ESTRO 2024 - Abstract Book

S5405

Radiobiology - Tumour biology

ESTRO 2024

To construct the tumor environment, we employed PDAC spheroids immersed in a 3D environment containing pancreatic-specific stromal cells, the Pancreatic Stellate Cells (PSCs).

We first optimized the technique to obtain the spheroids by using a mixture of a tumor cell line (MIA PaCa-2) and dermal Human fibroblast (FBs). We identified the best technique to obtain these spheroids among different methods ranging from liquid overlay to hanging drop, taking into account the addition of methyl-cellulose to the media. For the hanging drop method, we suspended 20 µl of cell suspension to obtain spheroids ranging from 0.5 to 10 k cells, considering different MIAPaCa-2:FB ratios (1:0, 1:1, 2:1, 1:2). We tested this technique with media (DMEM, 10% FBS, 1% L-Glu, 1% PS) both with and without 0.24% methylcellulose. Alternatively, we used the Liquid Overlay Technique. A 96-well plate with agarose coating was prepared by adding 100 µl of 1.5% agarose solution in deionized water to each well.

Once the technique was optimized, tumor spheroids were also obtained by mixing MIAPaCa-2 with PSCs.

The vascular compartment replicates pancreatic-specific microvasculature using Pancreatic Endothelial Cells (PECs) and PSCs. We tested different conditions (final concentrations: PECs 3-6 M/ml, PECs:PSCs ratio 0.5-2). The microvascular network developed within a week, leading to connected vessels.

Results:

The designed geometry allows independent seeding of the two compartments (Figure 1).

Regarding the method selection for spheroid generation, no spheroids were obtained by hanging drop after 72 hours, regardless of the methyl-cellulose concentration considered (Figure 2). In contrast to hanging drop, LOT spheroids were formed after 72 hours. Monoculture (MIA PaCa-2: FB ratio of 1:0) led to the formation of either singular cell suspensions or loose aggregates. The mean value of Aspect Ratio (AR) for hanging drop was 1.215 ± 0.222, while aggregates' AR, with the addition of methylcellulose, was 1.729 ± 0.591 (p < 0.0001). Contrarily, the inclusion of methylcellulose in the media did not significantly alter both the LOT spheroids' circularity and aspect ratio (Figure 2). LOT spheroids exhibited AR values closer to 1 (LOT = 1.257 ± 0.063, HD = 1.543 ± 0.123, p < 0.001) and greater circularity values (LOTC = 0.241 ± 0.048, HDC = 0.07 ± 0.02, p < 0.001). As a result, the most appropriate method for spheroid generation was found to be the standard LOT, with pancreatic cancer cells and stroma cells in a ratio of 2:1.