Establishment of an In Vivo Model for Pediatric Ewing Tumors by Transplantation into NOD/ scid Mice

Abstract

Ewing tumors are a clinically heterogeneous group of childhood sarcomas that represent a paradigm for understanding solid tumor biology, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level. However, the biologic organization of the tumor, especially the processes that govern proliferation, differentiation, and metastasis of primitive tumor stem cells is poorly understood. Therefore, to develop a biologically relevant in vivo model, five different Ewing tumor cell lines and primary tumor cells from three patients were transplanted into immune-deficient mice via intravenous injection. NOD/scid mice that carry a complex immune deficiency and thus nearly completely lack the ability to reject human cells were used as recipients. Overall, 26 of 52 mice (50%) transplanted with VH-64, WE-68, CADO-ES1, TC-71, and RM-82 cells and 4 of 10 mice (40%) transplanted with primary tumor cells engrafted. Moreover, primary cells that did not grow in vitro proliferated in mice. The pattern of metastasis was similar to that in patients with frequent metastases in lungs (62%), bone marrow (30%), and bone (23%). Using limiting dilution experiments, the frequency of the engraftment unit was estimated at 1 Ewing tumor-initiating cell in 3 ϫ 10 5 VH-64 cells. These data demonstrate that we have been able to establish an in vivo model that recapitulates many aspects of growth and progression of human Ewing tumors. For the first time, this model provides the opportunity to identify and characterize primitive in vivo clonogenic solid tumor stem cells. This model will, therefore, be instrumental in studying many aspects of tumor cell biology, including organ-selective metastasis and tumor angiogenesis. The family of Ewing tumors is a clinically heterogeneous group of tumors including Ewing's sarcoma of the bone, Askin tumors of the thoracic wall, and PNET affecting children, adolescents, and young adults. Despite this clinical heterogeneity, Ewing tumors represent a paradigm for understanding the biology of human solid tumors, as they are the first group of sarcomas for which a chromosome translocation has been characterized at the molecular level (1, 2). This translocation, t(11;22), leads to the fusion of the EWS gene at 22q12, encoding for a putative RNA binding protein, to FLI1 at 11q24, an ETS-related gene coding for a transcription factor. Different breakpoints occur with in-frame fusion of EWS exons 7-10 to FLI1 exons 4 -9 (3). The two most common combinations are EWS exon 7/FLI1 exon 6 (termed type 1 fusion) and EWS exon 7/FLI1 exon 5 (type 2 fusion). The biologic relevance of the different breakpoints is indicated by two retrospective analyses demonstrating that patients with nonmetastatic disease and a type 1 fusion had a better clinical outcome than patients with other exon combinations (4, 5). In another 5%-10% of Ewing tumor patients, EWS is fused to others members of the ETS family, most frequently ERG at 21q22 (6) or more rarely ETV1, E1AF and FEV (7-10). O...

Sequence-Specific Knockdown of EWS-FLI1 by Targeted, Nonviral Delivery of Small Interfering RNA Inhibits Tumor Growth in a Murine Model of Metastatic Ewing's Sarcoma

“…Over the next few hours, the bioluminescent signal gradually disappeared as the cells dispersed and reemerged 1 to 2 weeks later at various locations where tumors developed. The most common engraftment sites were lung, vertebral column, pelvis, femur, and soft tissue, similar to the most frequently observed sites for metastases in EFT patients (45). The locations of the engraftments were confirmed by MRI (Fig.…”

Animal Models of Bone Metastasis

“…Ewing sarcoma cell lines include (1) TC-71, derived from a humeral Ewing sarcoma recurrence, and (2) VH-64, derived from the pleural effusion of a patient with primary Ewing sarcoma of the metatarsal bone. 155,157,166 Intravenous administration of the cell lines in NOD/SCID mice results in a pattern of metastasis similar to clinical patients; however, only 23% of mice developed bone metastasis. 157 Intravenous injection of TC-71 cells in nude mice resulted in bone metastases …”

Primitive Neuro Ectodermal Tumor (PNET) in infant diagnosed by histopathology and immunohistochemistry techniques

“…Malignant lymphoma shows immunoreactivity for LCA (Leukocyte Common Antigen). 3,6,7,8 In our case, the immunostaining shows positive staining for vimentin, there for malignant lymphoma can be excluded. Since the tumor which arises from or showed neuronal differentiation they may show positive immunostaining for neuroendocrine markers like Neuro-Specific Enolase (NSE), chromogranin and neuronal marker.…”