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Ewing sarcoma (ES) is the second most common bone cancer in children, accounting for 2% of pediatric cancer diagnoses. Patients who present with metastatic disease at the time of diagnosis have a dismal prognosis, compared to the >70% 5-year survival of those with localized disease. Here, we utilized single-cell RNA sequencing (scRNA-seq) to characterize the transcriptional landscape of primary ES tumors, and to identify circulating tumor cells (CTCs) in peripheral blood at the time of diagnosis in order to further understand ES transcriptional heterogeneity and factors that drive metastasis. Methods: Viably frozen primary tumor and peripheral blood samples were obtained from 7 ES patients at the time of diagnosis and prior to the initiation of treatment. Tumors were dissociated into a single cell suspension and sorted for viability using FACS (fluorescence activated cell sorting) whereas peripheral blood samples were subjected to a size-based selection with the CellSieve microfiltration system. ScRNA-seq was performed using the 10xChromium platform and count matrices were generated using 10x Genomics Cell Ranger software. Quality control, integration, and cluster analysis was performed with Seurat. Results: Cluster analysis of integrated, primary tumor samples demonstrated that candidate ES cell clusters express FLI1 and ES marker NKX2-2 and cluster separately from immune cell populations. Additionally, using inferCNV, ES clusters were demonstrated to harbor chromosomal copy number alterations known to be associated with ES and that were previously identified clinically on preliminary pathology reports for each patient; further GSEA analysis showed significant overlap between published ES gene sets and genes upregulated in ES clusters. Further analysis of ES cells demonstrated that cell-cycle phase determined a cluster enriched in pro-proliferation gene signatures, and overall heterogeneity of expression of previously known therapeutic targets. Candidate ES CTCs were identified among the peripheral blood samples among clusters which corresponded with distinct immune cell populations. The candidate ES CTCs expressed NKX2-2 and demonstrated enrichment in oncogenic gene signatures. Conclusion: ScRNA-seq of primary ES tumors is feasible and demonstrates that ES tumor cells are largely homogeneous in nature; and candidate ES CTCs can be identified in peripheral blood at the time of diagnosis in ES patient and warrant further investigation as to their utility as a biomarker of metastatic disease. Citation Format: Sarah K. Nelson-Taylor, Avery Bodlack, Andrew Goodspeed, Amy Treece, Nathan Donaldson, Carrye Cost, Tim Garrington, Brian Greffe, Sandra Luna-Fineman, Jenna Sopfe, Masanori Hayashi. Single cell RNA sequencing of primary Ewing sarcoma tumors and identification of circulating tumor cells in patient-matched peripheral blood samples [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 1681.

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.

Undifferentiated round cell sarcomas, which carry the BCOR-CCNB3 fusion gene, have traditionally been called “Ewing-like sarcomas” and have been treated with Ewing sarcoma treatment regimens. While there has been emerging evidence that these BCOR-CCNB3 sarcomas (BCS) are molecularly distinct from Ewing sarcomas, the biology of these tumors is poorly understood, and patients have few options when they do not respond to Ewing sarcoma-based therapies. Here, we report the establishment of a novel BCS cell line, which we utilized to identify a unique BCS-specific therapeutic vulnerability using a hybrid screening approach consisting of a high-throughput drug screen combined with a CRISPR/Cas9-based knock out genetic screen. The BCOR-CCNB3-positive sarcoma cell line NBC-1 was established from the tumor tissue obtained from an 18-year-old male with a soft tissue tumor of the buttock with lymph node metastasis, and the expression of the fusion BCOR-CCNB3 transcript was confirmed by RT-PCR. An in vitro high-throughput screening was performed using the L1300 library of 2570 drugs, where the cells were incubated for 48 hours with drug before cell viability was measured. This drug screen identified 148 drugs, which included 84 FDA-approved drugs. While these drugs predictably included a significant number of conventional cytotoxic chemotherapy drugs currently used to treat BCS, such as vincristine and doxorubicin, several other drug classes emerged, identifying twenty tyrosine kinase inhibitors, proteasome inhibitors such as Carfizomib, Ixazomib, and Bortezomib, and a histone deacetylase inhibitor, Panobinostat, all of which had distinctly higher cytotoxicity compared to the Ewing sarcoma cell controls. Next, we utilized a custom-made CRISPR panel consisting of 9771 sgRNAs targeting 1221 genes where we were able to identify a specific pharmacologic inhibitor in the literature. Using this library, we carried out a genetic loss-of-function screen to identify genes that are required for the proliferation of BCS. Based on these initial findings, we were able to demonstrate that BCS tumors have distinct vulnerabilities from Ewing sarcoma, suggesting that a BCS-specific therapeutic approach is necessary. Citation Format: Naoki Oike, Hiroyuki Kawashima, Akira Ogose, Takashi Ariizumi, Yudai Murayama, Hajime Umezu, Molishree Joshi, Colin Sempeck, Deandra Warker, Avery Bodlak, Naoto Endo, Masanori Hayashi. High-throughput drug screening combined with a druggable target CRISPR drop-out screen identifies therapeutic vulnerabilities of BCOR-CCNB3 fusion positive sarcomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3411.

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 inability to effectively treat hematogenous metastasis remains the largest challenge of sarcoma therapy. The ability to isolate, quantify, and study circulating tumor cells (CTC) in sarcomas has the potential to enhance our understanding of the biology of metastasis, altering the way we treat patients with high grade sarcomas. Using a size based filtration device, CellSieve™, we were able to successfully identify and quantify CTC in a metastatic xenograft model as well as in blood samples from patients with high grade sarcomas. In mice, we utilized our orthotopic implantation/amputation model of metastasis. Using a TdTomato-expressing Ewing sarcoma cell line and a cocktail of fluorescently labeled antibodies to vimentin and CD45, we were able to detect both CTC and circulating tumor clusters (CTCL) in mice with growing tumors and with metastatic disease, but not in mice immediately post-amputation. These experiments were augmented with samples collected from patients with high grade sarcomas. In 18 samples from 15 patients, we were able to identify both CTC and CTCL. Furthermore, we were also able to validate the presence of CTC by PCR amplification of sarcoma-associated chromosomal translocations. Future work will use this system to explore the biology of sarcoma metastasis by studying genetic, transcriptional, and epigenetic characteristics of CTC and CTCL and comparing these cells to those in the bulk primary tumor. We will also further explore the possibility that CTC and/or CTCL might serve as a biomarker of disease burden, response to treatment, or metastatic risk. Citation Format: Masanori Hayashi, Peixuan Zhu, Catherine M. Albert, Diana Steppan, Gregory McCarty, Kyle W. Jackson, Cha-Mei Tang, David M. Loeb. Detection of sarcoma circulating tumor cells using a size based microfiltration device. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B37.

Sarcoma metastasis is a multi-step process that includes migration, intravasation into the circulation, extravasation and invasion to distant tissue, and distant tumor growth, and because commonly used xenograft models do not account for every step in the process, we sought to develop a novel metastasis model system. Our technique involves implanting small fragments of patient-derived xenografts into either the tibia or gastrocnemius muscle of NOD/SCID/IL-2Rγ knock-out mice and amputating the affected limb when the tumor reaches at least 1 cm in diameter. Mice are then followed for the development of distant metastases. We found that Ewing sarcoma, osteosarcoma, and rhabdomyosarcoma xenografts all undergo distant metastasis when implanted in an orthotopic location, but do not metastasize after subcutaneous implantation, even when the subcutaneous tumor is excised and mice are observed long-term post-excision. We also investigated whether the difference in metastatic potential is related to cell intrinsic or cell extrinsic factors. Analysis by immunofluorescence revealed that tumors are infiltrated by macrophages regardless of site of implantation, but that the polarization of these macrophages differs. We also found differences in expression of arginase and NOS2 based on site of xenograft implantation. Thus, we have demonstrated that sarcoma patient-derived xenografts undergo spontaneous distant metastasis when implanted in an orthotopic location, but not when implanted in a heterotopic location, highlighting the essential role of the tumor microenvironment in determining metastatic potential. This model has significant benefits compared with the more commonly used tail vein injection model of metastasis, which bypasses the requirements for local invasion and vascular intravasation, processes that are amenable to study using our model. We have begun to use this model to investigate the mechanisms by which tumor microenvironment influences metastasis, and also are using this model for preclinical drug evaluation to determine a therapy's effect not only on growth of the primary tumor, but also on development of metastasis. Citation Format: Seth D. Goldstein, Masanori Hayashi, Catherine M. Albert, Kyle W. Jackson, David M. Loeb. An orthotopic xenograft model of sarcoma metastasis demonstrates essential role of tumor microenvironment for metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3231. doi:10.1158/1538-7445.AM2015-3231