Figure 1.
Growth curve of A549 cell derived tumors.
Human pulmonary adenocarcinoma A549 cells were implanted in severe combined immunodeficiency (SCID) mice to form solid tumors. Note tumor volumes increased over time, with about 600 mm3 at 30-day implantation and about 4000 mm3 at 60-day implantation.
Figure 2.
Cellular removal evaluation for PAA, Trypsin-Triton, Tris-Trypsin-Triton and SDS treated samples compared with the native samples.
(A) Gross appearance showing solid and opaque for native and PAA treated tumor samples but translucent for Trypsin-Triton, Tris-Trypsin-Triton and SDS treated samples. (B) Hematoxylin–eosin (H&E) and (C) Hoechst staining showing complete cellular removal for Tris-Trypsin-Triton treated or SDS treated samples compared with samples of the other three groups. (D) Immunohistochemical staining for CK7 and (E) α-SMA showing complete removal of epithelial and mesenchymal cell components for Tris-Trypsin-Triton treated or SDS treated samples compared with samples of the other three groups. (F) DNA content analysis of native, PAA, Trypsin-Triton, Tris-Trypsin-Triton and SDS treated tumor samples. All presented results were mean ± SD (n = 10, *p<0.05 versus the native group; †p<0.05 versus the PAA group; #p<0.05 versus the Trypsin-Triton group).
Figure 3.
Extracellular matrix (ECM) structure and component investigation of native, Tris-Trypsin-Triton treated and SDS treated tumor sheets.
(A) Scanning electron microscopy (SEM) examination for Tris-Trypsin-Triton treated and SDS treated samples showing lack of cellular components and having more open spaces than the native samples. (B) Collagen fibril stained by Masson's trichrome showing similar distribution of collagen fibrils for Tris-Trypsin-Triton treated samples but more sparse distribution of collagen fibrils for SDS treated samples compared with the native samples. (C) Glycosaminoglycan (GAG) staining with Scott's alcian blue presenting mostly retaining of GAGs for Tris-Trypsin-Triton treated sheets but less GAGs for SDS treated sheets compared with their native counterparts. (D) Collagen and (E) GAG content analysis of native, Tris-Trypsin-Triton treated and SDS treated tumor sheets. All presented results were mean ± SD (n = 10, *p<0.05 versus the native group; †p<0.05 versus the Tris-Trypsin-Triton group).
Figure 4.
Porosities and biomechanical tests of native, Tris-Trypsin-Triton treated and SDS treated tumor sheets.
(A) Increase of porosity for Tris-Trypsin-Triton treated or SDS treated samples compared with the native samples. (B) Separation of stress-strain curves for the three groups. (C) Less elastic moduli shown for Tris-Trypsin-Triton treated or SDS treated tumor sheets compared with the native samples. All presented results were mean ± SD (n = 6 for porosity test and n = 10 for biomechanical test, *p<0.05 versus the native group; †p<0.05 versus the Tris-Trypsin-Triton group).
Figure 5.
Cell viability and growth factor secretion assays for breast cancer MCF-7 cells.
(A) Cell viability detected by MTT assay for MCF-7 cells cultured in 2D, Matrigel, Tris-Trypsin-Triton and SDS group over time. (B) Growth factor (IL-8, bFGF, and VEGF) secretion determined by ELISA assay for MCF-7 cells cultured in 2D, Matrigel, Tris-Trypsin-Triton and SDS group at 10-day culture. Graph represents mean ± SD of three independent experiments (n = 5 for MTT assay and n = 7 for growth factor secretion assay,*p<0.05 versus the 2D culture; †p<0.05 versus the Matrigel culture; #p<0.05 versus the Tris-Trypsin-Triton group).
Figure 6.
Representative light microscopy of MCF-7 cells cultured in 2D and different 3D scaffolds over time.
(A) Representative images of MCF-7 cells grown in 2D culture, showing more and denser cells over time. (B) MCF-7 cells cultured in the Matrigel group, showing formation and enlargement of multicellular tumor spheroids (MCTS) over time and the growth pattern looked like the classic “growth on top” method. (C) MCF-7 cells cultured in the Tris-Trypsin-Triton group, showing gradual decrease of the light transmittance of scaffolds. (D) MCF-7 cells cultured in the SDS group, also showing gradual decrease of the light transmittance of scaffolds, but presenting more transmittance of light at each time point compared with the Tris-Trypsin-Triton treated scaffolds.
Figure 7.
Representative fluorescence microscopic images of MCF-7 cells double-stained by CFDA-SE and PI for 2D and different 3D scaffolds over time.
(A) Representative photomicrographs of MCF-7 cells grown in 2D culture, showing great increasing of dead cells from 4- to 13-day culture. Note CFDA-SE stained viable cells green and PI stained dead cells red. (B) MCF-7 cells cultured in the Matrigel group, showing distribution of dead cells in the core of MCTS. (C) MCF-7 cells cultured in the Tris-Trypsin-Triton group, showing higher proportion of live cells at 7-, 10- and 13-day culture and formation of cell clusters at 13-day culture (arrowhead). (D) MCF-7 cells cultured in the SDS group, showing more dead cells but less live cells compared with the Tris-Trypsin-Triton treated scaffolds at 7-, 10- and 13-day culture.
Figure 8.
H&E-stained histological cross-sections of MCF-7 cells repopulated in the two acellular scaffolds over time.
(A) MCF-7 cells cultured in the Tris-Trypsin-Triton group, showing increase of cell numbers and infiltration depths over time. (B) MCF-7 cells cultured in the SDS group, showing less cell numbers and infiltration depths compared with the Tris-Trypsin-Triton treated scaffolds at 7-, 10- and 13-day culture.