Fig 1.
Spheroid integrity is lost after irradiation therapy.
CRC cell lines were grown in matrigel as 3D spheroids and irradiated with an Elekta Versa HD accelerator. (A) CaCo2 cells form sharp demarcated and perfect spherical structures. (B-D) Colo205, SW480, or HCT116 spheroids showed a granular, grape-like structure. Six days after irradiation all spheroids showed a loss of compact morphology, membrane fringing and apoptotic vesicles (scale bar 200μm).
Fig 2.
Histological analyses confirm loss of integrity and proliferation.
CRC cell lines were grown in matrigel as 3D spheroids and irradiated with an Elekta Versa HD accelerator. Six days after irradiation, spheroids were fixed with 4% PFA, embedded in paraffin, and stained with (A) H&E or (B) Ki-67 (scale bar 100μm).
Fig 3.
3D spheroids show decreased apoptosis rates after irradiation compared with 2D cultures.
(A) CRC cells were irradiated with 1, 4, and 10 Gy. Six days after irradiation, cells were stained with annexin V/ PI, and analysed by flow cytometry. Significance was calculated using a two-sided, unpaired Student’s T test. (B) CaCo2 and HCT116 cells were irradiated with 0 and 1 Gy. Six days after irradiation, cells were fixed with 4% PFA, embedded in paraffin and stained for TUNEL assay. Significance was calculated using a two-sided, unpaired Student’s T test. (C) Representative TUNEL stainings for CaCo2 and HCT116 2D and 3D cultures six days after irradiation with 0 and 1 Gy. Scale bar 50 μm.
Fig 4.
2D and 3D cultures respond differently to chemotherapeutic drugs.
(A) 2D and (B) 3D CRC cultures were treated with the indicated chemotherapeutics for 72h. Afterwards, cell viability was analysed by RT-Glo assay. The red line indicates IC50 thresholds.
Table 1.
IC50 values of different chemotherapeutic drugs in 2D and 3D CRC cultures.
Fig 5.
Upon 5-FU treatment the DNA repair pathway is activated stronger in 2D cultures.
2D and 3D cultures of HCT116 and SW480 cell lines were treated with 5-FU at the indicated concentrations for 24h. Afterwards, cells were harvested and analysed by immunoblotting. The size of the proteins is shown on the left. Figure shows representative blots from three independent experiments.