Legends to figures
Figure 1. SEM images at lower (a, c, e) and higher (b, d, f) magnification of the A-C hydrogels at different weight ratios: a-b) 0.5%-0.02%; c-d) 0.25%-0.02%; e-f) 0.125%-0.025%. Scale bar is 100 µm.
Figure 2. a) FTIR spectra of a hydrogel of only agarose (green curve), collagen (pink curve) and a 0.25%-0.02% A-C hydrogel (red curve). b) Swelling behaviour of the A-C hydrogels kept in PBS at 37 °C. c-d) Degradation curves of the A-C hydrogels in PBS and in DMEM medium with 10% FBS, respectively, up to 2 weeks. e-f) Diffusion tests performed with Hyaluronic acid-FITC and Transferrin-TRITC, respectively, up to one week.
Figure 3. Representative stress-strain curves of A-C hydrogels subjected to unconfined compression with a displacement rate of 0.01 mm/s, until 80 % strain (A); Compressive moduli of A-C hydrogels at 0, 1, 4, 8 days of incubation in PBS at 37 °C, in humified atmosphere with 5% CO2 (B). Values represent mean ± SD, where n = 3.
Figure 4. The upper panels show the morphology and the size of the spheroids obtained with MCF-7 and MDA-MB-361 cells grown in 0.25%-0.02% and 0.125%-0.02% A-C% hydrogels, respectively, from 2 to 14 days. The graphs report the growth trend of both cell lines.
Figure 5. Images of the tumor spheroids obtained with MCF-7, MDA-MB-361 and SH-SY5 after 8 days growth in 0.125%-0.02% A-C% (upper panels) and 0.25%-0.02% hydrogels (lower panels). The arrows point to the disseminating cells of the spheroids grown in the soft matrix. Scale bar is 100 µm.
Figure 6. Mitochondrial labelling of living spheroids after 5 days growth with Mitotracker red. Spheroids of MCF-7 and MBA-MB-361 were stained and compared. Scale bar is 50 µm.
Figure 7. Staining of E-cadherin in cellular spheroids of MCF-7 and MBA-MB-361 after 5 days growth in A-C hydrogels. Scale bars correspond to 57.6 µm.
Figure 8. Live dead assay performed with the MCF-7 spheroids embedded into the A-C hydrogels after 24 h incubation with cisplatin. The upper panels refer to the 0.25%-0.02% A-C hydrogels, while the lower panels to those with 0.125%-0.02% A-C ratio. The graph reports the number of live and dead cells in each sample estimated through the distribution of the fluorescence signals of Calcein AM and Ethidium Homodimer, respectively.
Figure 9. Images of MCF-7 spheroids grown within 0.25-0.02% (upper panels) and 0.125-0.02% (lower panels) A-C matrices, respectively, recovered after Agarase treatment, fixed and stained with DAPI.