3D versus 2D 5-FU effect at 48h.

<p><b>(A)</b> 3D cell survival of HCT116, SW480 and HT29 MCTS 48 hours after 5-FU treatment ranging from 0 to 100 μM. Survival represents the ratio of MCTS volume at 48h for a given drug condition to the volume of control MCTS (n = 7–12). <b>(B)</b> 2D cell survival of HCT116, SW480 and HT29 cell lines 48 hours after 5-FU treatment ranging from 0 to 100 μM. Survival represents the ratio of the normalized cell area at 48h for a given drug condition to the control normalized cell area (no drug) (n = 3). Error bars: SD.</p

Typical phase contrast images of the MCTS growth for the three CRC lines taken at 0h, 24h and 48h in the absence of drug.

<p><b>(A-C)</b> HT29, <b>(D-F)</b> HCT116 and <b>(G-I)</b> SW480. Scale bars: 200 μm.</p

Volume of the cohesive core of MCTS under drug treatment after transfer to new wells.

<p>Left. Typical images of spheroid cores at 48h for the three CRC cell lines after administration of 0 <b>μM (A, E, I)</b>, 10 μM <b>(B, F, J)</b> or 100 μM <b>(C, G, K)</b> 5-FU. The dotted circle represents the size of the control MCTS which is reported as a guide for the eye on other panels. Right. Time series of the mean MCTS volume relative to initial volume at day 0 for each particular condition after 5-FU treatment ranging from 0 to 100 μM (only 3 drug concentrations are reported for clarity). <b>(A-D)</b> HT29, <b>(E-H)</b> HCT116 and <b>(I-L)</b> SW480. Error bars: SEM (n = 7–12 for each cell line). Scale bar: 200 μm.</p

IC₅₀ values for 5-FU for the three cell lines at 24h and 48h in 2D versus 3D spheroids.

<p>A range of IC₅₀ values (e.g., 1–2 μM) indicates that the precise IC₅₀ was in between two concentrations tested.</p

Number of dead cells within the cohesive core of MCTS under drug treatment after transfer to new wells.

<p><b>(A)</b> Typical fluorescence images at 48h of living cells in green (calcein +) and dead cells in red (PI+) in control SW480 MCTS or in presence of 5-FU (300 μM). <b>(B)</b> Quantification of PI+ dead cells per unit volume for each cell line as a function of the 5-FU concentration. Error bar = SEM (standard error of the mean), (n = 12 for each cell line). Scale bar: 200 μm.</p

Nature of cells in the outer layerof the SW480 spheroids at 48h.

<p><b>(A-D)</b> Phase contrast and <b>(E-H)</b> corresponding fluorescent images of living cells in green (calcein +) and dead cells in red (PI+) treated with 0, 2, 10 and 100 μM of 5-FU. Compared to control <b>(A,E)</b>, a gradual disaggregation of peripheral cells appears clearly since 2 μM of 5-FU <b>(B,F)</b> to 100 μM <b>(D,H)</b>. Cells in the outer layer are equally dead and living cells. Scale bar: 200 μm.</p

An outer loose layer of cells breaks away from the MCTS under 10 μM of 5-FU treatment.

<p>Typical images of MCTS of the three different CRC cell lines, HT29 (A-C), HCT116 (D-F) and SW480 (G-I) at three different times after drug administration ([5-FU] = 10μM): 24h (A, D, G), 32h (B, E, H) and 48h (C, F, I). In the absence of transfer, numerous dead cells appear on the diffuse outer cell layer for the HCT116 and SW480 MCTS. Scale bar: 200 μm.</p