Oncogene (2016), 1–12 © 2016 Macmillan Publishers Limited All rights reserved 0950-9232/16 www.nature.com/onc

ORIGINAL ARTICLE

Non-migratory tumorigenic intrinsic cancer stem cells ensure breast cancer metastasis by generation of CXCR4+ migrating cancer stem cells S Mukherjee1, A Manna1, P Bhattacharjee1, M Mazumdar1, S Saha1, S Chakraborty1, D Guha1, A Adhikary1, D Jana2, M Gorain3, SA Mukherjee4, GC Kundu3, DK Sarkar2 and T Das1 Although the role of metastatic cancer stem cells (mCSCs) in tumor progression has been well documented, our study reveals a hitherto unidentified role of tumorigenic intrinsic CSCs (iCSCs) in breast cancer metastasis. We show that unlike highly migratory mCSCs residing in the breast tumor disseminating/peripheral regions, iCSCs populate the inner mass of the tumor and are nonmigratory. However iCSCs, via paracrine signaling, induce conversion of non-stem cancer cells to CSCs that (i) are identical to the previously reported mCSCs, and (ii) in contrast to iCSCs, express chemokine receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), which is crucial for their metastatic potential. These mCSCs also demonstrate high in vivo tumorigenicity. Physical non-participation of iCSCs in metastasis is further validated in vivo, where only mCSCs are found to exist in the metastatic sites, lymph nodes and bone marrow, whereas the primary tumor retains both iCSCs and mCSCs. However, iCSCs ensure metastasis since their presence is crucial for deliverance of highly metastatic CXCR4+ mCSCs to the migrating fraction of cells. Cumulatively, these results unveil a novel role of iCSCs in breast cancer metastasis as parental regulators of CXCR4+ mCSCs, and highlight the therapeutic requisite of targeting iCSCs, but not CXCR4+ mCSCs, to restrain breast cancer metastasis from the root by inhibiting the generation of mCSCs from iCSCs. Considering the pivotal role of iCSCs in tumor metastasis, the possibility of metastasis to be a ‘stem cell phenomena’ is suggested. Oncogene advance online publication, 29 February 2016; doi:10.1038/onc.2016.26 INTRODUCTION Increasing evidence suggests that cancer development is due to a rare population of cells, termed cancer stem cells (CSCs)1 that uniquely initiates and sustains disease. Growth of primary tumors is usually not always life threatening, unlike occurrence of metastatic lesions.2 CSCs are believed to be key players in the metastatic process.3,4 In fact, a distinct subpopulation of metastatic CSCs (mCSCs) distinct from tumor-initiating intrinsic CSCs was identified residing in the periphery of the tumor, depletion of which resulted in abrogation of metastatic activity of the entire tumor.5–7 Interestingly, epithelial–mesenchymal transition (EMT) induces generation of CSC-like cells8 with enhanced expression of invasion- and metastasis-related genes9 and increased chemoresistance.10 All these informations have raised a controversy whether only mCSCs undergo metastasis or tumorigenic ‘intrinsic CSCs’ (iCSCs) also participate in the same. To get a complete picture, focus of this study was to explore the individual roles of iCSCs and mCSCs in breast cancer metastasis. Breast tumors are composed of a distinct population of multipotent CSCs having the ability to self-renew,11 presence of which has also been identified in breast cancer cell lines.12 In the present study, we have designated the CSC pool that is stably present in the inner core mass of primary human breast cancer tissue samples and pre-residing CSCs in human breast cancer cell line, MCF-7 (under non-induced conditions), as iCSCs, whereas those present in the outer tumor disseminating end of primary tumors as mCSCs5 and have discreetly focused on the individual 1

roles of iCSCs and mCSCs in the process of breast cancer migration. Our results show that the iCSCs do not take part in tumor progression directly but they pre-reside over the entire metastatic cascade by stimulating surrounding non-stem cancer cells (NSCCs) by shedding various pro-migratory factors and thereby converting these NSCCs to ‘induced CSCs’. Subsequently, these induced CSCs actually actively participate in the process of breast cancer metastasis. We have also shown that these induced CSCs represent the previously described mCSC population and are distinguishable from iCSCs by the expression of the chemokine receptor, CXCR4 (chemokine (C-X-C motif) receptor 4). Therefore, to our knowledge, we show for the first time that although CXCR4-expressing-induced CSC/mCSC subset undergoes migration, this subpopulation is not independent of the pool of iCSCs that act as the mastermind or root cause of metastasis by parenting the pool of mCSCs, an essential pre-requisite for initiating the metastasis cascade. Taken together, while the apparently inactive members of tumor cell migration, iCSCs, reside in the primary tumor, they add highly migratory mCSCs to the migrating pool, possibly to empower the migrating fraction for better enduring the rigors of the metastatic voyage. RESULTS mCSCs are highly migratory whereas iCSCs are physically non-participants of the metastatic phenomenon It is unclear whether or not both iCSCs and mCSCs have identical roles in tumor progression, differing only in their origins.13

Division of Molecular Medicine, Bose Institute, P-1/12, Calcutta Improvement Trust Scheme VII M, Kolkata, India; 2Department of Surgery, SSKM Hospital, Kolkata, India; Laboratory of Tumor Biology, Angiogenesis and Nanomedicine Research, National Centre for Cell Science (NCCS), NCCS Complex, Pune, India and 4Department of Physiology, Bankura Sammilani Medical College, Kenduadihi, Bankura, India. Correspondence: Professor T Das, Division of Molecular Medicine, Bose Institute, P-1/12 CIT Scheme VII M, Kolkata 700 054, India. E-mail: [email protected] Received 16 June 2015; revised 2 November 2015; accepted 27 November 2015 3

Tumorigenic cancer stem cells promote metastasis S Mukherjee et al

2 We purified CSC populations from the inner core mass and outer tumor disseminating region of breast tumor samples from patients5 (Supplementary Figure S1). Interestingly, CSCs of outer

regions demonstrated high migration efficiency and mesenchymal phenotype in contrast to CSCs of inner core mass (Figure 1a; Supplementary Videos S1 and S2). Further analyses revealed

Figure 1. Detection of physical participation of iCSCs and mCSCs in breast cancer migration. (a) Left panel depicts automated tracking of iCSCs and mCSCs illustrating their migratory potentials (colors of tracks generated randomly using track Mate plugin, NIH ImageJ) (n = 3). Right panel graphically illustrates the mean trajectory length of cells. (b) Hematoxylin and Eosin staining of inner mass and outer migrating front of patient-derived breast tumors. (c) Immunohistological staining of patient-derived tumor samples for the epithelial markers— E-cadherin and cytokeratin 19, mesenchymal marker vimentin and counterstaining with hematoxylin illustrate the inner non-invasive tumor mass and outer migrating front. (d) Schematic representation of the experiment showing spheroid formation from GFP-iCSCs and NSCCs of MCF-7 cells followed by their migration (left panel). Right panel shows phase contrast fluorescent images depicting spheroid in non-adherent condition followed by its two-dimensional migration under adherent conditions. (e) Left panel depicts tumorigenicity of iCSCs, NSCCs and MCF-7 cells in NOD/SCID xenograft model. Right panel graphically represents the increase in tumor volumes with time post-inoculation (n = 4 mice per group). Error bars denote the standard deviation derived from four mice per group. Data are presented as mean ± s.e.m. or representative of three independent experiments. Oncogene (2016) 1 – 12

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Tumorigenic cancer stem cells promote metastasis S Mukherjee et al

3 higher expression of the mesenchymal marker vimentin, and lower expression of epithelial markers E-cadherin and cytokeratin, in the outer disseminating end of primary tumors than the inner core mass thereby confirming the outer end of the tumor as its invasive front (Figures 1b and c). These findings indicating that the highly migratory CSCs represent mCSCs whereas the CSCs with relatively negligible migration efficiency represent iCSCs, raised the possibility that iCSCs are physically non-participants in metastatic phenomenon. In order to validate this possibility, the mammospheres generated from MCF-7 cells using green fluorescent protein (GFP)labeled iCSCs were allowed to migrate under adherent conditions in presence of proliferation blocker, Mitomycin C. Whereas GFPiCSCs resided deep within the spheroids and did not physically participate in migration, migrating population was non-fluorescent, comprised of NSCCs (Figure 1d). Before this experiment, the tumorigenicity of MCF-7-derived iCSCs as compared with NSCCs and parental MCF-7 cells was verified in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice (Figure 1e).

iCSCs induce migratory potential in NSCCs and not vice versa To confirm above results, we re-examined the migratory potentials of iCSCs, mCSCs and NSCCs purified from primary breast tumor samples, where each of iCSC and NSCC subpopulations exhibited lower migration efficiencies, whereas mCSCs migrated significantly (Figure 2a). Interestingly, a combination (1:1) of iCSCs and NSCCs furnished several fold increase in the migration potential when compared with those of individual populations (Figure 2a), thereby pointing towards the possibility of altered migratory behavior of iCSC and/or NSCC population while in combination. At this point, to overcome the shortcomings of conventional migration and invasion assay systems, we developed a modified migration assay system for studying the migration property of tumor subpopulations within three-dimensional extracellular matrix (Figure 2b; Supplementary Figure S2). Interestingly, while iCSCs failed to show significant migration in response to either NSCCs or total pool of cells, they significantly promoted migration of NSCCs as well as the total pool of cells (Figures 2c and d)

Figure 2. iCSCs alter migratory behavior of NSCCs and not vice versa. (a) Representative images from the transwell migration assay of patientderived breast tumor cells, showing Giemsa-stained iCSCs, NSCCs, NSCCs+iCSCs and mCSCs (upper panel). Lower panel shows quantitative percent cell migration (n = 3). (b) Cell migration assay setup (original image, left panel), experimental model (right panel). (c, d) Representative fluorescent images (cells represented by nuclear 40 ,6-diamidino-2-phenylindole (DAPI) staining) showing the migratory behavior of iCSCs, mCSCs, NSCCs and total breast cancer cells of patient-derived primary breast cancer cells towards each other and its graphical quantification (n = 3). Data are presented as mean ± s.e.m. or representative of three independent experiments. © 2016 Macmillan Publishers Limited

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4 thereby indicating that the altered migration property of iCSCs and NSCCs in combination had resulted from the modulation of the metastatic behavior of NSCCs by iCSCs and not vice versa. However, the migration-promoting effect of mCSCs on other tumor subpopulations was very low as compared with that of iCSCs. These findings led us to envisage the role of non-migratory iCSCs as initiators of breast cancer metastasis. iCSCs induce migration in NSCCs by secreting pro-migratory factors At this juncture, reports suggesting that CSCs secrete microenvironmental factors,14–16 directed us to hypothesize that probably iCSCs and mCSCs secrete pro-migratory factors in the tumor microenvironment. To validate our hypothesis we

compared the migration-stimulating ability of primary breast tumor-derived iCSCs and mCSCs on NSCCs after blocking a plethora of potent pro-migratory factors17 within iCSC/mCSC medium. In the first case, although each of these blocking antibodies decreased the migration-promoting effect iCSCs to a varying degree, a cocktail of blocking antibodies significantly blocked the migration. Furthermore, brefeldin A completely abrogated the migration-promoting effect of iCSCs (Figure 3a). In the second case, we found that mCSCs also exerted migrationpromoting effects on NSCCs by secreting pro-migratory factors as observed in iCSCs. However, the migration-promoting effect of mCSCs on NSCCs was significantly lower in all the cases, when compared with the effect of iCSCs (Supplementary Figure S3). As these findings indicated that iCSCs exert highest migrationpromoting potential in breast cancer cells, hereafter we focused

Figure 3. iCSCs induce migration in NSCCs by secreting a plethora of pro-migratory growth factors and cytokines. (a) Left panel shows representative fluorescent images (cells represented by nuclear DAPI staining) showing migration-promoting effect of iCSCs on NSCCs from patient-derived breast tissue samples under the effect of the following conditions: (a) control (serum-free DMEM); (b) iCSCs+serum-free DMEM; (c) +anti-EGF; (d) +anti-TGF-β1; (e) +anti-VEGF; (f) +anti-interleukin (IL)-6; (g) +anti-bFGF; (h) +anti-TNF-α (tumor necrosis factor); (i) +anti-IL-8; (j) +anti-IL-4; (k) +anti-PDGF antibodies; (l) +antibody cocktail (of all nine antibodies used); and (m) secretion blocker brefeldin A. Right panel graphically illustrates percent migration of NSCCs towards iCSCs under the above-mentioned conditions (n = 3). (b) EGF, TGF-β1, VEGF and IL-6 levels in the spent media of primary breast tissue (n = 5) and MCF-7-derived iCSCs and NSCCs, determined by enzyme linked immunosorbent assay. Data are presented as mean ± SEM or representative of three independent experiments unless otherwise mentioned. Oncogene (2016) 1 – 12

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Tumorigenic cancer stem cells promote metastasis S Mukherjee et al

5 on the mechanisms responsible for iCSC-induced migration of breast cancer cells. Among nine pro-migratory factors studied, blocking epidermal growth factor (EGF) in iCSC fraction affected migration of primary breast cancer cells maximally thereby indicating the major contribution of EGF in iCSC-mediated breast cancer migration (Figure 3a, right panel). Among the most potent pro-migratory factors, EGF, transforming growth factor-beta1 (TGF-β1), vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6), EGF levels were several folds higher in both primary breast tumor- and MCF-7 cell-derived iCSCs as compared with NSCCs. (Figure 3b; Supplementary Figure S4a). Furthermore, the migratory response of MCF-7-derived NSCCs was found to be highest towards EGF among these four factors (Supplementary Figure S4b) when exogenously added at concentrations as obtained by our enzyme linked immunosorbent assay (Figure 3b). Consistent result was obtained when a constant dose (100 pg/ml) of all the four factors was used in another set of migration assay of NSCCs from MCF-7 cell line (Supplementary Figure S4c). Taken together, our results clearly establish that iCSCs induce migration in surrounding NSCCs by secreting a plethora of pro-migratory factors, EGF being the most potent one. Probability of the involvement of additional factors has not being ruled out. iCSCs induce migration in NSCCs by generating EMT and stemness through de-differentiation: contribution of iCSC-shed EGF Next, MCF-7-derived NSCCs grown in presence of iCSC-spent medium (in 1:1 ratio with complete Dulbecco’s modified Eagle’s medium (DMEM) showed increase in vimentin and decrease in E-cadherin indicating EMT (Figure 4a; Supplementary Figure S5a and b). In line with several reports suggesting that ‘gain of stemness’ occurs during EMT induction,8,18,19 we observed presence of 13.7% CSCs within NSCCs population grown in iCSC-spent medium but not in control media (Figure 4b). Moreover, significant increase in the expression levels of dedifferentiation markers Oct4, Sox-2 and Nanog in this ‘NSCC’ population as compared with the control NSCCs (Figure 4c) indicated that the ‘gain of stemness’ in iCSC-induced NSCCs resulted from de-differentiation of NSCCs. Spent medium of EGF-silenced iCSCs (Supplementary Figure S5c) resulted in significant abrogation of EMT, ‘gain of stemness’ and de-differentiation of NSCCs, thereby confirming a major role of iCSC-shed EGF in inducing migration and stemness in NSCCs (Figures 4a and c). As the spent medium of iCSC fraction of MCF-7 cells contained around 5 ng/ml EGF (Figure 4d), we exogenously treated MCF-7derived NSCCs with 2.5 ng/ml EGF (as iCSC-spent medium was added in 1:1 ratio with control medium) for experiments of Figures 4a–c. Our results furnished generation of CSCs (5.89%), increase in de-differentiation markers, migration and EMT properties in treated NSCCs (Figures 4e–g). These results firmly established that iCSCs initiate migration in the surrounding tumor cells (NSCCs) by parallel induction of EMT and stemness among them. iCSC-induced CSCs physically participate in migration Next, we performed migration assay under conditions similar to Figure 1d. Although GFP-iCSCs remained confined to the inner core of mammospheres, non-GFP tumor cells that disseminated from the spheroids showed presence of non-GFP CSCs (Figure 4h). These results identified newly generated non-GFP CSCs, but not iCSCs, as actual physical participants of the metastatic cascade. Next experiment using cell culture inserts showed that when NSCCs+GFP-iCSCs were placed in the upper well keeping iCSC-spent medium or complete DMEM in the lower well, after 48 h the migrated cells revealed the presence of ~ 13% and ~ 5% non-GFP CSCs in the migratory fractions of MCF-7 and primary © 2016 Macmillan Publishers Limited

breast tumor cells, respectively, rest being NSCCs, in response to iCSC-spent medium. These migratory non-GFP CSCs (Figure 4i, Supplementary Figure S5d), were generated de novo from NSCCs. These findings indicated that iCSCs generate migratory CSCs de novo from NSCCs and induce migration by secreting promigratory factors. Induced CSCs are distinguishable from iCSCs by cell surface expression of CXCR4 and are identical to mCSCs We next checked whether CXCR4 could be a distinguishing marker for these iCSC-induced migrating breast CSCs. Our results showed lower CXCR4 expression in human breast cancer specimens of stage I than of stages II and III (Figure 5a). Additionally, primary breast tumor cells with higher CXCR4 exhibited higher migration potential in vitro than those with lower CXCR4 (Figure 5b). Importantly, inhibiting CXCR4 expression in these primary cells by AMD3100 resulted in significant abrogation of migration (Figure 5b). These findings indicated that CXCR4 expression is exclusively related with the migrating pool of breast cancer cells and is required for migration. Furthermore, mCSCs isolated from tumor peripheral regions were almost entirely CXCR4+ whereas no significant CXCR4 expression was found on iCSCs residing in the inner tumor mass (Figure 5c). In addition, high CXCR4 expression at the tumor disseminating end of primary tumor but not at the inner tumor mass (Figure 5d) signified CXCR4 as a probable marker for breast mCSCs and distinguished latter from iCSCs. We next isolated breast CSCs (ESA+/CD44+/CD24−/low) from axillary lymph nodes of patients, a primary site for breast cancer metastasis,20 and observed that ESA+ breast cancer cells were present in metastatic lymph nodes. Moreover, the CSCs (exclusively mCSCs) present in these lymph nodes were also CXCR4+ (Figure 5e; Supplementary Figure S6). That CXCR4 is responsible for the aggravated migration potential of mCSCs was validated by our migration assay whereby silencing CXCR4 in axillary lymph node-derived mCSCs significantly abrogated their migration potential (Figure 5f). Next, we wanted to ascertain if CXCR4 expression also distinguished the newly generated migrating CSCs from the iCSCs. Migration assay of MCF-7 cells revealed the presence of significantly greater content of CSCs in the migrating fraction of iCSC-induced set than in the control set (Figure 5g, left panel). These results indicated that this increased pool of CSCs might represent iCSC-induced CSCs, the CSCs present in the migrating fraction of the control set being mCSCs. Interestingly, unlike iCSCs, both these mCSCs and iCSC-induced CSCs were CXCR4+ (Figure 5g, right panel). These results highlighted that CXCR4 is a specific marker for induced CSCs and mCSCs. Contribution of CXCR4 in CSC migration was confirmed when CXCR4 inhibition significantly abrogated the migratory potential of iCSC-induced MCF-7 cells while failing to alter their in vitro tumorigenic potential (Figure 5h). These findings signified that CXCR4 was specifically responsible for the enhanced metastatic property of breast cancer. Cumulatively, our results suggest that both induced CSCs and mCSCs are distinguishable from iCSCs by the cell surface expression of CXCR4. Next to understand whether iCSC-induced CXCR4+ CSCs represent the mCSC subset of CSCs we took two approaches: (i) following 24 h migration assay the migrated population of primary breast cancer cells and MCF-7 cell line showing the presence of 3% and 8% CXCR4+ mCSCs, respectively, were separated from the non-migrated one, containing 1.5% and 2% CXCR4− iCSCs (Figure 6a), respectively, using 8 μm cell culture insert (Supplementary Figure S6); (ii) using this non-migrated fraction of cells containing non-migratory NSCCs and iCSCs, migration assay was repeated for another 24 h (Supplementary Figure S7). Interestingly, the migrated fraction was again found to contain 2% and 7% CXCR4+ CSCs in cases of primary breast cancer Oncogene (2016) 1 – 12

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6

and MCF-7 cells, respectively (Figure 6b). These CXCR4+ CSCs now represented induced CSCs, as mCSCs initially present in the population were already separated out. These findings indicate

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that induced CSCs and mCSCs represent the same pool of migratory CXCR4+CSCs. Therefore, hereafter we have designated the total migratory CSC pool as ‘mCSCs’.

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Tumorigenic cancer stem cells promote metastasis S Mukherjee et al

7 Presence of iCSCs is crucial for the generation of mCSCs Next, our migration assay with (i) MCF-7 cells containing both NSCCs and iCSCs, and (ii) NSCC subpopulation alone showed that although the migrated fraction of MCF-7 cells contained ~ 12-14% mCSCs, similar fraction of the NSCC set showed insignificant mCSC content (Figure 6c). These results confirmed that iCSCs are the critical factors for the optimum generation of mCSCs. Next, after 48 h migration, the migrating fraction of MCF-7 cells and iCSCs of the non-migrated fraction were removed. Remaining nonmigrated NSCCs were allowed to migrate for another 48 h when the presence of around 3% mCSCs was detected in the migrated fraction. However, when the non-migrated fraction was again allowed to migrate for another 48 h, the migrated fraction furnished insignificant amount of mCSCs (Figure 6d, left panel). This could be due to the gradual loss of paracrine regulation of NSCCs by iCSCs after removal of the latter from the total cell population. Next, after removing the migrated fraction of MCF-7 cells following 48 h migration, the non-migrating fraction was again allowed to migrate in two different sets: (i) containing both iCSCs and NSCCs and (ii) containing only NSCCs. Interestingly, after 96 h, the migrating fraction of the set with both iCSCs and NSCCs was found to contain ~ 20% mCSCs, whereas that of NSCCs alone exhibited only 5–6% of mCSCs (Figure 6d, right panel). In all these cases, mCSCs and iCSCs were identified by the presence and absence of CXCR4 expression, respectively (Supplementary Figure S8). Cumulatively, these results clearly established that iCSCs are decisive factors for optimum generation of mCSCs. iCSC-induced mCSCs are not only metastatic but also tumorigenic We next aimed to validate the metastatic potential of the mCSCs in vivo as compared with NSCCs. For the same, we purified NSCCs and mCSCs from the migrated fraction of MCF-7 cells (Supplementary Figure S9a) and performed tail vein metastasis assay to assess their lung colonization potential. As evident from Figures 7a and b, unlike NSCCs, mCSCs were highly efficient in lung colonization. These findings validate high metastatic potential of mCSCs as compared with NSCCs. As tumor metastasis involves re-seeding of the tumor at the distant metastatic sites, this role has been associated with CSCs as they are the seeds of the primary tumor itself.4 These facts indicate that mCSCs should also be endowed with tumorigenic potential as observed in case of iCSCs. Our tumorigenicity assay using NOD/SCID mice revealed that the tumorigenic potential of mCSCs was very similar to that of iCSCs (Figures 7c and d). Since we have shown that the iCSCs are highly tumorigenic as compared with NSCCs (Figure 1e), it can be concluded that mCSCs are also highly tumorigenic as compared to NSCCs, hence establishing that the mCSCs seed metastasis with much higher efficiency as compared with NSCCs.

Validation of the physical involvement of mCSCs but not iCSCs in metastasis in a natural breast cancer model mimicking patient conditions Finally we used a natural tumor condition in presence of functional immune system in order to better mimic patient conditions. For the same we developed 7,12-Dimethylbenz(a) anthracene (DMBA)-induced breast tumors in Balb/c mice and analyzed primary tumor and bone marrow specimens of the same for murine CSC markers CD49fhi/CD24med 8 along with CXCR4 expression. Our flow cytometric analyses revealed that while the primary breast tumor comprised of both CXCR4− iCSCs and CXCR4+ mCSCs, the CSCs of bone marrow specimens were exclusively CXCR4+, indicating the presence of only mCSCs (Figures 7e and f; Supplementary Figure S9b and c). These in vivo findings further validated our hypothesis that iCSCs are stationary, that is, they do not take part in breast cancer metastasis physically. Intrinsic CSCs, therefore, reside at the crest of the entire metastatic cascade since they (i) initiate migration by inducing stemness in the surrounding NSCCs that then undergo tumor dissemination and metastasis (Figure 7g), and (ii) continuously generate mCSCs, thereby rendering the migrating fraction better equipped to battle against the bottlenecks of the metastatic process. DISCUSSION Primary breast tumor is a heterogenous population of cells where a distinct hierarchy is maintained between the tumor-initiating and terminally differentiated cells.11 However, it is undetermined whether the cells responsible for metastasis arise from the tumorigenic iCSCs and whether metastasis presents the same clonal dynamics and hierarchy as the primary tumor.21 Along such lines of study, we found that the tumorigenic iCSCs of breast tumor are at the crest of the metastatic hierarchy whereby they act as ‘master regulators’ of metastasis, initiating the metastatic cascade by endowing the migrating population with mCSCs having properties of both CSCs and migrating cells. In contrast to iCSCs, we found that the previously known ‘mCSCs’ residing in breast tumor invasive ends are not capable of significantly promoting migration of NSCCs as compared with iCSCs. Our study also suggests that this ‘mCSCs’ is not an independent category of CSCs, but are generated by the primary iCSCs, and are distinguishable from iCSCs by cell surface expression of CXCR4. Our in vivo experiments using NOD/SCID xenograft models re-confirmed the high metastatic potential of iCSC-induced mCSCs as compared to NSCCs. The in vivo tumorigenic potential of these mCSCs was similar to that of the iCSCs, thereby indicating that mCSCs are both metastatic and tumoriogenic. Interestingly, complete absence of iCSC subpopulation and presence of only NSCCs and CXCR4+ mCSCs were observed at the primary metastatic sites. These findings validated the physical inertness

Figure 4. iCSCs generate migration in NSCCs by inducing EMT and stemness through de-differentiation. (a) Western blot data (left panel) and comparative flow cytometric mean fluorescence intensities (MFI) in arbitrary units (a.u.; right panel), of EMT markers E-cadherin and Vimentin in presence of DMEM with 10% fetal bovine serum (FBS) medium or iCSC spent medium+DMEM with 10% FBS (1:1) or EGF-silenced iCSC spent medium+DMEM with 10% FBS (1:1) for 48 h. (b) Flow cytometric determination of percentage of CD44+/CD24  /low CSCs generated de novo (inCSCs) from NSCCs grown in the above-mentioned conditions. (c) Western blot data (left panel) and flow cytometric MFIs (in a.u.) (right panel) of de-differentiation markers, Sox-2, Nanog and Oct-4 in NSCCs grown in above-mentioned conditions. (d) EGF levels in the spent media of iCSCs, mammospheres, total cells and NSCCs derived from MCF-7 cell line determined by enzyme linked immunosorbent assay. (e and g) Western blotting and flow cytometric determination of and MFIs (in a.u.) of Oct-4, Sox-2, Nanog, E-cadherin and Vimentin of NSCCs grown in presence of serum-free DMEM or serum-free DMEM+2.5 ng/ml exogenous EGF (calculated from EGF secretion level of iCSCs present in the above used iCSC media). (f) Flow cytometric determination of percent count of inCSCs under above-mentioned conditions. (h) Representative fluorescent images depicting two-dimensional migration of spheroids derived from GFP-iCSCs and non-GFP NSCCs of MCF-7 cells and stained for CSC markers CD44 and CD24. (i) Percent count of non-GFP CSCs in the migrated fractions of MCF-7 and primary human breast cancer cells in presence or absence of iCSC-spent media. Data are presented as mean ± SEM or representative of three independent experiments. © 2016 Macmillan Publishers Limited

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8 of iCSCs in metastasis in contrast to mCSCs. Our study, therefore, reveals that iCSCs regulate tumor metastasis by inducing stemness in the surrounding NSCCs that undergo tumor cell dissemination and metastasis with the newly acquired CSC properties as mCSCs.

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Till date, all studies regarding the role of CSCs in initiating tumor migration have focused solely on the involvement of mCSCs, thereby eliminating any chances of iCSCs, seated deep inside the primary tumors, being involved in migration. These mCSCs are distinguishable from the iCSC pool by the presence of additional

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cell surface markers, like CD26+ in colorectal cancer,22 CXCR4+ in pancreatic cancer5 and CD44v6 in colon cancer.23 In line with these reports, we found a distinct population of CSCs, physically involved in the process of breast cancer metastasis which is distinguishable from tumorigenic primary iCSCs by CXCR4+ cell surface phenotype. Several reports demonstrated that EMT encourages NSCCs to enter into a CSC-like state8,24 implying that EMT confers on epithelial carcinoma cells the set of traits that empowers them to disseminate from primary tumors, and owing to their heightened resistance properties withstand the rigors of their voyage to distant sites where they seed metastasis.9 Such EMT program is considered to be instigated by the stromal cells surrounding the tumor.25 This information again ruled out the possibility of any role had by iCSCs in the metastatic process. These two pools of CSCs, therefore, appear to be totally independent of each other, each being responsible for their distinct roles within the tumor. This is further supported by the work of Hermann et al.,5 where they have shown that, although iCSCs are embedded deep inside the tumor, mCSCs dominantly reside at the peripheries where tumor cell dissemination is

9 observed. Targeting these iCSCs, therefore, can restrain tumorigenicity but not the development of secondary tumors or distant metastasis. However, not only iCSC niche regulates tumor function and maintains CSC pool,26 but also iCSCs secrete micro-environmental factors like IL-615 and VEGF.16 These reports tempted us to re-examine the role of iCSCs, if any, in metastasis. Our study reveals that iCSCs secrete a plethora of pro-migratory factors, the level of EGF being highest (probably additional factors are also involved), which concurrently induce EMT and stemness in surrounding NSCCs through paracrine signaling to initiate and maintain tumor metastasis by inducing generation of highly metastatic CSCs. Recent studies elucidated the importance of paracrine signaling between mesenchymal stem cells (MSCs) present in the surrounding stroma of the tumor, and carcinoma cells, in generating CSC niche via EMT.27 It is possible that tumorstroma interaction further aids tumor dissemination at the outer invasive front of the tumor which explains why NSCCs residing in the periphery of the tumor disseminate but not NSCCs situated in immediate proximity to iCSCs. Therefore, our work proposes

Figure 6. inCSCs represent mCSC subpopulation which is constantly replenished in the migrating fraction by iCSCs. (a) Percent count of CD44+/CD24−/low CSCs (left panel) and CD44+/CD24− /low/CXCR4+ CSCs (right panel) in the migrated and non-migrated fractions of primary human breast cancer (n = 3) and MCF-7 cells following 24 h transwell migration assay. (b) Percent count of CD44+/CD24 − /low CSCs (left panel) and CD44+/CD24 −/low/CXCR4+ CSCs (right panel) in the migrated and non-migrated fractions of non-migrated primary breast cancer and MCF-7 cells obtained from a following another 24 h transwell migration assay. (c) Percent count of mCSCs in the migrated fractions of MCF-7derived iCSCs+NSCCs and NSCCs alone after 48 h migration assay. (d) Percent count of mCSCs in the migrated fraction of MCF-7-derived NSCCs after 48 and 96 h of migration (left panel) and in the migrated fractions of iCSCs+NSCCs and NSCCs alone after 96 h of migration (right panel). Data are presented as mean ± SEM or representative of three independent experiments.

Figure 5. CXCR4 as a distinguishing marker for the newly generated migrating inCSCs. (a) Percent count of CXCR4-positive cells in different stages of primary breast tumor as determined by flow cytometry (n = 3 patients of each stage). (b) Graphical representation of percent cell migration of different stages of primary breast tumor in presence or absence of CXCR4 inhibitor, AMD3100 (n = 3 patients of each stage). (c) Percent count of CXCR4-positive CSCs in the inner tumor mass and outer migrating front of primary breast tumor as determined by flow cytometry (n = 3). (d) Immunohistological staining for CD44, CD24 and CXCR4 in inner tumor mass and outer migrating front of breast tumor samples. (e) Flow cytometric determination of the percent count of CXCR4-positive CSCs in metastatic axillary lymph nodes of breast cancer patients (lower left panel). Representative flow cytometric data shown in upper and right panels (n = 3). (f) Representative images from the transwell migration assay showing migration of mCSCs of metastatic axillary lymph nodes in presence or absence of CXCR4 inhibitor (left panel). Right panel graphically illustrates percent cell migration (n = 3). (g) Flow cytometric data showing percent count of CSCs (left panel) and CXCR4+ CSCs (right panel) in the migrating and non-migrating populations of MCF-7 cells, in presence or absence of iCSC induction. (h, i) Sphere forming efficiency and percent cell migration of MCF-7 cells in presence of iCSC-spent medium+DMEM with 10% fetal bovine serum (FBS; 1:1) or iCSC-spent medium+DMEM with 10% FBS (1:1)+CXCR4 inhibitor for 48 h. Representative images of sphere forming and migration efficiencies provided in the respective panels. Data are presented as mean ± s.e.m. or representative of three independent experiments. © 2016 Macmillan Publishers Limited

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Figure 7. mCSCs generated by iCSCs are tumorigenic and highly metastatic in vivo. (a, b) Macroscopic analyses (a) and Hematoxylin and Eosin staining (b) of lung specimens from NOD/SCID control mice and mice with tail vein injection of MCF-7-derived NSCCs or mCSCs for in vivo metastasis assay (n = 3 mice per group). (c) Representative gross morphology of tumor xenografts grown for 16 weeks from orthotopic implantation of iCSCs or mCSCs purified from MCF-7 cells, in NOD/SCID mice. (d) Size of xenograft tumors measured upto 65th day following orthotopic injection of MCF-7-derived iCSCs or mCSCs (n = 3 mice per group). (e) Representative gross morphology of carcinogen (DMBA)induced primary breast tumors in Balb/c mice grown for 12 weeks (n = 8 mice per group). (f) Graphical representation of percent count of CSCs and CXCR4+ CSCs in carcinogen-induced breast tumor and bone marrow (BM) specimens of Balb/c mice. (g) Schematic representation of our work, depicting the role of iCSCs in breast cancer metastasis. Data are presented as mean ± s.e.m. or representative of three independent experiments.

metastasis to be a ‘stem cell phenomena’ similar to the tumorigenic process and that the metastatic phenomena also represent specific hierarchy where iCSCs reside at the crest, similar to the tumorigenesis hierarchy. G protein-coupled receptor CXCR4 is a critical factor for tumor metastasis, including tumors of breast.28 It remains functional in nearly all stages of tumor metastasis.29 Increased abundance of CXCR4 associates with enhanced metastatic potential of breast cancer cells29 and causes increase in their self-renewal activity.30 Furthermore, overexpression of NANOG (known to promote cancer stem cell characteristics in tumor cells31) leads to significant up-regulation of CXCR4 receptor in breast cancer cells accompanied by a significant increase in drug resistance.31 But, there is no detail report demonstrating the inter-relationship between CXCR4 expression and metastatic potential of breast cancer stem cells although in pancreatic cancer, two populations of cancer stem cells have been identified: CD133+ cells that maintain primary tumor growth and migratory CD133+/CXCR4+ cells at the invasive edge of the tumor that promote metastatic growth.5 In line with these information our results revealed that iCSCs induce highly migratory mCSCs from NSCCs, and the degree of progression of the breast tumor increases with CXCR4 expression, whereas inhibiting CXCR4 expression in any stage of disease progression nearly abrogates tumor cell migration Oncogene (2016) 1 – 12

potential. All these information, as well as the gaps in the existing knowledge tempted us to explore the role of CXCR4 as a marker, if any, for iCSC-induced CSCs. We found that inhibiting CXCR4 expression significantly abrogated the high metastatic potential of the induced migratory CSCs. This finding is in line with several earlier studies which have shown that either the inhibition of CXCR4/SDF-1 interaction by selective antagonists or anti-CXCR4 antibody blocks breast cancer migration in vivo.32,33 Several reports suggest that CXCR4 may be key regulator of tumor invasiveness leading to local progression and tumor metastasis.5,34 Several reports demonstrate that NSCCs can convert to CSCs ‘spontaneously’.35 We have also observed that NSCCs secrete EGF, the level of the same being several folds higher by iCSCs. As a result, the percentage of CSCs generated from NSCCs in presence of iCSC spent medium is significantly higher than that generated by NSCCs spontaneously. Taken together, our results suggest that the dynamics of NSCC to CSC conversion is principally governed by iCSCs. Recent studies advocate that therapies directed at preventing NSCC to CSC conversion should be considered as essential components of adjuvant therapies for cancer patients.18 In keeping with this notion, the knowledge gathered from our study also show that targeting mCSC alone could likely be inefficient in controlling metastatic breast tumors, since they can be re© 2016 Macmillan Publishers Limited

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11 generated through NSCC to CSC conversion mediated by iCSCs. Therefore, targeting iCSCs might possibly emerge as a better alternative to help improve the therapeutic output of metastatic breast tumor patients.

Cytokeratin positive cells39,40 were selected. Thereafter, percentage of CSCs was determined in Cytokeratin+ bone marrow cells as Ck+/CD49fhi/CD24med. Thereafter, CXCR4 expression was checked in the CSCs of the bone marrow. The percentage of CSCs in primary breast tumor was determined by Lin-/CD49fhi/CD24med cell phenotype.

MATERIALS AND METHODS Cell lines and cell culture

Migration assay system developed in our laboratory

MCF-7 cell line was obtained from National Centre for Cell Science, Pune, India, and was authenticated by short-tandem repeat analysis. Cells were routinely maintained in DMEM medium as previously described.36 Experiments were performed using cells after 3-4 passages.

Primary human tissue culture Primary human breast cancer tissue samples and axillary lymph nodes were obtained with informed consent from all subjects from Department of Surgery, IPGME&R and SSKM Hospital, Kolkata, and Bankura Sammilani Medical College, Bankura, India, in accordance with the Research Oversight Committee of IPGME&R and Bankura Sammilani Medical College, and associated research and analyses were done in Bose Institute, Kolkata, in accordance with the Institutional Human Ethics Committee. These tumors were exclusively primary site cancers that had not been treated with either chemotherapy or radiation. Total 50 such cases of breast cancer were selected of stages I, II and III (Supplementary Table S1). All samples were immediately mechanically disaggregated, digested with collagenase as previously described37 and filtered through a 30-μm filter. Tumor cells were sorted after staining with allophycocyanin (APC)-anti-CD44, phycoerythrin (PE)-anti-CD24 and non-tumor cells were depleted with a cocktail of lineage marker antibodies (fluorescein isothiocyanate (FITC)tagged anti-CD2, anti-CD3, anti-CD16, anti-CD18, anti-CD31, anti-CD45, anti-CD10 and anti-CD34). The cells were sorted twice, and purity of sorted populations was verified by flow cytometry.

In vivo tumorigenicity and metastasis assay All procedures involving NOD/SCID mice and experimental protocols were approved by Institutional Animal Care and Use Committee (IACUC) of National Centre for Cell Science, Pune, India. Unsorted MCF-7 cells, sorted iCSCs, mCSCs and NSCCs cells were collected, and 106 cells were re-suspended in 100 μl of phosphate-buffered saline/Matrigel (BD Biosciences, San Jose, CA, USA) (1:1) mixture. Cells were then injected subcutaneously into the bilateral mammary pads of female NOD/SCID mice. The mice received an estradiol supplement (0.1 mg/kg) every 4 days until the appearance of the tumor after cell injection. The mice with or without tumors were examined visually every day. Tumor volumes were calculated using the formula π/6 ((d1 × d2)3/2), where d1 and d2 are the two perpendicular diameters. The tumor size was measured upto 16th day (for experiments involving MCF-7, iCSCs and NSCCs) or 65th day (for experiments involving iCSCs and mCSCs). For metastasis assay, 2 × 105 cells were injected into the tail veins. Mice were sacrificed after 16 weeks; lungs were removed, fixed in formalin, paraffin-sectioned and stained with Hematoxylin and Eosin staining.

Our migration assay system was developed with some modifications of the under agarose chemotaxis assay.41 Around 1 × 106 cells and/or antibodies/brefeldin A were transferred into the attractor/responder holes (channel 1 or 3) as per requirement. After incubating at 37 °C for 48 h in humidified 5% CO2 incubator, the cells were fixed, stained with 40 ,6diamidino-2-phenylindole (DAPI), images were taken under fluorescence microscope (Olympus, Tokyo, Japan) and their migration was quantified and expressed in terms of percent cell migration depending upon the distance migrated from the responder hole.

Cell migration assay Cell migration assay was performed using 8.0 μm cell culture inserts (BD Biosciences). Cells were seeded at 2.5 × 105 cells/well and allowed to migrate for 6/24/48 h as previously described.42 Migratory cells were stained with giemsa stain. To quantitate migratory cells, three independent fields of migratory cells per well were photographed under bright field microscope (Leica, Wetzlar, Germany). Data was analyzed using NIH ImageJ software (Bethesda, MD, USA).

Flow cytometry To study the cell surface expression of human breast CSC markers, CD44FITC/APC and CD24-PE antibodies (BD Biosciences) were used, whereas for identification of CSCs from axillary lymph nodes, ESA-APC (BD Biosciences) antibody was used in addition to CD44 and CD24 as described earlier.43 For the purpose of identification of CSCs in mouse mammary tumors or bone marrow samples, CD49f-FITC (BD Biosciences), CD24-PE (BD Biosciences) and Cytokeratin-APC (Thermoscientific, Waltham, MA, USA) antibodies were used. and EMT and de-differentiation phenomena were quantified flow cytometrically by measuring mean fluorescence intensities of EMT markers E-cadherin (e-biosciences, San Diego, CA, USA) and Vimentin (Santa Cruz, Paso Robles, CA, USA) tagged with secondary antibody Alexa Fluor-546 (Invitrogen Life Technologies, Carlsbad, CA, USA) or of dedifferentiation markers Oct-4-PerCP-Cy5.5, Nanog-PE and Sox-2-Alexa Fluor-647 (BD Biosciences). Intracellular levels of secretory proteins, EGF, VEGF (Santa Cruz), TGF-β1 (BD Biosciences) and interleukin-6 (R & D Systems, Minneapolis, MN, USA) were determined with respective primary antibodies conjugated with PE as described earlier.44

In vitro tumorigenicity assay and mammosphere culture Mammospheres were generated from MCF-7 cells as previously described.45 In vitro tumorigenicity or tumorsphere assay was performed as described by Fillmore and Kuperwasser.43

Development of DMBA-induced primary breast tumor

Phase contrast videomicrography

These animal experiments were performed following 'Principles of laboratory animal care’ (NIH publication No. 85–23, revised in 1985) as well as Indian laws on 'Protection of Animals’ under the provision of the Ethics Committee for the purpose of control and supervision of experiments on animals, Bose Institute. For each set of experiments, virgin female Swiss albino mice weighing 20–25 g and o5 weeks age were divided into two groups of eight mice each; first group containing control mice (non-tumor bearing) and second group comprised of DMBA-treated mice. Breast tumor was induced by orally gavaging the chemical carcinogen DMBA (1.0 mg in 0.2 ml of sesame oil) once per week for 6 weeks.38 The end point of the mice in the experiments was decided by measuring the tumor size. Mice were sacrificed after 12 weeks and mammary tumors and bone marrow specimens were collected. After single-cell preparation, flow cytometry was performed with both the specimen types. In case of bone marrow sample collection, the specimen was aspirated out from the tibia bone of the tumor-bearing mice, collected in phosphate-buffered saline. Thereafter, single-cell preparation was done as before, followed by sample preparation for flow cytometry. For the purpose of detection of total breast tumor cells in the bone marrow,

To assess migration potential, live-cell imaging was performed using Olympus BX700 inverted microscope equipped for time lapse differential interference contrast imaging. An environmental chamber (Tokai Heat, Iwata-shi, Shizuoka, Japan) was used to control temperature, humidity and CO2 during imaging. In each experiment, the dish was transferred to the microscopic stage after changing media to basal media (control), 10% fetal bovine serum. Migration activity was imaged for up to 12 h using a 40 × objective (non-immersion, 0.7 numerical aperture). Images were acquired at 10 min intervals. In order to track cell migration rate, time lapse sequences from single planes (one z-position) were extracted and tracked using Speckle Tracker J plugin (NIH ImageJ).

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Statistical analysis Values are shown as s.e.m. except otherwise indicated. Comparison of multiple experimental groups was performed by two-way Analysis of Variance test. Data were analyzed and, when appropriate, significance of the differences between mean values was determined by a Student’s t-test. Results were considered significant at P ⩽ 0.05. Oncogene (2016) 1 – 12

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CONFLICT OF INTEREST The authors declare no conflict of interest.

ACKNOWLEDGEMENTS We thank Gaurisankar Sa for his helpful discussions. Authors also acknowledge Uttam K. Ghosh and Ranjan Dutta for technical assistance. This work was supported by research grants from Council of Scientific and Industrial Research (CSIR), Department of Science and Technology (DST) and Department of Biotechnology (DBT), Government of India.

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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

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