Cryo-image analysis of tumor cell migration, invasion, and dispersal in a mouse xenograft model of human glioblastoma multiforme

Abstract

Purpose: The goals of this study were to create cryo-imaging methods to quantify characteristics (size, dispersal, and blood vessel density) of mouse orthotopic models of glioblastoma multiforme (GBM) and to enable studies of tumor biology, targeted imaging agents, and theranostic nanoparticles.

Procedures: Green fluorescent protein-labeled, human glioma LN-229 cells were implanted into mouse brain. At 20-38 days, cryo-imaging gave whole brain, 4-GB, 3D microscopic images of bright field anatomy, including vasculature, and fluorescent tumor. Image analysis/visualization methods were developed.

Results: Vessel visualization and segmentation methods successfully enabled analyses. The main tumor mass volume, the number of dispersed clusters, the number of cells/cluster, and the percent dispersed volume all increase with age of the tumor. Histograms of dispersal distance give a mean and median of 63 and 56 μm, respectively, averaged over all brains. Dispersal distance tends to increase with age of the tumors. Dispersal tends to occur along blood vessels. Blood vessel density did not appear to increase in and around the tumor with this cell line.

Conclusion: Cryo-imaging and software allow, for the first time, 3D, whole brain, microscopic characterization of a tumor from a particular cell line. LN-229 exhibits considerable dispersal along blood vessels, a characteristic of human tumors that limits treatment success.

Fig. 1

Blood vessel visualization using bright field images. a Functional diagram showing the steps to visualize blood vessels. b The particular 2D Gaussian (top) with σ=0.5 used to construct DoOG kernel (bottom) that enhances blood vessels in the x-direction.

Fig. 2

Tumor visualization using fluorescence images. Functional diagram showing the steps to visualize the main tumor mass and dispersed tumor cells.

Fig. 3

Steps for blood vessel visualization for tumor 1. a The raw color bright field block face image of a brain is shown. b Extracted green channel from the image shown in a used for further processing. c Application of the DoOG filters resulting in a high contrast, 2D image of blood vessel profiles. d 3D visualization of the image stack from this brain specimen, showing the detected vasculature of the brain (tumor 1, 20 days post-implantation).

Fig. 4

Steps for visualization of tumor and dispersing cells for tumor 2. a The raw color bright field block face image of a brain is shown. Arrow indicates approximate position of the LN-229-GFP tumor within this section. b Corresponding fluorescence image showing the LN-229-GFP tumor (green). c Higher magnification view of the main tumor shown in b with cells dispersing away from the tumor edge (arrows). d Deconvolved fluorescence image shown in c (tumor 2, 36 days post-implantation).

Fig. 5

Results of the dispersed cell detection algorithm for tumor 1. a 3D rendering of both the main tumor (green) and the dispersed cells (yellow). b, c Higher magnification views of dispersed cells shown in a (tumor 1, 20 days post-implantation).

Fig. 6

LN-229 tumor cell dispersal in 2D. b–d Histological section of a GFP-expressing tumor xenograft of LN-229 cells (green fluorescence) was immunolabeled with the endothelial cell specific antibody CD-31 and visualized with a secondary antibody conjugated to Texas Red. a Bright field image from the same section stained with hematoxylin and eosin is shown. b Dispersing cells were observed at varying distances from the main tumor mass, as indicated by expression of GFP. c, d Higher magnification views of the boxed regions in b indicate a close association of dispersing cells with blood vessels. Scale bar represents 100 μm (tumor 6, 25 days post-implantation).

Fig. 7

Projection of tumor growth along a blood vessel shown for tumor 5. a Dark blood vessels are shown in the color bright field image following perfusion with India ink. b The corresponding 2D fluorescence image clearly shows GFP-labeled tumor. c The vessel is segmented and a 2D fusion shows the projection of cells growing along the vessel. d A 3D visualization clearly shows a substantive (≈5% of total tumor volume) projection of the green tumor growing along the blood vessel (red) (tumor 5, 38 days post-implantation).

Fig. 8

Tumor cells dispersing along blood vessels shown for tumor 1. In a, cryo-images from a mouse brain containing a LN-229-GFP tumor were processed using the algorithms for visualization of main tumor mass (green), tumor cell dispersal (yellow), and blood vessel visualization (red). b Higher magnification image of tumor and surrounding vasculature viewed from a different angle. c Higher magnification image of small tumor cell clusters dispersing along blood vessels (arrows) (tumor 1, 20 days post-implantation).

Fig. 9

Dispersal distances from the main tumor mass. Histograms are shown with the number of cells normalized as a percentage. Specimens are analyzed at 20 (a) and 38 (b) days post-implantation. Clearly most dispersed cells are within 70 μm. Longer distances are recorded with increasing time post-implantation. Other tumors show a similar pattern of dispersal. Total number of dispersed clusters in these brains is 761 and 3,605 for tumors 1 and 5, respectively.