The Next Step in the Evolution of 3D Culture: Utilizing Extracellular Matrix to Enhance Multicellular Tumor Spheroid Models for

Proliferation and Invasion

Overview of methodology, tools and reagents for evaluating cell proliferation and invasion using multicellular tumor

spheroids.

TOOLS AND REAGENTS

3-D SPHEROID PROLIFERATION/VIABILITY

Calculate volumes for 10X Spheroid Formation ECM, Complete Growth Medium, and Cells (1 x 10^6 cells/ml):

Reagent 1 Well 3,000 Cells/Well

10X Spheroid Formation ECM (4 °C) 5 µl 5 µl

Cells (at 1 x 10^6 cells/ml) X µl 3 µl

Complete Growth Medium (4 °C) 45-X µl 42 µl

Total 50 µl 50 µl

Sample Preparation for Spheroid Formation

10X

Spheroid

Formation

ECM

Sample Preparation for Spheroid Formation

• Thaw 10X Spheroid Formation ECM on ice at 4 °C overnight.

• Divide into working aliquots; store at -80 °C for optimal stability, avoid freeze-thaws.

• Keep on ice throughout procedure.

• Dilute with cold (4 °C) complete growth medium, and pipet up and down to mix.

Complete Growth

Medium

Spheroid Formation ECM

• Culture cells to 80% confluence.

• Harvest cells, centrifuge, and aspirate medium.

• Resuspend at 1 x 10^6 cells/ml in complete growth medium.

• Add the calculated amount of cells to the tube containing Spheroid Formation ECM.

Cells at 80% Confluence Cells at

1 x 10^6 cells/ml

Spheroid Formation ECM

Cells in Spheroid Formation ECM

Harvest

Centrifuge Resuspend

Sample Preparation for Spheroid Formation

Sample Preparation for Spheroid Formation

• Add 50 µl of the cells in Spheroid ECM to the 96 Well Spheroid Formation Plate.

• Centrifuge at 200 x g for 3 minutes in a swinging bucket rotor.

• Incubate plate with cells at 37 °C, 5% CO2 for 72 hours.

Cells in Spheroid Formation ECM

96 Well Spheroid Formation Plate

Cells and Spheroid Formation ECM in 96 Well Spheroid Formation Plate

Sample Preparation for Spheroid Formation

• Cells assemble into one spheroid/well within 72 hours.

• Prepare solutions containing 2X treatment compounds in complete growth medium.

• Add 2X treatment (50 µl) to each well.

• Incubate at 37 °C, 5% CO2 for desired treatment period.

Spheroid formed within 72 hours

Add 50 µl of 2X treatment.

Incubate for desired treatment period.

Image Analysis Evaluation of Spheroid Growth

Image Analysis

3-D Culture Spheroid Proliferation/Viability

2 3 6 7 8

10 13 14 21 22

24 27 29 31 35

0

200000

400000

600000

800000

1000000

0 10 20 30 40

Are

a (µ

m2)

Days

MDA-MB-231 3-D Spheroid Growth

• 3-D spheroid cultures are large cell aggregates which exhibit physiological characteristics of small tumors .

• Incorporation of ECM proteins is necessary to promote in vivo cell behavior.

• This assay provides an in vitro model for evaluating tumor growth in response to pharmacological agents.

MDA-MB-231 Breast cancer cells in spheroid culture for 3 days form A) loose colonies in media and B) tight colonies in media + ECM.

A B

3-D Culture Spheroid Proliferation/Viability Image Analysis

y = 23918x + 163064 R² = 0.9941

0

50000

100000

150000

200000

250000

300000

0 1 2 3 4 5

Are

a (µ

m2)

Day

0 1 2 3 4

Spheroid growth of MDA-MB-231 breast cancer spheroids. Cells were seeded at the corresponding concentrations in the presence of spheroid formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid formation. At that time, 50 ul of complete medium was added to each well, and spheroids were incubated at 37 °C, 5% CO2. Spheroids were photographed every 24 hours, and images were analyzed using ImageJ software.

y = -0.143ln(x) + 0.7463 R² = 0.9992

0%

20%

40%

60%

80%

100%

120%

0.1 1 10 100

Are

a (

% u

ntr

eat

ed

)

Bleomycin (µM)

0.16 0.8 4 24

Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin. Cells were seeded at 3,000 cells/well in the presence of spheroid formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid formation. Spheroids were then treated with the corresponding doses of Bleomycin and incubated at 37 °C, 5% CO2 for 96 hours. Spheroids were photographed after 96 hours, and images were analyzed using ImageJ software

3-D Culture Spheroid Proliferation/Viability Analysis

Fluorometric

or

Colorimetric

Quantitation

Fluorometric Quantitation with Resazurin

3-D Culture Spheroid Proliferation/Viability Fluorometric Quantitation

y = 2935.7ln(x) - 10541 R² = 0.9973

0

2000

4000

6000

8000

10000

12000

14000

16000

10 100 1000 10000

RFU

Spheroid Seeding Density (Cells/Well)

Spheroid cell number corresponds to fluorescence output for MDA-MB-231 breast cancer spheroids. Cells were seeded at the corresponding concentrations in the presence of spheroid formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid formation. At that time, 50 ul of complete medium was added to each well, and spheroids were incubated at 37 °C, 5% CO2 for 96 hours and analyzed using Resazurin.

y = -0.086ln(x) + 0.8038 R² = 0.9877

0%

20%

40%

60%

80%

100%

120%

0.1 1 10 100

Via

bili

ty

Bleomycin (uM)

Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin. Cells were seeded at 3,000 cells/well in the presence of spheroid formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid formation. Spheroids were then treated with the corresponding doses of Bleomycin, incubated at 37 °C, 5% CO2 for 96 hours, and analyzed using Resazurin.

Colorimetric Quantitation with MTT

3-D Culture Spheroid Proliferation/Viability Colorimetric Quantitation

Spheroid cell number corresponds to absorbance for MDA-MB-231 breast cancer spheroids. Cells were seeded at the corresponding concentrations in the presence of spheroid formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid formation. At that time, 50 ul of complete medium was added to each well, and spheroids were incubated at 37 °C, 5% CO2 for 96 hours and analyzed using MTT.

Inhibition of MDA-MB-231 cell spheroid viability by Bleomycin. Cells were seeded at 3,000 cells/well in the presence of spheroid formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid formation. Spheroids were then treated with the corresponding doses of Bleomycin, incubated at 37 °C, 5% CO2 for 96 hours, and analyzed using MTT.

y = -0.118ln(x) + 0.7563 R² = 0.9979

-10%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

0.1 1 10 100 1000

Via

bili

ty

Bleomycin (µM)

y = -1E-09x2 + 4E-05x R² = 0.9961

-0.050

0.000

0.050

0.100

0.150

0.200

0.250

0.300

0.350

0.400

0 2,000 4,000 6,000 8,000 10,000 12,000 14,000

Ab

sorb

ance

(5

70

nm

)

Spheroid Seeding Density (Cells/Well)

3-D SPHEROID INVASION

Sample Preparation for Spheroid Invasion

• Cells assemble into one spheroid/well within 72 hours.

• Chill 96 Well Spheroid Formation Plate on ice at 4 °C for 5 minutes.

• Working on ice, add 50 µl of Invasion Matrix to each well.

• Centrifuge plate at 300 x g for 5 minutes at 4 °C (pre-chilled) in a swinging bucket rotor.

Invasion Matrix

Chill on ice at 4 °C for 5 minutes.

Add Invasion Matrix and centrifuge 300 x g for 5 min at 4 °C.

Spheroid in Spheroid Formation Plate

Thaw on ice at 4 °C overnight.

Sample Preparation for Spheroid Invasion

• Transfer the Spheroid Formation Plate to 37 °C, 5% CO2 for one hour to polymerize Invasion Matrix.

• Add 100 µl of complete medium with 2X invasion modulating agents (37 °C) to each well.

• Incubate at 37 °C, 5% CO2 for desired invasion period.

• Analyze plate.

Place Spheroid Formation Plate at 37 °C, 5% CO2.

Add 100 µl of 2X treatment (37 °C).

Incubate for desired treatment period.

3-D Culture Spheroid Invasion

Resuspend cells in spheroid formation ECM

and add to round bottom wells.

Cells assemble into compact spheroids.

Add Invasion Matrix and medium containing

chemoattractants and/or invasion modulating

compounds.

Cell invade into the surrounding matrix in

Response to chemoattractants

3 days

1-2 hours

2-4 days

1-2 hours

Total Time: 5-7 days

DESCRIPTION TIME

Bright field RFP

PKH67 PKH26

3-D Culture Spheroid Invasion Analysis

Morphology of 3D cell invasion over a four day period. Cells were seeded at 3,000 cells/well in the presence of Spheroid Formation ECM, and incubated at 37 °C, 5% CO2 for 72 hours to form spheroids. Spheroids were then embedded in 50 µl Invasion Matrix, and incubated at 37 °C, 5% CO2 for one hour for polymerization. Then 100 µl of DMEM, 10% FBS was added to

each well as a chemoattractant. Non-invasive cells (MCF-7) remain as cell aggregates and do not invade into the surrounding invasion matrix; whereas, invasive cells (MDA-MB-231) invade into the surrounding invasion matrix as spindle-like protrusions.

3-D Culture Spheroid Invasion Analysis

Quantitative analysis of surface area for non-invasive (MCF-7) and invasive (MDA-MB-231) cell lines over a four day period.

3-D Culture Spheroid Invasion Analysis

PC-3

MDA-MB-231

HT-1080

U-87-MG

Cell Line 0 1 2 3 4

Days Post Imbedding

Different cell lines exhibit different invasive potentials and morphologies

Invasion of cancer cells out of MCTS and into the surrounding invasion matrix in a time-dependent manner. Cancer cells invade into an invasion matrix composed of 10 mg/ml BME and 1 mg/ml collagen I in response to cell culture medium containing 10% FBS; wells were photographed every 24 hours over a 96 hour period.

0 0.5 1 5 10 20 No Matrix

0100000200000300000400000500000600000700000800000900000

0 0.5 1 5 10 20 NoMatrix

Sulforaphane (uM)

Are

a (µ

m2)

MDA-MB-231 3-D Invasion Day 4

Sulforaphane (µM)

3-D culture spheroid

invasion may be used

to evaluate

modulators of

metastasis and EMT.

3-D Culture Spheroid Invasion Analysis

MDA-MB-231, human breast cancer cells, were induced to form spheroids (72 hours) and embedded in Invasion Matrix. Serial dilutions of Sulforaphane were added to the DMEM, 10% FBS invasion modulating solution as indicated, and spheroid invasion was conducted over a 96 hour period. Sulforaphane inhibited MDA-MB-231 spheroid Invasion in a dose-dependent manner.

Summary

• 3-D cultures provide suitable environments for cells to self-assemble into organotypic structures mimicking the morphology and function their tissue of origin.

• Spheroids provide ideal tumor models based on morphology, size, physiological gradients, and the formation of heterogeneous cell populations.

• Spheroids exhibit an extracellular matrix which is instrumental in self-assembly of cancer cells.

• The addition of ECM proteins promotes spheroid formation and growth.

• ECM hydrogels provide physiological scaffolds for cancer cells to invade out of tumor spheroids, mimicking early events in metastasis.

Tools for 3-D Culture Spheroid Proliferation/Viability and Invasion

Description Size Catalog #

Cultrex® 96 Well 3-D Spheroid BME Cell Invasion Assay 96 samples 3500-096-K

Cultrex® 96 Well 3-D Spheroid Fluorometric Proliferation/Viability Assay 96 samples 3510-096-K

Cultrex® 96 Well 3-D Spheroid Colorimetric Proliferation/Viability Assay 96 samples 3511-096-K

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