Sensitivities of anti-cancer drugs to cancer spheroid growth in the cell arrayTM
three-dimensional culture system Cell-able Oncology
Koichi Yokota, Tomoko Jomura, Emiko Ozeki and Takashi Ikeya. Transparent Inc., 4-2-1, Wakahagi, Inzai, Chiba 270-1609

highlow
Increased CD44 /CD24 cell population in spheroids

Abstracts
Recently, the cell biology field has come to appreciate the dissimilarity between two- (2D) andthree-dimensional (3D) environments in which cells routinely operate in vivo. Efficacy of most anticancer drugs have been evaluated in 2D environment but it must be desirable to evaluate it in 3Denvironment. We developed a novel 3D cell culture system named Cell-able™ Oncology utilizing
photo-sensitive materials that change to hydro-gel after UV irradiation and the optimized
molecular design not to allow the cells adhere to the hydro-gel. In this study, we demonstrated
that the cancer spheroids formed on the Cell-able™ Oncology in serum-free and serum-containing
media showed chemo-resistant, high expression of stem cell markers and drug transporters
compared to that on the 2D monolayer plate. Thus, the spheroids grown on the Cell-able™
Oncology could provide novel insight to the pharmacodynamics and pharmacokinetics in the anticancer drug research and development.

103

CD 44

Human prostate cancer cell line, DU145 were cultured on the Cell-able™ or collagen-coated plates
as the 2D environment in RPMI-1640 containing 10% FCS (serum media) or serum-free ReproFF2(ReproCELL) (stem media). The Cell-able™ Oncology has arrayed cell attachment areas of circle 100
μm in diameter (see below) on each well of the culture plate.

102

10101110-1
10-1

10-1
10-1

1103

102

10110310210103

2D culture

0.6%

Cell-able™

62.2%

102103

CD 44

Stem media

103

Cell-able™

18.8%

Cell culture in 2D and 3D environments

100 µmSerum media

2D culture

16.6%

102

102

10101

110-1

10-1
10-1

DLD-1 human colon cancer cell line

The cancer cells were seeded on the Cell-able™ and cultured for 5 days. The spheroid formed on
the circular cell-attachment areas and the morphology was quite different from that of 2D plates asshown below;

10-11103

102

101

CD 24

10310210CD 24

The presence of CD44 and CD24 surface markers of DU145 spheroid grown on Cell-able™ werecompared to 2D culture by Fluorescence-activated cell sorting (FACS) analysis. Cells werecultured in serum and stem media and labeled with anti-human CD24-FITC antibody(Invitrogen) and anti-human CD44-APC antibody (BD Biosciences). Samples were analyzed withMACSQuant® Analyzer (Miltenye Biotec).
100μm

High expression of mRNA for stem cell markers in spheroids

Experimental protocols

0.02

0.015
0.01
0.005
03 daysDrug sensitivity
FACS analysismRNA expression
CDFDA exclusion

Cell counts %2D culture

50100

110100

1000

0.1

1

Gemcitabine (nM)

Stem media

10

Stem media

Cell-able

150

2D culture

1005001001000

Cell-able
2D culture

110100

Gemcitabine (nM)

Cell-able

2D

Serum media

Cell-able

2D

Stem media

Cell-able™

Serum Media

Stem Media

2000

2000

1500

1500

1500
1000

6.4%

93.6%

69.7%

1000

500

30.3%

1000

500

82.5%

17.5%

0500

500

250

500

750

1000

Hoechst 33342
fluorescence intensity

0250

500

750

1000

Hoechst 33342
fluorescence intensity

2507501000

Hoechst 33342
fluorescence intensity

1000

The fluorescence intensity of DU145 spheroid grown on Cell-able ™ stained by Hoechst33342
was compared to 2D culture by Fluorescence-activated cell sorting (FACS) analysis. Cells werecultured in serum and stem media and stained with Hochst33342 (sigma). Samples wereanalyzed with MACSQuant® Analyzer (Miltenye Biotec).

10050Conclusions

00.10

Paclitaxel (nM)

Cell counts %Cell counts %1500.001

5000.10.002

Spheroids are stained weakly by Hoechst33342

000.003

Stem media

Cell-able
2D culture0.004

2D

Serum Media

Serum media

1500.005

High expression of mRNA for stem cell markers, Nanog and Abcg2 in spheroids grown on Cellable™ compared with the 2D culture. Total RNA was extracted from the cultivated cells using(Takara Bio). Complementary DNA was synthesized from total RNA using Mix. Quantitative realtime polymerase chain reaction (qRT-PCR) analysis was performed using a Real time PCR with
TaqMan Gene Expression Assay reagent and probes (Applied Biosystems). The probe: Nanog(Hs04260_366_g1); Abcg2 (Hs0105370_m1).

Cell number

Cell-able100Cell-able

2D culture

Drug sensitivities of cancer spheroids
Serum media

2DSerum media

Groups;
1. Cell-able™ culture in RPMI-1640 supplemented with 10% FCS (serum media)
2. 2D culture in RPMI-1640 supplemented with 10% FCS (serum media)
3. Cell-able™ culture in ReproFF2 (stem media)
4. 2D culture in ReproFF2 (stem media)

Cell counts %0.025

Cell-able

1 or 2 days

150

0.03

Drug or vehicle

Seeding DU145

ABCG2Relative mRNA expression(/GAPDH)

100μm

Relative mRNA expression(/GAPDH)

NANOG

0.1

1

101001000Paclitaxel (nM)

Chemo-resistance of the spheroids grown on the Cell-able™ against gemcitabine (left) andpaclitaxel (right) in the serum and stem media compared to 2D culture. Each drug was treatedfor 3 days and the cell counts were determined by ATP luciferase method (for serum media)
or DAPI staining quantified by high content screening assay (for stem media)• The spheroid formation on Cell-able™ lead to the chemo-resistance against gemcitabine andpaclitaxel.
• The chemo-resistance was associated with high population of stem cells (CD44high/CD24low) andhigh expression of stem cell markers, NANOG and ABC transporters ABCG2 and ABCB1.
• The combination of Cell-able™ and the stem media conferred high stem cell population, which
may be suitable for drug research and development of drugs targeting the cancer stem cells.
• The pharmacodynamic and pharmacokinetic studies using the cancer stem cell-rich spheroids
could provide meaningful information relevant to the clinical conditions.