This document describes experiments testing several known anti-cancer compounds in three assays: cytotoxicity in 2D cell culture, cytotoxicity in 3D spheroid culture, and a scratch (wound healing) assay. The assays were automated in a 96-well plate format. Results showed that cytotoxicity EC50 values were higher in 3D culture compared to 2D, due to the time needed for compounds to reach the spheroid center. The scratch assay could distinguish between compounds that cause cell cycle arrest and cell death. Automating the 3D cytotoxicity and scratch assays in a high-throughput format allows efficient testing of compounds for poly-effective anti-cancer activity.

1. Abstract
To find anti-cancer drugs, different cellular modifications can be looked at: cell death effect on proliferating cells either in 2D culture or on
spheroids (3D culture), anti-mitotic, anti-migration/invasion, anti-angiogenesis effects… Different assays can be performed to follow these
different effects. High content screening is a multi-parametric technology allowing searching for poly-effects drugs.
In this poster, several well known compounds were tested in 3 different assays: cytotoxicity in 2D culture, cytotoxicity in spheroids (3D
culture) and scratch assay (also named wound healing). These 3 models were automated in 96-well plates.
Methods
Results
Conclusions & Perspectives
 Automation on cytotoxicity assay on 3D culture in 96-well microplate and scratch assay (would healing) was successful.
 EC50 results on cytotoxicity assay in 3D culture show a shift towards higher concentrations compared to 2D culture. This is due to the
time required for the compound to reach the center of the spheroid. Depending on this time, the shift may be higher or lower. These 3D
results are more relevant compared to the in vivo situation. Nevertheless, to be the most relevant as possible to the in vivo tumor, the
spheroid needs to reach a consistent size. However, a necrotic core appears from the 3rd day in this condition (seeding: 2000 cells/well).
 Perspective: the relevance of this 3D model would be increased by the addition of angiogenesis step with co-culture of HUVEC.
 Scratch assay is another assay which proliferation/migration is followed and depending of the profile of the results, cell cycle arrest
inducers (as colchicine) can be distinguished from cell death inducers. The two actions of Vinblastine (cell cylce arrest between 0.01 and
0.1 µM and cell death inducer at higher concentration) can be better observed in this assay than in the cytotoxicity assay.
Cell culture: Hela (HT29, HepG2 or Caco-2: data not shown) were routinely maintained in DMEM (v/v) supplemented with 10% FBS. Depending of the cell line,
cells were seeded between 2 500 and 10 000 cells/well in 96-well plates for 2D culture (Corning® cell-bind or Greiner® plates) or at 2000 cells/well in 96-well
Corning® Spheroid microplates for 3D culture. Then, cells were incubated at 37 °C in 5 % CO2 for plating. The medium was changed every 3 days if needed.
Cytotoxicity assay on 2D and 3D culture: After 48h of plating, cytotoxicity assay was performed by replaced medium in each well by fresh medium with or
without different anti-cancer drugs at different concentrations added by Hoechst (1/1000) and propidium iodide (1/1000).
Wound healing assay: When cells are at confluence, a scratch was performed in each well on a robotic platform with a 96-well head (Caliper ALH3000, Perkin
Elmer). Medium was changed and replaced as previously. Then cells were incubated at 37 °C in 5 % CO2 for several days.
Live cells imaging: Each day, pictures were taken on operetta (Perkin Elmer). Analysis and visualization were performed on Columbus (Perkin Elmer).
Cytotoxicity assay on 2D & 3D culture
Colchicine
Automated wound healing on 96-well plates
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Cmax - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cmin CTRL
Cisplatine
Colchicine
Cyclophosphamide
Etoposide
Paclitaxel
Staurosporine
Vinblastine
Compound X
T0 T48h
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Blue: Hoecsht, Yellow: Propidium iodide
%Surfacescratched/T0
[Colchicine] (µM)
CTRL T0 CTRL T48h
%Surfacescratched/T0
[vinblastine] (µM)
Blue: Hoecsht, Yellow: Propidium iodide
Heatmap of T48h plate
CTRL cells Vinblastine 1 µM
EC50=106 nM
EC50=149 nM
2D
3D
100 µM 30 µM 10 µM 3 µM 1 µM 300 nM 100 nM 30 nM 10 nM 3 nM CTRL
2D
3D
Vinblastine
2D
2D
3D
CTRL cells Etoposide 1 µM
100 µM 30 µM 10 µM 3 µM 1 µM 300 nM 100 nM 30 nM 10 nM 3 nM CTRL
Etoposide
30nM> EC50 >100nM
EC50> 100 nM
High Content Screening of automated wound healing and
cytotoxicity assays in 2D and 3D culture
MAUBON N.1*, ROUDAUT M.1, NDOYE A.2 & BURSZTYKA J.1
1 HCS Pharma, 6 rue Pierre Joseph Colin, 35000 Rennes
2 Perkin Elmer , 16 av Québec, Bât Lys, 91140 Villebon sur Yvette
1*nathalie.maubon@hcs-pharma.com
Vinblastine
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etoposide
Time and dose effect on
3D culture