Table 1.
Experimental mice for the development of an orthotopic model of bone malignancies.
Figure 1.
Prominent reactive bone formation following transplantation of Ewing sarcoma cell lines VH-64 (A–E) and TC-71 (F–I).
A, Representative CT image of a VH-64 transplanted femur 42 days after transplantation showing a “sunburst” pattern of reactive bone formation. B, Representative Maximum Intensity Projection CT image of a mouse with a VH-64 tumour 52 days after transplantation showing a “sunburst” pattern of reactive bone formation on the transplanted femur (between arrows, and extending proximally along the femur). C, Extensive reactive bone (arrows) arcing through a VH-64 tumour accompanied by foci of cartilage (arrowheads). Tumour is partly necrotic (N) (H&E, original magnification 20×). D, Reactive bone emanating from femoral cortex (top) within a VH-64 tumour (H&E, original magnification 100×). E, Reactive new bone formation partially lined by osteoblasts (arrows) amongst VH-64 tumour cells. A single multinucleate osteoclast (arrowhead) is also present (H&E, original magnification 400×). F, Reactive bone and a focus of cartilage (arrow) amongst VH-64 tumour cells (H&E, original magnification 200×) G, Representative CT image of a TC-71 transplanted femur 15 days after transplantation showing cortical destruction (arrow) and possible reactive new bone formation (arrowhead). H, Marked reactive new bone formation within a TC-71 tumour, beneath the raised periosteum (arrow). Cortical destruction is seen distally (arrowhead) (H&E, original magnification 40×). I, Reactive new bone in a TC-71 transplanted mouse, lined by osteoblasts (arrow) clearly distinct from infiltrating tumour cells (arrowhead) (H&E, original magnification 400×).
Figure 2.
“Malignant” bone formation within the tumour mass after SaOS-2 transplantation.
A, Representative CT image 70 days after intrafemoral transplantation of SaOS-2 cells showing “malignant” bone formation within the extraosseous tumour mass (arrow). B, Representative Maximum Intensity Projection CT image of a mouse bearing a SaOS tumour 70 days after transplantation, showing “malignant” bone formation within the extraosseous tumour mass (arrow). C, “Malignant” bone forming amongst tumour cells in the medullary cavity, showing appositional deposition (scaffolding) on existing bone trabeculae (“T”) (H&E, original magnification 200×). D, Left panel: malignant tumour cells amongst pale eosinophilic osteoid which is partially mineralised (arrow) (H&E, original magnification 400×). Right panel: fine, randomly arborising strands of mineralised osteoid (“malignant” bone) amongst SaOS-2 tumour cells (H&E, original magnification 400×).
Figure 3.
Osteolytic lesions caused by injection of PC3M.
A, Representative CT image 29 days after transplantation of PC3M cells, showing lytic destruction of the femur (arrow) (sagittal section). B, Lytic destruction of the femoral cortex associated with numerous osteoclasts (short arrow) and focal reactive bone formation (long arrow) (H&E, original magnification 200×). C, Lysis and virtual destruction of the bone by numerous osteoclasts on the periosteal surface (long arrows), with osteoblastic reaction on the medullary surface (short arrow) (H&E, original magnification 200×). D, Lysis of bone by osteoclasts in resorption pits on the medullary surface (arrows), clearly distinct from intrafemoral tumour (H&E, original magnification 400×).
Figure 4.
PET-CT imaging of primary tumour.
A, PET/CT image of a mouse with a VH-64 tumour following intrafemoral transplantation. Image taken 52 days after transplantation, tumour marked with a white arrow (Maximum Intensity Projection CT image overlaid with FDG-PET image, dorsal view, ketamine/medetomodine anaeshesia). B, PET/CT image of a mouse injected with medium alone, also 52 days after i.f. injection, no tumour visible (Maximum Intensity Projection CT image overlaid with FDG-PET image, dorsal view, ketamine/medetomodine anaeshesia).
Figure 5.
Comparison of different anaesthetic techniques.
Representative FDG-PET images of mice anaesthetized with isofluorane (right mouse) versus ketamine/medetomidine (left mouse) showing a marked reduction in cardiac FDG uptake in the latter.
Figure 6.
MR imaging of primary tumour with measurement of tumour volume.
Measurement of a VH-64 Ewing sarcoma of the injected femur with a large extra-osseous tumour component. The regions of interest (within red markings) were measured in sequential 2 mm thick slices that showed tumour, and by adding the volumes of individual slices the total tumour volume was calculated. In the depicted case the estimated total volume was 492 mm3 (tumour in 6 slices; region of interest per slice: 22 mm2, 42 mm2, 57 mm2, 59 mm2, 52 mm2, 14 mm2; total area = 246 mm2; slice thickness = 2 mm; estimated total volume = 492 mm3).
Figure 7.
MR images of liver deposits and lungs using respiratory gating.
MRI imaging performed 47 days after injection of a mouse with VH-64 cells, providing an example of respiratory gating to obtain good quality images. 2 out of 25 adjacent 2 mm slices (selected slices 12 mm apart) are presented showing upper abdomen (left panel), revealing a large liver tumour, and lungs (right panel). Despite slight movement artifacts on the thoracic images there is good visualisation of the lung fields. L: liver; K: kidney; S: stomach.
Figure 8.
Bioluminescent imaging of Rag2−/− γc−/− mice intrafemorally transplanted with transduced TC-71 cells.
Weekly bioluminescent imaging of two Rag2−/− γc−/− mice transplanted with 5×105 transduced TC-71 cells in a volume of 30 µl (25% EGFP positive cells, top row and sorted 100% EGFP positive cells, bottom row).
Figure 9.
Bioluminescent imaging of of Rag2−/− γc−/− mice intrafemorally transplanted with low numbers of transduced TC-71 cells.
Weekly bioluminescent imaging of 3 Rag2−/− γc−/− mice transplanted with 1×103 (top row), 5×103 (middle row) or 1×104 (bottom row) transduced TC-71 cells (EGFP sorted 1 week prior to injection). Injected cells were suspended in a volume of 10 µl. The images obtained on day 7 did show a low signal over the right femur for all of the mice, but due to the chosen radiance settings to enable comparison with subsequent iamges, they do not appear on this panel. On some images of mice who were not imaged individually towards the end of the experiment (i.e. middle row day 27 and 41), the reflection of a signal emitted from a neighbouring mouse is unfortunately projected onto the left femur (day 27) or left side (day 41) of that mouse. All mice developed distant disease, either in lungs (top row), liver/abdomen (middle and bottom row) or contralateral leg (middle row).
Table 2.
Summary of TC-71 bioluminescent signal detection in live mice and post-mortem (after dissection).
Figure 10.
Timecourse of bioluminescent signal in mice injected with low numbers of transduced TC-71 cells.
Depicted is the development of signal intensity for 5 animals injected with either 1×103, 5×103 or 1×104 cells in a volume of 10 µl. The experiment was performed with EGFP sorted cells one week prior to injection.
Figure 11.
Examples of bioluminescent imaging of tumour spread to bone, post mortem imaging of organs, and.the histology of tumour spread.
A, Example of a mouse transplanted with 1×105 25% EGFP positive TC-71 cells with metastatic spread to the jaw, and post mortem IVIS imaging of individual organs (dissected head, lungs and liver) demonstrating the signal from the jaw and additional weak signals from both lungs, all of which were confirmed by histology. B, TC-71 tumour deposit contained completely within liver parenchyma as an example of visceral tumour spread (H&E, original magnification 40×). C, TC-71 tumour deposit (T) within the mandible as an example of spread to distant osseus sites. Tng: tongue; arrowheads: teeth (H&E, original magnification 40×). D, Cluster of TC-71 tumour cells within a small arterial vessel adjacent to a bronchiole as an example of haematogenous spread (H&E, original magnification 400×). E, Cluster of TC-71 tumour cells within a lymphatic vessel adjacent to an arterial vessel and bronchiole as an example of intralymphatic spread (H&E, original magnification 400×).