Fig 1.
Invasion/chemotaxis of breast cancer cells towards different bone cell types.
(A) A schematic representation of the invasion assay on IC-Chip is shown. On the right, a zoom-in inset of the imaging area between two posts is shown. Arrows indicate the direction of invasion. (HMC: Homing Matrix Channel, MC: Medium Channel) (B) Representative Z-stack projection images showing invasion of MDA-MB-231 breast cancer cells (red) towards hFOB (osteoblast), D1 (bone marrow-derived mesenchymal stem cells), Saos2 (osteosarcoma cell line with osteoblastic properties) and HS5 (bone marrow-derived stromal cell) embedded in GFR-Matrigel. The dashed line represents the starting line of invasion/chemotaxis. (Scale bar: 100 μm) The distance of each bright pixel to the starting line was calculated and the data was normalized to Day 1. (C) Distribution and (D) mean of distance values were plotted (n = 3). * p< 0.01, ** p<0.005.
Fig 2.
Invasion/chemotaxis of breast cancer cells towards the combination of different bone cell types.
(A) Representative Z-stack projection images showing invasion of MDA-MB-231 breast cancer cells (red) towards microenvironments formed by the combination of cell lines embedded in GFR-Matrigel. Dashed lines represent the starting line of invasion/chemotaxis. (Scale bar: 100 μm) The distance of each bright pixel to the starting line was calculated and the data was normalized to Day 1. (B) Distribution and (C) mean of distance values were plotted (n = 3). * p<0.005.
Fig 3.
Rheological measurements of collagen I-based matrices.
Storage modulus (G’) of 3 mg/mL collagen I only, 3 mg/mL collagen I supplemented with 2.5 mg/mL agarose, and 3 mg/mL collagen I supplemented with 10 mg/mL chitosan both with (red) and without (black) bone cells cultured for 5 days, are shown.
Fig 4.
Viability of bone cell types in collagen I-based 3D matrices.
(A) 3D merged images of hFOB, HS5, and U937 cell lines 3 days after they were cultured in hydrogels composed of collagen I only, collagen I with agarose and collagen I with chitosan. The green color represents dead cells, blue color shows all the nuclei. (Scale bar: 200 μm) (B) Percentage of viability for each condition is shown (n = 3). * p<0.01.
Fig 5.
Invasion/chemotaxis of bone- and lung-specific metastatic clones of MDA-MB-231 breast cancer cells towards different bone-like microenvironments.
(A) Representative Z-stack projection images showing invasion of bone-specific (BoM 1833) and lung-specific (LM2) clones of MDA-MB-231 breast cancer cells (red) towards HS5, hFOB, and U937-laden collagen I only, collagen I and agarose, or collagen I and chitosan hydrogels. Dashed lines represent the starting line of invasion/chemotaxis. (Scale bar: 100 μm) The distance of each bright pixel to the starting line was calculated and the data was normalized to day 1. (B) Distribution and (C) mean of distance values were plotted (n = 6). * p<0.01, ** p<0.005.
Fig 6.
Extravasation of bone- and lung-specific metastatic clones of MDA-MB-231 breast cancer cells towards different bone-like microenvironments.
(A) A schematic representation of the extravasation assay on EX-Chip is shown. On the bottom, a zoom-in inset of the imaging area between two posts, side (i) and back (ii) point of view of the endothelial layer (green) and extravasating cancer cells (red) are shown. Arrows indicate the direction of extravasation. (EMC: Endothelial Monolayer Channel, HMC: Homing Matrix Channel, MC: Medium Channel) (B) Representative 3D images showing extravasation of bone-specific (BoM 1833) and lung-specific (LM2) clones of MDA-MB-231 breast cancer cells (red) through HUVEC-C endothelial layer (green) towards HS5, hFOB and U937-laden collagen I only (Col), collagen I and agarose (Col + Ag), or collagen I and chitosan (Col + Ch) hydrogels. Arrow heads show extravasated and arrows show associated cancer cells. (Scale bar: 100 μm) (C) Number of extravasated and associated bone-specific (B) and lung-specific (L) MDA-MB-231 cells are shown. Each black dot represents one experiment, and the red dots represent mean values. (n = 2) * p<0.01, ** p<0.005.