Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease

Abstract

The development of novel anti-cancer strategies requires more sensitive and less invasive methods to detect and monitor in vivo minimal residual disease in cancer models. Bone marrow metastases are indirectly detected by radiography as osteolytic and/or osteosclerotic lesions. Marrow micrometastases elude radiographic detection and, therefore, more sensitive methods are needed for their direct identification. Injection of cancer cells into the left cardiac ventricle of mice closely mimics micrometastatic spread. When luciferase-transfected cells are used, whole-body bioluminescent reporter imaging can detect microscopic bone marrow metastases of approximately 0.5 mm(3) volume, a size below the limit in which tumors need to induce angiogenesis for further growth. This sensitivity translates into early detection of intramedullary tumor growth, preceding the appearance of a radiologically evident osteolysis by approximately 2 weeks. Bioluminescent reporter imaging also enables continuous monitoring in the same animal of growth kinetics for each metastatic site and guides end-point analyses specifically to the bones affected by metastatic growth. This model will accelerate the understanding of the molecular events in metastasis and the evaluation of novel therapies aiming at repressing initial stages of metastatic growth.

Figure 1.

Fate of MDA-231-B/Luc+ cells injected into the left cardiac ventricle. Bioluminescent photon emission was externally imaged from the ventral projection of the same mouse 10 minutes (A), 24 hours (B), and 28 days (C) after injection. Signals are displayed as pseudo-color image (blue least intense, white most intense) at a 0- to 4-bit sensitivity range merged with the gray-scale body image of the mouse.

Figure 2.

BRI monitoring in vivo of the kinetics of bone metastatic growth. Bioluminescent photon emission was externally imaged 24 days after injection of MDA-231-B/Luc+ cells into the left cardiac ventricle. A–D: Ventral projections of four different mice. Signals are displayed as pseudo-color image at the most sensitive 0- to 3-bit range.

Figure 3.

BRI monitoring in vivo of the kinetics of bone metastatic growth. Bioluminescent photon emission was externally imaged 33 days after injection of MDA-231-B/Luc+ cells into the left cardiac ventricle. Ventral (A, C, E) and dorsal (B, D, F) projections of the three mice shown in Figure 2 ▶ ; B, C, and D, respectively. Signals are displayed as pseudo-color image at a 0- to 5-bit range, less sensitive than that of Figure 2 ▶ , to optimize the anatomical localization.

Figure 4.

BRI and radiographic monitoring in vivo of the kinetics of bone metastatic growth. Bioluminescent photon emission was externally imaged 38 days after injection of MDA-231-B/Luc+ cells into the left cardiac ventricle. Ventral (A), dorsal (B), and lateral (C) projections of the mouse shown in Figure 3, E and F ▶ . Signals are displayed as pseudo-color image at a 0- to 6-bit range, less sensitive than that of Figure 4 ▶ , to optimize anatomical localization. D: Whole body radiograph. R, right side; and L, left side of the mouse. The two regions delimited by the rectangular frames on the proximal humerus (e) and on the distal femur (f) are magnified in E and F, respectively, showing details of the bone density. Arrowheads point at osteosclerotic rims delineating areas of discrete osteolysis. G: Growth curve obtained by quantification in the same mouse of the bioluminescent signal localized over the left femur (▪) at day 24, as shown in Figure 2D ▶ , and over the central portion of the spine (•) and the distal regions of the right (♦) and left (★) tibia at day 33, as shown in Figure 3, E and F ▶ , and at day 38 (A and B).

Figure 5.

Histological and immunohistological identification of MDA-231-B/Luc+ cells in two representative sites of metastasis. H&E staining of sections from the left humerus (A) and the left calcaneus (B). Cytokeratin immunohistochemistry of adjacent sections from C, the same humerus, and D, the calcaneus. Cells immunoreactive with the anti-human pan-cytokeratin antibody are stained in brownish-red. Scale bars, 100 μm.

Figure 6.

BRI and radiographic monitoring in vivo of the growth kinetics of 2 × 10 MDA-231-B/Luc+ cells implanted in the marrow cavity of the right tibia. A: Bioluminescent photon emission was recorded immediately (day 0) and at various time points in the 5 weeks after implantation. Signals are displayed as pseudo-color image at the most sensitive 0- to 3-bit range. Arrows at days 4, 7, and 11 highlights the initial photon accumulation over the area of implantation. B: Radiographs of the corresponding tibia starting from day 14. Small arrows at days 14 and 18 point at the cortical hole that results from surgical drilling. Arrowheads at day 18 highlight the initial area of osteolysis. C: Growth curve obtained by quantification of the bioluminescent signal over the area of implantation during the 5-week recording period.