Cell Innovation Center, Oslo University Hospital, Norwegian Radium Hospital, 0310 Oslo, Norway; dCell and Developmental Biology, Department of Biology, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark; eLife Sciences Division, Berkeley National Laboratory, Berkeley, CA 94720; fDepartment of Pathology, Rigshospitalet, DK-2100 Copenhagen Ø, Denmark; gDepartment of Biostatistics, Institute of Basic Medical Science, University of Oslo, 0317 Oslo, Norway; hDepartment of Pathology and Hutchison/MRC Research Centre, University of Cambridge, Cambridge CB2 0X2, United Kingdom; and iK. G. Jebsen Center for Breast Cancer Research, Institute for Clinical Medicine, Faculty of Clinical Medicine, University of Oslo, 0318 Oslo, Norway Contributed by Mina J. Bissell, February 28, 2012 (sent for review January 10, 2012) The majority of human breast cancers exhibit luminal epithelial these contradictions is the concept of tumor cell plasticity, i.e., the differentiation. However, most aggressive behavior, including possibility that differentiated luminal cells must acquire basal-like invasion and purported cancer stem cell activity, are considered traits to become malignant (10–14). We set out to examine whether characteristics of basal-like cells. We asked the following questions: the above explanation always must be true or whether differenti- Must luminal-like breast cancer cells become basal-like to initiate ated luminal-like breast cancer cells within a basally initiated hi- tumors or to invade? Could luminally differentiated cells within a erarchy could be aggressive and stem-like in their own right. basally initiated hierarchy also be tumorigenic? To answer these To address these questions, we used two mutually exclusive questions, we used rare and mutually exclusive lineage markers to markers, milk mucin (MM) and CD271, which identify subtypes of isolate subsets of luminal-like and basal-like cells from human cells with either luminal-like differentiation or basal-like activity, respectively. MM was detected by the M18 antibody, which rec- breast tumors. We enriched for populations with or without prom- NTR inent basal-like traits from individual tumors or single cell cloning ognizes branched glycans (15), whereas CD271/p75 was detected by the ME20.4 antibody (16). We show that frankly dif- from cell lines and recovered cells with a luminal-like phenotype. ferentiated luminal-like cells without acquiring appreciable basal- Tumor cells with basal-like traits mimicked phenotypic and func- like traits can be aggressive and invasive when serially transplanted tional behavior associated with stem cells assessed by gene ex- into NOD SCID gamma (NSG) mice or tested for invasiveness in pression, mammosphere formation and lineage markers. Luminal- Boyden chambers. Furthermore, that luminal-like cells derived like cells without basal-like traits, surprisingly, were fully capable from a stem-like, basal hierarchy cannot only be tumorigenic, but of initiating invasive tumors in NOD SCID gamma (NSG) mice. In that they can also be more aggressive than their progenitors. fact, these phenotypically pure luminal-like cells generated larger and more invasive tumors than their basal-like counterparts. The Results tumorigenicity and invasive potential of the luminal-like cancer CD271 and MM, Two Distinct Differentiation Markers of Normal GCNT1 cells relied strongly on the expression of the gene , which Human Breast, Identify Distinct Subsets of Cells in Primary Tumors encodes a key glycosyltransferase controlling O-glycan branching. and Cell Lines. fi fi In a search for distinct and rare candidates in both These ndings demonstrate that basal-like cells, as de ned cur- basal and luminal compartments, we used multicolor imaging of requirement rently, are not a for breast tumor aggressiveness, normal breast tissue, primary breast carcinomas and established “ ” and that within a single tumor there are multiple stem-like cells cell lines stained with antibodies against a panel of markers, from with tumorigenic potential casting some doubt on the hypothesis which we selected two rare markers, CD271 (p75NTR) and MM of hierarchical or differentiative loss of tumorigenicity. (branched glycans, here abbreviated MM for ‘milk mucin’). These two markers were selected carefully based on their lineage clonal isolation | prospective | signatures specificity in normal breast, cell surface location, trypsin in- sensitivity and expression in cultured cells, features that made he two most frequent subtypes of human breast cancer are the them ideal for cell sorting and cloning applications (Fig. 1A In- Tluminal and the basal-like, named after their resemblance to set). Our initial staining of 53 biopsies of primary breast tumors + the two major lineages in the normal human breast (1). Luminal revealed that CD271 cells were present in distinct single cells or differentiation independently of estrogen receptor expression, small foci in 28 of 53 (53%) biopsies, of which 20 of 28 also typically includes MUC1 and simple epithelial keratins such as K19 contained distinct and nonoverlapping populations of MM+ cells − (MUC1+/K19+/ER+/ ), whereas basal-like activity is marked by expression of basal keratins K5, K14, and K17, and/or the transcription factor p63, on an estrogen receptor-low or -negative Author contributions: J.K., R.V., L.R.-J., M.J.B., and O.W.P. designed research; J.K., R.V., background (K5+/K14+/K17+/p63+/ERlo/-)(2–4). Despite the T.S., L.F., S.Z.G., F.R., and V.T.W. performed research; J.K., R.V., T.S., A.J.F., I.K., F.R., V.T.W., tempting extrapolation to luminal vs. basal cells-of-origin for lu- H.S., P.A.W.E., A.-L.B.-D., L.R.-J., M.J.B., and O.W.P. analyzed data; and J.K., R.V., I.K., L.R.-J., minal and basal-like breast cancer, respectively, there is increasing M.J.B., and O.W.P. wrote the paper. evidence to suggest that both subtypes originate from the luminal The authors declare no conflict of interest. epithelial lineage (5–7). However, once cancer is established, the Freely available online through the PNAS open access option. only cancer stem cells so far described are essentially basal-like, 1Deceased January 28th, 2011. and the majority of luminal cells within the tumors have been 2To whom correspondence may be addressed. E-mail: [email protected] or mjbissell@ characterized as more differentiated and less malignant than basal lbl.gov. cells (8, 9). Adding more complexity is the report that metastatic This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lesions are enriched in luminal cells (9). A popular explanation for 1073/pnas.1203203109/-/DCSupplemental. 6124–6129 | PNAS | April 17, 2012 | vol. 109 | no. 16 www.pnas.org/cgi/doi/10.1073/pnas.1203203109 Downloaded by guest on September 24, 2021 Primary tumors Tumor cell lines Tumor cell line A (MCF7) ER /PR/ ErbB2lo CD271 A PT1 B MCF7 CD271 MM ER /PR/ ErbB2hi MM Nuclei Parent population C PT2 D BT474 1 27 71 lo D ER /PR/ ErbB2 C CD2 E PT3 F BT549 MM MM Fig. 1. CD271 and MM, two distinct differentiation markers of normal + + human breast identify distinct subsets of cells in primary tumors and cell CD271 -derived clone MM -derived clone lines. (A–F) CD271 and MM staining define separate populations in cryostat B sections of tumors (A, C, and E) and in cultures of breast cancer cell lines (B, 60 60 D, and F) regardless of the subtype. Tumors and established cell lines rep- ± − − 50 50 resentative for ER+/PR+/ErbB2lo (A and B), ER /PR /ErbB2hi (C and D), and ER / %) 40 − lo ( 40 s PR /ErbB2 (E and F) breast tumors were stained with MM (red), CD271 l l e 30 30 (green), and nuclei (blue). (A Inset) Staining of normal breast tissue for c ive comparison. Irrespective of subtype, there was no overlap in staining be- t 20 20 tween MM+ and CD271+. (Scale bar, 25 μm.) Posi 10 10 0 0 CELL BIOLOGY ) 7 9 ry 9 1 a F7 4 aryC 474 474 5 im CF T T54 m C T M B ri TC1) M B Pr (TS B P (S BT (Table S1); 14 of 53 (26%) tumor biopsies exhibited foci of + MM cells only, and 11 of 53 (21%) showed neither CD271 nor Fig. 2. CD271+-derived clones contain MM+ populations, but freshly iso- MM staining. The above set of positive populations were present lated MM+ clones do not contain CD271+.(A) Representative FACS profiles of + + lo ± − hi − − lo in ER /PR /ErbB2 ,ER /PR /ErbB2 , and ER /PR /ErbB2 a tumor cell line (MCF7) stained with CD271 and MM. Note the L-shaped FACS subtypes of breast cancers and the corresponding sample cell profile of the parent population including CD271+ (green shade), MM+ (red − − lines chosen for further study (MCF7, BT474, and BT549) (Fig. shade) and double-negative CD271 /MM (no shade). Reanalysis of parent- 1). Multicolor imaging revealed that whereas MM was part of derived clones (encircled) reveal that the CD271+-derived clone regenerates a MUC1+/ K19+ compartment, CD271 often costained with p63, both cell types, whereas the MM+-derived clone does not generate CD271+. K17 and K5 albeit with the latter two exhibiting a more wide- (B) Frequency of CD271+ (green) and MM+ (red) cells after reanalysis of + + spread pattern of staining in general, but usually in an essentially CD271 - and MM -derived cells enriched or cloned from primary tumor (STC1) ER-and MM-negative background (Fig.