109426 2 merged 1374505435 - cancer research · 16 cytokines (8). these cells are present in vivo...

ADFs, a novel tumor-promoting cell type

1

Adipocyte-Derived Fibroblasts promote tumor progression and contribute to the desmoplastic reaction in 1

breast cancer. 2

Ludivine Bochet 1,2,3,#, Camille Lehuédé1,2,3#, Stéphanie Dauvillier 1,2, Yuan Yuan Wang 1,2,3, Béatrice Dirat 1,2,3, 3

Victor Laurent 1,2, Cédric Dray 1,3, Romain Guiet 1,2, Isabelle Maridonneau-Parini 1,2, Sophie Le Gonidec 1,3, 4

Bettina Couderc4, Ghislaine Escourrou 5, Philippe Valet 1,3 and Catherine Muller 1,2 * 5

6

1 Université de Toulouse, UPS, F-31077 Toulouse, France. 7

2 CNRS ; Institut de Pharmacologie et de Biologie Structurale F-31077 Toulouse, France. 8

3 Institut National de la Santé et de la Recherche Médicale, INSERM U1048, F-31432 Toulouse, France. 9

4 Institut Claudius Regaud, EA3035, F-31052 Toulouse, France. 10

5 Service d’Anatomo-Pathologie, Hôpital de Rangueil, 31403 Toulouse, France. 11

# The two first authors contributed equally to this work 12

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Running title: ADFs, a novel tumor-promoting cell type 14

Key words: Adipocytes - Breast cancer – Fibroblasts – Invasion - Wnt/β-catenin 15

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Financial Support : Studies performed in our laboratories were supported by the French National Cancer 17

Institute (INCA PL 2006–035 and INCA PL 2010-214 to PV, GE and CM), the ‘Ligue Régionale Midi-Pyrénées 18

contre le Cancer’ (Comité du Lot, de la Haute-Garonne et du Gers to CM), the Fondation de France (to PV and 19

CM) and the University of Toulouse (AO CS 2009 to CM). In addition, the Fondation pour la Recherche 20

Médicale, ANR Migre Flame 2010 and ARC #2010-120-1733 supported this work (RG and IMP). CL is a 21

recipient of a PhD fellowship from the Ligue Nationale Contre le Cancer. 22

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Corresponding author: Pr Catherine Muller, IPBS, 205 route de Narbonne, 31077 Toulouse Cedex – Tel: 33-24

561-17-59-32; Fax: 33-561-17-59-94; e-mail: [email protected] 25

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Conflict of Interest : The authors declare no conflict of interest 27

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on May 30, 2020. © 2013 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on July 31, 2013; DOI: 10.1158/0008-5472.CAN-13-0530

http://cancerres.aacrjournals.org/


2

Abstract 1

Cancer-associated fibroblasts (CAFs) comprise the majority of stromal cells in breast cancers, yet their precise 2

origins and relative functional contributions to malignant progression remain uncertain. Local invasion leads to 3

the proximity cancer cells and adipocytes, which respond by phenotypical changes to generate fibroblast-like 4

cells termed here adipocyte-derived fibroblasts (ADFs). These cells exhibit enhanced secretion of fibronectin 5

and collagen I, increased migratory/invasive abilities and increased expression of the CAF marker FSP-1 but not 6

�SMA. Generation of the ADF phenotype depends on reactivation of the Wnt/��catenin pathway in response to 7

Wnt3a secreted by tumor cells. Tumor cells co-cultivated with ADFs in 2D or spheroid culture display increased 8

invasive capabilities. In clinical specimens of breast cancer, we confirmed the presence of this new stromal sub-9

population. By defining a new stromal cell population, our results offer new opportunities for stroma-targeted 10

therapies in breast cancer. 11

12





3

Introduction 1

One of the features of breast cancer is the presence of a dense collagenous stroma, the so-called desmoplastic 2

response, comprising of fibroblast-like cells know as Cancer-Associated Fibroblasts (CAFs) (1). Accumulating 3

experimental evidence has shown that CAFs play an active role in breast cancer progression through the release 4

of growth factors and chemokines, and by contributing to the remodeling of the extracellular matrix ((2) for 5

review see (3), (4). CAFs are “activated” fibroblasts and were first identified upon morphological criteria and � 6

Smooth Muscle Actin (�SMA) expression, in the same manner as wound healing myofibroblasts (5). However, 7

this marker is not ubiquitous highlighting that CAFs is a heterogeneous cell population (6). Indeed, CAFs are 8

alternatively reported to stem from resident local fibroblasts, bone marrow derived progenitor cells, or trans-9

differentiating epithelial/endothelial cells (for review, see (4, 7)). At present, neither the origin of these cells, nor 10

the extent to which their origin determines their contribution to tumor progression can be unanimously agreed 11

upon. 12

We have recently demonstrated that invasive cancer cells have a dramatic impact on surrounding adipocytes. 13

Both in vitro and in vivo, these adipocytes exhibit a decrease in lipid content, a decreased expression of 14

adipocyte markers and an activated state indicated predominantly by the overexpression of proinflammatory 15

cytokines (8). These cells are present in vivo at the tumor invasive front and retain an adipocyte-like rounded 16

morphology. We named them Cancer-Associated Adipocytes (CAAs) (8, 9), reviewed in (10). CAAs also 17

display over-expression of ECM (such as collagen VI) and ECM related molecules (11), (12), (13), events 18

contributing to breast cancer progression. As the origin of CAFs is unclear, one attractive hypothesis is that 19

during tumor evolution, peritumoral adipocytes undergo “de-differentiation” first into CAAs then into fibroblast-20

like cells that may account for a part of the CAFs population present in the desmoplastic reaction of breast 21

cancers. Although suggested in several reviews (14) (15), this hypothesis has never been directly demonstrated 22

experimentally using combined in vitro and in vivo approaches. Independent of the effect of cancer cells, several 23

recent studies have shown that mature adipocytes are in fact plastic cells capable of “dedifferentiation” towards 24

fibroblast-like cells (16). Although limited data exists on the mechanisms that could potentially be involved in 25

this effect, the role of soluble factors such as TNF-α, Wnt3a (17), or specific mitochondrial uncoupling (18) has 26

been described. In vitro, recombinant MMP-11 is also able to “dedifferentiate” mature adipocytes (11). 27

However, the existence of this phenomenon in human pathology remains elusive. In the present study, we use 28

both in vitro and in vivo (including human tumors) approaches to demonstrate that breast cancer cells are able to 29

force mature adipocytes towards fibroblast-like cells, named Adipocyte-Derived Fibroblasts (ADFs), in a Wnt-30

dependent manner. We also extensively describe the distinctive phenotype of these new stromal cells, expressing 31

some CAFs markers like Fibroblast-Specific Protein 1/S100A4, but not αSMA, and underline their ability to 32

promote the spread of breast cancer cells. 33

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4

Materials and Methods. 1

Antibodies, probes and drugs. All the primary antibodies used in this study are presented in Supplementary 2

Table S1. The Wnt/�-catenin inhibitor, ICG-001 (19) was from Enzo Life Sciences and was used at 2.5 and 5 3

μM final concentrations. The bodipy lipid probe (used at 1μg/mL) and TO-PRO-3 (used at 1 μM final 4

concentration) were obtained from Invitrogen. 5

6

Cell Culture, cell lines and isolation of primary adipocytes. The murine 3T3-F442A pre-adipocytes cell line 7

was differentiated as previously described (20). To estimate the adipocyte phenotype, triglyceride content was 8

quantified using a colorimetric kit (Wako Chemicals), and Red Oil staining was performed as previously 9

described (20). The murine 4T1 breast cancer cell line (provided by Dr S. Vagner, INSERM U563, Toulouse, 10

France) was cultured in DMEM medium supplemented with 5 % Fetal Calf Serum (FCS). The human breast 11

cancer cell line SUM159PT (provided by Dr J Piette, IGMM, Montpellier, France) was grown in Ham F12 12

(50:50) medium complemented with 5 % FCS, 1 μg/ml hydrocortisone (Sigma, Saint-Quentin les Ulysses, 13

France) and 0. 2 UI / mL insulin. All the cell lines were used within 2 months after resuscitation of frozen 14

aliquots. Cell lines were authenticated based on viability, recovery, growth and morphology. The expression 15

status of E-Cadherin, N-cadherin and vimentin using immunofluorescence approaches, as well as their invasive 16

behavior, was confirmed before they were used in the experiments (8). Coculture of tumor cells and adipocyte 17

was performed using a Transwell culture system (0.4 μm pore size, Millipore) as previously described (8). In 18

certain experiments, cells genetically modified by lentiviral vectors encoding either eGFP (Genethon, Evry) 19

(3T3-F442A) or Hc-Red (vector core ICR, Toulouse) (SUM159PT) were used. The isolation of mammary 20

adipocytes either from tumorectomy (TU) or reduction mammoplasty (RM) was performed as previously 21

described by our group (8). 22

23

In vivo GFP-Subcutaneous Fat Pad Formation and tumor injection. 1x107 confluent GFP-3T3-F442A 24

preadipocytes were injected into the flank of athymic nude mice, as described (21). Fat pad formation was 25

allowed to proceed for 5 weeks, after which fat pads were injected with 4x104 4T1 breast cancer cells. Mice with 26

fat pad alone or tumor cells alone were used as controls. Five mice were used in each group. After 10 days, 27

mice were sacrificed and fat pad, tumors and fat pads containing tumors were embedded in paraffin after 48 h of 28

fixation in buffered formalin. The local Institutional Animal Care and Use Committee approved the experimental 29

protocol used in our study. 30

31

Immunofluorescence, immunohistochemistry. Adipocytes differentiated on coverslips grown alone, or 32

cocultivated for the indicated times with breast cancer cells were processed for immunofluorescence as 33

previously described (8). Fluorescence images were acquired with a confocal laser microscopy system (Leica 34

SP2). The image resolution was 512 x 512 pixels and scan rate was 400 Hz. For each sample, over 100 cells 35

were examined in at least three independent experiments. Immunohistochemistry was performed as previously 36

described (11). Adjacent sections were stained with Haematoxylin & Eosin or Masson's trichrome to visualize 37

collagen fibers (murine fat pad experiments). Images were captured on a Leica DMRA2. 38

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5

Western blot analysis. Whole cell extracts and immunoblots were prepared as previously described (22). All the 1

antibodies used in this study are presented in Supplementary Table S1. 2

3

RNA extraction and quantitative PCR (qPCR). RNA extraction and qPCR were performed as previously 4

described (23). All the primers used in this study are presented in the Supplementary Table S2. 5

6

Migration and invasion assays (2D). After 3 or 8 days of coculture in the presence of SUM159PT cells, 7

adipocytes were trypsinized and used to perform migration or Matrigel invasion assays as previously described 8

(23). Similar Matrigel invasion assays were performed with tumor cells grown alone or cocultivated with ADFs 9

or with mature adipocytes during 3 days (8). In these assays, FCS (10%) was used as a chemoattractant, whether 10

similar experiments were run in parallel in the absence of serum (no chemoattractant, negative control). 11

12

Spheroids formation and invasion in a 3D collagen matrix. The hanging-drop method has been adapted to 13

produce spheroids of similar diameter (24). Drops (20 μl) containing 1000 tumor cells alone or mixed with 1000 14

ADFs or preadipocytes were suspended on the lid of 2% agar coated 24-well dishes containing culture media. 15

After the 7 days required for cell aggregation, the spheroids were transferred to the bottom of a well containing 16

culture medium. Multicellular spheroids were then allowed to grow for 14 days and the spheroids used in the 17

invasion experiments have an average diameter of 500 μm. The spheroid invasion in collagen I matrix was 18

performed as previously described (25). 19

20

Glucose uptake experiments. Differentiated adipocytes cocultivated or not during 3 and 8 days with 21

SUM159PT cells were used to measure the uptake of 2-deoxy[3H]glucose in the presence or not of insulin as 22

previously described (26). 23

24

Statistical analysis. The statistical significance of differences (at least three independent experiments) was 25

evaluated using Student's t test or ANOVA using Prism (GraphPad Inc.). P values below 0.05 (*), <0.01 (**) and 26

<0.001 (***) were deemed as significant while “NS” stands for not significant. 27

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6

Results and Discussion 1

Breast tumor cells are able to cause mature adipocytes to revert to fibroblast-like cells in vitro and in vivo. 2

To test our main hypothesis in vitro, we took advantage of the coculture model recently set-up in our laboratory. 3

As previously described, mature 3T3-F442A adipocytes cocultivated in the presence of breast cancer cells 4

exhibited a decrease in the number and size of lipid droplets (8). We show here that this decrease is time-5

dependent and, after 8 days of coculture, these cells were almost devoid of lipid droplets (fig. 1A, upper panel). 6

By contrast to CAAs (typically obtained after 3 days of coculture) which retain their rounded morphology (8), 7

adipocytes that have been cocultivated for longer times in the presence of tumor cells took on a highly elongated, 8

fibroblastic shape (fig. 1A, middle panel). Preadipocyte factor 1 (Pref-1) expression, a preadipocyte 9

transmembrane protein that inhibits adipogenesis (27), is progressively detected in cocultivated adipocytes (fig. 10

1A, lower panel). After 8 days of coculture, we also observed that mature adipocytes exhibit a loss in adipose 11

markers expression that was more accentuated than in CAAs (8). An overall dramatic reduction of all the tested 12

adipogenic markers including Adipisin, Adiponectin (APN), Ap2, HSL, as well as C/EBPa and PPARγ2 mRNA 13

was observed by qPCR analysis (reduced by more than 70% for all tested genes, p<0.001, fig 1B). These results 14

were confirmed by Western blots for HSL, Adiponectin, PPARγ2 and C/EBPα expression, whose expression 15

was no longer detected in cocultivated adipocytes (fig. S1A). Similar results were obtained by cocultivating 16

adipocytes with the human ZR 75.1, HMT3522-T4.2 or mouse 4T1 breast cancer cell lines (fig. S1B) as well as 17

when human mammary adipocytes derived from ex vivo differentiation of progenitors purified from mammary 18

adipose tissue of healthy donors undergoing reduction mammoplasty were used (fig. S1C). At 8 days, 19

cocultivated adipocytes exhibited a marked decrease in GLUT4 expression and a concomitant increase in 20

GLUT1 expression (fig. 1C), and became resistant to insulin (fig. 1D). No gain of proliferative ability was 21

observed in these cocultivated adipocytes exhibiting insulin resistance and a fibroblast-like shape. Indeed, the 22

cell number, as well as the cell cycle distribution, remained unchanged during the coculture period (see fig. S2). 23

We next investigated whether this process might be observed in vivo. To address this critical issue, we used 24

GFP-tagged confluent 3T3-F442A preadipocytes implanted subcutaneously into athymic BALB/c nude mice, 25

where they developed into typical fat pads after 5 weeks (21). Murine breast cancer cells 4T1 were then either 26

injected into the newly formed fat pads or used alone as a control, whilst untreated fat pads also provided a 27

separate control. Immunohistochemical staining for GFP was performed in fat pads removed 10 days after the 28

injection (fig. 2A). Tagged-mature adipocytes were used to ensure that the cells with a fibroblast-like shape 29

present in the tumors did in fact arise from local adipocytes and not from other sources. The fat pads derived 30

from 3T3-F442A confluent preadipocytes exhibited histological features of adipose tissue. Adipocytes were 31

positive for GFP whereas tumor cells were not (fig. 2B, lower panel). In the case of tumors injected into the fat 32

pads, adipose tissue at the tumor periphery was composed of adipocytes of small size and elongated cells 33

containing many small lipid droplets – all cells that are GFP positive (fig. 2B, lower panels, indicated by 34

arrows). Within the tumor, GFP-positive cells with fibroblast-like shape appear to interdigitate with tumor cells 35

(fig. 2B, lower panels, indicated by asterisks). Staining of the tissue sections with Masson’s trichrome revealed a 36

significant amount of collagen fibrils in the tumor grown within the fat pad whereas few collagen fibrils were 37

seen in tumor grown alone (fig. 2B, upper panel). Taken together, these results are consistent with the fact that 38

breast tumor cells induce phenotypical changes in mature adipocytes leading to cells exhibiting low (if any) lipid 39

content and profound morphological changes associated with a marked increase in the expression of matrix 40





7

proteins in the tumors. Therefore, our results compellingly suggest that cells with a fibroblast-like shape could be 1

derived from mature adipocytes in a tumor-context both in vitro and in vivo. 2

3

Adipose-Derived Fibroblasts (ADFs) overexpress FSP-1, but not �SMA, and exhibit an activated 4

phenotype. We then investigated the expression of major markers, previously used to identify CAFs, in these 5

cells (6). As shown in fig. 3A, adipocytes cocultivated with tumor cells exhibit a gradual increase in FSP-1 6

expression, as opposed to �SMA or FAP (Fibroblast Activated Protein) expression that remains very low. We 7

confirmed that mature adipocytes cocultivated for 8 days with tumor cells overexpressed FSP-1 mRNA by 3-8

fold, compared to mature adipocytes grown alone (p< 0.01) whereas the difference in mRNA levels of αSMA 9

between these two conditions was not statistically significant (fig. 3B). These results were confirmed with 10

human mammary adipocytes (fig. S3). Functionally activated fibroblasts surrounding tumors are highly 11

contractile and motile and also display increased extracellular matrix (ECM) expression (3). As shown in fig. 3C, 12

increased expression of the ECM proteins type I collagen and fibronectin was also observed during adipocyte 13

conversion. The fibrotic network produced by these cells in vitro is in accordance with the results obtained with 14

Masson’s trichrome staining in vivo (see fig. 2). As shown in fig. 3D (upper panel), coculture of adipocytes with 15

tumor cells was associated with a profound reorganization of actin cytoskeleton, marked by the occurrence of 16

stress fibers and the formation of punctuate spots of vinculin at the ends of actin bundles. Accordingly, 17

cocultivated adipocytes, as they acquire a fibroblast-like phenotype, progressively exhibit increased migratory 18

(fig. 3D, left bottom panel) and invasive (fig. 3D, right bottom panel) abilities. Therefore, our compelling results 19

demonstrate that tumor cells can cause mature 3T3-F442A adipocytes, as well as human mammary adipocytes, 20

to revert to an activated FSP-1 fibroblastic cell population. Due to the common characteristics of CAFs and these 21

cells, highlighting that they represent a subpopulation of CAFs, we named them Adipocyte-Derived Fibroblast 22

(ADFs). In vitro, the CAAs to ADFs transition is time dependent (fig. 1 and 3) and intermediate forms of 23

adipocyte phenotypical changes (from small adipocytes to fibroblast-like cells that may or may not still contain 24

small lipid droplets) could be observed in vitro and in vivo (fig. 1 and 2). Once modified at the tumor invasive 25

front, where the cross-talk between breast tumor and adipocytes is established (8), our data strongly suggests that 26

ADFs are able to join the center of the tumor and participate to the desmoplastic reaction by exhibiting increased 27

migratory and invasive abilities (fig. 3), as shown in the murine fat pad experiments (fig. 2). 28

29

The Wnt/�-catenin pathway is reactivated in ADFs and contributes to the occurrence of the ADFs 30

phenotype. Among the many signaling networks involved in relaying information from extracellular signals 31

during adipogenesis, the Wnt/β-catenin pathway plays a prominent role (28). This pathway negatively regulates 32

precursors commitment and differentiation along the adipocyte lineage (28) and its activation has recently been 33

shown to also induce a “dedifferentiated” state in mature adipocytes (17). As shown in fig. 4A (upper panel), 34

adipocytes cocultivated with tumor cells exhibit a gradual increase in β-catenin expression associated with its 35

redistribution from the cell membrane to the cytoplasm. During activation of this pathway by Wnt ligands (the 36

so-called canonical pathway), the �-catenin is hypo-phosphorylated and translocated into the nucleus where it 37

contributes to the activation of Wnt target genes (28). A progressive increase in active β-catenin in the nucleus of 38

cocultivated adipocytes was detected (fig. 4A, lower panel). In accordance, cocultivated adipocytes exhibit an 39

upregulation of all Wnt target gene mRNAs analyzed as described for Dishevelled-1 (Dvl1), Wnt10b, 40





8

Endothelin-1 (EDT1) and metalloprotease-7 (MMP7) (fig. 4B). Taken together, our results show that the 1

canonical Wnt/�-catenin pathway is reactivated during coculture and is likely to contribute to the occurrence of 2

the ADFs. Given our results, we reasoned that targeted inhibition of this signaling using ICG-001, a unique small 3

molecule that selectively inhibits TCF/β-catenin transcription in a CBP-dependent fashion (19) might be able to 4

mitigate the tumor-induced changes in adipocytes. As shown in fig. 4C, exposure to ICG-001 during 3 days 5

significantly inhibits the increase in Wnt10b expression induced by coculture (about 3-fold decrease in 5μM 6

ICG-001 treated cells compared to untreated cells, p<0.05) and partially restores lipid accumulation in 7

cocultivated adipocytes in a dose-dependent manner. We next investigated the signal emanating from tumor cells 8

that could be able to reactivate the canonical Wnt/β-catenin pathway. Soluble Wnt3a or TNF-α has been recently 9

demonstrated to induce the “dedifferentiation” of murine and human adipocytes through activation of the Wnt/β-10

catenin pathway (17). TNF-α was undetectable in the supernatant of all breast cancer cell lines that were 11

cultivated with or without adipocytes. By contrast, Wnt3a was expressed and secreted by the breast cancer cells, 12

as demonstrated in fig. 4D for the SUM159PT cells. The levels of Wnt3a expression and secretion were not 13

regulated by coculture. Similar results were obtained with the human breast cancer cell lines ZR 75.1 and MDA-14

MB231 (fig. S4). As shown in fig. 4E (left panel), in the presence of Wnt3a blocking antibody (Ab), we observe 15

a clear inhibition of β-catenin up-regulation and redistribution induced by coculture, suggesting that Wnt3a is 16

indeed upstream of the β-catenin stabilization. Of note, cells that have been treated with Wnt3a blocking Ab 17

exhibit a significant decrease of Wnt10b mRNA expression (1.6-fold decrease in Ab treated cells compared to 18

untreated cells, p<0.05) and conversely an increase in lipid content as compared to untreated cells (fig. 4E, right 19

panel). Therefore, the occurrence of ADFs is associated with activation of the Wnt/β-catenin pathway and 20

blocking this pathway partially reversed the ADFs phenotype (fig. 4C and 4E). Elevated levels of cytoplasmic 21

and/or nuclear β-catenin are detectable by immunohistochemistry in the majority of breast tissue samples and 22

high β-catenin activity significantly correlated with poor prognosis of the patients (29). This activation might 23

rely on Wnt ligands since several studies have described overexpression of Wnt protein themselves, including 24

Wnt3a, in breast cancer cells lines as well as breast tumors relative to normal tissue (30). Expression of these 25

ligands is generally thought to activate the canonical Wnt/β-catenin pathway in tumor cells by an autocrine 26

mechanism (31) to favor tumor growth, migration, inhibition of apoptosis and potentially stem cell renewal in 27

certain tissues (32). We propose here a new and paracrine effect of Wnt ligands in promoting the occurrence of a 28

sub-population of CAFs. 29

30

ADFs stimulate tumor cells invasive phenotype in 2D and 3D coculture systems. Mature adipocytes were 31

cocultivated with breast cancer cells for 5 days to obtain ADFs, which were then used to perform 2D coculture 32

experiments or to produce mixed spheroids with “naïve or unprimed” tumor cells. In 2D coculture experiments, 33

ADFs strongly stimulated the invasive properties of SUM159PT cells (increase of 2.4-fold compared to tumor 34

cells grown alone, p<0.01) (fig. 5A), this effect being more pronounced in the presence of ADFs than in the 35

presence of mature adipocytes (1.5-fold increase as compared to tumor cells grown alone, p< 0.05; 1.6-fold 36

increase in cells cocultivated with ADFs as compared to mature adipocytes, p<0.05). In addition to the increased 37

cell invasive abilities, we have previously demonstrated that cocultivated SUM159PT exhibit morphological 38

changes along with the reorganization and polarization of vimentin (8). As shown in fig. 5B, after 3 days of 39

coculture, ADFs induce similar but more pronounced effects on vimentin expression that was associated with 40





9

profound actin reorganization leading to the occurrence of stress fibers. As 3D invasion models more closely 1

follow the cell behavior observed in vivo (33), we confirmed our results using a spheroid invasion model within 2

a collagen matrix (25) (see fig. 5C, left panel, for a representative experiment). We found that mixed 3

ADFs/tumor cells spheroids exhibit higher invasive capacities compared to spheroids of tumor cell alone 4

(p<0.01), yet the presence of preadipocytes had no effect on 3D tumor cells invasion (fig. 5C, right panel) in 5

accordance with the results previously obtained in 2D (8). HcRed-SUM159PT cells, GFP-preadipocytes and 6

GFP-ADFs were used to investigate the invasive behavior of each cell sub-population. Interestingly, ADFs (and 7

preadipocytes) remained in the center of the spheroids in contrast to tumor cells that invaded the collagen matrix 8

(fig. 5D). These combined results indicate that ADFs stimulate the invasive behavior of tumor cells. 9

10

Reorientation of mature adipocytes into ADFs is observed in human tumors 11

We next investigated whether ADFs are present in primary human tumors. To address this critical issue, we first 12

conducted a histological analysis of human breast tumors to investigate the presence of cells exhibiting ADF 13

traits. As defined in vitro and in vivo in murine fat pads, ADFs originate from progressive changes of the 14

morphology of adipocytes giving rise to small adipocytes and of elongated cells containing many small lipid 15

droplets. As shown in figure 6A, we observed a profound reorganization of the adipose tissue (AT) at the tumor 16

invasive front (see left panel for the AT aspect at distance of the tumors) with numerous cells exhibiting the 17

features of intermediate forms of ADFs. Of note, elongated cells with lipids droplets could be observed within 18

the dense desmoplastic reaction surrounding the tumors (fig. 6A, lower panel, indicated by arrows). 19

To further characterize these cells, we investigated FSP-1 expression in the breast cancer surrounding adipocytes 20

at variable distances from the tumor center. As shown in fig. 6B, the mature adipocytes further from the tumor 21

are negative for FSP-1 expression whereas as closer to the tumor exhibit FSP-1 expression. FSP-1 positive 22

fibroblasts were present in the center of the tumors as previously described (6). Therefore, as observed in vitro, 23

we consistently observed a gradient of FSP-1 expression in mature adipocytes exhibiting first CAA then ADF 24

phenotype (fig. 6B). Finally, we investigated the differential expression of both αSMA and FSP-1 in tumor-25

surrounding adipocytes by immunohistochemistry. In fact, one of the key findings of our study is that ADFs, at 26

least in vitro, express FSP-1 but not αSMA (see fig. 3A and B). Interestingly, using experimentally generated 27

tumors in mice, the group of R. Kalluri found unique FSP-1-expressing CAFs, which are clearly distinct from 28

the αSMA positive myofibroblasts (6). This subpopulation of CAFs is very important functionally, since FSP-1 29

positive fibroblasts promote tumor metastasis (34). Mammary carcinoma cells injected into FSP1-/- mice 30

showed significant delay in tumor uptake and decreased tumor incidence (34). Co-injection of carcinoma cells 31

with FSP1+/+ mouse embryonic fibroblasts partially restored the kinetics of tumor development and the ability 32

to metastasize, underlying the role of this specific subset of stromal population in invasive processes (34). As 33

shown in fig. 6C (left panel), we found that αSMA expression was not up-regulated in adipocytes close to 34

tumors (TU) when compared to normal breast adipose tissue (reduction mammoplasty, RM) whereas in the same 35

sample an up-regulation of FSP-1 expression was found. To further assess the expression of these markers by a 36

quantitative approach, adipocytes were isolated from fat samples associated with TU or RM. A series of 16 37

samples, with 8 samples in each group (Supplementary Table S3 for the clinical characteristics of the patients), 38

was used, matched with respect to age (mean: 54.5 ± 10.4 vs 51.1 ± 8.3 years) and Body Mass Index (BMI) 39

(mean: 26.1 ± 3.5 vs 24.9 ± 2.3 kg/m2). Adipocytes isolated from tumor samples overexpressed FSP-1 mRNA 40





10

when compared with adipocytes isolated from normal mammary glands, whereas the mRNA levels of αSMA 1

were not statistically different between the two groups (fig. 6C, right panel). Therefore, the phenotype described 2

here both in vitro and in vivo for ADFs is clearly reminiscent of the one of FSP-1 positive CAFs including the 3

absence of �SMA expression and the invasion-promoting effect. Because mature adipocytes represent the most 4

abundant cellular type in breast cancer stroma, the process of adipose “dedifferentiation” is likely to provide a 5

large part of this CAFs subpopulation. Interestingly, the impact of tumor cells on various component of adipose 6

tissue might also participate to the heterogeneity of CAFs phenotype. In fact, it has been demonstrated both in 7

vitro and in vivo that breast cancer cells activate adipose stem cells towards a CAFs-like phenotype associated 8

with �SMA overexpression (35), (for review see, (10). Therefore, mature and precursor adipose cells could 9

contribute to divergent CAFs subpopulation, whose relative contributions in cancer progression remain to be 10

determined in lean and obese conditions. 11

To conclude, the proposed mechanism to explain the occurrence and the role of ADFs in breast cancer is 12

summarized in fig. 7. In our model, early local tumor invasion in breast cancer results in immediate proximity of 13

tumor cells and adipocytes. Cancer cells secrete soluble factors, including Wnt3a, that are able to activate Wnt/�-14

catenin pathway in mature adipocytes leading to adipocytes “dedifferentiation”. Adipocytes will undergo 15

morphological changes, first marked by a decrease in adipocytes size and lipid content (CAAs), then by 16

acquiring a fibroblast-like morphology (ADFs), yet still containing small lipid droplets. At the start of this 17

process, tumor-primed adipocytes express FSP-1. ADFs exhibit increased migratory and invasive abilities and 18

will be able to join the center of the tumor, where they significantly promote tumor invasion. In the center of the 19

tumor, these cells no longer express adipose markers nor exhibit lipid droplets, which render them 20

indistinguishable from other CAFs cell population. Studying the process of occurrence of ADFs in breast cancer 21

is of major clinical importance. In fact, the occurrence of specific stromal cells, such as ADFs, is critical to the 22

aggressiveness of nearby tumor cells and they need to be identified in order to develop targeted therapeutic 23

strategies inhibiting their development and/or tumor-promoting activity. Implicating adipose tissue in this 24

crosstalk is also of major interest since several studies pointed out that obese women exhibit higher propensity 25

for local invasion and distant metastasis (10). Obesity condition is also associated with an increase in adipose 26

tissue fibrosis (15). Therefore, further studies should address whether ADFs are overrepresented in the breast 27

tumor stroma of obese patients in which they would provide an invasion-promoting population. Such studies will 28

provide unique opportunities to set up specific strategies for the treatment of the subsets of patients exhibiting 29

aggressive diseases. 30

31

Acknowledgements 32

The authors would like to acknowledge Pr Laurence Nieto for helpful technical advices for qPCR, Guillaume 33

Andrieu, Emilie Mirey and Nathalie Ortega for critical reading of the manuscript. The authors would like to 34

thank Jamie Brown for the English editing of the manuscript and Françoise Viala for iconographic assistance. 35

36

References 37

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22

Figures legends. 23

Figure 1. Breast cancer cells revert mature adipocytes into fibroblast-like cells in vitro. A, mature adipocyte 24

cultivated in the presence (C) or in the absence (NC) of tumor cells during the indicated times were stained with 25

Bodipy (top), Pref-1 antibody (bottom) (scale bars 20μm) or observed through phase contrast microscopy 26

(middle, scale bar 50μm). Nuclei were labeled with TO-PRO-3. Similar experiments were performed with 3T3-27

F442A preadipocytes used as a control. B, expression of the indicated genes analyzed by qPCR in mature 28

adipocytes cocultivated for 8 days in the presence (C) or in the absence (NC) of SUM159PT tumor cells. C, Left: 29

GLUT4 and GLUT1 gene expression in mature adipocytes cultivated in the presence (C) or in the absence (NC) 30

of SUM159PT cells during the indicated times. Right: analysis of Glut1 and Glut4 protein expression done by 31

Western blot with extracts from mature adipocytes cultivated in the presence (+) or in the absence (-) of 32

SUM159PT cells for the indicated times. D, glucose uptake assay in the presence of the increasing 33

concentrations of insulin performed in adipocytes cultivated in the presence (C) or in the absence (NC) of tumor 34

cells during 3 and 8 days. For the fig. 1, bars and errors flags represent the means ± SD of at least three 35

independent experiments; statistically significant by Student’s t-test, ** p<0.01, *** p<0.001 relative to NC cells 36

or to untreated cells for glucose uptake experiments. 37

38

Figure 2. The phenotypic changes of mature adipocytes towards fibroblast-like cells are observed in vivo. 39

A, GFP-tagged confluent 3T3-F442A preadipocytes were implanted subcutaneously into athymic BALB/c nude 40

mice, where they develop into fat pads after 5 weeks. Murine breast cancer cells 4T1 were then injected into the 41

newly formed fat pads or assayed separately. Immunohistochemical staining for GFP was performed in fat pads 42

removed 10 days after the injection, tumor cells alone being also used as a control. B, Upper panel: 43

Representative Masson’s Trichrome stained sections from fat pads alone, tumors grown alone or tumors grown 44

into the fat pads with collagen stained in blue, and nucleus in red. Middle panel: Representative images of the 45

expression of GFP (in brown) visualized in fat pads alone, tumors grown alone or tumors grown into the fat pads 46





13

(lipids in white, nucleus in blue). Arrows indicate GFP positive elongated cells with small lipid droplets present 1

at the tumor periphery whereas asterisks identify GFP-fibroblast-like cells within the tumor, scale bars 50μm. 2

The bottom panel shows a 2-fold magnified view of the framed areas. 3

4

Figure 3. Adipose-Derived Fibroblasts (ADFs) overexpress FSP-1, but not �SMA, and exhibit an activated 5

phenotype. A, 3T3-F442A mature adipocytes were grown on coverslips and cultivated in the presence (C) or in 6

the absence (NC) of tumor cells during 3, 5 and 8 days (D3, D5 and D8). After this period, cells were fixed and 7

stained with the indicated antibodies. 3T3-F442A preadipocytes served as a control. Nuclei were labeled with 8

TO-PRO-3, scale bars 20μm. B, �SMA and FSP-1 gene expression was evaluated by qPCR in adipocytes 9

cultivated in the presence (C) or in the absence (NC) of tumor cells during 8 days with tumor cells. Bars and 10

errors flags represent the means ± SEM of at least three independent experiments; statistically significant by 11

Student’s t-test, ** p < 0.01 relative to NC cells. NS, non significant. C, fibronectin and collagen 1 expression 12

were assessed by immunofluorescence in adipocytes cultivated in the presence (C) or in the absence (NC) of 13

tumor cells during 3, 5 and 8 days (D3, D5 and D8). 3T3-F442A preadipocytes served as a control. Nuclei were 14

labeled with TO-PRO-3, scale bars 20μm. D, Upper panel, 3T3-F442A mature adipocytes were grown on 15

coverslips and cultivated in the presence (C) or in the absence (NC) of tumor cells during 3, 5 and 8 days (D3, 16

D5 and D8). After this period, cells were fixed and stained with the indicated antibodies. 3T3-F442A 17

preadipocytes served as a control. Nuclei were labeled with TO-PRO-3, scale bars 20μm. Lower panel, 18

adipocytes grown alone (NC) or in the presence of tumor cells (C) for 3 (D3) and 8 (D8) days. Adipocytes were 19

then trypsinised and used for migration (left panel) or Matrigel invasion (right panel) assays towards a medium 20

containing either 0% (white bars) or 10% FCS (black bars). Bars and errors flags represent the means ± SEM of 21

three independent experiments; statistically significant by Student’s t-test, * p < 0.05, ** p<0.01, *** p<0.001 22

relative to NC cells. 23

24

Figure 4. The Wnt/�-catenin pathway is reactivated in ADFs and contributes to the occurrence of this new 25

stromal cell population. A, adipocytes cultivated in the presence (C) or in the absence (NC) of tumor cells 26

during 3, 5 and 8 days (D3, D5, D8) and 3T3-F442A preadipocytes were fixed and stained with the indicated 27

antibodies. Nuclei were labeled with TO-PRO-3, scale bars 20μm. B, mRNA expression (qPCR) of the indicated 28

Wnt/β-catenin targets was evaluated in adipocytes cultivated in the presence (C) or in the absence (NC) of tumor 29

cells during 3, 5 and 8 days (D3, D5, D8). C, Wnt10b mRNA expression (right panel) and Bodipy accumulation 30

or morphology observed through phase contrast microscopy (left panel, scale bars 20μm) were evaluated in 31

adipocytes cultivated in the presence (C) or in the absence (NC) of tumor cells for 3 days with tumor cells in the 32

presence of increasing concentrations of ICG-001. D, Wnt3a expression and secretion was measured in the 33

SUM159PT cells cultivated in the presence (C) or in the absence (NC) of adipocytes. E, Adipocytes were 34

cultivated in the presence (C) or in the absence (NC) of breast tumor cells in the presence (+) or absence (-) of a 35

Wnt3a blocking Ab. In these adipocytes, the expression of Wnt10b mRNA was measured by qPCR, (right panel) 36

and the β-catenin expression and lipid accumulation was evaluated by immunofluorescence. Nuclei were labeled 37

with TO-PRO-3, scale bars 20μm (left panel). Bars and errors flags represent the means ± SEM of three 38

independent experiments; statistically significant by Student’s t-test or ANOVA, * p < 0.05, ** p<0.01, *** 39

p<0.001 relative to NC cells. 40





14

1

Figure 5. ADFs enhance tumor cells invasion in 2D and 3D coculture models. A, tumor cells cultivated in 2

the presence (C) or in the absence (NC) of mature adipocytes or ADFs for 3 days were used for Matrigel 3

invasion assay toward a medium containing either 0 (white bars) or 10 (black bars) % FCS. Representative 4

images of Matrigel invasion assay performed towards a medium containing 10% FCS are shown below each 5

graph, scale bar 50 μm. Bars and errors flags represent the means ± SD of three independent experiments; 6

statistically significant by Student’s t-test, * p < 0.05, ** p<0.01 relative to NC cells. B, immunofluorescence 7

experiments using mAb against vimentin or rhodamin phalloidin were performed on tumor cells cultivated in the 8

presence (C) or in the absence (NC) of mature adipocytes or ADFs for 3 days. Nuclei were labeled with TO-9

PRO-3, scale bar 20μm. C, A representative picture of spheroid invasion in a collagen matrix (left, scale bar 10

250μm and measurement of the invasion distance (in μm) 48h after collagen embedding (right panel). Bars and 11

errors flags represent the means ± SEM of three independent experiments, statistically significant by Student’s t-12

test, ** p<0.01 relative to spheroids composed by tumor cells alone (SUM), NS, non significant. D, Confocal 13

microscopy analysis of spheroids invasion 48h after collagen inclusion, spheroids being composed by HcRed-14

SUM, by mixed HcRed-SUM + GFP-3T3-F442A or by HcRed-SUM159PT + GFP-ADF, scale bar 100μm. 15

16

Figure 6. ADFs are found in human tumors. A, Histological examination of human breast cancers showing 17

distant adipose tissue (left panel) and tumor tissue containing reorientated adipocytes (middle panel) after H&E 18

staining (original magnification x200, scale bar 50μm.). The right panels show a 2-fold magnification of the 19

framed area. Results of representative experiments were obtained using tumors issued from 2 independent 20

donors. B, Histological examination of adipose tissue (lipids in white, nucleus in blue) in a representative breast 21

tumor after anti FSP-1 staining (in brown). The results obtained in different areas from the adipose tissue away 22

from the tumor to the tumor center are shown (scale bar 50μm). C, Left panel, the expression of FSP-1 and 23

αSMA was visualized (in brown) in adipocytes (lipids in white, nucleus in blue) located adjacent to invading 24

cancer cells (TU) as compared with normal adipose tissue (RM). Right panel, the expression of the indicated 25

genes was analyzed by qPCR in mature adipocytes obtained from RM or TU (8 independent samples per group), 26

statistically significant by Student’s t-test, * p<0.05. NS, Non Significant. 27

28

Figure 7. Mechanism of ADFs occurrence and role in tumor progression. Schematic representation of the 29

described bidirectional crosstalk established between breast cancer and adipose cells underlying the presence of 30

ADFs in the desmoplastic reaction of breast cancers. 31

32

33

34

35

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Published OnlineFirst July 31, 2013.Cancer Res Ludivine Bochet, Camille Lehuede, Stephanie Dauvillier, et al. contribute to desmoplastic reaction in breast cancer.Adipocyte-Derived Fibroblasts promote tumor progression and

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