antonio ruiz-vela´s project at cipf

Specific Unsaturated Fatty Acids Enforcethe Transdifferentiation of Human Cancer Cellstoward Adipocyte-like Cells

Antonio Ruiz-Vela & Cristóbal Aguilar-Gallardo &

Ana M. Martínez-Arroyo & Mario Soriano-Navarro &

Verónica Ruiz & Carlos Simón

Published online: 16 April 2011# Springer Science+Business Media, LLC 2011

Abstract Differentiation therapy pursues the discovery ofnovel molecules to transform cancer progression into lessaggressive phenotypes by mechanisms involving enforcedcell transdifferentiation. In this study, we examined theidentification of transdifferentiating adipogenic programs inhuman cancer cell lines (HCCLs). Our findings showed thatspecific unsatturated fatty acids, such as palmitoleic, oleic andlinoleic acids, trigger remarkable phenotypic modifications ina large number of human cancer cell lines (HCCLs), includinghepatocarcinoma HUH-7, ovarian carcinoma SK-OV-3,breast adenocarcinoma MCF-7 and melanoma MALME-3M. In particular, we characterized a massive biogenesis oflipid droplets (LDs) and up-regulation of the adipogenicmaster regulator, PPARG, resulting in the transdifferentiationof HCCLs into adipocyte-like cells. These findings suggestthe possibility of a novel strategy in cancer differentiationtherapy via switching the identity of HCCLs to anadipogenic phenotype through unsaturated fatty acid-induced transdifferentiation.

Keywords Transdifferentiation . Adipocytes . LipidDroplets . PPARG . Unsaturated Fatty Acids

Introduction

The transdifferentiation process of one differentiated celltype into another lineage has received significant attentionbecause it could have considerable applications in cellularand cancer therapy. Experiments conducted in a number ofdifferent species have shown that lineage conversion can beaffected simply by the introduction of defined transcriptionfactors [1]. Recently, Deepak Srivastava’s group documenteda direct transdifferentiation of fibroblasts into functionalcardiomyocytes, where only three genes (Gata4, Mef2c andTbx5) were needed to generate functional cardiomyocytes[2]. Similarly, Wernig’s group were able to transdifferentiatefibroblasts into functional neurons by combinations of threetranscription factors (Ascl1, Brn2 and Myt1l) [3]. Theseexamples show that enforced cell transdifferentiation canbe accomplished by the overexpression of the appropriatecocktail of genes.

Furthermore, enforced cell transdifferentiation throughmolecules is a promising alternative to conventionalchemotherapy for cancer. In this sense, the most remarkabledifferentiation therapy approach has been the usage of all-trans retinoid acid (ATRA) for the treatment of acutepromyelocytic leukemia (APL) [4]. This form of leukemiatransdifferentiates into mature granulocytes after ATRAtreatment [4]. Although ATRA has been proven to inducecell transdifferentiation and to provide clinical benefit topatients with APL [5], there is no treatment based on celldifferentiation therapy for human solid tumors.

These precedents prompted us to investigate whethernovel transdifferentiating molecules secreted by humanembryonic stem cells (hESCs) could prevent cancerprogression [6]. Initially, we found that culturing distincthuman cancer cell lines, HCCLs (ovarian carcinoma,

Stem Cell Rev and Rep (2011) 7:898–909DOI 10.1007/s12015-011-9253-7

Antonio Ruiz-Vela and Cristóbal Aguilar-Gallardo have contributedequally to this work.

Electronic supplementary material The online version of this article(doi:10.1007/s12015-011-9253-7) contains supplementary material,which is available to authorized users.

A. Ruiz-Vela : C. Aguilar-Gallardo :A. M. Martínez-Arroyo :M. Soriano-Navarro :V. Ruiz :C. Simón (*)Valencia Node of the Spanish Stem Cell Bank,Prince Felipe Research Centre (CIPF),Avda. Autopista del Saler, 16-3,46012 Valencia, Spaine-mail: [email protected]

http://dx.doi.org/10.1007/s12015-011-9253-7

hepatocarcinoma, breast adenocarcinoma and melanoma),with embryonic microenvironments conditioned with andwithout hESCs resulted in HCCL transdifferentiation intoan adipocyte-like cells, irrespective of the presence ofhESCs. Further analysis of human embryonic stem cellgrowth media (HES) identified the fatty acid-rich albumin(AlbuMAX) fraction as being responsible for triggeringdevelopment of adipogenic features, including the biogen-esis of lipid droplets (LDs) and the induction of PPARG.We further screened the ability of unsaturated fatty acids toinduce transdifferentiation in HCCLs and discovered thatthe poly- and monounsaturated fatty acids associated withalbuMAX (linoleic, oleic, and palmitoleic acids) trigger thebiogenesis of LDs. Our data also revealed that selectiveunsaturated fatty acids cocktails are capable of inducing aneven more efficient adipocyte-like cells transdifferentiationof HCCLs.

Results

Knock-out serum replacement (KOSR) triggersgranulogenesis of human cancer cell lines (HCCLs)

In order to gain insight into the role of human embryonicstem cells (hESCs) conditioned media as a potential sourceof differentiation factors in human cancer cell lines(HCCLs) [6], we examined whether hESC-conditionedmedia might trigger cellular modifications in HCCLs(MCF-7, MALME-3M, HUH-7 and SK-OV-3) by search-ing for alterations in the threshold of forward scatter (FSC)and side scatter (SSC) parameters [7]. We cultured MCF-7and MALME-3M cells with HES medium containing 20%of KOSR (HES+20%KOSR) conditioned both in thepresence and in the absence of hESCs (Fig. 1a). As negativecontrols, MCF-7 and MALME-3M cells were cultured withRPMI-1640 containing 10% of fetal bovine serum (RPMI+10%FBS). This experiment demonstrated remarkable alter-ations in the threshold of SSC of MCF-7 and MALME-3Mcells (≥ 2-fold induction at 48 h) upon incubation with HES+20%KOSR, irrespective of the presence of hESC (Fig. 1a).Such dramatic changes in the SSC parameter were notobserved when MCF-7 and MALME-3M cells were grownin RPMI+10%FBS (Fig. 1a). This indicates that themodifications in the SSC parameter are a result of culturingthe cells with HES+20%KOSR.

In order to cross validate this finding, we examined theeffect of HES+20%KOSR on both HUH-7 and SK-OV-3cells (Fig. 1b). HCCLs were cultured in HES+20%KOSR(Fig. 1b). In addition, KOSR was replaced with 10% FBS(HES+10%FBS) and resulted in no significant alterations.We received similar results when HCCLs were cultured inthe presence of RPMI+10%FBS (Fig. 1b). Changes in the

SSC parameter were not detected when HCCLs were grownin the presence of FBS, indicating that KOSR itself is thefactor responsible for triggering alterations in the SSCpatterns of HCCLs. These results strongly suggest thatKOSR might induce granulogenesis in HCCLs [8].

KOSR induces the biogenesis of lipid droplets (LDs)in HCCLs

Next, we used electron microscopy (EM) to analyze theultrastructural features or subcellular modifications respon-sible for the observed modifications in the SSC parameterinduced by KOSR in HCCLs. MALME-3M and MCF-7cells were cultured in HES+20%KOSR, whereas controlcells were cultured in RPMI+10%FBS. EM studiesdemonstrate a massive accumulation of spherical structureswhen cells are cultured with HES+20%KOSR (Fig. 2b-d, fand h), which were not detected in the control cells culturedin RPMI+10%FBS (Fig. 2a, e and g). Such sphericalstructures are considered typical hallmarks of lipid droplets(LD) [8], indicating that the main observable ultrastructuralfeature was the biogenesis of LDs. These results are inagreement with the increased values obtained in the SSCparameter (Fig. 1a and b), which established granulogenesis[7, 9]. The LD core is composed of neutral lipids, such astriacylglycerols (TGAs), surrounded by a monolayer ofphospholipids [8]. TGAs are produced in the glycerol-phosphate pathway in which fatty acid moieties are sequen-tially added to a glycerol backbone [8]. In the final step ofTGA synthesis, diacylglycerol and fatty acyl CoAs areconverted into TGAs by acyl CoA: diacylglycerol acyltrans-ferases (DGAT), which are localized in mitochondria-associated membranes [8]. We detected an accumulation ofmitochondria in close proximity to LDs (Fig. 2h), suggestingthe biosynthesis of TGAs upon KOSR incubation.

To corroborate a KOSR-dependent incubation accumu-lation of LDs, we employed the Oil-RED dye thatspecifically binds to neutral lipids [10]. HCCLs werecultured with HES+20%KOSR or HES+10%FBS for48 h and were then stained by Oil-RED and hematoxylin(Fig. 3a-o). As a positive control, we used the preadipocytecell line (3 T3-L1) that underwent accumulation of LDsafter incubation with KOSR (Fig. 3j-l). Human embryonicstem cell (hESC) VAL9 was used as the negative controland did not show a detectable accumulation of LDs afterKOSR incubation (Fig. 3m-o). Furthermore, we quantifiedthe accumulation of LDs in HCCLs in response to 48 hincubation with HES+20%KOSR by means of fluorescentdye Nile-RED [11] and flow cytometry analysis. A markedincrease in FL2 intensity was obtained when compared tothe controls (Fig. 3p). Notably, MALME-3M cellsresponded very efficiently to LDs accumulation, showingmore than a 3-fold induction after incubation with HES+

Stem Cell Rev and Rep (2011) 7:898–909 899

20%KOSR. Accumulation of LDs in cancer cell lines hasbeen previously described [12], however, our resultsunequivocally demonstrate for the first time that HCCLsaccumulate LDs in response to KOSR incubation. Based onour findings, we hypothesize that HCCLs could trans-differentiate toward adipocyte-like cells in response toKOSR since a massive accumulation of LDs in the cell isa hallmark of adipocyte differentiation [13].

Adipogenic transdifferentiation of HCCLs is associatedwith PPARG1 induction

The transcriptional cascade required for adipocyte differen-tiation centers on the expression and activation of PPARG1,

a lipid-activated nuclear hormone receptor. PPARG1 servesas the master transcriptional regulator of adipogenesisis[14]. To better understand the molecular mechanism ofKOSR-induced LD biogenesis in HCCLs, we examined theexpression of several PPARG1-downstream effector genes(such as PPARG1 itself, ADIPOQ, MPZL2, LPL, PRMD16,ELOVL3, MLYCD and PPARA) [15]. We cultured MCF-7cells with HES+20%KOSR with (w) or without (w/o)KOSR, and examined the expression of the adipogenicgenes by RT-PCR techniques (Fig. 4). Notably, an up-regulation of PPARG was detected after KOSR incubationwith no apparent effect on the expressions of ADIPOQ,LPL, MPZL2, PPARA and MLYCD (Fig. 4). Furthermore,we performed a quantitative RT-PCR analysis of the

Fig. 1 KOSR induces modifications in the FSC and SSC parametersof HCCLs. (a). FSC and SSC plots from the HCCLs cultured withconditioned media. MCF-7 and MALME-3M cells were grown on 6-well plates (105 cells/well) and cultured in HES+20%KOSR condi-tioned with VAL9 for 48 h. As a negative control, HES+20%KOSRwas conditioned with the feeder only, and in the absence of VAL9.Cancer cells were also cultured in RPMI+10%FBS (considered time=0). FSC and SSC parameters were assessed and quantified by flowcytometry. Fold induction indicates the ratio between treated con-

ditions and the untreated condition (time=0). (b). FSC and SSC plotsfrom the HCCLs cultured with KOSR. MALME-3M, MCF-7, SK-OV-3, and HUH-7 cells were cultured with HES+20%KOSR for 24 hat a concentration of 105 cells/well in 6-well plates. As controls,cancer cells were cultured in RPMI-1640 (considered time=0) or in aHES medium containing 10% of FBS. Quantification in the FSC andSSC parameters was assessed using the FlowJo software. Foldinduction was estimated by calculating the ratio between treatedconditions and the untreated condition (time=0)

900 Stem Cell Rev and Rep (2011) 7:898–909

PPARG1 expression. This qRT-PCR exhibited a mean 3.34-fold induction (SD=0.47) in PPARG1 in comparison tocells cultured HES w/o (Supplementary Fig. 1a and b). It issignificant to note that the PPARG1 up-regulation wascross-validated in the MALME-3M cells in time-courseexperiments (Supplementary Fig. 2). These experimentsindicate that the PPARG1 expression is induced with 20%KOSR, therefore, PPARG is up-regulated in HCCLs afterKOSR incubation. As PPARG1 is responsible for thetransdifferentiation of committed cells into adipocyte-likecells [16], our data suggest that KOSR could trigger theadipogenic transdifferentiation of HCCLs.

Transdifferentiation of HCCLs into adipocyte-like cellsinduced by the fatty acid-rich albumin fraction (albuMAX)

Next, we evaluated the nature or origin of the trans-differentiating agents present in the KOSR formulationresponsible for triggering the adipogenic transdifferentia-tion in HCCLs. The fatty acid-rich albumin fraction(albuMAX) represents the major component of KOSR

[17]. It is important to note that a genetic defect of thehuman albumin gene (ALB) causes lipodystrophy, charac-terized by a lack of adipose tissue [18]. Consequently, theprimary candidate to be tested as an adipogenic inductorwas albuMAX [17]. To examine the effect of albuMAX onHCCLs, we used MALME-3M cells because this cell typeresponds very efficiently to both KOSR-induced LDaccumulation (Figs. 2 and 3) and KOSR-induced PPARG1up-regulation (Supplementary Fig. 2). MALME-3M cellswere cultured in HES+20%KOSR with 1.6% of albuMAXand without albuMAX [17]. As a negative control, we alsocultured HCCLs in the presence of RPMI+10%FBS. Aftera 48 hours incubation, the alterations in the SCC profilewere assessed (Fig. 5a) and an analysis of LD accumulationwas performed using Oil-RED (Fig. 5b–d) and Nile-REDstaining (Fig. 5e-h). AlbuMAX-induced transdifferentiationwas also quantified by flow cytometry and showed anaccumulation of LDs in MALME-3M cells (Fig. 5i).Furthermore, PPARG1 was also found to be up-regulatedin a time-course analysis after incubating MALME-3Mcells with albuMAX (Supplementary Fig. 3). This result

Fig. 2 Electron Microscopy of LDs biogenesis in the HCCLs culturedwith KOSR. (a-h). Electron microscopy (EM) of HCCLs: MALME-3M (a-d), MCF-7 (e-f) and HUH-7 (G-H) was performed as indicatedin the Materials and Methods section. Cells were grown in the

presence of HES+20%KOSR for 48 h (b, c, d, f and h). As a negativecontrol, MALME-3M (a), MCF-7 (e) and HUH7 (g) cells were grownwith RPMI-10%FBS


confirmed that albuMAX incubation enforces the adipo-genic transdifferentiation of MALME-3M cells. All analy-ses were also performed in MCF-7, HUH-7 and SK-OV-3(data not shown). In general, these results indicate thatalbuMAX works as an efficient adipogenic transdifferenti-ating agent in HCCLs.

Linoleic, oleic, palmitoleic and petroselinic unsaturatedfatty acids are responsible for the adipogenictransdifferentiation in HCCLs

AlbuMAX is considered a fatty acid-albumin complex [17]formed by unsaturated fatty acids [19]. These data, togetherwith the role of nitro-linoleic and hydroxy-oleic acids asPPARG ligands [20, 21], led us to hypothesize thatunsaturated fatty acids could be crucial to transdifferentia-

tion. To confirm this, we initially screened 10 mono-andpolyunsaturated fatty acids and their ability to induce LDsin MALME-3M cells. Accumulation of LDs was quantifiedby Nile-RED staining after incubating cells with mono-andpolyunsaturated fatty acids. As a negative control, weincubated HCCLs in the presence of RPMI+10%FBSwithout unsaturated fatty acids and with the vehicle DMSO.Cells grown in the presence of albuMAX were used as apositive control. After a 48 hours incubation period, LDaccumulation was induced by palmitoleic, oleic, petrose-linic, elaidic, erucic, and linoleic acids (Fig. 6), but not bydocosahexaenoic, nervoic, arachidonic and linolenic acids.One important finding was that all the monounsaturatedfatty acids tested in MALME-3M cells induced LDs,whereas the all of the polyunsaturated fatty acids, exceptfor linolenic acid, induced only a marginal accumulation of

Fig. 3 Oil red and Nile red quantifications of neutral lipids inducedby KOSR. (a-o). Oil-red staining of HCCLs cultured with KOSR.HCCLs: MALME-3M (a-c), SK-OV-3 (d-f), and MCF-7(g-i) werecultured as described in Fig. 1b. Oil-red and hematoxylin stainingswere performed as described in the Materials and Methods section. Asa negative control, KOSR was replaced with 10% of FBS (c, f, i, l, o).VAL9 was grown on HES+20%KOSR and considered a negativecontrol (n). The preadipocyte 3 T3-L1 cell line was grown under the

same conditions as HCCLs, and was considered a positive control forLDs (K). Magnification was x 40. (p). HCCLs (MALME-3M, MCF-7, SK-OV-3) were cultured as described in Fig. 1b, and nile-REDstaining was performed as described in the Materials and Methodssection. Quantification was assessed using the FlowJo software. Foldinduction was estimated by calculating the ratio between treatedconditions and the untreated condition (control)


LDs (Fig. 6). Furthermore, only linoleic (L), oleic (O),palmitoleic (P) and petroselinic acids (T) efficientlyinduced the generation of LDs (≥ 6 fold induction)(Fig. 6). Oleic acid has been previously used to inducelipid droplets in macrophages [22] and monocytes [9].However, our experiments show for the first time thatspecific unsaturated fatty acids (L, O, P and T) triggerefficient LD accumulation in melanoma cells.

In order to search for an even more efficient trans-differentiating cocktail of unsaturated fatty acids, wecombined L, O, P and T, and tested them in MALME-3Mand HUH-7 cells. The results obtained indicate that thecombination of P, O and L (POL) in MALME-3M and P, Oand T (POT) in HUH-7 proved to be the most efficientcocktails (Supplementary Figs. 4 and 5) after an 8 hoursincubation period as they induce a ≥5- and ≥2.75-foldinduction, respectively. These cocktails (POT and POL)were also tested in breast adenocarcinoma MCF-7 andpreadipocyte 3 T3-L1 and induced a ≥5- and ≥4-foldinduction, respectively (data not shown). These experi-ments indicated that POT is the most efficient adipogenictransdifferentiation cocktail in both HUH-7 and MCF-7

Fig. 4 PPARG up-regulation inMCF-7 cells after KOSRtreatment. RT-PCR analysis ofMCF-7 cultured with KOSR.Gene expression analysis forPPARG1, ADIPOQ, LPL,MLYCD, MPZL2 and GAPDH(loading control). MCF-7 cellswere cultured with (w) andwithout (w/o) 20% of KOSR for48 h. The expressions of thesegenes were also analyzed inVAL9 (negative control) andwhite adipose tissues (WAT,positive control, +). (−) denotesRT-PCR w/o RNA and in thepresence of H2O. PPARG1 wasused as a positive control of theKOSR-induced lipogenesis ineach gene testing. The resultsare representative of at leastthree independent experimentsfor each gene

Fig. 5 LDs accumulation induced by albuMAX in MALME-3Mcells. (a). FSC and SSC plots from the HCCLs cultured withalbuMAX. The MALME-3M cells cultured on 6-well plates’05 cells/well) were cultured for 48 h w and w/o 1.6% of albuMAX. Cells werealso cultured with RPMI-10%FBS and considered negative controls.Quantification in the FSC and SSC parameters was assessed by flowcytometry and further analyzed by the FlowJo software. Foldinduction indicates the ratio between treated conditions and theuntreated condition (control). (b-d). The MALME-3M cells culturedon 6-well plates were cultured for 48 h w (d) and w/o (c) 1.6% ofalbuMAX. Controls (time=0) were considered when cells werecultured with RPMI+10%FBS (b). Pictures showing Oil-REDstaining are provided. Magnification was x 40. (e-h). Confocalmicroscopy analysis of the Nile-Red stained-MALME-3M cellscultured either in the presence (F, H) or absence (E, G) of 1.6% ofalbuMAX. The cells fixed with 4% of paraformaldehyde were stainedwith Nile-RED, as indicated in the Materials and Methods (E, F). Nextcells were mounted with Prolong antifade containing DAPI for nuclearstaining (G, H). Images (x 63) are shown. (i). MALME-3M cells werecultured as described in Fig. 1b and nile-RED staining was performedas described in the Materials and Methods section. Quantification wasassessed by the FlowJo software. Fold induction was estimated bycalculating the ratio between treated conditions and the untreatedcondition (control)

b


cells, whereas POL appeared to be the most efficient inMALME-3M cells and preadipocyte 3 T3-L1. Thesedifferences in adipogenic responses may reflect thedifferences in the downstream effector pathways trig-

gered by the distinct unsaturated fatty acids. It isinteresting to note that linoleic acid, but not linolenicacid, triggers LD accumulation in MALME-3M andHUH-7 (Fig. 6, and Supplementary Figs. 4 and 5). These


findings are important since linoleic acid structurallyresembles linolenic acid, but lacks only one unsaturationat the position ω3 (http://www.lipidmaps.org/), indicatingthe specificity of the transdifferentiation process inducedby linoleic acid.

Since P, O and L have been shown to associate directlywith albumin [19, 23, 24], we assayed the POL cocktail tosee if it merges with human recombinant albumin to induceLDs. MALME-3M cells were incubated in the presence ofPOL, recombinant albumin only, or in combination withboth POL and albumin. After a 12 hours incubation period,the neutral lipids contained in the LDs were quantified byNile-RED. The POL cocktail induced a similar foldinduction to that generated by the combination of POLplus recombinant albumin, while albumin itself presentedno adipogenic properties (data not shown). EM studiesfurther demonstrate a massive multilocular, and an eventualunilocular LDs formation, in close proximity to mitochon-dria and the endoplasmic reticulum in HUH-7 andMALME-3M upon incubation with unsaturated fatty acidcocktails (Fig. 7a–h). This data confirms that specific fattyacids, but not albumin itself, are responsible for HCCLstransdifferentiation into adipocyte-like cells.

Discussion

Despite the important accomplishment of differentiationtherapy in the identification of novel genes/molecules totrigger terminal transdifferentiation of human cancer cells,many key questions remain unanswered. In this study, lipid-albumin complexes (albuMAX) were found to simplyinduce the transdifferentiation of HCCLs towardadipocyte-like cells and led us to the provider of transcrip-tional signaling linked to PPARG1 up-regulation. Thesefindings indicate that the connection between albuMAXand pluripotency [17] is surprisingly more complex thanpreviously anticipated. In addition to the albuMAX func-tion as a factor required for maintaining self-renewal andpluripotency in hESC [17], a role for albumin [18] and theassociated lipids [19, 23, 24] has been discovered inadipogenesis in other cell types [20]. At this point, it isnot clear whether albuMAX-induced pluripotency is due toa direct target or if it is possibly a specific effect on hESCs.How all the targets of albuMAX are sensed in hESC versusHCCLs is a piece of this intricate puzzle.

AlbuMAX efficiently triggers the biogenesis of the LDsin HCCLs; as detected by several techniques including

Fig. 6 Oleic, linoleic, palmitoleic and petroselinic acids, but notalbumin, are the transdifferentiating lipids of HCCLs to an adipocyte-like phenotype. MALME-3M cells were cultured in the presence of10 μg/ml of docosahexaenoic acid, 100 μg/ml of nervonic acid,100 μg/ml of erucic acid, 10 μg/ml of arachidonic acid, 100 μg/ml oflinolenic acid, 100 μg/ml of linoleic acid, 100 μg/ml of oleic acid,100 μg/ml of palmitoleic acid, 100 μg/ml of elaidic acid, or 100 μg/mlpetroselinic acid for 12 h. Fatty acids were dissolved in DMSO;

therefore the cells cultured with RPMI+10%FBS and treated withDMSO only were considered negative controls. The Nile-Red analysiswas performed as described in the Material and Methods section.Quantification in the FL2 parameter was assessed by the FlowJosoftware. Fold induction was estimated by calculating the ratiobetween treated conditions and the untreated condition (control). Theresults are representative of three independent experiments


http://www.lipidmaps.org/

electron microscopy and oil-RED staining, and as quanti-fied by flow cytometry using the nile-RED dye. AlbuMAX-induced transdifferentiation was also accompanied byPPARG1 up-regulation. We demonstrated the specificity ofalbuMAX-induced PPARG1 up-regulation by showing thatthe other genes associated with adipogenesis (i.e., ADIPOQ,MPZL2, LPL, PRMD16, ELOVL3, MLYCD and PPARA)were not up-regulated along with PPARG1. These findingsare important because most of the adipogenesis-associatedgenes have been discovered in murine cells and our resultsindicate important differences between adipogenesis in miceversus humans.

Encouraged by the results obtained with albuMAX inHCCLs, we decided to determine the specific moleculesresponsible for the adipogenic transdifferentiation process.Given that several fatty acids form a complex with albumin[17, 19, 23, 24], we screened the unsaturated fatty acidfamily and its ability to induce adipogenic transdifferentia-tion. With this approach, we discovered that linoleic, oleicand palmitoleic acids, but not albumin, induce LD

accumulation efficiently, which is in agreement with therole of linoleic, oleic and palmitoleic as PPARG ligands[25–27]. Thus, our results indicate that certain HCCLsexhibit a surprisingly high degree of plasticity in responseto linoleic, oleic and palmitoleic acids, which suggestsadipocyte-like cells transdifferentiation. In addition, ourresults indicated that the use of poly- and monounsaturatedfatty acids cocktails improved their efficiency and sug-gested the possibility of inducing terminal differentiation(Supplementary Fig. 6) more efficiently and blockingcancer progression.

Since the transdifferentiating lipids in cancer (e.g., alltrans-retinoic acids [28] or the linoleic acid isomer 9Z-11E[29]) mediate their functions together with nuclear recep-tors, pathologic processes such as human cancer should betreated by considering a complex network of interconnectednuclear receptors. Thus, we may consider a “systembiology approach” in which enforced transdifferentiationof human cancer cells should be activated by a givencombination of transdifferentiating lipids. The elucidation

Fig. 7 Electron Microscopy of LDs biogenesis in both the HUH-7and MALME-3M induced by the POT and POL fatty acids cocktails.Cells were cultured in the presence of the indicated fatty acidscocktails at a concentration of 100 μg/ml for 48 h (b-d, f-h). As anegative control, HUH-7 and MALME-3M were grown with RPMI-

10%FBS (a, e). Bar scales: 10, 5 and 1 μm. The presence of massivemultilocular (in MALME-3M) or unilocular (in HUH-7) LDs can beobserved with LDs in close proximity to mitochondria and the roughendoplasmic reticulum, even with a polarization pattern that furtherdemonstrates the specificity of this effect


of these novel connections, along with the roles played inadipogenic transdifferentiation, should provide a solidfoundation to open up new cell biology avenues for cancertherapy.

Methods

Human cancer cell lines (HCCLs)

MALME-3M melanoma (ATTC# HTB-64), HUH-7 hep-atocarcinoma, MCF-7 breast adenocarcinoma (ATTC#HTB-22) and SK-OV-3 ovarian adenocarcinoma (ATTC#HTB-77). HCCLs were split into growth media containingRPMI-1640, 10% fetal bovine serum (FBS) and 2 mMglutamine following a standard 3 T3 protocol [30].

Human embryonic stem cells

VAL9 [http://www.isciii.es/htdocs/terapia/pdf/Documento_Deposito_VAL_9.pdf] was split onto a monolayer ofirradiated human foreskin fibroblasts (FSK) (ATTC#CRL-2429). hESCs were cultured at a density of 104

cells/cm2 in HES growth media (KO-DMEM, 20% knock-out serum replacement-KOSR, 2 mM glutamine, 1% non-essential amino acids, 50 μM β-mercaptoethanol and10 ng/ml human recombinant bFGF). VAL9 colonies weremanually expanded using Pasteur pipettes tips every 4 days.40 hESC colony fragments (containing 10,000 cells/fragment) were split into new plates (6-well plates) andgrowth media were replaced daily.

Electron microscopy

HCCLs were seeded on Permanox slides (Nalge NuncInternational, Naperville, IL) at a density of 2000 cells/well.Cells were fixed with 3.5% glutaraldehyde (1 h, 37°C),treated with 2% osmium tetroxide (1 h, RT) and stainedwith 2% uranyl acetate away from light (2 h, 4°C). Cellswere rinsed in sodium phosphate buffer (0.1 M), dehy-drated in ethanol, and infiltrated overnight in araldite resins(Durcupan, Fluka, Buchs SG, Switzerland). After polymer-ization, embedded cultures were detached from the cham-ber slide and glued to Araldite blocks. Semi-thin sections(1.5 μm) were cut with an Ultracut UC-6 (Leica, Heidelberg,Germany), mounted onto slides and stained with 1% toluidineblue. Semi-thin sections were glued to araldite blocks anddetached from the glass slide by freezing-thawing cyclesusing liquid nitrogen. Ultrathin sections (0.07 μm) wereprepared with an Ultracut and then stained with lead citrate.Finally, photomicrographs were obtained under an FEImicroscope (Tecnai Spirit G2) using a digital camera (SoftImaging System, Olympus).

Nile-RED staining

Neutral lipid quantification was performed with Nile-Red[11]. Cells (105) were washed twice in PBS and thenincubated with 25 μg/ml of Nile-Red (Invitrogen) in PBS(15 min, 4°C). After incubation, cells were filtered througha 30 μm sieve (CellTrics, Partec). Fluorescent emission(525 nm) was registered in the FL2 channel by a BeckmanCoulter Flow Cytometer (Cytomics™ FC 500). Data fileswere analyzed using the FlowJo flow cytometer analysissoftware.

Oil-RED staining

Lipid droplets were stained by Oil-red [10]. A stock of Oil-red solution was prepared in 2-propanol (0.3%), vortexedand filtered through a 0.8 μm sieve before staining. Thecells (105) grown on the 6-well plates were washed twice inPBS and fixed with 4% of formaldehyde (Panreac) for30 min. After fixation, cells were washed 3 times in PBSand stained with Oil-red (60 volumes of stock solution: 40volumes of water). Cells were washed with cold PBS (atleast 3 times) and then stained in filtered hematoxylin(Sigma-Aldrich) for 2 min. After staining, cells werewashed with PBS and pictures were taken under a lightcontrast microscope (Leica DMIL) using the Leica Applica-tion Suite version 2.4.0 R1 software (Leica Microsystems,Switzerland).

Reverse transcriptase PCR (RT-PCR)

Gene expression was performed by RT-PCR techniques.RNA was isolated by using RNeasy® (Qiagen) accord-ing to the manufacturer’s instructions. RNA quantifica-tion was measured by a Nanodrop platform (ThermoScientific). Then, 500 ng of total RNA was convertedinto cDNA with oligo-dT by using the Advantage® RT-for-PCR kit (Clontech) and following the manufacturer’sprotocol. Next, 5 μl of cDNA were subjected to PCRamplification using Biotaq™ DNA polymerase (Bioline)following the manufacturer’s instructions. The PCRamplification cycles included [(94°C, 5′); 30 cycles at(94°C, 20″)(56°C, 20″)(72°C, 40″); (72°C, 1′)]. Theprimers for each gene were designed using Primer3,(http://frodo.wi.mit.edu/primer3/). Subsequently, a blastanalysis was performed to test primer specificity usingthe Primer-Blast tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). PCR products were resolved on 1.5%agarose gels (Agarose D-1, low EEO-GQT, Pronadisa),stained with 0.1 μg/ml of ethidium bromide (Sigma-Aldrich), and visualized in an UV-transilluminator (BioRad).Band densitometry was calculated by using the Quantity Onesoftware.


http://www.isciii.es/htdocs/terapia/pdf/Documento_Deposito_VAL_9.pdf

http://www.isciii.es/htdocs/terapia/pdf/Documento_Deposito_VAL_9.pdf

http://frodo.wi.mit.edu/primer3/

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

http://www.ncbi.nlm.nih.gov/tools/primer-blast/

qRT-PCR

The qPCR analysis was performed using a LightCycler®platform including all the kit components (Roche). qPCRcycles included: [(94°C, 10′); 40 cycles at (95°C, 10″)(65,6″)(72°C, 10″)]. RNA normalization was performed withglyceraldehydes-3-phosphate dehydrogenase (GAPDH) 32.Fold induction was calculated by the equation: 2-ΔΔCt,ΔΔCt=[(Ct gene of interest-Ct internal control) sample A-(Ct gene of interest-Ct internal control) sample B.

Confocal microscopy

Images were acquired with a Leica TCS SP2 AOBS (LeicaMicrosystems Heidelberg GmbH, Mannheim, Germany)inverted laser scanning confocal microscope using a 63 xPlan-Apochromat-Lambda Blue 1.4 N.A. oil objective. Theexcitation wavelengths for fluorochromes used were514 nm (Nile-red) and 405 nm (DAPI). The emissionapertures for fluorescence detection were (580–680 nm forNile-red) and (420–470 nm for DAPI). Two-dimensionalpseudo color images were obtained with a size of1024x1024 pixels (step side=0.5 μm). All the confocalimages were acquired using the same settings, while thedistribution of fluorescence was analyzed using the LeicaConfocal “Leica Lite” software, version 2.61.

Acknowledgments We would like to thank Dr. Alicia Martinez forher technical assistance in flow cytometry (Cytomics Laboratory atCIPF), the Confocal Microscopy Laboratory (CIPF), AlejandroRincón-Bertolín for his technical assistance, all the Valencia Node ofthe Spanish Stem Cell Bank members for their technical support andhelpful discussions, Dr. José Manuel García-Verdugo (CellularMorphology Laboratory) for technical assistance in electron micros-copy, and Dr. Pérez-Payá (Protein Chemistry Laboratory) and Dr. JuanSaus (Cellular Pathology laboratory) for critically reading thismanuscript. This work has been supported by the RegenerativeMedicine Program, The Regional Valencian Ministry of Health, andthe Carlos III Institute of Health.

Disclosures The authors indicate no potential conflicts of interest.

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