Cellular mechanisms of organ-specific metastasis of Ewing's sarcoma

Ewing's sarcoma is the second most common bone tumour in children and adolescents. The prognosis is mainly influenced by the occurrence of primary metastasis. Although great improvement in treatment has been achieved, still only 2/3 of patients with localized disease can be cured. Furthermore, the 3-year event free survival in patients with lung metastases is only ~50%, and is less than 20% in patients with bony metastases. Metastatic models of Ewing’s sarcoma developed in this study using cell lines in immunocompromised mice show a pattern of disease spread similar to that found in patients, providing a suitable system for studying the metastatic process likely occurring in the course of Ewing’s sarcoma. The comparison of microarray gene expression patterns revealed interesting candidate genes for diagnosis and identified putative metastasis-specific targets that might be exploited in the development of new treatment approaches. However, it will be necessary to additionally analyse these patterns in primary material. One gene that formerly has been shown to play a role in the metastasis to bones in a variety of cancer types is CXCR4, which encodes for the cytokine receptor of CXCL12 (SDF-1), and which plays a role in the metastasis to bones in a variety of other cancer types. As Ewing’s sarcoma cells express CXCR4, a shRNA vector was constructed, transduced and stably expressed to investigate the role of the CXCR4/CXCL12 axis in Ewing’s sarcoma cells via RNA interference. This stability provides the possibility of an in vitro and furthermore an in vivo use for investigations. In order to investigate the biology of bone malignancy and especially the interaction of tumour cells with cells of the microenvironment of the bone directly, an orthotopic model for Ewing’s sarcoma was developed. Additionally, osteosarcoma as a further primary bone sarcoma and prostate carcinoma as a cancer type with frequent bone metastases were tested in this model. The previously described technique of intrafemoral transplantation was used in this model. Using small animal imaging techniques such as nano computed tomography and magnetic resonance imaging in combination with histology it could be shown that the transplanted cells led to the development of orthotopic tumours presenting a comparable picture to the clinical situation. This model will be further used for research projects performed in the Northern Institute for Cancer Research on the effectiveness of drugs targeting Ewing’s sarcoma cells.EThOS - Electronic Theses Online ServiceDeutsche Krebshilfe : Bone Cancer Research Trust : North of England's Children's Cancer Research Fund : Newcastle Healthcare CharityGBUnited Kingdo

Development of a Preclinical Orthotopic Xenograft Model of Ewing Sarcoma and Other Human Malignant Bone Disease Using Advanced <i>In Vivo</i> Imaging

<div><p>Ewing sarcoma and osteosarcoma represent the two most common primary bone tumours in childhood and adolescence, with bone metastases being the most adverse prognostic factor. In prostate cancer, osseous metastasis poses a major clinical challenge. We developed a preclinical orthotopic model of Ewing sarcoma, reflecting the biology of the tumour-bone interactions in human disease and allowing <i>in vivo</i> monitoring of disease progression, and compared this with models of osteosarcoma and prostate carcinoma. Human tumour cell lines were transplanted into non-obese diabetic/severe combined immunodeficient (NSG) and Rag2^−/−/γc^−/− mice by intrafemoral injection. For Ewing sarcoma, minimal cell numbers (1000–5000) injected in small volumes were able to induce orthotopic tumour growth. Tumour progression was studied using positron emission tomography, computed tomography, magnetic resonance imaging and bioluminescent imaging. Tumours and their interactions with bones were examined by histology. Each tumour induced bone destruction and outgrowth of extramedullary tumour masses, together with characteristic changes in bone that were well visualised by computed tomography, which correlated with post-mortem histology. Ewing sarcoma and, to a lesser extent, osteosarcoma cells induced prominent reactive new bone formation. Osteosarcoma cells produced osteoid and mineralised “malignant” bone within the tumour mass itself. Injection of prostate carcinoma cells led to osteoclast-driven osteolytic lesions. Bioluminescent imaging of Ewing sarcoma xenografts allowed easy and rapid monitoring of tumour growth and detection of tumour dissemination to lungs, liver and bone. Magnetic resonance imaging proved useful for monitoring soft tissue tumour growth and volume. Positron emission tomography proved to be of limited use in this model. Overall, we have developed an orthotopic <i>in vivo</i> model for Ewing sarcoma and other primary and secondary human bone malignancies, which resemble the human disease. We have shown the utility of small animal bioimaging for tracking disease progression, making this model a useful assay for preclinical drug testing.</p></div

PET-CT imaging of primary tumour.

<p><b>A</b>, 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>B</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).</p

Bioluminescent imaging of of Rag2^−/− γc^−/− mice intrafemorally transplanted with low numbers of transduced TC-71 cells.

<p>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).</p

“Malignant” bone formation within the tumour mass after SaOS-2 transplantation.

<p><b>A</b>, Representative CT image 70 days after intrafemoral transplantation of SaOS-2 cells showing “malignant” bone formation within the extraosseous tumour mass (arrow). <b>B</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). <b>C</b>, “Malignant” bone forming amongst tumour cells in the medullary cavity, showing appositional deposition (scaffolding) on existing bone trabeculae (“T”) (H&E, original magnification 200×). <b>D</b>, 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×).</p

Osteolytic lesions caused by injection of PC3M.

<p><b>A</b>, Representative CT image 29 days after transplantation of PC3M cells, showing lytic destruction of the femur (arrow) (sagittal section). <b>B</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×). <b>C</b>, 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×). <b>D</b>, Lysis of bone by osteoclasts in resorption pits on the medullary surface (arrows), clearly distinct from intrafemoral tumour (H&E, original magnification 400×).</p

Timecourse of bioluminescent signal in mice injected with low numbers of transduced TC-71 cells.

<p>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.</p

Prominent reactive bone formation following transplantation of Ewing sarcoma cell lines VH-64 (A–E) and TC-71 (F–I).

<p><b>A</b>, Representative CT image of a VH-64 transplanted femur 42 days after transplantation showing a “sunburst” pattern of reactive bone formation. <b>B</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). <b>C</b>, 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×). <b>D</b>, Reactive bone emanating from femoral cortex (top) within a VH-64 tumour (H&E, original magnification 100×). <b>E</b>, 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×). <b>F</b>, Reactive bone and a focus of cartilage (arrow) amongst VH-64 tumour cells (H&E, original magnification 200×) <b>G</b>, Representative CT image of a TC-71 transplanted femur 15 days after transplantation showing cortical destruction (arrow) and possible reactive new bone formation (arrowhead). <b>H</b>, 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×). <b>I</b>, 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×).</p

Experimental mice for the development of an orthotopic model of bone malignancies.

<p>VH-64 and TC-71: Ewing sarcoma cell lines; SaOS-2: osteosarcoma cell line; PC3M: prostatic adenocarcinoma cell line; Medium: mice were injected with medium alone. N.A.: not applicable. All mice were NSG mice unless stated otherwise.</p

MR imaging of primary tumour with measurement of tumour volume.

<p>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).</p