Comparative assessment of the in vitro efficacy of doxorubicin against BT-20 triple-negative breast carcinoma cells in a 2D and 3D cellular model

Keith Ncube, Werner Cordier, Duncan Cromarty

Department of Pharmacology, Faculty of Health Sciences, University of PretoriaArcadia 0007, South Africa

TNBC occurs in 12-17% of breast cancer casesBreast cancer is a global problem

Estrogen receptor

Progesterone receptor

HER2

Triple-negative breast cancer (TNBC)1.7 million diagnosed

522,000 deaths

Clinical trials

Patient care

Regulatory approval

Basic researchBasic research

1. Discovery 2. Development 3. Delivery

Pre-clinical studies should provide accurate predictions

Cell culture

Animal models

Cell culture

2D cell culture3D cell culture

There are 2 models used for in vitro pre-clinical drug tests

Three dimensional spheroids are used as an alternative

Medium

Glass/ plastic surface

Gel

Monolayer cell culture provides insufficient predictions

Spheroids optimally mimic the tumour microenvironment

Proliferative zone

Necrotic core

Quiescent zone

2D3D

12

3

This study has three main objectives

Cell suspension (200 µL) added(40 000 cells/well)

Plates pre-coated with 50 µL of 1% agarose

Liquid overlay was used to generate spheroids

Imaged at day 4, 7, and 10 using light microscopy

Dense spheroids stabilised in size at day seven

Phase contrast images of spheroids at day 4, day 7, day 10, and monolayers at 24 h

Day 4 Day 7

MonolayerDay 10 Spheroids: 5X objective lens

Monolayers: 10X objective lens

Scale bar = 100 µm

SpheroidSizer and ImageJ were used to automate diameter

SpheroidSizer ImageJManual

Diameter decreased significantly from day 4 to day 7

= SpheroidSizer

= ImageJ

Day 4 vs 7: Mann Whitney t-test

SpheroidSizer vs manual vs ImageJ: 2-way ANOVA

Day 4 n = 52 spheroidsDay 7 n = 33 spheroids

Change in spheroid diameter from day 4 to day 7 measured using SpheroidSizer and ImageJ

= Manual

Volume decreased significantly from day 4 to day 7

Day 4 vs 7: Mann Whitney t-test

Day 4 n = 52Day 7 n = 33

95% Confidence level

Change in spheroid volume from day 4 to day 7

Sectioned into 3 µm sections, and fixed onto glass slides

Fixed in 4% formaldehyde, embedded in paraffin wax

H&E staining was used for spheroid histochemical analysis

Stained with haematoxylin (10 min) & eosin (dip)

H&E staining suggested possible spheroid compaction

Day 7

Haematoxylin-eosin sections of spheroids at day 4 and day 7

Day 4

Haematoxylin: stains nucleiEosin: stains cytoplasm

20X magnificationScale bar = 100 µm

Samples (5 µL) added to 195 µL BCA reagent, 40 min, 60°C

Spheroid (8) pooled, and lysed in 100 µL RIPA buffer

Protein content was analysed using the BCA assay

Protein spectrophotometrically compared to BSA standard curve(562 nm)

Protein content decreased slightly on day 7

Day 4 vs 7: Mann Whitney t-test

n = 24 spheroids

95% Confidence level

Change in the protein content per spheroid from day 4 to day 7

Stained with FDA (4 µg/mL) and PI (5 µg/mL) solution

Spheroids harvested into 6-well plate

Live/Dead staining was used to assess viability in spheroids

Washed and viewed under filters for different stains

FDA

PI

Live/Dead staining indicated a centralised necrotic core

=FDA

=PI

Fluorescence images showing a spheroid and monolayer cells stained with FDA (green) and PI (red)

Spheroid Monolayer

Resorufinfluorescent

Metabolic activity was evaluated with the resazurin assay

Resazurinnon-fluorescent

Incubated for 2 hours and fluorescence measured

Medium (100 µL) replaced with 60 µM resazurin

Excitation : 530 nmEmission : 590 nm

Metabolic activity increased over the growth period

Change in spheroid metabolic activity from day 4 to day 7

Day 4 vs 7: Mann Whitney t-test

Day 4 n = 30 spheroidsDay 7 n = 36 spheroids

95% Confidence level

Cytotoxicity was assessed with the SRB assay

FIX WASH

Half logs of 32 µM doxorubicin

Fixed with 50% cold TCA overnight

Washed and stained with 0.057% SRB

Absorbance measured at 540/630 nm

Doxorubicin reduced cell density at 24h and 72h

24 h 72 h

IC10: 204 nMIC25: 555 nMIC50: 1316 nM

IC10: 216 nMIC25: 456 nMIC50: 600 nM

Cell density (expressed as a percentage of the negative control) after 24 and 72 hour exposure to half-log dilutions of 32 µM doxorubicin on 2D cells

n = 4

R2 = 0.79n = 5

R2 = 0.90

DXR did not significantly alter spheroid morphology

Negative control IC25 IC50 IC70IC10

Sphe

roid

s M

onol

ayer

s

Images of spheroids (A to E) and monolayers (F to J) after a 72 hour exposure to different concentrations of doxorubicin

A B C D

JIHGF

Spheroids: 5X objective, phase contrast Monolayers 10X objective, DAPI fluorescenceScale bar = 100 µm

E

There was a slight decrease in spheroid volume at the IC50

NC vs IC50: One-way ANOVA

NC = negative control

n = 4

Change in spheroid volume after 72 h exposure to doxorubicin

Metabolic activity was reduced more in monolayers

= Monolayers

= Spheroids

2D vs 3D: 2-way ANOVA

Bonferroni post test

n = 4

Change in spheroid and monolayer metabolic activity after 72 h exposure to doxorubicin

A brief discussion of our findings

BT-20 spheroids resist apoptosis by internalising death receptorsDense spheroids resistant to doxorubicin compared to monolayers

Imamura et al,. 2015Chandrasekaran et al,. 2015

3D- cultured BT-20’s

Conclusion

• More resistant to doxorubicin

• Better representation of in vivo architecture

• Potential future screening platform

• Sensitive to doxorubicin

2D- cultured BT-20’s

www.up.ac.za/phamacology

Department of Pharmacology

Faculty of Health Sciences

Denkleiers • Leading Minds • Digkopolo tša Dihlalefi

Thank you Acknowledgements

Miss S. Arbi Histology

Miss T. Hurrell Technical advice