Fig 1.
Different steps for gels preparation.
GBM: Glioblastoma multiforme; Alg 1%: Alginate hydrogel 1%; Alg/Chit-NPs CXCL12: Alginate/Chitosan-Nanoparticles loaded with CXCL12; Alg:M: Alginate:Matrigel (50:50); CXCL12: C-X-C motif chemokine 12.
Fig 2.
(a) Schematic representation of the perfusion bioreactor system for GBM cells migration assay. (b) The perfusion bioreactor assembly. GBM: Glioblastoma multiforme; Alg 1%: Alginate hydrogel 1%; Alg/Chit NPs-CXCL12: Alginate/Chitosan-Nanoparticles loaded with CXCL12; Alg:M: Alginate:Matrigel (50:50); PDMS: Polydimethylsiloxane; 3D: three-dimensional.
Table 1.
Forward and reverse primer sequences used in the qPCR.
Fig 3.
(a) Hydrogels scans of mCherry-F98 GBM cells migration at t = 0 h, t = 24 h and t = 72 h with and without CXCL12 (0.33 μg) under a flow rate of 0.5 μL/min. (b) Hoechst staining of the hydrogels at t = 72h. (c) DNA quantification of the different hydrogel sections at the end of the experiment (N = 15, n = 3). Bars represent the mean ± SD and the squares are zoomed regions. Statistical analysis was performed using a Mann-Whitney t-test, * p ≤ 0.05, ** p ≤ 0.01. The scale bar represents 2000 μm. NPs-Empty: Alginate/Chitosan-Nanoparticles unloaded; NPs-CXCL12: Alginate/Chitosan-Nanoparticles loaded with CXCL12; Alg:M: Alginate:Matrigel (50:50); Alg 1%: Alginate 1% hydrogel.
Fig 4.
(a) Hydrogels scans of mCherry F98 GBM cells migration at t = 0 h, t = 24 h and t = 72 h with and without CXCL12 at a flow rate of 3 μL/min. (b) Hoechst staining of the hydrogels at t = 72 h. (c) DNA quantification of the different hydrogel sections at the end of the experiment (N = 9, n = 3). Bars represent the mean ± SD and the squares are zoom regions. Statistical analysis was performed using a Mann-Whitney t-test, * p ≤ 0.05, ** p ≤ 0.01. The scale bar represents 2000 μm. NPs-Empty: Alginate/Chitosan-Nanoparticles unloaded; NPs-CXCL12: Alginate/Chitosan-Nanoparticles loaded with CXCL12; Alg:M: Alginate:Matrigel (50:50); Alg 1%: Alginate 1% hydrogel.
Fig 5.
(a) Hydrogels scans of U87-GFP GBM cells migration at t = 0 h, t = 24 h and t = 120 h with and without CXCL12 at a flow rate of 0.5 μL/min. (b) Hoechst staining of the hydrogels at t = 120 h. (c) DNA quantification of the different hydrogel sections at the end of the experiment (N = 9, n = 3). Bars represent the mean ± SD and the squares are zoom regions. Statistical analysis was conducted using a Kruskal-Wallis test followed by Dunn’s multiple comparison post-hoc test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. 1: (Alg:M + cells NPs-Empty vs. Alg:M + cells NPs-CXCL12) *, (Alg:M + cells NPs-Empty vs. Alg:M + cells NPs-CXCL12+) *, (Alg:M + cells NPs-CXCL12 vs. Alg:M + cells NPs-CXCL12+) ns. 2: (Alg:M NPs-Empty vs. Alg:M NPs-CXCL12) ns, (Alg:M NPs-Empty vs. Alg:M NPs-CXCL12+) **, (Alg:M NPs-CXCL12 vs. Alg:M NPs-CXCL12+) ns. 3: (Alg:M NPs-Empty vs. Alg:M NPs-CXCL12) ns, (Alg:M NPs-Empty vs. Alg:M NPs-CXCL12+) ***, (Alg:M NPs-CXCL12 vs. Alg:M NPs-CXCL12+) ns. 4: (Alg:M + cells NPs-Empty vs. Alg:M + cells NPs-CXCL12) ns, (Alg:M + cells NPs-Empty vs. Alg:M + cells NPs-CXCL12+) ns, (Alg:M + cells NPs-CXCL12 vs. Alg:M + cells NPs-CXCL12+) **. The scale bar represents 2000 μm. NPs-Empty: Alginate/Chitosan-Nanoparticles unloaded; NPs-CXCL12: Alginate/Chitosan Nanoparticles loaded with CXCL12; Alg:M: Alginate:Matrigel (50:50); Alg 1%: Alginate 1% hydrogel.
Fig 6.
(a) Hydrogels scans of GFP U87 GBM cells migration at t = 0 h, t = 24 h and t = 120 h with and without CXCL12 at a flow rate of 3 μL/min. (b) Hoechst staining of the hydrogels at t = 120 h. (c) DNA quantification of the different hydrogel sections at the end of the experiment (N = 9, n = 3). Bars represent the mean ± SD and the squares are zoom regions. Statistical analysis was conducted using a Kruskal-Wallis test followed by Dunn’s multiple comparison test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. 1: (Alg:M + cells NPs-Empty vs. Alg:M + cells NPs-CXCL12) ns, (Alg:M + cells NPs-Empty vs. Alg:M + cells NPs-CXCL12+) **, (Alg:M + cells NPs-CXCL12 vs. Alg:M + cells NPs-CXCL12+) ns. 2: (Alg:M NPs-Empty vs. Alg:M NPs-CXCL12) ns, (Alg:M NPs-Empty vs. Alg:M NPs-CXCL12+) **, (Alg:M NPs-CXCL12 vs. Alg:M NPs-CXCL12+) ns. 3: (Alg:M NPs-Empty vs. Alg:M NPs-CXCL12) ns, (Alg:M NPs-Empty vs. Alg:M NPs-CXCL12+) ***, (Alg:M NPs-CXCL12 vs. Alg:M NPs-CXCL12+) ns. 4: (Alg:M + cells NPs-Empty vs. Alg:M + cells NPs-CXCL12) ns, (Alg:M + cells NPs-Empty vs. Alg:M + cells NPs-CXCL12+) ns, (Alg:M + cells NPs-CXCL12 vs. Alg:M + cells NPs-CXCL12+) **. The scale bar represents 2000 μm. NPs-Empty: Alginate/Chitosan-Nanoparticles unloaded; NPs-CXCL12: Alginate/Chitosan-Nanoparticles loaded with CXCL12; Alg:M: Alginate:Matrigel (50:50); Alg 1%: Alginate 1% hydrogel.
Fig 7.
Relative CXCR4 mRNA levels in F98 and U87 cells determined by semiquantitative qPCR, *P ≥ 0.01, (N = 1, n = 3).
Fig 8.
Assessment of CXCR4 receptor activity in (A) F98 and (B) U87 cells by measuring the increase in intracellular calcium flow, (N = 8; n = 3).