ANTICANCER RESEARCH 33: 5301-5310 (2013)

Silicate Fiber-based 3D Culture System for Anticancer Screening

YOSHIE YAMAGUCHI1*, DAWEI DENG1*, YOSHINORI SATO1, YUNG-TE HOU1, RIE WATANABE2, KOHEI SASAKI2, MASAAKI KAWABE2, EIICHI HIRANO1 and TETSUO MORINAGA1

1Japan Bio Products Co., Ltd., Aikawa, Kurume, Fukuoka, Japan; 2Japan Vilene Company Ltd., Koga, Ibaraki, Japan

Abstract. Background: Three-dimensional (3D) in vitro they can provide various high-throughput applications (4, 5). cultures can recapitulate the physiological in vivo However, it is necessary to develop a more biomimetic in microenvironment. 3D Modeling techniques have been vitro model of tumor tissue for prediction of drug efficiency. investigated and applied in anticancer drug screening. Cell–cell and cell-extracellular matrix (ECM) interactions Materials and Methods: A silicate fiber scaffold was used for form a complex communication network of biochemical and 3D cell cultures, and used to model the efficacy of anticancer mechanical signals that are crucial for in vivo tumor , such as mytomicin C and . Results: A physiology. Three-dimensional (3D) cultures are known to unique 3D was observed in 13 human tumor cell closely mimic properties of tumor tissues (6), and many tools lines on scaffold, and these cells exhibited higher drug are now available to develop 3D cultures in vitro (7). One of resistance than cells in two-dimensional (2D) cultures. the most critical factors in 3D cultures is the ‘scaffold’, which Furthermore, the production of lactate and expression of the serves as an ECM substrate for cell growth and formation of nuclear factor-kappa B (NF-κB)-regulated genes B cell 3D . Scaffolds for 3D culture heve been developed lymphoma-2 (BCL2), cyclooxygenase-2 (COX2), and using natural scaffolds, such as collagen (8) and hyaluronic vascular endothelial growth factor (VEGF) were higher in acid (9), synthetic polymers such as poly(lactide-coglycolide) 3D cultures than in 2D cultures. Conclusion: These findings (10), and polymer microparticles formed from poly(D,L- suggest that a 3D model using a silicate fiber scaffold can lactic-co-glycolic) acid and polylactide (11). These models mimic features of , and is also a suitable model for the are convenient; however, their applications are limited evaluation of anticancer drugs in vitro. because they do not permit regulation of 3D structure or reproduction of 3D culture (12). The ECM has a predilection In anticancer , dominantinly, screening and to adhere to hydrophobic surfaces; therefore, control of initial characterizations of potential anticancer drug substrate hydrophobicity may be an effective approach for candidates is performed using tumor cell monolayer cultures modulating cellular adhesion and proliferation. [two-dimensional (2D) cultures]. However, many Recently, silicate fibers (SNFs) prepared by therapeutics that demonstrate efficiency in vitro fail to electrospinning technology via a sol-gel process have been present the same effect in vivo (1) because the 2D culture developed as a scaffold for tissue engineering (13-15). does not accurately reflect the physiological properties of Electrospinning using the sol-gel method is useful for tumors in vivo (2, 3). Most cancer researchers have to depend generating SNFs, as it enables the control of fiber diameter on 2D cultures at the initial step of drug discovery because from several microns down to 100 nm. The resulting SNFs have a large surface area-to-volume ratio, high fraction of void space, and small pore sizes. In SNFs treated at 500˚C, the hydroxyl group content decreases to 1% or less, and the *These Authors contributed equally to this work. surface of the SNFs becomes hydrophobic (15). Therefore, it is expected that SNFs treated in this manner may improve Correspondence to: Tetsuo Morinaga, Ph.D., Japan Bio Products cell adhesion and cell proliferation while facilitating control Co., Ltd., 1488-4 Fukuoka Bio Factory 204, Aikawa, Kurume, and in vitro reproduction of the 3D structure. Our hypothesis Fukuoka, 839-0861, Japan. Tel: +81 942342216, e-mail: [email protected] is that the use of an SNF scaffold-based 3D tumor model may simulate in vivo conditions and thus be more effective Key Words: Three-dimensional (3D) culture, silicate fiber scaffold, than conventional 2D culture for determining the efficacy of , anticancer drug screening. anticancer drug candidates.

0250-7005/2013 $2.00+.40 5301 ANTICANCER RESEARCH 33: 5301-5310 (2013)

In the present study, we developed in vitro SNF-scaffold streptomycin and allowed to attach for 24 h. Medium containing 3D cultures using various types of tumor cell lines. The drugs was exchanged every 24 h for 96 h. Measurement of cell anticancer effects of chemotherapeutic agents were studied, growth inhibition was calculated based on the amount of DNA, which was determined with a DNA Quantity kit (Primary Cell, and their cytotoxicity was compared to that determined in Hokkaido, Japan) according to the manufacture’s instructions. conventional 2D cultures. Furthermore, we evaluated the uptake of anticancer drugs in 3D culture, and compared the Scanning electron microscope (SEM). Paraformaldehyde-fixed effects of anticancer drugs on lactate production (tumor samples were rinsed in phosphate-buffered saline (PBS) thrice and metabolic marker) and alternations of nuclear factor-kappa then post-fixed with osmium tetroxide. Dehydration was B (NF-κB)-regulated gene B cell lymphoma-2 (BCL2; accomplished using a graded series of ethanol (10, 50, 60, 70, 80, antiapoptosis), cyclooxygenase-2 (COX2; cell growth), and 90 and 100%), and then the samples were transferred to t-butyl vascular endothelial growth factor (VEGF; angiogenic factor) alcohol. After the samples were frozen, dried samples without sputter coating were viewed in an ES-2030 Scanning electron expression in 2D and 3D cultures. microscope (Hitachi Ltd., Tokyo, Japan) at 15 kV.

Materials and Methods Doxorubicin uptake. Seven days after growing, media were removed and 3D cells were incubated with fresh media containing 10 μM Chemicals. Anticancer drugs were purchased as follows: mitomycin doxorubicin. The indicated incubation period (3, 6 and 24 hour), 3D C from (Nacalai Tesque, Kyoto, Japan) and doxorubicin from cells were washed with PBS twice to remove unbound free (Sigma, St. Louis, MO, USA). SNFs were supplied from Japan doxorubicin, and were observed with a fluorescence microscope at Vilene Company Ltd. (Ibaraki, Japan). excitation and emission wavelengths of 534 nm and 602 nm for doxorubicin. 2D cell culture. The following human cancer cell lines were To establish cellular drug uptake profiles, HT-29 cells were purchased from Health Science Research Resources Bank (Osaka, plated onto 24-well plates at densities of 5.0×105 cells/well at Japan) or RIKEN (Tsukuba, Japan) or the American Type Culture 37˚C. Seven days later, doxorubicin (10 μM) was added to each Collection (Rockville, MD, USA): A549 (lung adenocarcinoma), well to initiate cellular drug accumulation. At different time MCF-7 (breast cancer), MDA-MD-453 (breast cancer cell), HeLa intervals (3, 6 and 24 h), the supernatant was removed and cells (ovarian carcinoma), NIH:OVCAR-3 (ovarian carcinoma), HT-29 were washed with ice-cold PBS (pH 7.6) and sonicated with PBS (colon adenocarcinoma), DLD-1 (colon carcinoma), DU145 containing 2 mM EDTA. Doxorubicin in the cell lysates was (prostate cancer), HepG2 (hepatoma), Huh-7 (hepato cellular measured with an FP-8300 Spectrofluorometer (JASCO, Easton, carcinoma), HLE (hepatoma), SUIT-2 (pancreatic cancer) and MIA MD, USA) at an excitation wavelength of 480 nm and an emission PaCa-2 (pancreatic cancer). The cells were cultured in RPMI or wavelength of 560 nm. To adjust for background fluorescence from Dulbecco’s modified Eagle’s medium (DMEM) supplemented with cellular components or non-specific binding to SNFs, doxorubicin 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin standard curves were also prepared using cells or SNF lysates. and 100 μg/ml streptomycin at 37˚C in humidified atmosphere concentration was determined by BCA assay according to the manufacture’s instructions (Pierce, Rockford, IL, USA). containing 5% CO2.

2D cell growth inhibition. Briefly, the cells were plated at an Lactate production assay. To determine the glycolytic activity in 2D appropriate density in 96-well plates in medium with 10% fetal bovine and 3D culture, the amount of produced lactate was measured. The serum, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml cells were cultured until confluent or 3D form in regular medium. streptomycin and allowed to attach for 24 h. The inhibition of cell The medium was then changed, and the cells were incubated in growth was assessed by measuring changes in total cellular protein in Hank’s balanced salt solution for 2 h at 37˚C in humidified a culture of each cell line after 72 h of drug treatment by using of a atmosphere containing 5% CO2. The lactate in each sample was sulforhodamine B (SRB) assay (16). Concentrations that inhibited cell measured by EnzyChrom Lactate Assay Kit (BioAssay Systems, Hayward, CA, USA). growth by 50% (IC50) and 80% (IC80) after 72 h of treatment were calculated based on the survival rate compared against untreated cells. Total RNA extraction and reverse transcription (RT). Isogen RNA 3D culture. Three-dimensional culture using SNFs was described by extraction kit was purchased from Nippon Gene (Toyama, Japan). Yamaguchi et al. (13). Briefly, SNFs were sterilized by autoclave, The quality of RNA was verified by the OD260:OD280 absorption and transferred to 24-well plates. Culture media as same as 2D ratio. Total RNA (0.5 μg) isolated from 2D or 3D cells was culture containing human cancer cells were poured into each well converted to single-strand cDNA using PrimeScript RTase (Takara, with SNFs of 24-well plates. Cells were cultured at 37˚C in Kyoto, Japan) using 50 pM random hexamer primers (TaKaRa, Kyoto, Japan) according to the manufacturer’s instructions. humidified atmosphere containing 5% CO2. Forty-eight hours later, each SNF with cells was transferred to new 24-well plates, and fresh media were poured into each well. The media were exchanged every LightCycler real-time polymerase chain reaction (PCR). For other day. LightCycler reaction, a master mix of the following reaction components was prepared to the indicated end-concentration: 3 μl water 3D cell growth inhibition. The cells were plated at an appropriate (PCR grade), 1 μl forward primer (10 μM), 1 μl reverse primer (10 μM) density in 24-well plates in medium with 10% fetal bovine serum, and 10 μl LightCyler FastStart Essential DNA Green Master (Roche, 2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml Tokyo, Japan). cDNA (5 μl) was amplified by using a standardized RT-

5302 Yamaguchi et al: Silicate Fiber Scaffold as In Vitro Tumor Model

Figure 1. 3D structure formation of HT-29 cells on silicate fibers (SNFs). A: Generation of 3D structure of HT-29 cells on SNFs. Time-lapse scanning electron micrograph of HT-29 cells growing on SNFs. B: Cross section of 3D structure of HT-29 cells on SNFs at day 7. C: Amount of DNA in growing cultures (reflective of cell number).

PCR protocol with FastStart Essential DNA Green Master kit (Roche) Table I. Morphology of 3D cancer cell lines on silicate fibers (SNFs). in a LightCycler instrument (Roche). The melting curve analysis program of the LightCycler was used to identify specific PCR products. Tissue Cell line Morphology The expression levels were normalized by that of 28S. The primers were as follows: BCL2: F, 5’-GGCTGGGATGCCTTTGTG-3’ and R, Lung A549 Loose aggregate 5’-GCCAGGAGAAATCAA ACAGAGG-3’; COX-2: F, 5’-CTTCACG Breast MCF-7 Tight aggregate CATCAGTTTTTCAAG-3’ and R, 5’-TCACCGTAAATATGATTT MDA-MD-453 Loose small spheroidal aggregate AAGTCCAC-3’; VEGF: F, 5’-CCGCAGACGTGTAAATGTTCCT-3’ Cervix HeLa Loose small spheroidal aggregate and R, 5’-CGGCTTGTCA CATCTGCAAGTA-3’; 28S: F, 5’- Ovarian NIH:OVCAR-3 Loose aggregate Colon HT-29 Tight aggregate CCGCTGCGGTGAGCCTTGAA-3’ and R, 5’-TCTCCGGGATC DLD-1 Tight aggregate GGTCGCGTT-3’. The presence of the expected PCR products after Prostate DU145 Loose aggregate quantitative real-time RT-PCR reactions (64 bp for BCL2, 96 bp for Liver HepG2 Loose aggregate COX2, 95 bp for VEGF and 312 bp for 28S) was confirmed by an Huh-7 Loose aggregate agarose gel electrophoresis (data not shown). HLE Loose aggregate Pancreas SUITt-2 Rod aggregate Statistical analysis. All data are presented as the mean±SD for at MIA PaCa-2 Rod aggregate least three replicate experiments and were analyzed with Student’s t-test.

Results cross-section showed that HT-29 cells on SNFs formed tight Generation of unique 3D structures by tumor cell lines on aggregates (Figure 1B). As shown in Figure 1C, the amount SNFs. To determine whether tumor cells form 3D structures of DNA (used as a proxy for cell number) in HT-29 cells on on SNF scaffolds, HT-29 colorectal adenocarcinoma cells SNFs increased commensurately with the formation of the were used. SEM showed that HT-29 cells grew gradually and 3D structure, reaching a plateau at day 8. Twelve other tumor had tight cell-cell adhesions where individual cell margins cell lines were also found to form unique 3D structures on were hardly detectable at day 10 (Figure 1A). The SEM SNFs (Table I), and these 3D cultures were grouped into four

5303 ANTICANCER RESEARCH 33: 5301-5310 (2013)

Figure 2. Representative unique 3D cell morphology of four types of tumor cell lines. Unique 3D structures were generated from 5.0×105 cells (HT-29, HepG2 and MDA-MB-453) and 2.5×105 cells (Suit-2) on silicate fibers (SNFs) and cultured for seven days. 3D cell morphology: HT-29 (A), HepG2 (B), SUIT-2 (C), MDA-MB-453 (D), cross section shows 3D cell morphology: HT-29 (E), HepG2 (F), SUIT-2 (G), and MDA-MB-453 (H) cells.

categories: tight aggregate, loose aggregate, small spheroidal not observed in 3D culture with SNFs (data not shown). aggregate, and loose small spheroidal aggregate. As shown Similar effects were also observed for other anticancer drugs in Figure 2, HT-29, DLD-1, and MCF-7 cells on SNFs were (camptothecin, doxorubicin, , and ; data tightly aggregated without cell margins, whereas cell margins not shown). We therefore determined the IC80 in 2D cultures were observed in A549 cells and ovarian, prostate, and liver exposed to pulses every 24 h (Figure 3A). Cell viability was cancer cells. The aggregates appeared to be undistinguished similar in 2D and 3D cultures at the time of treatment, rod aggregates (SUIT-2 and MIA PaCa-2) with loose small relative to the untreated control. Treatment of 3D-cultured spheroidal aggregates (MDA-MB-453 and HeLa). Cross- cells with resulted in some disruption of 3D sections showed that in rod aggregates, small spheroidal structure relative to the untreated control by SEM (Figure units were linearly-arrayed in a vertical direction whereas 3B), particularly in MDA-MD-453 cells on SNFs. A cell these units were completely randomly distributed in the loose viability assay showed a significant difference between 2D small spheroidal aggregates (Figure 2G and H). and 3D cultures with regard to anticancer drug sensitivitiy: resistance to mitomycin C in 3D cultures was increased by Antiproliferative activity of anticancer drugs in 2D culture 4.1-, 2.2-, 4.5-, and 1.8-fold for HT-29, HepG2, SUIT-2, and vs. 3D culture. The response to mitomycin C was evaluated MDA-MB-453 cells, respectively (Figure 3C). Increased in 2D and 3D cultures of HT-29, HepG2, SUIT-2, and MDA- drug resistance to camptothecin was also observed for HT- MB-453 cells. The IC50 determined in initial 2D experiments 29, HepG2, and MDA-MB-453 cells, and to sorafenib for was then applied to 3D culture; however, cytotoxicity was Huh-7 cells (data not shown).

5304 Yamaguchi et al: Silicate Fiber Scaffold as In Vitro Tumor Model

Figure 3. Anti-proliferative activity of anticancer drugs in 3D model of HT-29, HepG2, SUIT-2 and MDA-MB-453 cells. A: Anticancer drug administration protocol for 3D culture. Arrowhead shows time of anticancer drug administration. B: Images by scanning electron microscopy of HepG2, Suit-2, MDA-MB-453, and HT-29 cells growing on SNF cultures and the effect of mitomycin C (MMC). C: Comparative cytotoxicity of MMC in 2D and 3D cultures. **p<0.001.

Doxorubicin uptake by HT-29 cells on SNFs. It is possible inhibition of glycolysis in tumor cells leads to increased drug that the aggregated morphology may have restricted drug sensitivity (18, 19). Lactate production was evaluated to penetration and reduced drug exposure in cells in the interior determine the glycolytic activity in 2D and 3D cultures. As of the 3D structure; therefore, the penetration of doxorubicin anticipated, lactate levels were significantly higher in 3D into 3D cultures was evaluated. The fluorescence intensity of cultures relative to 2D cultures (Figure 5). Moreover, a the cultures was measured at 3, 6, and 24 h after drug similar increase in lactate production was observed in HepG2, exposure. Over time, the fluorescence intensity of SUIT-2, and MDA-MB-453 cells on SNFs (data not shown). doxorubicin increased, with maximum fluorescence being observed at 24 h (Figure 4A). The cells were still alive after Genes regulated by the transcriptional factor NF-κB in 2D 24 h of incubation with doxorubicin (data not shown). Only culture vs. 3D culture. NF-κB is a central transcriptional 7.5% of the total doxorubicin added (10 μM) penetrated into factor that influences oncogenesis, , and anticancer HT-29 cells on SNFs (Figure 4B). drug sensitivity in colorectal cancer. NF-κB is involved in cell survival, proliferation, and angiogenesis in multiple Glycolysis in 2D culture vs. 3D culture. Cancer cells tumor types (20, 21). To evaluate the molecular mechanism generally exhibit increased glycolysis for ATP generation, underlying anticancer drug resistance in 3D culture, the which is known as the Warburg effect (17). This increased expression of NF-κB-regulated genes was studied. glycolysis is achieved in part by mitochondrial respiration Expression of BCL2 (survival), COX2 (proliferation), and injury and hypoxia, which are frequently associated with VEGF (angiogenesis) was significantly higher in 3D than in resistance to anticancer drugs. It has been shown that 2D cultures (Figure 6).

5305 ANTICANCER RESEARCH 33: 5301-5310 (2013)

Figure 4. Doxorubicin uptake in a 3D model (HT-29 cells) on silicate fibers (SNFs). A: Representative images of doxorubicin uptake in HT-29 cells growing on SNFs. Images were taken at 3-, 6-, and 24-h time points. B: Relative fluorescence intensitiy of doxorubicin uptake.

Discussion

Three-dimensional culture platforms present different cell differentiation and behavior relative to conventional 2D culture (1, 3, 22-23). Given the benefit of 3D culture models, particularly with regard to anticancer drug discovery, we have developed an in vitro model using various types of tumor cells with SNFs prepared by electrospinning via a sol- gel process. 3D Culture systems facilitate the expression of hallmark features of cancer cells, such as unlimited proliferation, self-sufficiency in growth signals, resistance to anti-growth signals, anti-apoptosis, invasion, and angiogenesis (12, 24). Conventionally, naturally-occurring or organic synthetic scaffolds formed from collagen, hyaluronic acid poly(lactide-coglycolide), and poly(D,L-lactic-co- glycolic), or polylactide polymer microparticles have been used in 3D culture systems (8-11). Figure 5. Glycolysis in 2D and 3D culture on silicate fibers (SNFs). The amount of lactate produced by 2D and 3D tumor models was measured In the present study, we found that an inorganic scaffold by lactate assay. Representative data (mean±SD, n=3) were normalised generated using SNFs could be adapted to 3D culture. to their DNA content. Experiments were performed in triplicate. Although SNF scaffolds have been used for the 3D culture of several cell lines, such as MG63, CHO-K1, and HepG2 (13-15), they have not yet been used for other human tumor cell lines. In the present study, we found that at least 13 Previous studies have shown that various factors, including tumor cell lines can be cultured in 3D culture using SNF the affinity of the cell to the scaffold surface, contribute to scaffolds. The effective formation of 3D structures and the formation of 3D structures, and that scaffold subsequent culture of these cancer cells depend to a large hydrophobicity is a particularly important factor in this extent on the features of the SNFs, including hydrophobicity. process, likely because the ECM facilitates adhesion to

5306 Yamaguchi et al: Silicate Fiber Scaffold as In Vitro Tumor Model

Figure 6. Analysis of nuclear factor-kappa B (NF-κB)-regulated genes in 2D and 3D culture on silicate fibers (SNFs). Relative expression of NF-κB- regulated genes, B cell lymphoma-2 (BCL2), cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) in 2D and 3D culture. All data (mean±SD, n=4-6) were normalized by 28S RNA. *p<0.005, **p<0.001.

scaffold surfaces with hydrophobic composition (25, 26). In developed for direct quantitative assessment of the penetration the present study, the cultured cells formed four classes of of anticancer drugs into solid tumors (28, 29). The structural unique 3D structures, partly according to their original features of HT-29 and DLD-1 cells on SNFs should allow tissues. The reason for such different 3D structures in culture their adaptation to such an assay system, thereby resulting in of cancer cell lines remains unclear. Among the cell lines, a novel model for assessment of anticancer drug penetration. 3D structural differences were not observed in colon, liver, Moreover, although the 3D structure of HT-29 and DLD-1 and pancreas cancer cells. In contrast, breast cancer cell lines cells on SNFs is similar to that of multicellular spheroids had a completely different 3D structure. However, other cell (30), their planar structure allows flux through the cultures to lines must be evaluated to exclude the possibility that these be more easily measured (31-32). Consequently, the 3D SNF differences may reflect the individual characteristics of each system will likely provide more predictive data, allow for cancer cell line. reduced animal testing, and reduce the costs and time for It has been reported that MCF-7 cells form spheroids in required drug discovery, in addition to enabling a faster time Matrigel (27), whereas they induce the formation of tight to the markets. aggregates on SNFs. Spheroid cultures have been In this study, we observed a critical difference in the predominantly used in in vitro 3D culture systems but are response to anticancer drugs in 2D and 3D culture systems. associated with limitations, such as those related to culture To investigate the disparity in the effective drug dose in our handling, control of spheroid size, and adaptation of the re- tumor model, we compared drug uptake and gene expression circulation experiment, for example, with respect to the in conventional 2D and 3D culture systems. Firstly, we studied bioreactor system which is for assessment of the penetration of the penetration efficiency of doxorubicin into 3D-cultured anticancer drugs through a solid tissue environment. The use cells on SNFs. Only 7.5% of the doxorubicin penetrated inside of SNFs can overcome these drawbacks and may thus enable the 3D cells, which is consistent with the well-known feature the development of a novel bioreactor system. In addition, of insufficient penetration of drugs in 3D tumor models (12, regarding the four different unique 3D conformations, human 28, 33). Moreover, the poor penetration of anticancer drugs colon adenocarcinoma HT-29 and DLD-1 cells formed the into an in vitro tumor may explain clinically-observed same type of 3D structure, that is, a tight aggregate, not anticancer drug resistance, as a similar environment is present demonstrated by other cell lines. in vivo (34). Poor drug penetration results in a requirement for Many solid tumors are poorly-vascularized, with variable high doses of anticancer drugs during therapy. We speculate rates of blood flow and larger intercapillary distances than in that the poor drug penetration observed in a 3D culture may be normal tissues. The requirement for anticancer drugs to why many anticancer drug candidates screened in 2D tumor penetrate multiple layers of tissue may pose a barrier to the models fail in in vivo tests or clinical trials. effective therapy of solid tumors, and penetration of Another possible factor underlying anticancer drug anticancer drugs into tumor cells distant from the vascular resistance in 3D culture is increased glycolytic activity. system is necessary for efficacy of cancer Persistent glycolysis is a characteristic of cancer cells and a against solid tumors. Recently, an in vitro model was hallmark of advanced cancer. Lactate is associated with tumor

5307 ANTICANCER RESEARCH 33: 5301-5310 (2013) progression (35), and lactate accumulation in tumors is 3 Labarbera DV, Reid BG and Yoo BH: The multicellular tumor associated with metastasis and poor survival (36, 37). We spheroid model for high-throughput cancer drug discovery. observed that lactate accumulation was higher in 3D cultures Expert Opin Drug Discov 9: 819-830, 2012. than in 2D cultures. In addition, in the media of the 3D 4 Newman MJ, Rodarte JC, Benbatoul KD, Romano SJ, Zhang C, Krane S, Moran EJ, Uyeda RT, Dixon R, Guns ES and Mayer cultures, the color of the phenol red dye changed from red to LD: Discovery and characterization of OC144-093, a novel yellow within only 24 h, i.e. between pre-confluency and post- inhibitor of P-glycoprotein-mediated multidrug resistance. confluency, whereas this change was not observed in the media Cancer Res 60: 2964-2972, 2000. of 2D cultures, even after confluency (data not shown). The 5 Torrance CJ, Agrawal V, Vogelstein B and Kinzler KW: Use of rapid acidification of the 3D culture likely resulted from the isogenic human cancer cells for high-throughput screening and secretion of lactate by the cells, as supported by the higher drug discovery. Nat Biotechnol 19: 940-945, 2001. lactate production in 3D cultures relative to that in 2D cultures. 6 Sutherland RM: Cell and environment interactions in tumor microregions: the multicell spheroid model. Science 240: 177- These findings further suggest that 3D culture on SNFs may 184, 1988. be better representative of the in vivo tumor environment. 7 Lin RZ and Chang HY: Recent advances in three-dimensional Furthermore, the disparity of anticancer drug sensitivity in multicellular spheroid culture for biomedical research. 2D and 3D cultures may be associated with altered gene Biotechnol J 3: 1172-1184, 2008. expression. We found that gene expression of BCL2, COX2, 8 Holliday DL, Brouilette KT, Markert A, Gordon LA and Jones and VEGF was higher in 3D cultures than in 2D cultures. All JL: Novel multicellular organotypic models of normal and these genes are known to be regulated by NF-κB (20, 21). The malignant breast: Tools for dissecting the role of the microenvironment in breast cancer progression. Breast Cancer up-regulation of the anti-apoptic marker BCL2 in 3D cultures Res 11: R3, 2009. is consistent with findings from other 3D culture systems 9 Gurski LA, Jha AK, Zhang C, Jia X and Farach-Carson MC: showing that a decrease in apoptosis alters gene expression Hyaluronic acid-based hydrogels as 3D matrices for in vitro (38, 39). It was previously found that COX2 is up-regulated evaluation of chemotherapeutic drugs using poorly adherent in colorectal adenoma and colorectal cancer (40, 41). prostate cancer cells. Biomaterials 30: 6076-6085, 2009. Considering that COX2 inhibitors can induce apoptosis and 10 Fischbach C, Chen R, Matsumoto T, Schmelzle T, Brugge JS, enhance the cytotoxicity of anticancer drugs (42), COX2 may Polverini PJ and Mooney DJ: Engineering tumors with 3D play a role in colorectal cancer. COX2 overexpression has scaffolds. Nat Methods 4: 855-860, 2007. 11 Sahoo SK, Panda AK and Labhasetwar V: Characterization of been reported to confer resistance to apoptosis by up- porous PLGA/PLA microparticles as a scaffold for three regulation of BCL2 (43, 44). VEGF as a survival factor for dimensional growth of breast cancer cells. Biomacromolecules tumor cells is also regulated by NF-κB, and it has been 6: 1132-1139, 2005. reported that COX2 expression in cancer cells stimulates the 12 Horning JL, Sahoo SK, Vijayaraghavalu S, Dimitrijevic S, Vasir JK, production of VEGF (45). In addition, VEGF up-regulates Jain TK, Panda AK and Labhasetwar V: 3-D tumor model for in BCL2 and inhibits apoptosis of human cancer cells (46). Up- vitro evaluation of anticancer drugs. Mol Pharm 5: 849-862, 2008. regulation of BCL2 may be mediated by COX2 and VEGF, 13 Yamaguchi T, Sakai S and Kawakami K: Application of silicate electrospun nanofibers for cell culture. J Sol-Gel Sci Technol 48: thereby inducing anticancer drug resistance. 350-355, 2008. In conclusion, our study demonstrates that an in vitro 3D 14 Sakai S, Yamaguchi T, Putra RA, Watanabe R, Kawabe M, Taya tumor model using SNF scaffolds prepared by M and Kawakami K: Controlling apatite microparticles formation electrospinning technology via a sol-gel process can be used by calcining electrospun sol-gel derived ultrafine silica fibers. J as a novel system for assessment of the efficacy of potential Sol-Gel Sci Technol 61: 374-380, 2012. anticancer drugs. This method allows for direct quantitative 15 Yamaguchi T, Sakai S, Watanabe R, Tarao T and Kawakami K: assessment of the penetration of anticancer drugs through a Heat treatment of electrospun silicate fiber substrates enhances solid-tissue environment. Furthermore, this 3D tumor model cellular adhesion and proliferation. J Biosci Bioengin 109: 304- 306, 2010. can be used in screening angiogenic factor-targeted therapies. 16 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica In addition, the fact that SNF scaffolds can create different D, Warren JT, Bokesch H, Kenney S and Boyd MR: New unique 3D structures, with each type being tumor-dependent, colorimetric cytotoxicity assay for anticancer-drug screening. J can help to understand tumor cellular morphological Natl Cancer Inst 82: 1107-1112, 1990. evolution more precisely. 17 Warburg O: On the original of cancer cells. Science 123: 309- 314, 1956. References 18 Zhou M, Zhao Y, Ding Y, Liu H, Liu Z, Fodstad O, Riker AI, Kamarajugadda S, Lu J, Owen LB, Ledoux SP and Tan M: 1 Griffith LG and Swartz MA: Capturing complex 3D tissue Warburg effect in chemosensitivity: targeting lactate physiology in vitro. Nat Rev Mol Cell Biol 7: 211-224, 2006. dehydrogenase-A re-sensitizes taxol-resistant cancer cells to 2 Cukierman E, Pankov R, Stevens DR and Yamada KM: Taking taxol. Mol Cancer 9: 33, 2010. cell-matrix adhesions to the third dimension. Science 294: 1708- 19 Le A, Cooper CR, Gouw AM, Dinavahi R, Maitra A, Deck LM, 1712, 2001. Royer RE, Vander Jagt DL, Semenza GL and Dang CV:

5308 Yamaguchi et al: Silicate Fiber Scaffold as In Vitro Tumor Model

Inhibition of lactate dehydrogenase A induces oxidative stress 35 Stubbs M, McSheehy PM, Griffiths JR and Bashford CL: Causes and inhibits tumor progression. Proc Natl Acad Sci USA 107: and consequences of tumour acidity and implications for 2037-2042, 2010. treatment. Mol Med Today 6: 15-19, 2000. 20 Aggarwal BB: Nuclear factor-κB: The enemy within. Cancer 36 Brizel DM, Schroeder T, Scher RL, Walenta S, Clough RW, Cell 6: 203-208, 2004. Dewhirst MW and Mueller-Klieser W: Elevated tumor lactate 21 Kunnumakkara AB, Diagaradjane P, Anand P, Harikumar KB, concentrations predict for an increased risk of metastases in head- Deorukhkar A, Gelovani J, Guha S, Krishnan S and Aggarwal and-neck cancer. Int J Radiat Oncol Biol Phys 51: 349-353, 2001. BB: Curcumin sensitizes human colorectal cancer to 37 Walenta S, Salameh A, Lyng H, Evensen JF, Mitze M, Rofstad by modulation of cyclin D1, COX2, MMP9, VEGF EK and Mueller-Klieser W: Correlation of high lactate levels in and CXCR4 expression in an orthotopic mouse model. Int J head and neck tumors with incidence of metastasis. Am. J. Cancer 125: 2187-2197, 2009. Pathol 150: 409-415, 1997. 22 Kim JB: Three-dimensional tissue culture models in cancer 38 Godugu C, Patel AR, Desai U, Andey T, Sams A and Singh M: biology. Semin Cancer Biol 15: 365-377, 2005. AlgiMatrix™ based 3D cell culture system as an in-vitro tumor 23 Pampaloni F, Reynaud EG and Stelzer EH: The third dimension model for anticancer studies. PLoS One 8: e53708, 2013. bridges the gap between cell culture and live tissue. Nat Rev Mol 39 Yang TM, Barbone D, Fennell DA and Broaddus VC: BCL2 Cell Biol 8: 839-845, 2007. family contribute to apoptotic resistance in lung cancer 24 Hanahan D and Weinberg RA: The hallmarks of cancer. Cell multicellular spheroids. Am J Respir Cell Mol Biol 41: 14-23, 100: 57-70, 2000. 2009. 25 Karikusa F and Sawasaki Y: The restoration of the functions of 40 Eberhart CE, Coffey RJ, Radhika A, Giardiello FM, Ferrenbach serially passaged calf hepatocytes by spheroid formation. In S and DuBois RN: Up-regulation of cyclooxygenase 2 gene Vitro Cell Dev Biol Anim 32: 30-37, 1996. expression in human colorectal adenomas and adenocarcinomas. 26 Curran JA, Chen R and Hun JA: The guidance of human Gastroenterology 107: 1183-1188, 1994. mesenchymal stem cell differentiation in vitro by controlled 41 Koki AT, Leahy KM and Masferrer JL: Potential utility of COX2 modifications to the cell substrate. Biomaterials 27: 4783-4793, inhibitors in chemoprevention and chemotherapy. Expert Opin 2006. Investig Drugs 8: 1623-1638, 1999. 27 Ivascu A and Kubbies M: Diversity of cell-mediated adhesions 42 Hida T, Kozaki K, Muramatsu H, Masuda A, Shimizu S, in breast cancer spheroids. Int J Oncol 31: 1403-13, 2007. Mitsudomi T, Sugiura T, Ogawa M and Takahashi T: 28 Kyle AH, Huxham LA, Chiam AS, Sim DH and Minchinton AI: Cyclooxygenase-2 inhibitor induces apoptosis and enhances Direct assessment of drug penetration into tissue using a novel cytotoxicity of various anticancer agents in non-small cell lung application of three-dimensional cell culture. Cancer Res 64: cancer cell lines. Clin Cancer Res 6: 2006-2011, 2000. 6304-6309, 2004. 43 Tsujii M and DuBois RN: Alterations in cellular adhesion and 29 Grantab R, Sivananthan S and Tannock IF: The penetration of apoptosis in epithelial cells overexpressing prostaglandin anticancer drugs through tumor tissue as a function of cellular endoperoxide synthase 2. Cell 83: 493-501, 1995. adhesion and packing density of tumor cells. Cancer Res 66: 44 Sun Y, Tang XM, Half E, Kuo MT and Sinicrope FA: 1033-1039, 2006. Cyclooxygenase-2 overexpression reduces apoptotic susceptibility 30 Suntherland RM: Cell and environment interactions in tumor by inhibiting the cytochrome c-dependent apoptotic pathway in microregions: The multicell spheroid model. Science 240: 177- human colon cancer cells. Cancer Res 62: 6323-6328, 2002. 184, 1988. 45 Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M and DuBois 31 Kyle AH and Minchinton AI: Measurement of delivery and RN: Cyclooxygenase regulates angiogenesis induced by colon metabolism of tirapazamine to tumour tissue using the cancer cells. Cell 93: 705-716, 1998. multilayered cell culture model. Cancer Chemother Pharmacol 46 Pidgeon GP, Barr MP, Harmey JH, Foley DA and Bouchier- 43: 213-220, 1999. Hayes DJ: Vascular endothelial growth factor (VEGF) up- 32 Hicks KO, Ohms SJ, van Zijl PL, Denny WA, Hunter PJ and regulates BCL2 and inhibits apoptosis in human and murine Wilson WR: An experimental and mathematical model for the mammary adenocarcinoma cells. Br J Cancer 85: 273-278, 2001. extravascular transport of a DNA intercalator in tumours. Br J Cancer 76: 894-903, 1997. 33 Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK and Krishnakumar S: A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs. Mol Vis 18: 1361- 1378, 2012. 34 Kyle AH, Huxham LA, Yeoman DM and Minchinton AI: Limited Received October 22, 2013 tissue penetration of : A mechanism for resistance in solid Revised November 7, 2013 tumors. Clin Cancer Res 13: 2804-2810, 2007. Accepted November 8, 2013

5309