Polymer Thin Film Induced Tumor Spheroids Acquire Cancer ...(KAIST),Daejeon,RepublicofKorea.3CenterforNano-BioMeasurement,Korea Research Institute of Standards and Science (KRISS),

Convergence and Technologies

Polymer Thin Film–Induced Tumor SpheroidsAcquire Cancer Stem Cell–like PropertiesMinsuk Choi1, Seung J. Yu2, Yoonjung Choi1, Hak R. Lee2, Eunbeol Lee1,Eunjung Lee2, Yumi Lee1, Junhyuk Song1, Jin G. Son3, Tae G. Lee3, Jin Y. Kim4,Sukmo Kang1, Jieung Baek2, Daeyoup Lee1, Sung G. Im2, and Sangyong Jon1,4

Abstract

Although cancer stem cells (CSC) are thought to be respon-sible for tumor recurrence and resistance to chemotherapy,CSC-related research and drug development have been ham-pered by the limited supply of diverse, patient-derived CSC.Here, we present a functional polymer thin film (PTF) plat-form that promotes conversion of cancer cells to highlytumorigenic three-dimensional (3D) spheroids without theuse of biochemical or genetic manipulations. Culturing var-ious human cancer cells on the specific PTF, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4),gave rise to numerous multicellular tumor spheroids within24 hours with high efficiency and reproducibility. Cancer cellsin the resulting spheroids showed a significant increase in the

expression of CSC-associated genes and acquired increaseddrug resistance compared with two-dimensional monolayer-cultured controls. These spheroids also exhibited enhancedxenograft tumor-forming ability and metastatic capacity innude mice. By enabling the generation of tumorigenic spher-oids from diverse cancer cells, the surface platform describedhere harbors the potential to contribute to CSC-related basicresearch and drug development.

Significance: A new cell culture technology enables high-ly tumorigenic 3D spheroids to be easily generated fromvarious cancer cell sources in the common laboratory.Cancer Res; 78(24); 6890–902. �2018 AACR.

IntroductionSince the initial discovery of stem cell–like cancer cells in acute

myeloid leukemia (1), increasing evidence has supported thepresence of a minor population of cancer cells in bulk tumorsthat are mainly responsible for tumor recurrence and drug resis-tance (2). These cells, termed cancer stem cells (CSC) or tumor-initiating cells, share many of the common characteristics ofnormal stem cells (1), including self-renewal capacity (3), intrin-sic drug resistance (4), anddifferentiation ability (3).Hence, CSCshave attracted considerable interest in the fields of cancer researchand drug development (5). CSCs are usually isolated frompatient-derived tumor tissues through standard cell-sorting pro-cesses based on characteristic surface markers (6) and cell isola-

tion based on spheroid-forming ability (7). However, the supplyof patient-derived CSCs is limited, creating a bottleneck for CSCresearch (1, 3, 8). As an alternative, attempts have been made toisolate CSCs from conventional cancer cell lines, but only a smallsubpopulation (<1%–2%) of cancer cells expresses CSC-relatedsurface markers, making such approaches also impractical forobtaining sufficient amounts of CSCs (2). A recent reportshowed that soft fibrin gels with well-controlled stiffness canselectively promote the growth of tumorigenic cells fromamong a pool of cancer cells, providing a useful method forCSC isolation based on tumorigenic behavior rather thanputative CSC surface markers (9).

There is currently substantial and growing interest in develop-ing methods that facilitate the formation of cancer cell spheroidsbecause these three-dimensional (3D) structures are thought tobetter mimic in vivo tumor environments than two-dimensional(2D) monolayer cultures (10). Such spheroids, which have beenemployed for drug screening and efficacy tests, have been gener-ated using a variety of methods, including seeding cells onhydrophilic ultra-low-attachment (ULA) surfaces (11) or onagarose gels with a concave shape (U-bottom; ref. 12), or byinserting cells into holes of hanging-drop culture plates (13).However, the tumor spheroids generated from cancer cell linesusing the conventional methods do not systematically result inCSC enrichment (7, 14). In this context, there is a need for a facilemethod or simple technique that enables generation of tumor-igenic spheroids from conventional human cancer cells with highefficiency and reproducibility. A few recent reports have suggestedthe possibility of bidirectional conversion between nontumori-genic cancer cells and CSCs (15). In light of the possibility of sucha bidirectional conversion, we hypothesized that it may be pos-sible to transform cancer cells to tumorigenic CSC-like cells if

1Department of Biological Sciences, Korea Advanced Institute of Science andTechnology (KAIST), Daejeon, Republic of Korea. 2Department of Chemical andBiomolecular Engineering, Korea Advanced Institute of Science and Technology(KAIST), Daejeon, Republic of Korea. 3Center for Nano-Bio Measurement, KoreaResearch Institute of Standards and Science (KRISS), Daejeon, Republic ofKorea. 4Graduate School of Medical Science and Engineering, Korea AdvancedInstitute of Science and Technology (KAIST), Daejeon, Republic of Korea.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M. Choi, S.J. Yu, and Y. Choi contributed equally to this article.

CorrespondingAuthor:Sangyong Jon, KoreaAdvanced Institute of Science andTechnology (KAIST), 291 Daehak-ro, Daejeon 350-701, Republic of Korea.Phone: 82-42-350-2634; Fax: 82-42-350-4450; E-mail: [email protected]; andSung G. Im, Phone: 82-42-350-3936; Fax: 82-42-350-3910; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-18-0927

�2018 American Association for Cancer Research.

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appropriate stimuli (chemical or biological) are provided on aculture surface (16, 17). Here, we report a general platform thatpromotes the transformation of diverse cancer cells to tumori-genic CSC-like 3D spheroids by simply culturing the cells on aspecific functionalized surface.

Materials and MethodsFormationof diverse polymer thinfilms on cell culture plates orcover glasses via the initiated chemical vapordepositionprocess

The process for preparing pV4D4 thin films is described as atypical example; other polymer thinfilms (PTF) are also depositeddirectly on tissue culture plates (TCP) using the same protocol,with slightmodifications of deposition conditions, as needed (seeSupplementary Methods). For vaporization of the monomer,V4D4 (99%; Gelest) and tert-butyl peroxide (TBPO, 98%;Aldrich) were heated to 70�C and 30�C, respectively. VaporizedV4D4 and TBPOwere introduced into an initiated chemical vapordeposition (iCVD) chamber (DaekiHi-TechCo. Ltd.) atflow ratesof 1.5 and 1 standard cm3/min (sccm), respectively. The substratetemperature was maintained at 40�C, the filament temperaturewas kept at 200�C, and the iCVD chamber pressure was set to 180mTorr. The deposition rate of pV4D4 filmwas estimated to be 1.8nm/min. The thickness of pV4D4 films was monitored in situusing a He-Ne laser (JDS Uniphase) interferometer system.

Human cancer cell linesHuman ovarian cancer cell lines (SKOV3 and OVCAR3),

human breast cancer cell lines (MCF-7, T47D, and BT-474),human liver carcinoma cell lines (Hep3B and HepG2), humanglioblastoma cell lines (U87MG and U251), human colon cancercell lines (SW480, HT-29, HCT116, and Caco-2), human lungcancer cell lines (A549, NCI-H358, and NCI-H460), and humanprostate cancer (22RV1), cervical cancer (HeLa), melanoma(A375), and gastric cancer (NCI-N87) cell lines were purchasedfrom Korea Cell Line Bank. Cell lines were authenticated bystandard short tandem repeat DNA typing methodology. Allcancer cells were Mycoplasma-free, tested using an e-Myco Myco-plasma PCR Detection Kit (iNtRON Biotechnology).

Cell culture conditionsSKOV3, T47D, BT-474, SW480, HT29, 22RV1, A549, NCI-

H358, NCI-N87, OVCAR3, NCI-H460, and HCT116 cell lineswere cultured inRPMI-1640medium(Gibco) supplementedwith10%(v/v) FBS (HyClone), 1% (v/v) penicillin/streptomycin (P/S;Gibco), and 25 mmol/L HEPES (Gibco). MCF-7, Hep3B, HeLa,U251, and A375 cell lines were cultured in DMEM (Gibco)supplemented with 10% (v/v) FBS (HyClone) and 1% (v/v)P/S (Gibco). HepG2, U87MG, and Caco-2 cell lines were culturedin Minimum Essential Media (MEM; Gibco) supplemented with10%(v/v) FBS (HyClone) and 1%(v/v) P/S (Gibco). All cells weremaintained at 37�C in a humidified 5%CO2 atmosphere. The celllines were used within 10 to 12 passages after thawing.

Spheroid formation on pV4D4Cancer cells (1� 106)were seededonpV4D4-coated plates and

cultured in RPMI-1640, DMEM, orMEM, as appropriate, contain-ing 10% (v/v) serum replacement (Gibco), 1% (v/v) P/S (Gibco),and L-glutamine at 37�C inahumidified5%CO2atmosphere. Thespecific cell culture medium used for each cancer cell line isdescribed in detail in Supplementary Information. For optimumspheroid growth, culturemediumwas refreshed every 2 to 3 days.

Spheroid formation using conventional methodsHanging-drop 96-well plates (3D Biomatrix), U-bottom

96-well plates (S-Bio), and ULA 6-well plates (Corning) wereused for conventional spheroid formation. Cellswere plated at thefollowing densities: 1� 104 cells/50 mL for hanging drop, 5� 104

cells/250 mL for U-bottom, and 5 � 105 cells/2 mL for ULA. Foroptimum spheroid growth, culturemediumwas refreshed every 2to 3 days.

ImmunocytochemistrySKOV3 spheroids were transferred fromULA and pV4D4 plates

to a 1.5 mL tube and fixed by incubating with a 4% paraformal-dehyde solution (Sigma) for 30 minutes at room temperature.Fixed spheroids were incubated with 0.25% (w/v) Triton X-100(Sigma) in Dulbecco's phosphate-buffered saline (D-PBS) solu-tion for 10minutes at room temperature, washedwithD-PBS, andincubatedwithblocking solution (3%BSA inD-PBS). For lamininstaining, spheroids were incubated with rabbit anti-human lam-inin primary antibody (1:100, cat. no. 11575; Abcam) for 12hours at 4�C. After washing with D-PBS, the resulting spheroidswere stained with rhodamine red-X–conjugated anti-rabbit sec-ondary antibody (1:500, cat. no. R6394; Invitrogen) for 1 hour atroom temperature, and then with Hoechst 33342 for 10 minutes.For tenascin-C (TNC) staining, SKOV3 2D controls or SKOV3spheroids were incubated with rabbit anti-human TNC primaryantibody (20 mg/mL, cat. no. AB19011; Millipore) for 12 hours at4�C. After washing with D-PBS, cells and spheroids were stainedwith FITC-conjugated anti-rabbit secondary antibody (1:500, cat.no. sc-2012; Santa Cruz Biotechnology) for 1 hour at roomtemperature, and then with Hoechst 33342 for 10 minutes. Forb-catenin staining, SKOV3 2D controls and SKOV3-ssiCSCswere incubated with mouse anti-human b-catenin primary anti-body (1:100, cat. no. 13–8400; Invitrogen) for 1 hour at roomtemperature. After washing with D-PBS, cells were stained withTRITC-conjugated anti-mouse secondary antibody (1:1,000, cat.no. ab6786; Abcam) for 1 hour at room temperature, and thenwith Hoechst 33342 for 10 minutes. All fluorescent images werevisualized using a confocal laser-scanning microscope (LSM 780,Carl Zeiss).

Flow cytometry analysisMonolayer-cultured 2D control cancer cells and the corre-

sponding pV4D4-cultured ssiCSC spheroids or ULA-culturedSKOV3 spheroids were trypsinized and then individually dis-persed in buffer (D-PBS containing 1% FBS). SKOV3, MCF-7,Hep3B, and SW480 cancer cells were stained with allophyco-cyanin-conjugated anti-CD133 (1:100; eBioScience), FITC-conjugated anti-CD44 (1:200; BD Biosciences), phycoery-thrin-conjugated anti-CD90 (1:100, MACS; Miltenyi Biotec),and FITC-conjugated anti-CD133 (1:100; Miltenyi Biotec) pri-mary antibodies, respectively, and analyzed using a flow cyto-metry system (BD Calibur and BD LSR Fortessa).

For side population assays, 2D control cancer cells and ssiCSCswere dissociated using trypsin and then stained with Hoechst33342 (ThermoFisher Scientific) in DMEM containing 2% FBSand 10 mmol/L HEPES buffer for 90 minutes at 37�C. Cells werethen washed with HBSS containing 2% FBS and analyzed using aflow cytometry system (BD LSR Fortessa). Flow cytometry datahistograms and plots were analyzed using FlowJo software (TreeStar Inc.).

Surface Stimuli–Induced Cancer Stem Cell–like Cells

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RNA extraction and mRNA sequencingmRNAwas extracted fromSKOV3 spheroids cultured for 8 days

on pV4D4-coated plates and 2D control SKOV3 cells using aMagnetic mRNA Isolation Kit (NEB) according to the manufac-turer's protocol. A library was prepared from DNase-treatedmRNA using a NEXTflex Rapid Directional mRNA-Seq Kit(BIOO), as described by the manufacturer. Each library wassequenced on a HiSeq2500 system using the single-end method(50-bp reads). The sequenced reads were aligned to the humangenome (version: Hg19) using STAR aligner (v.2.4.0; ref. 18). TheHOMER software algorithm (19) andDESeq R package were usedto investigate differentially expressed genes (DEG). Heatmap andMA plots were visualized using the pheatmap function andplotMA function, respectively, of the R statistical programminglanguage v.3.3.0. (http://www.r-project.org/).

Gene ontology analysis and gene set enrichment analysisThe Gene Ontology (GO) analysis for up- and downregulated

genes in SKOV3-ssiCSCs was carried out using the Consensus-PathDB database (http://consensuspathdb.org/). The significancethresholdwas defined by a P value less than 0.01. TheGOanalysiswas repeated on a published expression dataset (GSE62905).Gene set enrichment analysis (GSEA; ref. 20)was performedusingGSEA software (version 3.0) with 1,000 phenotype permutationsand default values for other parameters. Gene sets used in thisstudy were selected from the MSigDB hallmark gene sets (http://software.broadinstitute.org/gsea/msigdb/collections.jsp).

Animals and xenograft tumor formationFemale BALB/c nude mice (6 weeks old) were obtained from

Orient Bio Inc. and housed under pathogen-free conditions in theanimal facility at Korea Advanced Institute of Science and Tech-nology. Mice were assigned randomly to experimental groups.The experiments themselves were not randomized, and investi-gators were not blinded to allocation during experiments andoutcome assessment unless each section specifically includedblind assessments. All surgeries were performed under isofluraneanesthesia, and every effort was made to minimize suffering. Allanimal procedures were reviewed and approved (approval num-ber: KA2014–21) by the Korea Advanced Institute of Science andTechnology's Institutional Animal Care and Use Committee forcompliance with ethical procedures and scientific care.

For preparation of the human ovarian cancer xenograft model,2D-cultured control SKOV3 cells or SKOV3-ssiCSCs dissociatedfrom the corresponding spheroids were mixed with 50%Matrigel (Corning) at different serial dilutions (106 to 102 cells)and then s.c. injected into 6-week-old female BALB/c nude mice.Tumor formation was monitored for up to 120 days, and theformation of tumors was recorded when tumor volumes reachedapproximately 50 mm3. For preparation of the human breastcancer xenograft model, different serially diluted numbers (107 to102) of 2D control cells or ssiCSCs from MCF7-Luc cancer cellswere subcutaneously inoculated into 6-week-old female BALB/cnude mice. b-Estradiol 17-valerate (2.5 mg; Sigma), dissolved in50 mL of sesame oil (Sigma), was subcutaneously administered toBALB/c nude mice via the neck every 10 days. For the humanglioma xenograft model, different serially diluted numbers (106

to 102) of 2D control U87MG cells, ULA-cultured U87MG spher-oids, or pV4D4-cultured U87MG-ssiCSC cells were mixed with50% Matrigel and s.c. injected into 6-week-old female BALB/cnude mice. Tumor formation from MCF7-Luc and U87MG cells

was monitored up to 90 days, and the formation of tumors wasrecorded when tumor volumes reached approximately 50 mm3.

Statistical analysisData were expressed as means � SD. Statistical analyses were

conducted using unpaired Student t tests with GraphPad Prismsoftware. A P value< 0.05 was considered statistically significant.

Data availability statementRNA sequencing data are available at the National Center for

Biotechnology Information Gene Expression Omnibus datarepository with the accession code GSE106848.

ResultsA PTF enables formation of 3D tumor spheroids from diversehuman cancer cells

To introduce various surface functionalities on cell cultureplates, we constructed a library of PTFs on conventional TCPsfromvariousmonomersusingan iCVDprocess (21)andexaminedthe ability of each PTF to promote formation of tumor spheroids(Fig. 1A). The chemical structures of a subset of the tested PTFsare shown in Supplementary Fig. S1A–S1F. When cells of theSKOV3 human ovarian cancer cell line were cultured on variousPTFs, only one PTF surface—poly(2,4,6,8-tetravinyl-2,4,6,8-tetra-methyl cyclotetrasiloxane), designated pV4D4—supported theformation of numerous multicellular spheroids within 24 hours.In contrast, these same cells grown on other PTFs exhibited a well-adhered, spread-out morphology similar to that of cells grownon TCPs (Fig. 1B). Encouraged by these preliminary findings,we further examined whether the spheroid-promoting ability ofpV4D4 could be extended to other cancer cell lines. As shownin Fig. 1C,most human cancer cell lines regardless of origin or typerapidly formed multicellular spheroids (�50–300 mm in diame-ter) within 24 hours, with high efficiency and reproducibility.Although the morphology of each spheroid varied from "clus-ter-of-grapes" appearance to densely packed spheres (Supplemen-tary Fig. S2), these observations are indicative of the versatility ofthe PTF platform. Unlike a conventional hydrophilic ULA surface(11), thepV4D4PTF surface, as characterized by Fourier transforminfrared spectroscopy and high-resolution X-ray photoelectronspectroscopy (Supplementary Fig. S3A–S3C and SupplementaryTable S1),was relatively hydrophobicwith awater contact angleofapproximately 90� (Supplementary Fig. S4) and had a smoothsurface with a roughness similar to that of conventional TCPs(SupplementaryFig. S5A). Inaddition, therewas littledifference inthe surface hardness between a pV4D4 PTF (200 nm in thickness)and a conventional TCP (0.510 vs. 0.517GPa; Supplementary Fig.S5B). Notably, variations in the thickness of pV4D4 PTFs, whichranged from50 to300nm,hadnoeffecton their spheroid-formingability (Supplementary Fig. S6), suggesting that a certain surfacefunctionality (chemical or biological stimulus) present on pV4D4rather than amechanical cue like surface stiffnessmight be respon-sible for spheroid formation.

Interestingly, individual cancer cells initially adhered to thepV4D4 surface, but soon began to spontaneously interact witheach other to form multicellular spheroids (SupplementaryVideos S1 and S2). As shown in the selected representative snap-shots for the spheroid-forming behaviors on pV4D4 surface atearly time points (Supplementary Fig. S7), cancer cells on thesurface are kept interacting with surface and move around whilebeing attached until theymeet and interact with neighboring cells

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http://www.r-project.org/

http://consensuspathdb.org/

http://software.broadinstitute.org/gsea/msigdb/collections.jsp

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Figure 1.

Formation and characterization of cancer cell spheroids generated on a pV4D4 thin film from diverse human cancer cell lines. A, Schematic illustration oftumorigenic spheroid formation on a specific PTF surface. Human cancer cell lines were seeded on various functional PTFs prepared by polymerization of thecorrespondingmonomers on conventional cell culture dishes using an iCVD process. During culture on the PTF surface, cancer cells becomeCSC-like cells, giving riseto tumorigenic multicellular spheroids. B, Morphologies of SKOV3 cells (2� 104 cells/cm2) cultured for 24 hours in RPMI-1640 medium containing 10% (v/v) serumreplacement on a conventional TCP and on various functional PTFs. Only the pV4D4 surface supported spheroid formation. C,Morphologies of cancer cell spheroidsgenerated from various human cancer cell lines (2� 104 cells/cm2) after culturing for 24 hours on a pV4D4 PTF surface. Cancer cell lines (and their tissue of origin):SKOV3 (ovarian), MCF-7 (breast), T47D (breast), BT-474 (breast), Hep3B (liver), HepG2 (liver), U87MG (brain), SW480 (colon), HT-29 (colon), 22RV1 (prostate),HeLa (cervical), A549 (lung), NCI-H358 (lung), andNCI-N87 (gastric).D, Shapes andmorphologies of SKOV3 spheroids prepared using three conventional spheroid-forming methods (hanging-drop, U-bottom, ULA surface) and a pV4D4 surface after culturing the cells for 24 hours. E, Representative images showingimmunostaining for laminin expression in 8-day SKOV3 spheroids cultured on a ULA or pV4D4 surface. A primary rabbit anti-human laminin antibody and arhodamine red-X–conjugated anti-rabbit secondary antibody were used for laminin staining. Hoechst 33342 was used for staining nuclei. F, Expression of ALDH1A1mRNA inday-8SKOV3 spheroids prepared byconventionalmethods (hanging-drop,U-bottom,ULA) andby culturing onapV4D4 surface, as quantifiedbyqRT-PCR.SKOV3 cells cultured as amonolayer on TCPs for 8 dayswere used as a control (n¼ 3 independent experiments; �, P < 0.05; #, P <0.01; and �� , P <0.005). Scale bars,100 mm (B and C) and 20 mm (D).






to form mini-spheroids. These active cell-to-surface and subse-quent cell-to-cell interactions on the pV4D4 surfaces were notobserved on the ULA surface in which SKOV3 cells seemed toprefer direct cell-to-cell interactions to cell-to-surface interactions(Supplementary Video S3).

Tumor spheroids generated on pV4D4 PTFs are enriched forCSC-like cells

We next compared the features of pV4D4 PTF–cultured cancercell spheroids at days 4 and 8 with those prepared using otherconventional spheroid-forming methods. Whereas hanging-dropandU-bottommethods caused SKOV3 cancer cells to form single,large, aggregated spheroids, the ULA and pV4D4 surfaces pro-duced multiple and much smaller spheroids; those cultured onpV4D4 were also more uniform and slightly smaller than thosecultured on ULA (Fig. 1D). An immunocytochemical analysisrevealed a stark difference between SKOV3 spheroids cultured for8 days on aULA surface versus those cultured on a pV4D4 surface,showing that considerable amounts of laminin, a major compo-nent of extracellular matrix (ECM; ref. 22), were present withinpV4D4-cultured spheroids, whereas much less amount of theECMprotein was deposited only at the periphery of ULA-culturedspheroids (Fig. 1E). Considering that tumor cells in vivo aresurrounded by and interact with densely packed ECMs in tumormicroenvironment (23), the deposition of abundant ECM pro-teins inside ssiCSC-tumor spheroids is a unique structural featuremimicking tumor tissue in vivo. Importantly, ECM in the tumormicroenvironment is suggested to play a pivotal role in thedevelopment of thedrug resistance, self-renewal, and tumorigenicproperties of tumor-resident CSCs (22, 24).

The abundant expression of ECMwithin spheroids only in cellscultured on pV4D4 prompted us to examine the expression ofCSC-associated genes. qRT-PCR analyses revealed that, amongspheroids formed from SKOV3 cells using different methods,only those grown on pV4D4 showed a dramatic increase in theexpression of aldehyde dehydrogenase 1 family member A1(ALDH1A1), a putative CSC marker (Fig. 1F; refs. 25–27). Alde-fluor assay further revealed that SKOV3 spheroids cultured onpV4D4 for 8 days showed a considerable increase in the ALDH-positive cell population compared with 2D-cultured control cells(17.5%vs. 3.8%; Supplementary Fig. S8). Spheroids grownon thepV4D4 surface also showed a striking increase in the expression ofthe typical self-renewal genes, OCT3/4, SOX2, and NANOG,compared with 2D-cultured SKOV3 controls grown on TCPs(Supplementary Fig. S9). These results suggest that the cancercells within spheroids acquired some of the essential features of"stemness" (28, 29). Wound-healing assays further showed thatcancer cells dissociated fromday-8pV4D4-cultured SKOV3 spher-oids were able to migrate and fill the lined gap much faster(Supplementary Fig. S10A), and Transwell-based invasion assaysshowed that these cells were also able to penetrate a gel matrixmore efficiently (�4-fold) than 2D-cultured control cells (Sup-plementary Fig. S10B), demonstrating significantly enhancedcapacity for cell migration and invasion. In addition, when cancercells dissociated from day-8 pV4D4-cultured SKOV3 spheroidswere seeded on conventional TCPs under the tumor sphere-forming conditions, they started to form spheroids spontaneous-ly, suggesting maintenance of the acquired CSC-like properties(Supplementary Fig. S11). Collectively, thesefindings suggest thatthe pV4D4 surface provides certain stimuli that activate andtransform SKOV3 cancer cells, giving rise to tumor spheroids that

are significantly enriched for CSC-like cells. We have designatedthese CSC-like cells, surface stimuli–induced cancer stem cells(ssiCSC).

ssiCSC spheroids exhibit robust drug resistanceTo test the generalizability of the pV4D4 method, we

prepared various ssiCSC spheroids from other cancer cell linesand assessed CSC-related characteristics. Four human cancer celllines with different tissue origins—SKOV3, MCF-7 (humanbreast cancer), Hep3B (human liver cancer), and SW480 (humancolon cancer)—were chosen for the generation and analysis ofssiCSCs. Previously reported specific surfacemarkers were used toidentify putative CSCs for each cell line: ALDH1A1 for SKOV3(26), CD44 (cluster of differentiation 44) for MCF-7 (30, 31),CD90 for Hep3B (32), and LGR5 (leucine-rich repeat-containingG-protein-coupled receptor 5) for SW480 (33). CD133 was usedas a common putative CSC marker for all cell lines (6, 9, 27).Expression of CSCmarker genes in ssiCSC spheroids was assessedby qRT-PCR after culture on pV4D4 surface for 4 and 8 days, andcompared with that of the corresponding 2D controls culturedon TCPs. Genes for each cell-type–specific CSC marker weresignificantly upregulated in the corresponding spheroids, andexpression of the common marker CD133 was increased in allssiCSC spheroids (Fig. 2A). Interestingly, gene expression levelsincreased with time in culture, suggesting the CSC-like character-istics become strengthened over time. RT-PCR analyses alsoshowed far higher expression of various CSC-related genes in allssiCSC spheroids compared with 2D-cultured control cancer cells(Supplementary Fig. S12). Next, we used flow cytometry toquantify the fraction of putative CSCmarker–positive cancer cellsin the resulting spheroids at day 8 in culture. Flow cytometryrevealed roughly a 10-fold increase in the expression of cell-type–specific CSC-associated surface markers (expressed as genecounts) in SKOV3, Hep3B, and SW480 ssiCSC spheroids com-pared with the corresponding 2D-cultured controls, except forCD44 in MCF-7 cells, which was upregulated to a lesser degree(Fig. 2B). Furthermore, although both 2D-cultured controlSKOV3 cells and ULA-cultured SKOV3-spheroids showed only0.1% and 1.9%of CSC-like CD133þ cell population, respectively,pV4D4-cultured SKOV3-ssiCSC spheroids, as expected, resultedin a dramatic increase of approximately 28.6% in the cell pop-ulation (Supplementary Fig. S13), indicating a distinct differencein the CSC-generation ability between pV4D4 and the conven-tional spheroid-forming surfaces.

Another key feature of CSCs is that they possess intrinsic oracquired resistance against chemotherapeutics owing to theirability to extrude drugs (34, 35). We, therefore, assessed thedrug-efflux capacity of individual cancer cells dissociated fromday-8 ssiCSC spheroids using a Hoechst dye-based side-popula-tion assay. These assays, performed using flow cytometry, showeda significant increase in the fraction of drug-efflux–positive cells inall four types of ssiCSCs compared with 2D-cultured controls.Specifically, the efflux-positive fraction was increased from 0% toapproximately 13.8% for SKOV3 cells, 0.59% to approximately9.6% for MCF-7 cells, 0.58% to approximately 9.2% for Hep3Bcells, and 0.1% to approximately 10% for SW480 cells (Fig. 2C).Next, we evaluated the drug resistance of ssiCSCs against doxo-rubicin (Dox), awidely used anticancer drug (36, 37). Each type ofday-8 ssiCSC spheroids was dissociated into single cells, whichwere then cultured as 2D monolayers on a conventional TCPsurface and treated with different concentrations of Dox for 24

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hours. Cell viability measurements using a WST-1 assay showedthat, compared with 2D controls, ssiCSCs were highly resistant toDox, even at concentrations as high as 50 mmol/L (Fig. 2D).Remarkably, both SKOV3- and SW480-ssiCSCs were highly resis-tant to Dox, and the latter exhibited even higher cell viability thanuntreated control cancer cells. Moreover, SW480-ssiCSCs main-tained their drug resistance even after two subcultures on a TCPsurface (Supplementary Fig. S14), implying that the originalcancer cells were transformed to CSC-like cells. In contrast, thedissociated SKOV3 cells fromULA-cultured spheroids weremuchmore sensitive to Dox compared with SKOV3-ssiCSC spheroids,showing IC50 value of 0.79 and 12.23 mmol/L, respectively(Supplementary Fig. S15).

Drug-efflux ability is mediated by a family of ATP-bindingcassette (ABC) proteins (35, 38, 39). Accordingly, we analyzedexpressionof apanel ofmajormultidrug-resistant (MDR)genes—ABCB1, ABCB2, ABCB5, ABCC1, and ABCG2 (40)—in SKOV3-

ssiCSCs by qRT-PCR. All five MDR-related genes were highlyupregulated in ssiCSCs compared with 2D-cultured controls;these increases were particularly striking for ABCB1 and ABCB5(Fig. 2E). This significant upregulation of MDR genes in ssiCSCswas well correlated with our side-population assay results (Fig.2C) and tests of Dox resistance (Fig. 2D). Taken together, ourmolecular and functional analyses of four types of ssiCSC spher-oids suggest that, upon exposure to certain stimuli present onpV4D4 surfaces, cancer cells can be transformed to CSC-like cellsthat exhibit strong expression of CSC-related genes and robustdrug resistance.

SKOV3-ssiCSC spheroids express stem cell–related genes on agenome-wide scale

To examine whether ssiCSC spheroids have typical geneticsignatures of CSCs, we performed high-throughput mRNAsequencing (RNA-seq) on 8-day SKOV3-ssiCSC spheroids and

Figure 2.

Characterization of the CSC-like properties of various ssiCSC spheroids. A, Expression of CSC-associated markers in SKOV3-, MCF-7-, Hep3B-, and SW480-ssiCSCspheroids culturedonapV4D4surface for4 and8days, asquantifiedbyqRT-PCR (n¼ 3 independent experiments). TheexpressionofputativeCSC–relatedmarkerswassignificantly increased for each type of ssiCSC compared with the corresponding 2D-cultured controls, and also gradually increased with time in culture. B, Flowcytometric analysisof theCSC-associatedmarker–positive cell fraction fromday-8 ssiCSCsand their corresponding2D-culturedcontrols. PutativeCSCmarkers:CD133 forSKOV3, CD44 for MCF-7, CD90 for Hep3B, and CD133 for SW480. C, Representative flow cytometry plots for side-population (SP) discrimination assays usingHoechst 33342 in 2Dcontrol cells and ssiCSCs fromday-8 spheroidsof SKOV3,MCF-7, Hep3B, and SW480 cells.D,Drug resistanceof ssiCSCs fromday-8 SKOV3,MCF-7,Hep3B, and SW480 cell spheroids was assessed after treatment with different concentrations of Dox for 24 hours (n ¼ 4–6 independent experiments; � , P < 0.05;#, P < 0.01; and �� , P < 0.005 for ssiCSCs vs. 2D controls). E, Expression of drug-efflux ABC transporter–related genes in SKOV3-ssiCSCs and 2D controlsfrom day-8 cultures, as quantified by qRT-PCR (n ¼ 3 independent experiments; � , P < 0.05; #, P < 0.01; ��, P < 0.005; n.s., not significant).






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2D-cultured SKOV3 controls on a genome-wide scale. Using anadjusted P value of 0.05, we identified 2,086 genes with at least4-fold differential expression (Fig. 3A). Interestingly, amajority ofsignificantly upregulated genes (1,547 of 2,086) were found to becell–cell adhesionmolecules and ECM-associated genes (Fig. 3B),an observation that may explain the abundant expression oflaminin in SKOV3-ssiCSC spheroids (Fig. 1E). To analyze theexpression pattern of ECM genes in more details, we generated aheatmap displaying normalized expression levels of the geneslisted in Fig. 3B (n ¼ 56; Fig. 3C). Among the ECM genes whoseexpression increased (i.e., collagen, integrin, matrix metallopro-teinase, and adhesion molecules), genes encoding LAMA1 (lam-inin subunit alpha 1), a constituent of laminin, andWnt3a, whichpromotes tumorigenicity and stemness through Wnt/b-cateninsignaling (41), were also highly upregulated. We next examinedthe 10 most highly expressed genes encoding cell–cell adhesionmolecules listed in Fig. 3B. Notably, CLDN2, which is not only atarget gene of Wnt/b-catenin signaling, but also encodes a keyfactor in cancer cell migration and invasion (42, 43), showed asignificant increase in expression level (Fig. 3D). Collectively,these findings suggest that ECM–cell and cell–cell interaction-associated signaling pathways, such as Wnt/b-catenin, may con-tribute to the de novo acquisition of tumorigenicity and stemnessproperties by cancer cells in ssiCSC spheroids.

To assess representative features of gene expression that reflectactual biological behavior in SKOV3-ssiCSCs, we used GSEA (Fig.3E). These ssiCSCs showed marked upregulation of gene setsrelated to inflammation, STAT3, and KRAS. Inflammation pro-motes tumor progression by facilitating angiogenesis, invasion,andmetastasis (44–47). In addition, the STAT3 signalingpathwayenforces the maintenance of CSCs and plays an important role inKRAS-induced tumorigenesis (48, 49). This analysis thus furthersupports the conclusion that SKOV3-ssiCSC spheroids possess thegenetic traits of CSCs. Not surprisingly, we found that SKOV3-ssiCSCs exhibited considerable downregulation of mitotic cell-cycle–related genes (Fig. 3F). A GO analysis of downregulatedgenes (n ¼ 539) revealed over-representation of multiple GOterms related to cell-cycle control, including mitotic cell cycle,mitotic nuclear division, organelle fission, and sister chromatidsegregation. To investigate the semantic similarity of these GOterms,we compared transcripts assigned to eachGOcategory (Fig.3G). A Venn diagram identified 49 genes that were commonlyassociatedwith the fourGO terms; a heatmapof these intersectinggenes showed a substantial reduction in their expression (Fig.3H). Intriguingly, cancer cells within ssiCSC spheroids seemed tobe in a quiescent state, which is an intrinsic property of stem cellsthat contributes to drug resistance. Next, we performed RNA-seqfor both ULA-cultured SKOV3 spheroids and CD133þ cell pop-ulation isolated from 8-day pV4D4-cultured SKOV3-ssiCSC

spheroids and compared the gene expression with that of 8-daySKOV3-ssiCSCs (Fig. 3). As shown in Supplementary Fig. S16A,with an adjusted P value of 0.05 and at least 4-fold differentialexpression, the ULA-cultured spheroids shared merely a part ofgenes with the pV4D4-cultured ssiCSC spheroids (134 of 1,547genes in upregulated genes and 40 of 539 genes in downregulatedgenes relative to 2D control, respectively). In contrast, the RNA-seq data of the isolated CD133þ cancer cell population weresubstantially overlapped with those of the original ssiCSC spher-oids before separation (583 of 1,547 genes in upregulated genesand 185 of 539 genes in downregulated genes, respectively).A heatmap displaying normalized expression levels of typicalECM genes (upregulation) and cell-cycle–related genes (down-regulation) further revealed the distinct difference between ULA-cultured spheroids and the isolated CD133þ cancer cells (Sup-plementary Fig. S16B). This result suggests that the CD133þCSC-like cells contribute largely to the overall gene expression patternof ssiCSC spheroids that contain both CSC-like cells and non-transformed cancer cells. Furthermore, we identified five majorclasses of subpopulation cells in 8-day–cultured SKOV3-ssiCSCspheroids by performing single-cell RNA-seq (Supplementary Fig.S17A). The single-cell transcriptome data reflected distinct geneexpression patterns between each class of cells, many of which areinvolved in cell cycle, hypoxia, and metabolic processes (Supple-mentary Fig. S17B). Taken together, these genome-wide geneexpression analyses support the conclusion that ssiCSCs possessthe general molecular signature of CSCs and pV4D4-based spher-oid-forming method is distinctly different from the conventionalspheroid-forming ULA surface.

ssiCSC spheroids are highly tumorigenic in vivoWe next examined the in vivo tumorigenic capacity of ssiCSCs.

SKOV3-derived ssiCSC spheroids were dissociated into singlecells, serially diluted (102 to 106) inMatrigel, and subcutaneouslyinoculated into BALB/c nude mice (Fig. 4A). Xenograft tumorformation from the spheroid-dissociated cells was monitored for120 days and compared with that for 2D TCP-cultured SKOV3controls (Table 1). We found that 2D controls formed no tumors(0/5 mice) at doses of 105 cells per mouse or less, and formedtumors at a frequency of only 50% (2/4) at a dose of 106 cells permouse (Table 1). In contrast, much smaller doses of ssiCSC-derived cells were capable of forming tumors at much higherfrequencies. Specifically, the resulting frequencies of tumor for-mationwere approximately 60% (3/5) for 105 cells, approximate-ly 80% (4/5) for 104 cells, and approximately 20% (1/5) for 103

cells (Table 1). Considering how difficult it is to get xenografttumors from human ovarian cancer (SKOV3) cells to grow inathymic nude mice without using SCID mice, the demonstratedtumorigenicity of SKOV3-ssiCSCs in vivo is impressive.

Figure 3.Genome-wide gene expression profiling in SKOV3-ssiCSCs. A, MA plot showing log2-fold change of gene expression between SKOV3-ssiCSCs and 2D controlcells. Golden-yellow marks indicate genes exhibiting fold changes greater than 2, as determined by DESeq analysis (adjusted P value < 0.05). Transcriptswith reads per kilobase per million mapped (RPKM) values less than 0.03 were removed. B, GO terms associated with biological processes of upregulated genesin SKOV3-ssiCSCs. The negative log10 P value is plotted on the x axis. C,Heatmap of ECM genes listed in B. Expression levels were expressed as relative values (log2)normalized to control signals. Red, high expression; blue, low expression. D, Bar graph of the 10 genes exhibiting the greatest increase in expression among cell–celladhesion molecules. Values represent log2-fold change relative to 2D-control cells; results are presented as mean of two biological replicate samples. E, GSEAof the transcriptome of SKOV3-ssiCSCs compared with that of 2D-controls cells. The enrichment score (ES; y axis) reflects the degree to which a gene set wasupregulated in ssiCSCs. NES, normalized enrichment score; FDR, false discovery rate. F, GO terms associated with biological processes of downregulatedgenes in SKOV3-ssiCSCs. The negative log10 P value is plotted on the x axis. G, A four-set Venn diagram showing the overlap between gene sets assigned to cell-cycle–related GO categories—mitotic cell cycle, mitotic nuclear division, organelle fission, and sister chromatid segregation—presented in F. H, Heatmap ofintersecting genes (n ¼ 49) in G. Expression levels were presented as log2-fold change values normalized to control cells.






Notably, we found that livers from ssiCSC-inoculated micewere filled with metastatic nodules, a strikingly abnormalappearance compared with livers of mice inoculated with2D SKOV3 controls, which appeared normal (Fig. 4B). Histo-logic analyses of abnormal livers showed numerous metastaticlesions throughout the tissue, with a clear delineation betweennormal and tumorous areas. As expected given their normalappearance, the livers of mice injected with 2D controlcancer cells showed no evidence of metastasis (Fig. 4C). Nota-bly, mice inoculated with as few as 102 SKOV3-ssiCSC–derivedcells also showed a high frequency of liver metastasis (4/5mice; Supplementary Fig. S18; Table 1), indicating the enor-mously enhanced metastatic ability and tumorigenicity ofSKOV3-ssiCSCs. Moreover, an IHC examination of liver metas-tases for the expression of TNC, a major component of thecancer-specific ECM and an essential component of the met-astatic niche (50), revealed significant localization of TNCaround the tumor boundary region at the interface with nor-mal tissue (Fig. 4D), suggesting that tumor nodules found inthe liver resulted from the metastasis of subcutaneously inoc-ulated SKOV3-ssiCSCs.

Next, we tested the tumorigenicity of other cancer cell line–derived ssiCSCs. ssiCSCs derived from luciferase-transfectedMCF-7 (MCF7-Luc) cells and U87MG human glioblastomacells showed significant increases in tumor-forming abilitycompared with their corresponding 2D-cultured control cells(Supplementary Tables S2 and S3). Whereas 2D-culturedMCF7-Luc cells formed no tumors, even at a dose of 106 cellsper mouse, its corresponding ssiCSCs formed tumors at a doseof 105 cells per mouse with high frequency (4/5 mice; Supple-

mentary Table S2). Similarly, tumors formed from U87MG-ssiCSCs at a frequency of approximately 60% (3/5), even at 104

cells per mouse; in contrast, no tumors formed from ULAsurface–cultured U87MG spheroids (Supplementary TableS3), indicating a stark difference in tumorigenicity betweenULA- and pV4D4-cultured spheroids. Collectively, these resultssuggest that the pV4D4-cultured ssiCSCs possess dramaticallyenhanced tumor-forming ability compared with the originalcancer cells and are further applicable to the preparation ofvarious human xenograft tumor models that are notoriouslydifficult to form in athymic nude mice.

Tumorigenicity of ssiCSC spheroids is associated withactivation of Wnt/b-catenin signaling

To explore the cellular and molecular mechanisms responsiblefor the stem cell–like characteristics of ssiCSCs, we turned ourattention to several key signaling pathways related to tumorige-nicity and stemness of CSCs, namely Notch, Hedgehog, andWnt/b-catenin (51). Given that our genome-wide gene expressionstudy of SKOV3-ssiCSCs revealed activation of theWnt/b-cateninsignaling pathway (Fig. 3), we first examined the expression ofWnt target genes (n¼ 46). Figure 5A shows that the expression of30 of 46Wnt/b-catenin target genes increased more than 1.5-foldin SKOV3-ssiCSCs together with a marked reduction in theexpression of Dickkopf-related protein 1 (DKK1), a key inhibitorof theWnt signaling pathway. qRT-PCR analyses also confirmed adramatic reduction in DKK1 mRNA expression in 1-, 4-, and 8-day–cultured SKOV3-ssiCSC spheroids (Fig. 5B), indicative ofactivation of Wnt/b-catenin signaling pathways at an early timepoint in spheroid formation. qRT-PCR further revealed that this

Figure 4.

Tumor-forming and metastatic ability of human ovarian cancer cell–derived ssiCSCs. A, Illustration of the overall process of tumor formation and liver metastasisafter subcutaneous inoculation of serially diluted (102 to 106 cells/mouse) 2D-cultured control or SKOV3-ssiCSC spheroid-derived cells into the dorsal areaof BABL/c nude mice. Prior to inoculation, day-8 ssiCSCs were dissociated from the corresponding spheroids into single cells and mixed with Matrigel. B,Representative images of livers dissected frommice that received either 2D control SKOV3 cells (1� 106 cells/mouse; n¼ 5) or SKOV3-ssiCSC spheroid-derived cells(1 � 105 cells/mouse; n ¼ 5) when palpable tumors were detected (30 days). C, Representative hematoxylin and eosin–stained images of liver tissuesshown in B. Yellow arrows in the �4 magnification image indicate metastatic lesions in the liver, and the dotted line in the �20 magnification image delineatesthe boundary between normal tissue and tumor metastasis. D, IHC analysis of metastatic tumors in the liver depicted in B showed intense staining for TNC(arrowheads) near the boundary of the tumor and the invasive front. Images were obtained at �4 and �20 magnification.

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reduction in DKK1 expression was directly associated with sig-nificant increases in the expression of the Wnt/b-catenin down-stream target genes, AXIN2 (axis inhibition protein 2) andMMP2(Fig. 5B). Moreover, although qRT-PCR showed no evidence ofchanges in the level of b-catenin mRNA in ssiCSC spheroids,

Western blot analyses indicated a significant reduction in phos-phorylated b-catenin protein (Fig. 5C). In addition, immunos-taining revealed substantial translocation of b-catenin into thenucleus of ssiCSCs, confirming activation of Wnt/b-catenin sig-naling pathways; by contrast, 2D-cultured SKOV3 cells showed

Figure 5.

Activation of Wnt/b-catenin signaling pathways in SKOV3-ssiCSC spheroids. A, Heatmap of Wnt target genes (n ¼ 46). Expression levels were expressed asrelative values (log2) normalized to control signals.B, Expression of DKK1 in SKOV3-ssiCSCs (days 1, 4, and 8) and AXIN2, andMMP-2mRNAs in SKOV3-ssiCSCs (days 4and 8) was quantified by qRT-PCR (n ¼ 3 independent experiments; � , P < 0.05; #, P < 0.01; �� , P < 0.005). Primers used are listed in Supplementary Table S4. C,Westernblot analysis ofphosphorylatedb-catenin and totalb-catenin in 2Dcontrols andSKOV3-ssiCSCs (days4and8).GAPDHwasusedasan internal protein standard.Cells were incubated first with primary antibodies against phosphorylated b-catenin (rabbit), b-catenin (mouse), and GAPDH (rabbit) and then with horseradishperoxidase–conjugated IgG or horseradish peroxidase-conjugated anti-mouse IgG secondary antibodies to detect the amount of each protein. D, Confocal imagesshowing localization of b-catenin in 2D controls and SKOV3-ssiCSCs, detected by immunocytochemistry. Mouse anti-human b-catenin primary antibodies and TRITC-conjugated anti-mouse secondary antibodieswere used forb-catenin staining, andHoechst 33342was used to stain nuclei. Scale bar, 20mm.E,Confocal images showingimmunostaining for TNC expression in 2D control cells and SKOV3-ssiCSC spheroids. Primary rabbit anti-human TNC antibody and FITC-conjugated anti-rabbitsecondary antibody were used for immunostaining, and Hoechst 33342 was used to stain nuclei. Scale bar, 100 mm.

Table 1. Tumor formation and metastasis of SKOV3 in BALB/c nude micea

Number of tumor formation (liver metastasis)/number of injected animalsb

SKOV3 100 1,000 10,000 100,000 1,000,000Tumor-initiating cell frequency

(95% confidence interval)P value

(vs. 2D control)

2D Control 0/5 (0/5) 0/5 (0/5) 0/5 (0/5) 0/5 (0/5) 2/4 (0/4) 1: 1.73 � 106 (1: 4.36 � 105–1: 6.87 � 106)ssiCSC 0/5 (4/5) 1/5 (4/5) 4/5 (4/5) 3/5 (5/5) 1: 4.11 � 104 (1: 1.55 � 104–1: 1.09 � 105) 1.39 � 10�7

aTumor formation and metastasis were monitored up to 120 days.bAll cells were dissociated into single cells and counted with a hemocytometer before s.c. injection.






little nuclear localization of b-catenin (Fig. 5D). Next, we furtherexamined the effect of DKK1 protein on spheroid-forming abilityand acquisition of CSC-like signature. Although numerous small-sized spheroids were observed at 6 hours and larger spheroidswere formed at 24 hours under the normal ssiCSC culture con-dition (no DKK1 addition), the presence of excess DKK1 proteinsupplied to the culture media hindered spheroid formation at thesame time frames and rather, formed a strange ameba-like cellnetwork (Supplementary Fig. S19A).Moreover, after culturing foradditional 3 days, we carried out qRT-PCR to examine the expres-sion of Wnt signaling–related genes. As shown in SupplementaryFig. S19B, the expression of Wnt3a, Wnt target genes AXIN2 andMMP-2, and a putative CSC marker ALDHA1 was significantlyreduced. This result supports the proposed mechanism thatDKK1-mediated activation of Wnt/b-catenin signaling pathwaysmight be responsible for the conversion of cancer cells to tumor-igenic CSC-like phenotypes by the pV4D4 surface.

Next, we searched for upstream signals that might have causedthe significant reduction in DKK1 in ssiCSC spheroids. Interest-ingly, it has been shown that TNC, which is abundantly present inliver metastases of SKOV3-ssiCSCs (Fig. 4D), activates Wnt/b-catenin signaling pathways by downregulating DKK1 (52).Thus, to explore a possible link between TNC and DKK1, weimmunostained day-8 SKOV3-ssiCSC spheroids for TNC. Asshown in Fig. 5E, TNC was abundantly present throughout thespheroids, suggesting possible downregulation of its target DKK1and thus activation ofWnt/b-catenin signaling pathways. ssiCSCsobtained fromMCF-7,Hep3B, and SW480 spheroids also showedconsiderable expression of TNC (Supplementary Fig. S20A),accompanied by a dramatic reduction in DKK1 gene expression(Supplementary Fig. S20B), suggesting that the same Wnt/b-cate-nin signaling pathwaysmay be involved in the generationof otherssiCSCs. Collectively, these findings strongly suggest that TNC-DKK1–mediated activation of Wnt/b-catenin signaling pathwayscould be responsible for the conversion of cancer cells to tumor-igenic CSC-like phenotypes by the pV4D4 surface. However, itremains unclear what specific stimuli (chemical or biological)present on the pV4D4 surface trigger the activation of Wnt/b-catenin signaling in cancer cells. Answering this importantquestion will require further studies.

DiscussionAs tumor-repopulating cancer cells, CSCs have been of intense

interest to oncologists, and considerable effort has been dedicatedto understanding their properties and developing candidate drugsthat target them. A robust and versatile platform method thatenables facile production of CSC-like cells would go a long waytoward expediting CSC-related research. In the present study, wesuccessfully developed a platform method capable of generatingtumorigenic CSC-like spheroids by simply culturing a variety ofconventional cancer cells on a PTF surface (pV4D4). No addi-tional biological growth factors, genetic transfections, or chemicaltreatments were involved in the transformation process. Never-theless, the usefulness and broad applicability of ssiCSC spher-oids depends on how closely they recapitulate the features ofaggressive tumors in vivo or patient-derived CSC spheroids. Asconfirmed by IHC imaging, the key ECM proteins, laminin andTNC, were abundantly expressed throughout ssiCSC spheroids,but were not present on tumor spheroids produced by conven-tionalmethods (Figs. 1E and 5E). Given that interactions between

cancer cells and the ECM are important for maintaining andpromoting CSC characteristics (22), we speculate that the pres-ence of ECMwithin ssiCSC spheroidsmay explain their enhancedtumorigenicity. In addition, ssiCSC spheroids were not onlysignificantly enriched for tumorigenic CSC-like cells, they alsocontained a certain fraction of nontumorigenic cancer cells. Suchunique features—the presence of ECM and enrichment of tumor-igenic CSC-like cells—suggests that our ssiCSC spheroidmay be abetter in vitromimic ormodel for highly aggressive andmalignanttumors in vivo.

Another key consideration is whether human cancer cell line–derived ssiCSCs obtained as described here are phenotypicallyand genetically similar to patient tumor-derived CSCs. We werenot able to directly compare the genetic traits of SKOV3-ssiCSCs(Fig. 3) with those of patient-derived CSCs owing to a lack ofgenome-wide gene expression and GO analyses of patients withovarian cancer CSCs. Instead, a comparison of DEG-driven GOterms between SKOV3-ssiCSCs and patient-derived liver CSCs(53) provides convincing evidence that our cancer cell line–derived ssiCSCs share several important features of gene expres-sion profiles, such as ECM-related genes, with the patient-derivedCSCs (Supplementary Fig. S21A and S21B). Although such directcomparisons between two cancer cells with different origins maynot be appropriate, the similarities in some key genetic traits ofCSCs suggest that our cell line–derived ssiCSCs have potential forfuture use as a feasible model for cancer research and drugdevelopment.

In conclusion, the findings presented here clearly demonstratethat a pV4D4-based cell-culture platform enables the conversionof conventional cancer cells to highly tumorigenic CSC-likespheroids with high efficiency, reproducibility, and versatility.In vitro molecular and functional analyses showed that pV4D4-cultured cancer cell spheroids are substantially enriched fortumorigenic cells that showdramatically increased drug resistanceagainst an anticancer drug compared with 2D-cultured controls.We also confirmed that the in vivo tumor-forming ability andmetastatic propensity of pV4D4-cultured CSC-like spheroids isgreatly enhanced; thus, this system could be used as a platform forpreparation of hard-to-form human xenograft tumor models innudemice. Taken together, our results suggest that by providing afacile method of generating CSC-like tumor spheroids fromdiverse cancer cells, the PTF platform described here will contrib-ute to CSC-related basic research and drug development.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Choi, S.J. Yu, Y. Choi, H.R. Lee, S.G. Im, S. JonDevelopment ofmethodology:M.Choi, S.J. Yu,H.R. Lee, E. Lee, E. Lee, S. Kang,J. Baek, S.G. Im, S. JonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Choi, S.J. Yu, Y. Choi, H.R. Lee, E. Lee, T.G. LeeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Choi, S.J. Yu, Y. Choi, E. Lee, J. Song, T.G. Lee,D. Lee, S.G. Im, S. JonWriting, review, and/or revision of the manuscript: M. Choi, S.J. Yu, Y. Choi,E. Lee, D. Lee, S.G. Im, S. JonAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):M. Choi, S.J. Yu, Y. Choi, Y. Lee, J. Song, J.Y. KimStudy supervision: E. Lee, D. Lee, S.G. Im, S. JonOther [identify differences in the samples used in the concept setting bysurface analysis (additional informationnot included in the article)]: J.G. Son


Choi et al.




AcknowledgmentsThis work was supported by a grant from the Samsung Research Funding

Center of Samsung Electronics (Project Number SRFC-MA1501-01).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 28, 2018; revised August 27, 2018; accepted October 18,2018; published first October 23, 2018.

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Choi et al.




2018;78:6890-6902. Published OnlineFirst October 23, 2018.Cancer Res Minsuk Choi, Seung J. Yu, Yoonjung Choi, et al.

like Properties−CellInduced Tumor Spheroids Acquire Cancer Stem−Polymer Thin Film

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