Mouse Model Of Ewing Sarcoma Research Articles

Abstract 5464: Galectin-3 expression in Ewing sarcoma: Modulation of the immune microenvironment in an immunocompetent model

Abstract Objective: Ewing sarcoma is an aggressive cancer of adolescents. Patients with metastatic Ewing sarcoma often receive radiation as part of their treatment protocol. We have demonstrated that both Ewing tumor cells and T cells infiltrating Ewing tumors express Galectin-3 (Gal3), an immunosuppressive protein in the tumor microenvironment. Here, we utilize an immunocompetent, humanized mouse model of Ewing sarcoma to investigate the role of Galectin-3 in the Ewing tumor immune microenvironment both at baseline and following radiation. Specifically, we sought to determine the influence of Galectin-3 on post-radiation inflammation in Ewing sarcoma. Methods: Human Ewing cells -/+ Gal3 expression were generated using CRISPR/Cas 9 technology. Cells -/+ Gal3 were orthotopically injected into humanized mice and allowed to established tumors over ~3 weeks. Tumors were then treated with radiation (or no radiation controls). To also address immune cell expression of Gal3, galectin inhibitors were utilized. Tumors were analyzed by bulk RNA sequencing and flow cytometry analysis (Cytek Aurora) for human CD45, CD3, CD4, CD20, CD8, CD56, CD14, CD16, etc. Peripheral blood controls were also collected and analyzed to ensure consistent baseline immune reconstitution. Results: CHLA10 and TC32 Ewing sarcoma cell lines both demonstrate Gal3 expression at baseline by western blot. CRISPR/Cas 9 knock-out of Gal3 in both cell lines showed successful knock down. Impact on growth and sensitivity to radiation in vitro was assessed by live cell Incucyte assays. Tumors in humanized mice were established utilizing these cells (or Gal3 expressing non-targeting controls) and changes in the post-radiation transcriptional profiles were assessed. Immune cells infiltrating these tumors were also quantified. Ongoing work focuses on additional characterization of these tumor infiltrating immune cell populations following radiation therapy and the impact of broader galectin inhibition on this effect. Conclusion: This work contributes to the understanding of radiation-induced tumor inflammatory responses in Ewing sarcoma by defining the role of tumor and T cell galectin-3 expression inn Ewing immunobiology. Next steps will explore translational opportunities based on these findings. Citation Format: Yunash Maharjan, Sreya Dey, Elina Mukherjee, Jessica Daley, Kelly M. Bailey. Galectin-3 expression in Ewing sarcoma: Modulation of the immune microenvironment in an immunocompetent model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5464.

A humanized orthotopic mouse model for preclinical evaluation of immunotherapy in Ewing sarcoma.

The advent of novel cancer immunotherapy approaches is revolutionizing the treatment for cancer. Current small animal models for most cancers are syngeneic or genetically engineered mouse models or xenograft models based on immunodeficient mouse strains. These models have been limited in evaluating immunotherapy regimens due to the lack of functional human immune system. Development of animal models for bone cancer faces another challenge in the accessibility of tumor engraftment sites. Here, we describe a protocol to develop an orthotopic humanized mouse model for a bone and soft tissue sarcoma, Ewing sarcoma, by transplanting fresh human cord blood CD34+ hematopoietic stem cells into young NSG-SGM3 mice combined with subsequent Ewing sarcoma patient derived cell engraftment in the tibia of the humanized mice. We demonstrated early and robust reconstitution of human CD45+ leukocytes including T cells, B cells, natural killer cells and monocytes. Ewing sarcoma xenograft tumors successfully orthotopically engrafted in the humanized mice with minimal invasive procedures. We validated the translational utility of this orthotopic humanized model by evaluating the safety and efficacy of an immunotherapy antibody, magrolimab. Treatment with magrolimab induces CD47 blockade resulting in significantly decreased primary tumor growth, decreased lung metastasis and prolonged animal survival in the established humanized model. Furthermore, the humanized model recapitulated the dose dependent toxicity associated with the CD47 blockade as observed in patients in clinical trials. In conclusion, this orthotopic humanized mouse model of Ewing sarcoma represents an improved platform for evaluating immunotherapy in bone and soft tissue sarcoma, such as Ewing sarcoma. With careful design and optimization, this model is generalizable for other bone malignancies.

Evaluation of geranylgeranyl diphosphate synthase inhibition as a novel strategy for the treatment of osteosarcoma and Ewing sarcoma.

Rab GTPases are critical regulators of protein trafficking in the cell. To ensure proper cellular localization and function, Rab proteins must undergo a posttranslational modification, termed geranylgeranylation. In the isoprenoid biosynthesis pathway, the enzyme geranylgeranyl diphosphate synthase (GGDPS) generates the 20-carbon isoprenoid donor (geranylgeranyl pyrophosphate [GGPP]), which is utilized in the prenylation of Rab proteins. We have pursued the development of GGDPS inhibitors (GGSI) as a novel means to target Rab activity in cancer cells. Osteosarcoma (OS) and Ewing sarcoma (ES) are aggressive childhood bone cancers with stagnant survival statistics and limited treatment options. Here we show that GGSI treatment induces markers of the unfolded protein response (UPR) and triggers apoptotic cell death in a variety of OS and ES cell lines. Confirmation that these effects were secondary to cellular depletion of GGPP and disruption of Rab geranylgeranylation was confirmed via experiments using exogenous GGPP or specific geranylgeranyl transferase inhibitors. Furthermore, GGSI treatment disrupts cellular migration and invasion in vitro. Metabolomic profiles of OS and ES cell lines identify distinct changes in purine metabolism in GGSI-treated cells. Lastly, we demonstrate that GGSI treatment slows tumor growth in a mouse model of ES. Collectively, these studies support further development of GGSIs as a novel treatment for OS and ES.

Abstract A004: Radiation-induced changes to the immune microenvironment in an immunocompetent mouse model of Ewing sarcoma

Abstract Background: Ewing sarcoma is a rare, aggressive, fusion oncoprotein-driven pediatric cancer. Patients with upfront metastatic or surgically unresectable disease commonly undergo radiation as part of standard of care therapy. Currently, little is known about the effect of radiation specifically on the immune microenvironment of Ewing tumors, as no routine biopsies or other clinical samples are acquired during radiation. Further, historically, the field has lacked an immunocompetent mouse model of Ewing sarcoma in which to study Ewing tumor-immune cell interactions. Given our interest in understanding the anti-tumor immune response specifically during times of DNA damage, we sought to model Ewing sarcoma in a humanized mouse model (where human immune cell interactions with human tumor cells can be examined) in order to examine the Ewing tumor immune microenvironment (TIME) during radiation. Here, we determine the likeness of our established humanized mouse model of Ewing sarcoma to that of human Ewing tumors at baseline and examine changes in the Ewing TIME upon delivery of radiation. Methods: Blood from humanized NSG mice was analyzed by flow cytometry to confirm human immune cell reconstitution. Human Ewing tumor cells were injected into humanized mice and allowed to grow for approximately three weeks. Tumors were treated with radiation (either single dose or fractioned doses x 5 days) using the MultiRad350 Precision irradiator and lead shields were used to isolate the tumor area in the radiation field. Immune cells infiltrating tumors with/without radiation exposure were subsequently analyzed by multiplexed immunohistochemistry, flow cytometry, PCR, and RNAseq analysis. Ewing tumor immune infiltrates (as baseline/without radiation) from humanized mice models were compared to that of patient Ewing tumors. Results: Ewing tumors from both established and primary cell lines were successfully established in humanized mice. Similar to patient primary Ewing tumor samples, local Ewing tumors in humanized mice demonstrate a proportion of T-cell infiltration, although the overall number of infiltrating immune cells is low. Macrophage populations in Ewing sarcoma are not as predominant as in tumors such as osteosarcoma and are represented in this model. Analyses detailing the spatial localization and transcriptional profiles of Ewing tumor immune infiltrates following radiation are ongoing. Conclusions: In the absence of syngeneic and transgenic models of Ewing sarcoma, the use of humanized mouse models is a feasible alternative to address specific questions regarding the Ewing sarcoma TIME. Understanding the Ewing immune microenvironment during radiation therapy, a commonly used treatment modality in Ewing sarcoma, provides clues as to promising agents that may be worthy of preclinical testing to enhance the anti-tumor immune response during radiation in high-risk patients. Citation Format: Jessica D. Daley, Elina M. Mukherjee, Anthony R. Cillo, Adriana C. Tufino, Nathanael G. Bailey, Tullia C. Bruno, Linda M. McAllister-Lucas, Dario A. Vignali, Kelly M. Bailey. Radiation-induced changes to the immune microenvironment in an immunocompetent mouse model of Ewing sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A004.

Abstract 957: Cadherin-11 contributes to the heterogenous and dynamic Wnt-β-catenin pathway activation in Ewing sarcoma

Abstract Introduction: Metastatic Ewing sarcoma (ES) has a dismal prognosis, and clarifying the molecular and cellular mechanism of metastasis is the most pressing need to improving prognosis. Recently, the heterogenous expression of the driving fusion gene EWS-FLI1, as well as canonical Wnt/β-catenin signaling activity has been increasingly recognized as a potential mediator of metastatic progression. However, targeting the pathway has been difficult due to its complexity and cross-activity, and not much is known of the mechanism of how ES cells maintain Wnt signaling heterogeneity. Cadherin-11 (CDH11) is a member of the cadherin super-family, which modulates calcium ion-dependent cell-cell adhesion and signal transduction, and is highly expressed in ES. CDH11 has been found to regulate the expression level of β-catenin, and play a key role in enhancing the stem cell, migration and invasion in other carcinomas, however the relationship between CDH11 and β-catenin in ES is not clear. Methods: Wnt/β-catenin dependent transcriptional activity of ES cells in vitro and in vivo were assessed by flow cytometry, immune fluorescence, as well as single cell RNA-seq. β-catenin and CDH11 knock-down cell lines were generated, and affected on invasion/migration, and metastatic progression was assessed in vitro and in vivo. Furthermore, the interaction of CDH11 with β-catenin, and the contribution of the protein-protein interaction to the heterogenous activation of Wnt/β-catenin was assessed by immunofluorescence, flowcytometry, Western blot, and co-immunoprecipitation. Results: At baseline, only a small fraction of ES cells have activated Wnt/β-catenin dependent transcriptional activity both in vitro and in vivo, which can respond rapidly with Wnt ligand stimulation. β-catenin knockdown reduced clonogenicity in vitro, and metastatic tumor burden in the orthotopic implantation amputation mouse model of ES. We further identified that most of the tested ES cells had abundant β-catenin bound to CDH11 in the cell membrane, which would rapidly be released through the cytoplasm and into the nucleus within 2 hours of adding exogenous Wnt ligand. CDH11 knockdown reduced β-catenin expression, rapid activation of signaling by Wnt ligands, and metastasis progression. Conclusion: Our data suggests that the cell surface CDH11- β-catenin complex contributes to the heterogenous and dynamic activation of the Wnt/β-catenin pathway in ES that drives ES metastasis. Targeting the CDH11- β-catenin complex can present a novel strategy to interfere with Wnt driven metastasis in ES. Citation Format: Ryota Shirai, Tyler Biebighauser, Sarah Nelson-Taylor, Avery Bodlak, Timothy Porfilio, Deandra Walker, Naoki Oike, Masanori Hayashi. Cadherin-11 contributes to the heterogenous and dynamic Wnt-β-catenin pathway activation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 957.

Abstract A28: Targeted therapy of Ewing sarcoma

Abstract Ewing sarcoma is a high-risk sarcoma with overall 50% survival, unchanged for decades. No effective new therapies have been introduced in that time frame. It is thus an ideal candidate for targeted therapy. It is also a tumor with several candidate targets for therapy. Since essentially all Ewing sarcomas express the cell surface antigen CD99, monoclonal antibodies can be used to deliver nanoparticle-encapsulated chemotherapeutic agents with high efficiency. Compared to conventional antibody-drug conjugates (ADCs), binding avidity and delivered dose per bound antibody is increased exponentially. Similarly, the tumor-specific EWS-FLI1 or equivalent gene translocation that drives oncogenesis is an attractive candidate target, so far untargetable with conventional small molecules. Both nanoparticle-delivered shRNA and CRISPR/Cas9 constructs targeting the fusion gene are candidate therapeutics that can be directed specifically to the fusion junction or the ets domain. Downstream targets of the fusion gene include a number of coding and noncoding genes, among them FEZF1-AS, a long noncoding RNA (lncRNA) that we have shown is both a direct target of the Ewing fusion gene and an oncogenic driver of tumor growth, invasion, and metastasis. While otherwise untargetable, lncRNAs can be effectively downregulated by ASOs (anti-sense oligonucleotides) with a proportional attenuation of their oncogenic effect. Here we present evidence that a novel CD99 targetable nanoparticle, termed hybrid polymerized liposomal nanoparticles, can effectively deliver small-molecule cytotoxics with complete tumor ablation in a nude mouse model of Ewing sarcoma, as well as EWS-FLI1 targeted CRISPR/Cas9 constructs and FEZF1-AS targeted ASOs, with proportional knockdown and oncogenic attenuation. Tumor uptake of the therapeutic nanoparticles is rapid, with over 90% of particles at therapeutic doses bound and internalized within 20 minutes in vitro. The IC50 for cytotoxics is improved 40-fold over free agent and 20-fold over untargeted nanoparticle formulations. Animals with Ewing sarcoma xenografts treated with CD99-targeted hPLNs containing irinotecan show complete tumor ablation with no tumor regrowth at 90 days, 20 days after cessation of treatment, unlike free irinotecan or untargeted irinotecan liposomal nanoparticle animals, all of whom died with progressive tumor growth. Surviving animals showed no toxicity. Ancillary studies identified no evidence of bone marrow, liver, or kidney toxicity at therapeutic doses. Over 70% of tumor cells express EGFP after internalizing targeted nanoparticles containing CRISPR/Cas9 plasmids with an EGFP construct. Similarly, FEZF1-AS lncRNA expression is reduced by over 70% after treatment with FEZF1-AS specific ASOs. Taken together, these results suggest that both targetable (cytotoxics delivered preferentially to tumor cells) and previously untargetable features (fusion genes, lncRNAs) can be effectively and specifically treated when conventional and novel therapeutics are delivered directly to tumor cells by CD99-targeted nanoparticles. The successful treatment of Ewing sarcoma xenografts and metastases in animal models by any of these approaches also suggests combined therapy with both conventional and unconventional agents is feasible, with no appreciable toxicity. Citation Format: Timothy J. Triche, Jon Nagy, Hyung Kang, Chris Denny, Sheetal Bajaj Mitra. Targeted therapy of Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A28.

Proteasome Addiction Defined in Ewing Sarcoma Is Effectively Targeted by a Novel Class of 19S Proteasome Inhibitors.

Ewing sarcoma is a primitive round cell sarcoma with a peak incidence in adolescence that is driven by a chimeric oncogene created from the fusion of the EWSR1 gene with a member of the ETS family of genes. Patients with metastatic and recurrent disease have dismal outcomes and need better therapeutic options. We screened a library of 309,989 chemical compounds for growth inhibition of Ewing sarcoma cells to provide the basis for the development of novel therapies and to discover vulnerable pathways that might broaden our understanding of the pathobiology of this aggressive sarcoma. This screening campaign identified a class of benzyl-4-piperidone compounds that selectively inhibit the growth of Ewing sarcoma cell lines by inducing apoptosis. These agents disrupt 19S proteasome function through inhibition of the deubiquitinating enzymes USP14 and UCHL5. Functional genomic data from a genome-wide shRNA screen in Ewing sarcoma cells also identified the proteasome as a node of vulnerability in Ewing sarcoma cells, providing orthologous confirmation of the chemical screen findings. Furthermore, shRNA-mediated silencing of USP14 or UCHL5 in Ewing sarcoma cells produced significant growth inhibition. Finally, treatment of a xenograft mouse model of Ewing sarcoma with VLX1570, a benzyl-4-piperidone compound derivative currently in clinical trials for relapsed multiple myeloma, significantly inhibited in vivo tumor growth. Overall, our results offer a preclinical proof of concept for the use of 19S proteasome inhibitors as a novel therapeutic strategy for Ewing sarcoma. Cancer Res; 76(15); 4525-34. ©2016 AACR.

Abstract 4119: Inhibition of PORCN prolongs metastasis free survival in a mouse model of Ewing sarcoma

Abstract Metastatic disease is the most important factor in determining the survival of patients with Ewing sarcoma (ES). Although intensive cytotoxic chemotherapy has improved the survival of patients with localized disease, the survival rate for patients with metastatic disease remains dismal, and the most pressing unmet need in ES is to prevent and treat metastasis better. The Wnt signaling pathway is a key regulator of a number of cellular functions associated with metastasis, including proliferation, motility, and stem cell self-renewal. A single enzyme, Porcupine (Porcn), mediates post-translational palmitoylation of Wnt ligands, and it's activity is required for secretion of all 17 Wnt ligands, making it an attractive target for a pan-Wnt inhibitor. We have studied the effect of LGK974, a potent, selective, Porcn inhibitor, on Ewing sarcoma metastasis. In vitro, we have observed that LGK974 does not affect proliferation or sarcosphere formation, but inhibits migration of ES cell lines through the suppression of expression of multiple transcriptional regulators of Epithelial-Mesenchymal Transition (EMT). In our orthotopic implantation/amputation mouse model, single agent LGK974 treatment leads to significant delay in formation of lung metastasis following amputation of the affected leg, without a significant effect on primary tumor growth. This delay leads to a significant post-amputation survival benefit in multiple xenografts. In LGK974 treated xenografts, the expression of multiple EMT related transcription factors were observed to be suppressed. Furthermore, in tail vein injection models, LGK974 did not produce a survival benefit, suggesting that LGK974 effects Ewing sarcoma metastasis by inhibiting early steps in the metastatic cascade, such as migration and invasion. Our findings strongly implicate Wnt signaling in the early steps of ES metastasis and demonstrate that LGK974 has the potential to significantly improve the survival of ES patients through the specific inhibition of metastasis. Citation Format: Masanori Hayashi, Alissa C. Baker, Seth D. Goldstein, Catherine M. Albert, Kyle W. Jackson, David M. Loeb. Inhibition of PORCN prolongs metastasis free survival in a mouse model of Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4119.

The Macrophage Inhibitor CNI-1493 Blocks Metastasis in a Mouse Model of Ewing Sarcoma through Inhibition of Extravasation

Metastatic Ewing Sarcoma carries a poor prognosis, and novel therapeutics to prevent and treat metastatic disease are greatly needed. Recent evidence demonstrates that tumor-associated macrophages in Ewing Sarcoma are associated with more advanced disease. While some macrophage phenotypes (M1) exhibit anti-tumor activity, distinct phenotypes (M2) may contribute to malignant progression and metastasis. In this study, we show that M2 macrophages promote Ewing Sarcoma invasion and extravasation, pointing to a potential target of anti-metastatic therapy. CNI-1493 is a selective inhibitor of macrophage function and has shown to be safe in clinical trials as an anti-inflammatory agent. In a xenograft mouse model of metastatic Ewing Sarcoma, CNI-1493 treatment dramatically reduces metastatic tumor burden. Furthermore, metastases in treated animals have a less invasive morphology. We show in vitro that CNI-1493 decreases M2-stimulated Ewing Sarcoma tumor cell invasion and extravasation, offering a functional mechanism through which CNI-1493 attenuates metastasis. These data indicate that CNI-1493 may be a safe and effective adjuvant agent for the prevention and treatment of metastatic Ewing Sarcoma.

The association between let-7, RAS and HIF-1α in Ewing Sarcoma tumor growth.

Ewing Sarcoma (ES) is the second most common primary malignant bone tumor in children and adolescents. microRNAs (miRNAs) are involved in cancer as tumor suppressors or oncogenes. We studied the involvement of miRNAs located on chromosomes 11q and 22q that participate in the most common translocation in ES. Of these, we focused on 3 that belong to the let-7 family.We studied the expression levels of let-7a, and let-7b and detected a significant correlation between low expression of let-7b and increased risk of relapse. let-7 is known to be a negative regulator of the RAS oncogene. Indeed, we detected an inverse association between the expression of let-7 and RAS protein levels and its downstream target p-ERK, following transfection of let-7 mimics and inhibitors. Furthermore, we identified let-7 as a negative regulator of HIF-1α and EWS-FLI-1. Moreover, we were able to show that HIF-1α directly binds to the EWS-FLI-1 promoter. Salirasib treatment in-vitro resulted in the reduction of cell viability, migration ability, and in the decrease of cells in S-phase. A significant reduction in tumor burden and in the expression levels of both HIF-1α and EWS-FLI-1 proteins were observed in mice after treatment.Our results support the hypothesis that let-7 is a tumor suppressor that negatively regulates RAS, also in ES, and that HIF-1α may contribute to the aggressive metastatic behavior of ES. Moreover, the reduction in the tumor burden in a mouse model of ES following Salirasib treatment, suggests therapeutic potential for this RAS inhibitor in ES.

Abstract A76: Ras pathway and HIF-1α contribute to the aggressiveness of Ewing sarcoma

Abstract Aim: Ewing sarcoma (ES) is the second most common and aggressive primary malignant bone tumor in children and adolescents. The hallmarks of the tumor are specific translocations; the most frequent involves the EWS gene on chromosome 22 and the FLI-1 gene on chromosome 11. Our aim was to identify microRNAs (miRs) and genes involved in the aggressiveness of ES. Out of the miRs located on these chromosomes, 3 belong to the let-7 family. Methods: We evaluated the expression levels of hsa-let-7a and hsa-let-7b by RQ-PCR in 57 primary ES tumors. We measured the protein levels of p-ERK, HIF-1a and EWS-FLI-1 following transfection with let-7a, let-7b or HIF-1a . The effect of a Ras inhibitor was evaluated in vitro and in vivo. Results: In the group of patients with localized disease, a significant correlation with outcome was observed. Progression free survival (PFS) at ten years for patients with high hsa-let-7b expression was 62% versus 29% for those with low hsa-let-7b expression level (p=0.029). Multivariate Cox regression analysis identified expression of let-7b as an independent prognostic factor in ES, with a 4.4 fold increased risk of relapse (p=0.021). hsa-let-7 is known to be a negative regulator of the RAS oncogene. Positive p-ERK immuno-staining was detected in 31 out of the 45 (69%) tumors analyzed, and a significant inverse correlation was identified between hsa-let-7a and p-ERK (p=0.037). One of the target genes of RAS is Hypoxia inducible factor 1 (HIF-1). We detected a high level of HIF-1α protein in ES cell lines, under normoxic conditions, which did not change following induced hypoxia. This implies for a mechanism that mimics hypoxia and probably contributes to the high frequency of metastasis and thereby aggressiveness of this tumor. Following transfection with hsa-let-7a and hsa-let-7b mimics (overexpression) the levels of RAS, p-ERK, HIF-1α and EWS-FLI-1 proteins were significantly reduced (60%, p=0.0019, 52%, p=0.003, 45%, p=0.0028 and 38%, p=0.006, respectively). Silencing of HIF-1α resulted in a decrease of 61% of EWS-FLI-1, suggesting that HIF-1α regulates EWS-FLI-1, and we were able to show that HIF-1α directly binds EWS promoter. This was accompanied by a reduction of 32% (p=0.009) in cell proliferation rate and by a significant decrease of 36% (p=0.0013) in cell migration ability. Using a RAS inhibitor, Salirasib, we observed a significant decrease of HIF-1α and EWS-FLI-1 protein levels in both in-vitro and in-vivo settings. In addition, a significant inhibition of tumor growth was demonstrated in the treated animals. Conclusions: These results imply that Ras and HIF-1α might contribute to the high frequency of metastasis and thereby aggressiveness of ES tumors. The reduction in tumor burden in a mouse model of ES following Salirasib treatment, demonstrates the therapeutic potential of this RAS inhibitor in ES. Citation Format: Michal Hameiri-Grossman, Adi Porat-Klein, Ian J. Cohen, Shifra Ash, Yoel Kloog, Ronit Haklai, Galit Elad-Sfadia, Avraham Weizman, Isaac Yaniv, Smadar Avigad. Ras pathway and HIF-1α contribute to the aggressiveness of Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A76.

Bone marrow cells participate in tumor vessel formation that supports the growth of Ewing’s sarcoma in the lung

An MHC-mismatch bone marrow (BM) transplant Ewing's sarcoma mouse model was used to investigate whether BM cells participate in the vessel formation that support Ewing's sarcoma lung metastasis. BM cells from H-2K(b/d) donor mice were transplanted into sublethally irradiated H-2K(d) recipient mice. Donor BM cells were identified using the H-2K(b) marker. Engraftment was confirmed by identifying the H-2K(b) IL-1β-type specific polymorphism. After engraftment highly lung metastatic TC71-PM4 cells were injected intravenously. Mice were sacrificed 10 weeks after tumor cell injection. Hematoxylin-and-eosin staining was performed to identify lung metastatic foci. These tumors were then evaluated using immunohistochemical analysis. H-2K(b)-positive cells were found in lung metastases but not in normal lung, liver or spleen tissues. Injection of CM-Dil-labeled BM cells into tumor bearing and control mice showed that nonspecific organ migration occurred at 24 h, but that these cells were absent 1 week later in control mice. These data suggest that the migration of the H-2K(b) BM cells to lung nodules was specific because these cells were observed 14 weeks after transplantation. Co-localization of H-2K(b) and CD31 or VE-Cadherin demonstrated that some endothelial cells were BM-derived. Co-localization of H-2K(b) and Desmin, smooth muscle actin (α-SMA) or PDGFR-β indicated that a fraction of pericytes was also BM-derived. These results suggest that BM cells participate in the vascular formation that supports the growth of Ewing's sarcoma lung metastases. BM cells migrated to the metastatic tumor and differentiated into endothelial cells and pericytes. These data indicated that targeting this process may have therapeutic potential.

Suppression of Ewing's Sarcoma Tumor Growth, Tumor Vessel Formation, and Vasculogenesis Following Anti–Vascular Endothelial Growth Factor Receptor-2 Therapy

We previously showed that bone marrow cells participate in new tumor vessel formation in Ewing's sarcoma, and that vascular endothelial growth factor 165 (VEGF(165)) is critical to this process. The purpose of this study was to determine whether blocking VEGF receptor 2 (VEGFR-2) with DC101 antibody suppresses tumor growth, reduces tumor vessel formation, and inhibits the migration of bone marrow cells into the tumor. An H-2 MHC-mismatched bone marrow transplant Ewing's sarcoma mouse model was used. Bone marrow cells from CB6F1 (MHC H-2(b/d)) mice were injected into irradiated BALB/cAnN mice (MHC H-2(d)). TC71 Ewing's sarcoma cells were s.c. injected 4 weeks after the bone marrow transplantation. Mice were then treated i.p. with DC101 antibody or immunoglobulin G (control) twice a week for 3 weeks starting 3 days after tumor cell injection. DC101 antibody therapy significantly reduced tumor growth and tumor mean vessel density (P < 0.05) and increased tumor cell apoptosis. Decreased bone marrow cell migration into the tumor was also shown after DC101 therapy as assessed by the colocalization of H-2K(b) and CD31 using immunohistochemistry. DC101 inhibited the migration of both human and mouse vessel endothelial cells in vitro. These results indicated that blocking VEGFR-2 with DC101 antibodies may be a useful therapeutic approach for treating patients with Ewing's sarcoma.