Figure 1.
Schematic illustration of 3D Col-Tgel tumor model.
A, Enzymatic crosslinking of collagen base gel by transglutaminase. B, Tumor cells suspended in soluble gel at desired densities. C, Gel was pipetted into a single well as a droplet, or multi droplets in single dish after addition of crosslinker, Tgase. D&E, Comparison of transparency of three different 3D matrices, Type I collagen, Matrigel, and Col-Tgel. F, The stiffness of Col-Tgel was manipulated by changing the gel concentrations.
Figure 2.
A, Dome model, 20 µl of gel was pipetted on the bottom of each well of 48-well plate. D, Plug model, on top of the dome, 100 ul of gel was added to fill to the height of the dome. MTT diffusion and reduction by MDA-MD-231 cells was monitored by inverted light microscope after 3 hours addition. Micrographs were obtained under light microscope on the edge (B) and center (C) of dome model or surface (E) and bottom (F) of plug model. Scale bar = 500 µm for fig. 2B and 2E, scale bar = 100 µm for fig. 2C and 2F.
Figure 3.
Formation of tumor spheroids in 3D gel.
A, Breast cancer cells (MDA-MB-231) were culture in 3D dome gel for 6 days and formed spheroids inside the gel. Cells started to migrate out of gel either as individual cell or as collective cell clusters. Migrated cells showed elongated branched morphology on 2D surface. B–E, Time course of spheroid formation (scale bar = 140 µm). MDA-MB-231 M cells were cultured in 3D Col-Tgel for 0, 2, 4, and 6 days and recorded observation under light microscope (B–E, scale bar = 140 µm). F, Prostate cancer C4–2B cells, Colon cancer HCT116 cells and Breast cancer MDA-MB-231 cells were stained with F-actin (red) and DAPI (blue) and observed under fluorescence microscope (scale bar = 100 µm). G, MDA-MB-231 cells cultured in 2D or 3D were IHC stained with E-cadherin, laminin 5 and β1 integrin (scale bar = 100 µm).
Figure 4.
3D tumor model reproduce tumor cell heterogeneous conditions.
A, Tumor cells formed quiescent or necrotic center; small to big spheroids from the innermost to the periphery region of the gel when MDA-MB-231 cells were cultured for 6 days (scale bar = 1000 µm). B, MDA-MB-231 cells stained with proliferation marker anti-Ki67 antibody at day 6 and showed strong to faint staining from edge to center (Scale bar = 300 µm). C, Oxygen concentrations were measured in the medium and in the gel. OM-1 oxygen meter probe was used to directly measure the O2 percentage in medium and in gel. *P<0.001, data mean ± standard deviation, n = 4.
Figure 5.
Tumor cells and mesenchymal cells showed different cell-cell and cell-matrix interaction in 3D Col-Tgel system.
A, Individual culture of human squamous cell carcinoma line, SCC-71 and bone marrow derived mesenchymal stem cells in 3D gel for 6 days. B, Chimera tumor spheroid were formed when co-culture of SCC-71 and bone marrow derived mesenchymal stem cells in 3D Col-Tgel system for 6 days. Top row: light microscope; bottom row: H&E staining from paraffin section. Fluorescence: MSC (green) and SCC-71 (red). Scale bar = 100 µm.
Figure 6.
Drug sensitivity test on MDA-MB-231 cells in 2D and 3D.
A, MDA-MB-231 cells were treated with paclitaxel at concentrations of 0, 0.2, 2, and 20 µM (from left to right, scale bar = 2000 µm) and analyzed with Live/Dead cytotoxicity/viability kit. Dead cells were in red and live cells in green. B, MDA-MB-231 cells displayed a dose response to paclitaxel in 2D and 3D. IC50 was 1.897µM for 2D monolayer culture and 7.318 µM for 3D culture. ** P<0.05, data mean ± standard deviation, n = 3.
Figure 7.
Xenograft tumor induction using Col-Tgel as a carrier.
A, Tumor cell and Col-Tgel mixture was injected at the desired site before gel curing. B, Initial tumor formation at the subcutaneous injection site 7-days post injection with gel border clearly defined (scale bar = 2 mm). C–D, Osteosarcoma SaoS-2 cells, 200 µl of Col-Tgel with 1.0×106 cells (dark arrow) or 100 ul of Col-Tgel with 0.5×106 cells (red arrow) were injected subcutaneously in animals (scale bar = 10 mm). Gross tumor formation by observed at day 3 (C) and day 20 (D). E, Retrieved tumors at day 20 exhibited positive correlation between tumor size and initial injection volume (scale bar = 10 mm). F, Histological micrograph of Osteosarcoma tumor with vascular invasion (scale bar = 180 µm). G&H, MDA-MB-231 cells formed clusters (green arrows) and blood vessel infiltration (hollow arrows) inside the Col-Tgel after 14 days of injection (G, scale bar = 550 µm). MDA-MB-231 cells (green arrow), host cells (blue arrows), and new blood vessels (hollow arrow) in the microenvironment created by co-delivery of tumor cells with Col-Tgel after 14 days of injection (H, scale bar = 180 µm). I, Tumor formation curves of different cancer cells using Col-Tgel as carrier (MDA-MD-231, HCT116 and CFPAC-1, scale bar = 10 mm). The plot is a representative value of six tumors.