Investigation of Cancer Spheroid Growth and Analysis Sydney Wade, Laura Abriola M.S.1 1 Yale Center for Molecular Discovery, West Haven, CT Abstract Three-dimensional aggregates of cancer cells—or spheroids—are a more accurate representation of the nature of tumors in vivo. Unfortunately, there has been little research into optimizing the cultivation and maintenance of spheroids. By testing several specialized microplates created for the cultivation of multidimensional spheroids, we were able to determine which plates were optimal for producing uniform and compact spheroids for individual cell lines. Additionally, these spheroids were treated with anti-cancer drugs to analyze the difference in drug response between 2D and 3D cancer cells. We found that the multidimensional spheroids were more resistant to traditional anti-cancer drugs in comparison to their two-dimensional counterparts. Additionally, we discovered that the Corning Spheroid Microplates and Elplasia Kuraray Microplates were the best culture plates for cultivating uniform and robust spheroids at all seeding densities. Introduction In conventional oncological research, two-dimensional monolayer cells have been cultivated and treated as models for in vivo tumor cells. Unfortunately, these two-dimensional cells are not accurate representations for in vivo tumors. Three-dimensional “balls” of cancer cells, or spheroids, serve as better models for in vivo cancerous tumors, but little research has been done to determine the best methods and protocols for producing the most uniform, spherical and compact spheroids for study. In this experiment, we wanted to test how well special microplates designed for spheroid formation actually produced the optimal spheroids that are advertised. Additionally, we wanted to learn if spheroid formation was cell line specific or if the microplates worked well or poorly for all cell lines. Moreover, we were interested in drug treatment and if 3D spheroids reacted to drugs differently than 2D monolayer cells. We had read of some prior research that had answered some of these questions, but they did not provide clear protocols and did not test more than a few plates or drugs

against their spheroids. They also did not provide any information about the general maintenance of spheroids beyond seeding the cells, like feeding spheroids. In this experiment, we wanted to test several methods of producing and analyzing spheroids in order to find the most optimal protocol for spheroid research. Materials and Methods Overview In this study, a series of six individual experiments was conducted to investigate more about the nature of cancer cell spheroids. The first experiment investigated how well two brands of spheroid microplates formed spheroids out of HCT-116 and A549 cell lines. This experiment was later replicated using DU145, MDA-MB-468 and MCF7 cell lines. Additionally, the transferability and feeding of spheroids were tested on spheroids grown on Corning 4516 Spheroid Microplates (see Transferring and Feeding section). Later, spheroids of one cell line were treated with drugs to compare the drug reaction of 3D spheroids to 2D monolayer cells (see Drug Treatment section) while DU145 spheroids were tested for self-disassembly (see DU145 Growth Analysis section). Cell Culture All cell lines were cultured in Dulbecco’s Modified Eagle’s Media [DMEM] phenol red cell media (Gibco) supplemented with 10% of Fetal Bovine Serum [FBS] to promote cell growth and health. Cells were incubated at 37°C with 5% CO2. The cell-passaging rate averaged twice per week. During passaging, the cells were washed with Dulbecco’s Phosphate Buffer Saline [DPBS] without calcium and magnesium (Gibco) as a buffer and detached with 0.25% Trypsin 1X EDTA (Gibco). Cells were stained with 0.18% trypan blue and counted with a hemocytometer before re-immersion. The cell lines used include HCT-116, A549, DU145, MDA-MB-468, and MCF7. Spheroid Formation Seven different spheroid microplates were tested with the five cells lines to determine how well

each plate produced uniform, spherical and compact spheroids (Table 1). For each plate, the standard protocol for plating spheroids was followed. The cells were cultured in DMEM with 10% FBS while plated in each microplate. For the Perfecta3D Hanging Drop Plates, only 40 uL of cells were plated per well and DPBS was used to fill the reservoirs. Assay Development and Analysis To analyze spheroid growth or lack thereof, equal volumes of the CellTiter-Glo 3D Luminescent Viability Assay were added to drug-treated wells. The plates were shaken vigorously at 1100 rpm for

5 minutes before incubating in a dark drawer for another 25 minutes. Luminescence readouts were conducted on an EnVision 2101 Multilabel Plate Reader. Imaging and Analysis All spheroids were imaged in Brightfield with the IN Cell Analyzer 2200 using the 4X objective lens. Spheroid measurements including area, median radius and perimeter were analyzed with the CellProfiler software. This data was then analyzed with GraphPad Prism. HCT-116 2D and 3D drug reaction data was analyzed with Activity Base.

Microplate Brand

Methodology

Spheroid

Number (per well)

Spheroid

Size

Uniform

Spheroids?

Consistent Results?*

Corning 3474 Ultra

Low Attachment Plate, 96-well

Ultra Low

Attachment surface

Several

Small

No

N/A

Corning 4515

Spheroid Microplate, 96-well

Ultra Low

Attachment surface, Rounded bottom

One

Large

Yes

Yes

Corning 4516

Spheroid Microplate, 384-well

Ultra Low

Attachment surface, Rounded bottom

One

Large

Yes

Yes

Elplasia SQ 200 100 NA Microplates, 96-

well

Square Grid

(discourages cell adhesion)

Several

Small

Yes

No

Nunclon Sphera

Microplate 174925, 96-well

Ultra Low

Attachment Surface, Rounded Bottom

One

Large

Yes

Yes

Perfecta3D Hanging

Drop Plates, 96-well

Hanging Drops

One

Small

No

No

SCIVAX

NanoCulture NCP-LH96-2

Hexagonal Grid

(discourages cell adhesion), Low

binding

Several

Small

No

N/A

Table 1: Microplates tested !

Transferring and Feeding To test how well spheroids maintained shape and size when transferred (moved to a new microplate) or fed (media replacement), HCT-116 and A549 spheroids were each cultured on two Corning 4515 Spheroid Microplates at six seeding densities created by serial dilutions (16000 to 500). After an incubation period of 72 hours, one plate of HCT-116 and A549 spheroids was fed while the other plate had its spheroids transferred. During feeding, 50% of the DMEM in each well was removed using a multichannel pipet and replaced with an equal volume of fresh media. Post-feeding, the spheroids were observed via a light microscope and imaged 24 hours later. During transferring, 50% of the DMEM in each well was removed with a multichannel pipet. The remaining 50% of the solution including the spheroids was then transferred with a multichannel pipet to a Corning 3474 Ultra Low Attachment (flat bottom) plate. The spheroids were immediately observed under a light microscope and imaged 24 hours later. All image data was then analyzed with CellProfiler to determine if the spheroids were damaged or absent from the wells. Drug Treatment In two experiments, HCT-116 spheroids were treated with anti-cancer drugs. Initially, HCT-116 cells were cultured on a Corning 4516 Spheroid Microplate (384-well) at six seeding densities created by serial dilutions (8,000 to 250) and incubated for 72 hours. After 72 hours, each row of spheroids was treated with one of seven anti-cancer drugs (Table 2). The last row contained the DMSO control. In a later experiment, HCT-116 cells were seeded and cultured on four Corning 4516 Spheroid Microplates (384-well). Each plate contained a different seeding density (8000, 4000, 2000, or 1000). The spheroids were incubated for 72 hours. At 48 hours, HCT-116 cells were seeded on a standard two-dimensional monolayer culture plate at 400 cells/well. After the 2D monolayer cells had incubated for 24 hours (and the 3D spheroid cells had incubated for 72 hours), all five plates were treated with the National Cancer Institute AOD VI oncology drug set using the Tecan Aquarius multichannel pipetting system. Each drug had a concentration of 10 uM. 42 uM of tamoxifen was manually added to the last two columns (23 and 24) with a multichannel pipet to serve as the positive control and to normalize the data to

Table 2: HCT-116 Anti-Cancer Drugs

Drug Name

Concentration

(uM)

Etoposide

50

Methotrexate

10

Puromycin

5

Staurosporine

10

Tamoxifen

60

Taxol

10

Tirapazamine

20

percent effect. All plates were spun at 500 rpm for 2 seconds following treatment before incubating for 72 hours. Post-incubation, the spheroid plates were imaged and assessed with CellTiter-Glo 3D assay. DU145 Growth Analysis Previous research suggested that DU145 spheroids do not maintain growth after 72 hours of incubation. To test this assertion, DU145 cells were seeded on three Corning 4515 Spheroid Microplates. Each plate had six different seeding densities (16000 to 500) with one seeding density per two columns. Each plate was imaged and assessed with the CellTiter-Glo 3D assay at either 24 hours, 72 hours or 144 hours to determine spheroid growth. Results and Discussion Spheroid Formation For the formation of one spheroid per well, both the Nunclon Sphera Microplates and Corning 4515 Spheroid Microplates produced optimal spheroids for all cell lines except the MCF7 spheroids. With the MCF7 cell line, the Corning Spheroid Microplates produced more compact and intact spheroids in comparison to the Nunclon Sphera Microplates (Figure 1). Additionally, the Corning Spheroid Microplates have black wells with clear bottoms, and therefore were better plates for running assays without well cross-talk. The Nunclon plates have clear wells. The Nunclon plates only come in the 96-well format while the Corning plates also come in the 384-well

format, which is better for screening. Because of these features, the Corning Spheroid Microplates are superior to the Nunclon Sphera Microplates in the development and analysis of 3D spheroids. For producing several small spheroids per well, the Elplasia Kuraray plates are superior to the SCIVAX Nanoculture plates because of their ability to produce more uniform spheroids. This could be due to the compartmentalization of the spheroids on the Elplasia plates that is not present on the SCIVAX plates. Additionally, the ability for Elplasia plates to produce multiple small, yet uniform spheroids could also be benefited for growing stem cells or neuro-spheroids that would better represent their in vivo counterparts. The Perfect3D hanging drop plates proved to be unreliable and inconsistent. After seeding the cells, it was evident that some cells did not fall into the hanging drop. Additionally, the plates required that the surrounding reservoirs were filled with either a liquid buffer or water. This presented handling problems. We had to be very careful not to hit, shake or tip the plates otherwise, the hanging drop would fall from the well or the surrounding buffer would contaminate the spheroids. Additionally, each cell line produced spheroids of different properties, suggesting that the creation of compact spheroids on each plate is cell line specific (Figure 2). Looking directly at the MDA-MB-468

cell line, the spheroids produced are large, yet loosely compact in comparison to the other cell lines with spheroids grown with the same initial cell count. MCF7 spheroids (not pictured) produced even less compact spheroids especially when cultured on Nunclon Sphera Microplates. This suggests that these cell lines may need additional supplements, like the addition of E-cadherin or Matrigel, in order to produce more compact spheroids. Transferring and Feeding Transferring and feeding spheroids proved to be infeasible and inconsistent. In the microplate with the transferred spheroids, only one of the spheroids of 16,000 seeding density was successful transferred. The other seven spheroids of the same seeding density were absent from their wells following the transfer. Additionally, only two spheroids at 8,000 seeding density were transferred successfully. However, as the seeding density decreased, the number of spheroids properly transferred increased (Figure 3). From this, we conclude that smaller spheroids are less likely to be damaged or removed during transfer. Feeding spheroids proved to be less difficult, yet still unpredictable. There were at least two spheroids from the 16,000 seeding density that were absent from their wells post-feeding.

Fig 1. Brightfield images of MCF7 spheroids on A) Nunclon Sphera Microplates and B) Corning Spheroid Microplates [SM]. The spheroids were seeded at 8,000 cells/well and incubated for 72 hours. Nunclon Sphera Microplates produced less compact spheroids than the Corning SM, and therefore had spheroids with larger areas.

AB

B 16000

8000

4000

2000 100

050

00

500000

1000000

1500000

Initial Seeding Density per Welln = 8 (per seeding density per cell line)

Are

a (u

m2 )

Corning vs Nunclon - Area

Corning SpheroidMicroplate

Nunclon Sphera

A

B

For the most consistent results, it is best to eliminate feeding or transferring spheroids. This makes Perfecta3D plates undesirable because these plates require spheroids to be transferred to an assay plate for analysis. Additionally, this means shorter incubation times are better for spheroid development. However, because the formation of spheroids is cell line dependent, not all cell lines can form compact spheroids quickly. For some cell lines, such as HCT-116, increasing the seeding density may help produce spheroids faster. Drug Treatment In the initial drug treatment, 60 uM of tamoxifen proved to be the most effective drug for killing the spheroids. This drug was used in the second drug treatment as the positive control. Unfortunately, due to the lack of tamoxifen in stock, only 42 uM of tamoxifen was used in the second treatment, but this concentration proved to be as effective as the 60 uM concentration. The primary goal for drug treatment was to determine if there were any drugs that targeted 3D spheroids over the 2D monolayer cells. Out of the 114 cancer drugs from the NCI compound plate and the tamoxifen, none of the drugs killed the spheroids more than the 2D monolayer cells. This leads us to believe that 3D spheroids are less sensitive to traditional anticancer drugs. However,

Fig 2. Brightfield images of A) HCT-116, B) A549, C) DU145 and D) MDA-MB-468 spheroids at 8,000 seeding density grown on Corning 384-well Spheroid Microplates for 144 hours. Based on the cell line, the spheroid characteristics were different. HCT-116 spheroids produced large yet compact spheroids. However, the MDA-MB-468 spheroids produced loose spheroids that resulted in larger areas.

A B

C D

500

1000

2000

4000

8000

1600

00

200000

400000

600000

Initial Seeding Density (cells)n varies per seeding density

Mea

nA

rea

(um

2 )

HCT116

Pre-Transfer

Post-Transfer

500

1000

2000

4000

8000

1600

00

100000

200000

300000

A549

Initial Seeding Density (cells)n varies per seeding density

Mea

nA

rea

(um

2 )

Pre-TransferPost-Transfer

Fig 3. Graphs of HCT-116 and A549 image data for pre- and post-transfer spheroids. Transferring proved inconsistent with many spheroids damaged or absent following transfer. No spheroids were present post-transfer for the seeding densities 16,000 and 2,000 for HCT-116 and A549, respectively.

because there were several wells that were missing spheroids, this experiment will be repeated to ensure more consistent results. DU145 Growth Analysis After analyzing both the spheroid viability data from the CellTiter-Glo 3D assay and the Brightfield images of the spheroids, only the spheroids seeded at 16,000 cells/well showed a decrease in both spheroid viability and spheroid area (Figure 4). Most of the other seeding densities showed slight decreases in spheroid area, but increases in luminescence. This suggests that as the spheroids matured, they became more compact even with the addition of new cells. From this data, we can speculate that once DU145 spheroids get to a certain size, they begin to disassemble over time. The cause of the decrease, however, is unknown. Outside factors such as media depletion could also have caused the spheroid size decrease. However, the experiment was strategically planned so that the spheroids grew for no longer than 144 hours in

order to eliminate the effect of the media depletion on the cells’ health. Another key factor to note is that this data contradicts earlier research on the growth of DU145 spheroids. Other literature has shown a decrease in DU145 spheroid viability at lower seeding densities and shorter time periods. Another work asserted that DU145 growth only slows after 72 hours but does not halt completely. This data however showed that DU145 spheroids at the highest seeding density exclusively not only stopped growing, but began to disassemble and even die. Further research will have to be conducted to gain consistent data about the growth pattern of DU145 spheroids. This knowledge will be crucial for completing further drug treatment research with this cell line. Conclusion In this paper, we investigate the development of uniform, spherical and compact spheroids for research use. We have found that the Corning Spheroid Microplates and Elplasia Kuraray Microwell Plates are optimal for the development and analysis of multidimensional spheroids. Additionally, we demonstrated that 3D spheroids are more resistant to conventional anticancer drugs than 2D monolayer cells. This discovery will effect how future in vitro anticancer drug screenings and treatments will be conducted especially in the search of more effective treatments. Acknowledgements I would like to thank Laura Abriola for all of her guidance and insight in developing this project. Additionally, I gratefully acknowledge Yulia Surovtseva for her assistance in spheroid imaging, Peter Gariess for his assistance in the spheroid drug treatment, and Sheila Umlauf and Janie Merkel for their support and guidance. References Costa, Elisabete C., Vítor M. Gaspar, Paula Coutinho, and Ilídio J. Correia. "Optimization of Liquid Overlay Technique to Formulate Heterogenic 3D Co-cultures Models." Biotechnol. Bioeng. Biotechnology and Bioengineering 111.8 (2014): 1672-685. 25 Feb. 2014. Web. 28 May 2015. Fennema, Eelco, Nicolas Rivron, Jeroen Rouwkema, Clemens Van Blitterswijk, and Jan De Boer. "Spheroid Culture as a Tool for Creating 3D Complex Tissues." Trends in Biotechnology 31.2 (2013): 108-15. ScienceDirect. Web. 28 May 2015.

Fig 4. Graphs of DU145 spheroids cultivated for 24, 72 and 144 hours. The data were analyzed with CellProfiler (top) and CellTiter-Glo 3D. Only the spheroids seeded at 16000 cells/well show both a decrease in spheroid growth and viability.

1600

080

0040

0020

0010

00 500

0

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150000

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Initial Seeding Density per Welln = 8 (per seeding density per cell line)

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a (u

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(cps

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24 HRS72 HRS144 HRS

Friedrich, J., W. Eder, J. Castaneda, M. Doss, E. Huber, R. Ebner, and L. A. Kunz-Schughart. "A Reliable Tool to Determine Cell Viability in Complex 3-D Culture: The Acid Phosphatase Assay." Journal of Biomolecular Screening 12.7 (2007): 925-37. SAGE Journals. Web. 3 June 2015. Ho, Wan Yong, Swee Keong Yeap, Chai Ling Ho, Raha Abdul Rahim, and Noorjahan Banu Alitheen. "Development of Multicellular Tumor Spheroid (MCTS) Culture from Breast Cancer Cell and a High Throughput Screening Method Using the MTT Assay." PLOS ONE 7.9 (2012): n. pag. PLOS One. Web. 28 May 2015. Nagelkerke, Anika, Johan Bussink, Fred C.g.j. Sweep, and Paul N. Span. "Generation of Multicellular Tumor Spheroids of Breast Cancer Cells: How to Go Three-dimensional." Analytical Biochemistry 437.1 (2013): 17-19. ScienceDirect. Web. 26 May 2015. Pardo, Ana Maria P., Katherine Strathearn, Nicolas Andre, and Terry Riss. Corning Spheroid Microplates and Promega CellTiter-Glo 3D Cell Viability Assay Provide a Novel Approach for High Throughput Screening of Multicellular Spheroids. Rep. Promega Corporation, 2014. Web. 29 May 2015. Vinci, Maria, Sharon Gowan, Frances Boxall, Lisa Patterson, Miriam Zimmermann, William Court, Cara Lomas, Marta Mendiola, David Hardisson, and Suzanne A. Eccles. "Advances in Establishment and Analysis of Three-dimensional Tumor Spheroid-based Functional Assays for Target Validation and Drug Evaluation." BMC Biology BMC Biol 10.1 (2012): 29. BioMed Central. Web. 16 June 2015. !