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

Abstract

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 in vivo 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 in vivo 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.

High neuropeptide Y release associates with Ewing sarcoma bone dissemination -in vivomodel of site-specific metastases

“…Although formation of distant bone metastases has been previously reported using other cell lines and animal models, the 50% frequency of their development observed in SK-ES1 orthotopic xenografts is one of the highest described [4, 6-8, 10, 35]. Dissemination to bone was also shown for TC71 cells after tail vein injection or intrafemoral transplantation [4, 6-8, 35].…”

“…Dissemination to bone was also shown for TC71 cells after tail vein injection or intrafemoral transplantation [4, 6-8, 35]. However, systemic injections of ES cells bypass the initial steps of the metastatic process and result in an inconsistent frequency of bone metastases, often as low as 16-25% [4, 6-8].…”

“…However, systemic injections of ES cells bypass the initial steps of the metastatic process and result in an inconsistent frequency of bone metastases, often as low as 16-25% [4, 6-8]. The intrafemoral injection of TC71 cells leads to development of distant bone metastases in 27% of mice [35]. The discrepancy between a lack of TC71 bone metastases in our model and their formation in other models may result from differences in the route of cell administration.…”

Molecular genetics of Ewing sarcoma, model systems and finding novel (immuno-) therapeutic targets

“…Earliest xenotransplantation models were injected either subcutaneously or intravenously in nude or NOD/scid mice [93][94][95] . Later murine models sought to more closely resemble the native tumour environment by performing orthotopic xenografts in rib bones [96] , femur [97] , pretibial space [98] , and gastrocnemius muscles [99] . Not only new compounds are tested but also novel drug delivery methods, such as silk gel to effectuate a sustained release of chemotherapeutics locally [100] .…”

Human Bone Xenografts: from Preclinical Testing for Regenerative Medicine to Modeling of Diseases

“…Thus generated, these models act as avatars to elucidate optimal drug regimes or as disease models for the evaluation of the role of oncogenic molecules in disease progression, including p53 [121], IL-6/ STAT3 [122], and ROCK1 [124]. Of these, orthotopic models are favored for better approximation to the osseous tumor microenvironment; multimodal positron emission tomography, computed tomography, magnetic resonance imaging, and bioluminescent imaging may be performed to achieve highresolution imaging of tumor interactions with bone [125]. Technical challenges of such approaches remain, however, such as mortality arising from the intrabone injections [123], technically demanding imaging techniques and difficulty in harvesting or retrieving the xenografted cells.…”