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. Author manuscript; available in PMC: 2024 Feb 1.
Published in final edited form as: Curr Protoc. 2023 Feb;3(2):e670. doi: 10.1002/cpz1.670

Modeling Ewing Sarcoma Lung Metastasis

Atreyi Dasgupta 1,*, Lyazat Kurenbekova 1, Tajhal D Patel 1, Kimal Rajapakshe 2, Gargi Ghosal 3, Bikesh Nirala 1, Cristian Coarfa 4,5, Jason Yustein 1,*
PMCID: PMC9942121  NIHMSID: NIHMS1864726  PMID: 36799651

Abstract

Ewing Sarcoma (EwS) is the second most common malignant bone tumor in adolescents and young adults. The single-most powerful predictor of outcome in EwS is presence of metastatic burden at the time of diagnosis. Patients with metastatic Ewing Sarcoma have an abysmal 5-year survival rate of 10–25%, which has not changed over the past 30–40 years. Thus, unraveling underlying mechanisms of EwS metastasis are imperative for developing effective therapeutic measures. Investigations towards this goal are limited by the lack of reliable genetically engineered mouse models and specialized metastatic models. Using two established cell lines, A673 and TC71, we generated lung specific metastatic cell lines by serial orthotopic intra-tibial injection followed by isolation of cells from lung metastases. The lung metastatic lines generated exhibit distinct differential molecular signatures from the parental cells when analyzed using a multi-omics approach. These signatures overlapped with EwS patient primary bone and metastatic lung specimens supporting the clinical relevance of these preclinical models of EwS.

Basic Protocol 1: (Intratibial Injection in Immunocompromised Mice)

Basic Protocol 2: (Development and Characterization of Lung Metastatic Cell Line)

Keywords: Ewing Sarcoma, Metastasis, Lung, Micro-environment, Intra-tibial

INTRODUCTION:

Metastasis, the process of dissemination of cancer cells from a primary organ to distal sites is a complex biological and molecular process. More than 90% of cancer related deaths are due to metastasis (Chaffer & Weinberg, 2011). Yet, we have very limited understanding of this process, particularly how certain cancers can metastasize only to specific organs, and how the microenvironment at the distal organ supports the growth of the lesion.

Approximately 25–30% of patients with Ewing Sarcoma (EwS) present with metastasis at diagnosis. Systemic chemotherapy, which includes a combination of vincristine, doxorubicin, and cyclophosphamide, alternating with ifosfamide and etoposide (VDC/IE) (Abboud et al., 2021; Womer et al., 2012) along with surgery and/or radiotherapy, has significantly increased the survival rate of patients with localized disease. While patients with metastatic EwS have an extremely poor 5-year survival rate of 10–25% (Cotterill et al., 2000; Shulman et al., 2022). This dismal prognosis has not changed over the past several decades. Thus, there is an urgent need to focus particularly on mechanisms of metastatic growth and progression and to identify the underlying molecular features to define therapeutic targets. In case of EwS, studying metastasis is further complicated by the lack of a reliable preclinical models. To develop functionally appropriate models for studying EwS lung metastasis, an approach was taken to integrate tumor intrinsic and stromal factors driving metastatic phenotypes by developing highly aggressive, lung specific metastatic cell lines.

To generate lung specific metastatic lines, a serial in vivo transplantation approach was used by repeated intra-tibial injection of two commonly used EwS cell lines (A673 and TC71), allowing metastatic foci to form followed by collecting and dissociating lung tumors and subsequent reinjection into the tibia. This process was repeated for 5 to 6 times to generate highly aggressive lung specific metastatic cell lines as shown in the schematic in Fig. 1A. While this model is cell-derived, it consistently develops lung metastatic foci with high specificity. Using these two well established cell lines, we observed upregulation of overlapping pathways when analyzed using the MSigDB, which has the databases for GO, Hallmark KEGG and Reactome (http://www.gsea-msigdb.org/gsea/msigdb/index.jsp) implicated in metastasis in the literature, including pathways involved in growth, vascularization, matrix remodeling, and epithelial-mesenchymal transition (Bakir, Chiarella, Pitarresi, & Rustgi, 2020; Bielenberg & Zetter, 2015; Hanahan & Weinberg, 2011; Nwabo Kamdje et al., 2017; Winkler, Abisoye-Ogunniyan, Metcalf, & Werb, 2020)

Figure 1. Intratibial Injection and Development of Lung Metastatic Line.

Figure 1.

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A Schematic showing the strategy of developing a lung metastatic line from TC71 Ewing Sarcoma cells by serial intra-tibial injection followed by isolation of lung lesion and dissociation. Parental TC71 cells were labeled with EGFP-C1, and TC71-Lm (lung-met) cells were labeled with mCherry. B. Steps and materials involved in intra-tibial injection demonstrated in NSG mice.

In this manuscript, the first protocol describes the basic method of developing an orthotopic xenograft EwS tumor in immunocompromised mice, specifically NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratories), and nude (J:NU, Jackson Laboratories). This is done by an intratibial injection in 4–6 weeks old mice (Fig. 1B). The second protocol describes the several iterations of injections and labeling of tumor cells to develop the lung metastatic EwS cell lines. Finally, in the comment section a brief characterization of the cell lines, the tumor tissues derived from them, and the comparison of these tissues to patient primary EwS and paired metastatic samples are described to illustrate the clinical relevance of this preclinical model of EwS lung metastases.

BASIC PROTOCOL 1

Intra-tibial Injection in NSG Mice:

Introductory paragraph:

This protocol describes how an orthotopic xenograft model of Ewing Sarcoma is generated. Immunocompromised mice, at 4–6 weeks of age are used to inject into the tibia with human EwS cells. Care should be taken that no air-bubbles are introduced during the injection as that can cause death due to pulmonary embolism.

Materials:

Animals

  • NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Jackson Laboratories OR J:NU, Jackson Laboratories, male or female of 4–5 weeks of age.

Material and Reagents

  • Syringes 28G1/2 (FisherScientific, #329461)

  • Isoflurane 100mL (Covetrus #11695-6777-1)

  • Matrigel Matrix (CORNING, #354230)

  • Meloxicam (COVERTUS, #11695-6936-1)

  • RPMI (Gibco, #11875-093)

  • 0.25% Trypsin-EDTA (Gibco, #25200-056)

  • Fetal bovine Serum, qualified, United States (Gibco, # 10437-028)

Equipment

  • Single Animal vaporizer unit (Veterinary Anesthesia Systems, model# M3000), fitted with an induction chamber and individual inhalation masks for mice.

  • Micro-dissection scissor (FST, catalog number: 14060-09)

  • Micro-dissection forceps (FST, catalog number: 11251-35)

Methods:

Cell maintenance and preparation

  1. Ideally, cells should be maintained in culture only briefly before inoculation (recommend less than 1 week or until you have enough cells for the experiment), and a new vial should be thawed if changes in cell growth rate occur.

  2. Trypsinize cells at 80–90% confluence with 0.25% Trypsin/EDTA.

  3. Remove cells from plate with 10mL medium into 15mL conical tube and centrifuge at 200x g for 5 min.

  4. Resuspend pellet in a 15mL conical tube in 10 mL PBS and count via hemocytometry

  5. Centrifuge at 200x g for 5 min to re-pellet and resuspend at 5×105–106 cells/10μL ice-cold PBS.

  6. Cell suspension is mixed in equal ratio with Matrigel at the time of injection (20μL cells + 10μL Matrigel).

Intratibial injection

  1. Prepare cell suspension mixed in Matrigel and draw in syringe, dispel out air bubbles and excess to have only 30μL in syringe. Keep syringe on ice (Fig 1B–I).

  2. 4–6 weeks old mice are used for the injection.

  3. Inject mouse with Meloxicam (0.1 mg/kg) or similar approved analgesic immediately before procedure.

  4. Anesthetize mouse using isoflurane, then move to nose cone and maintain anesthesia using vaporizer (Fig 1B–II).

  5. Clean legs with 10% povidone/iodine swab/solution, followed by ethanol, repeating 2 times. Depilate legs (with Nair or similar product) prior to cleaning if fur present (Fig. 1B–III).

  6. With forefinger and thumb gently grasp lateral malleolus, medial malleolus, and the lower half of tibia, then bend leg (combination of flexion and lateral rotation), such that the knee is visible and accessible.

    Get the skin wet with 70% EtOH to increase visibility of underlying patellar ligament, which is visible as a distinct, thick, white line. Firmly holding the leg of mouse, insert 28g ½ needle under patella. Needle should be 2–3mm deep in the middle of patellar ligament, and into the anterior intercondylar area in top of tibia (Fig. 1B–IV).

  7. With slight but steady drilling action, guide the needle carefully through growth plate. When markedly less resistance is felt, it would signify the tibial growth plate has been penetrated through.

  8. Use a gentle, lateral movement of needle to ensure needle is in tibia and through the growth plate. Movement will be limited if needle is in proper place in the tibia (Fig. 1B–V).

  9. To inject 30μL of cell solution (20μL cells + 10μL Matrigel), slowly depress plunger. Little to no resistance should be felt at this point.

  10. Extract needle with caution.

  11. Remove mouse from anesthesia and keep on heating pad until recovered.

  12. Monitor the mice over the next 3 days for morbidity.

  13. The body weight and tumor volume of the mice are recorded every three days.

  14. For all models, the diameters of the tumors are determined by caliper measurements.

  15. Tumor volumes are calculated by using the following formula: V= (Length × Width2)/2.

  16. Each model has a slightly different time course to develop palpable tumors. For intratibial injected mice it is between 3–5 weeks post-injection.

  17. Once mice develop tumors ≥1.5 cm, sacrifice by CO2 euthanasia with cervical dislocation.

  18. Remove tumors for further study. Section tumors, flash freeze and store for proteins, RNA. If needed process it for single cell suspensions for subsequent assays and tumor cell culture.

  19. Fix in Formalin for histopathological evaluation.

Evaluation of Lung Metastatic Lesions

Please note this step is omitted if cell lines are being generated from the metastatic lesion as described in BASIC PROTOCOL 2

  1. Examine lungs for distant metastasis. With fluorescent tagged cells, metastatic lesions can be readily observed under a stereo-microscope like Leica MZ10F. In the absence of a fluorescent tag, they can be visualized as discolored small (<1 mm) spots appearing as a single lesion or in clusters.

  2. Remove and store metastatic lung lesions directly by snap freezing in liquid nitrogen for proteomic and/or in RNA-later followed by freezing in liquid nitrogen for transcriptomic analysis.

  3. For lung tissue to be fixed and processed for histological assessment, expose trachea gently by removing muscles surrounding the trachea.

    Cut a straight opening just enough to insert the needle, approximately 1 mm across in the exposed trachea, without completely cutting through. The cut should be made close to the larynx and parallel to the tracheal rings.

  4. Inject ~ 0.5 ml 10% formalin into the exposed trachea bevel side up toward the lung using a 23-gauge needle.

  5. Excise the lung lobes from trachea and immerse fixed lung in formalin for 12–16 hours for subsequent paraffin embedding and/or OCT-freezing media per standard histological procedures.

  6. Evaluate metastatic burden in lungs by serial sectioning of FFPE lung tissue and H&E staining.

CAUTION: Animals should be exposed to isoflurane for as short a duration as possible, and at the same time care should be taken that they are fully anesthetized by checking response to pinching toes of mice. All animals should be treated as per institutional guidelines under IACUC.

CAUTION: For larger experiments, cell suspension should be made in batches, as letting the suspension sit on ice for more than 30 minutes to 2 hours or more, depending upon the cell line, can lead to clumping and will not ensure proper distribution. The temperature of PBS is important to prevent clumping of cells and subsequent embolism after injection. The cell suspension should remain on ice while performing injections and will be viable and remain un-clumped for 30 min.

BASIC PROTOCOL 2

Development and Characterization of Lung Metastatic Cell Lines:

Introductory paragraph:

This protocol is in continuation with Basic Protocol 1. This describes how a lung metastasis line is created by repeated intratibial injection after isolating the metastatic lesion from lungs, followed by single cell suspension and culture of the cell line. Cells were fluorescently labeled for specificity during isolation and tracking. After tissue isolation, all steps should be performed under sterile conditions.

Materials:

Material and Reagents

  • RPMI (GIBCO #11875-093)

  • 0.25%Trypsin-EDTA (GIBCO #25200-056)

  • Fetal Bovine Serum, qualified, United States (GIBCO #10437-028)

  • Gentle MACS (TM) C Tubes (Miltenyi Biotec #130-096-334)

  • Micro-dissection scissor (FST, catalog number: 14060-09)

  • Micro-dissection forceps (FST, catalog number: 11251-35)

  • Cell strainers 70μm

  • Tissue culture dishes

  • Tumor Dissociation Kit human (Miltenyi Biotec #130-095-929)

Equipment

  • RotoFlex Tube Rotator (Argos #R2000) in combination with an incubator at 37°C

  • Gentle Macs Dissociator (Miltenyi Biotec #130-093-235)

  • Tabletop high speed centrifuge

Methods:

Excision of Lung Lesions and Dissociation

  1. Prepare the enzyme mix according to the manufacturer’s manual using the Tumor Dissociation Kit for human tissue.

  2. Resect the distant metastasis nodules, taking care to leave out a margin to avoid stromal contamination. Place nodules in sterile petri dishes on ice in a sterile laminar flow cabinet.

  3. Clean tumor tissue nodules by gently swirling around with PBS.

  4. Remove necrotic areas, blood clots with forceps and scissors or crossed scalpels.

  5. Cut the tumor into very small pieces of 2–4mm.

  6. Transfer the tissue into gentle MACS C-Tube containing the enzyme mix.

  7. Tightly close C Tube and attach it upside down onto the sleeve of the GentleMACS Dissociator (make sure that the sample material is within the area of the rotator). This is done at room temperature.

  8. Choose an appropriate GentleMACS program according to manual (used m-imp Tumor-02-02 C-Tube).

  9. After termination of the program, detach C-Tube from the machine.

  10. Incubate tissue samples for 40 minutes under continuous rotation using the MACS mix Tube.

  11. Resuspend sample and apply the cell suspension to Rotator.

  12. Perform a short centrifugation step (300xg) at room temperature to collect the sample material at the bottom of the tube.

  13. Perform the following steps using sterile conditions, under a laminar flow cabinet.

  14. Place a cell strainer (70μm) on a 50mL tube.

  15. Add homogenized tissue sample to strainer. Wash cell strainer with 10mL of RPMI.

  16. Centrifuge cell suspension at 300xg for 7minutes. Aspirate supernatant completely.

  17. Wash the cell with PBS and pellet the cell suspension for 5 minutes at 300xg and carefully aspirate the supernatant without disturbing the cell pellet.

  18. Resuspend the pellet in 4–10 mL of cell culture medium depending upon the size of the lesion and subsequent cell pellet. Plate the cells at 0.2 ×106 cells/4 mL media in 60 mm or 1 × 106 cells/10 mL in 100 mm tissue culture treated plates.

  19. Incubate cells at 37°C tissue culture incubator with 5% CO2.

  20. Change medium the next day, and then change medium continuously once every other day until confluence is reached.

Cell Maintenance and Preparation for Injection

  1. Passage confluent culture and seed again. After two or three passages, these tumor cell lines can be cryogenically preserved in complete medium, supplemented with 5% DMSO and 20% FBS and store in a liquid N2 tank. Further characterize cells with appropriate staining and histological analysis.

  2. Cells can then be labeled with fluorescent tags for sorting and in vivo tracking. Fluorescent labeling was done with Lipofectamine3000 Plus (Cocchiararo, Cornut, Soldati, Bonavoglia, & Castets, 2022). For cell specific transfection, consult protocol at manufacturer’s website (https://www.thermofisher.com/us/en/home/brands/product-brand/lipofectamine/lipofectamine-3000.html).

  3. Prepare labeled lung metastatic cells as described under basic protocol 1 from step 2 and follow until the end.

  4. Dissect and observe lungs for fluorescent lesion and resect nodule and repeat procedure as described in the protocol.

  5. This should be done several iterations and checked by mixing with parental cells to confirm for specificity.

  6. Depending upon the cell line, within 3–5 iterations, an organ specific metastatic line would be formed for further characterization.

CAUTION: With subsequent injections, time to form metastatic lesion may shorten. This may lead to the animal being sick while externally the primary tumor may remain much smaller. Care should be taken, and behavior should be monitored closely to prevent death of animal.

COMMENTARY:

Background Information:

Although rare, Ewing Sarcoma (EwS) is the second most common bone tumor in children, adolescents and young adults (Riggi & Stamenkovic, 2007). EwS is characterized by EWS-FLI1 (EF), a pathognomonic translocated protein arising due to a fusion between EWSR1 and FLI1. Studying EwS metastasis in its physiological context is limited due to the lack of applicable model systems. The cell of origin and the driving molecular mechanisms by which EWS-FLI1 mediates tumor progression remains limited, thus complicating further the development of an animal model. Although historically there have been several attempts to developed conditional genetically engineered mouse models (Minas et al., 2017), they have resulted in embryonic lethality, site non-specific expression, promoter leakiness and developmental defects. Other experimental models include the use of a 3D tissue-engineered bone-like matrix (Molina et al., 2020), Drosophila (Molnar et al., 2022), or zebrafish, with the latter expressing the pathognomonic EWS-FLI1 in a p53 null background having more successful EwS-like tumor development (Leacock et al., 2012). The drawback to these models is that the applications of these models are limited for any translational purposes. To circumvent a spontaneous or syngeneic model, cell-based models have been developed by several groups as xenografts from established cell lines, or by patient derived xenograft (PDX) implantations (Carrabotta et al., 2022; Surdez, Landuzzi, Scotlandi, & Manara, 2021). These models are helpful as they support the formation and growth of tumor, but they lack having a key component, the immune system. They also miss out on tumor initiation, and many of them do not show any metastatic colonization and growth.

The strategy adopted in this protocol is similar to the one used in developing metastatic breast and osteosarcoma models (Bos et al., 2009; Fan et al., 2021; Hu, Kang, & Wang, 2009). Instead of using tail-vein injections typically used to develop metastasis, our adoption of intra-tibial injection to spontaneously metastasize to lungs is closer to a true metastatic model, hence with more translational relevance. However, it needs to be noted that since the implantations employ immunocompromised mice, the role of the immune system in promoting, or inhibiting tumor cell dissemination and growth cannot be fully evaluated.

Even within its limitation, this model allows for analyzing differential molecular signatures by identifying pathways and proteins that are key components of the metastatic cascade and ultimately reveal potential targets for therapeutic intervention.

Critical Parameters:

It is important to keep in mind that this model is as robust as the cell lines used. Both TC71 and A673 are aggressive Ewing Sarcoma cell lines that metastasize to lungs via orthotopic injection. Having viable, mycoplasma and microbial-free cells will help with the injection as well as in tumor growth. Special attention should be given when excising out the lung lesion, so that stromal components are not included. Excision and cell dissociation should be done quickly to propagate healthy live cells.

Troubleshooting:

Understanding Results:

The technique described here lays out a protocol to develop a powerful tool to study lung metastasis. As seen from our data, once developed, these cell lines are highly aggressive. Additionally, although parental cells could form in both the lung (at a delayed time point, 4–5 weeks compared to 1–2 in NSG mice) and liver, these cells were specific to lungs (Fig. 2A). Hematoxylin-Eosin staining revealed numerous foci formed in lungs within 2 weeks in NSG mice and in 4 weeks in nude mice (Fig. 2B). Principal component analysis (PCA) using gene expression data from parental TC71, and TC71-Lm5 (5th iteration of lung-metastatic cells derived from TC71) cells demonstrated separate clustering (Fig. 3A). Of note, we observed clustering of the transcriptomic signatures for the TC71 parental cell line and primary bone tumor as well as the TC71 Lm5 cell line and in vivo Lm5 lung tumors. These models are effective tools for investigating lung organotropism, steps in metastatic cascade such extravasation, intravasation, colonization, and colony growth. We demonstrate this by characterizing these cells with a multi-omics approach including proteomics and glycoproteomics, secretome, RNA-sequencing, and Nanostring technology. Integration of focused proteomics and glycoproteomics using the two independent EwS cell lines of parental cells, and derived lung met cells, revealed differential expression of several key genes involved in critical pathways. Upregulation in AXL, MEK6, Stat1, and Stat3 proteins and phosphoprotein in metastatic lesions compared to the primary tumors were observed. In addition, modulation in several EMT associated proteins (Lambert & Weinberg, 2021; Vergara et al., 2016) were observed. Focused RNA profiling with Nanostring technologies using formalin-fixed paraffin-embedded (FFPE) slides of matched patient tumor samples revealed increased expression of several other upstream genes that can signal through STAT1, including IL-6, IKK-β, TRAF-6, and Myd88. Additionally, modulation in factors involved in the tumor invasive front such as Furin, TIMP-2, MMP-2, and MMP-14 were also noted. A Heatmap using normalized enrichment scores from GSEA analysis (Fig. 3B) shows overall upregulation of gene signatures representing pathways involved in extracellular matrix modelling, immune and cytokine signaling, angiogenesis, locomotion, and invasion (FDR <0.1), thus allowing us to explore underlying mechanisms and pathways that are crucial in EwS metastasis.

Figure 2. Lung Specific Metastatic Cell Line.

Figure 2.

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A. Organ specificity of lung metastatic cells was demonstrated as both parental (green) and lung-metastatic (red) cells were mixed and injected into the tibia, but only lung-metastatic cells formed lung metastatic lesions. B. Hematoxylin-Eosin staining from lungs showing metastatic foci 2 weeks post injection in NSG mouse and 4 weeks post injection in Nude mouse.

Figure 3. Preliminary characterization of lung metastatic cells.

Figure 3.

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A. Principal Component Analysis (PCA) score plot of transcriptomic data. B. GSEA overview from gene signatures that were differentially expressed in lung cell line over parental cell line (left panel), primary bone tumors in mice over lung lesions in mice (middle panel), and in patient lung metastatic lesion over primary patient bone tumor (right panel). Heatmap shows normalized enrichment scores (NES) for select GSEA gene sets that were significantly changed, FDR < 0.1.

Time Considerations:

Depending upon the EwS cell line, palpable tumors will be formed with 2–3 weeks of injection. Typically lung lesions are found 4–6 weeks post intratibial injection, but with the aggressive lung-specific cell lines developed here, lung metastatic lesions were found within 2–3 weeks.

Table 1.

Troubleshooting Guide for Intra-tibial Injection

Problem Possible Cause Solution
Hard to inject Older mice or cell suspension too viscous Choose younger mice and check cell counts; if cell suspension is sticky, dilute further.
Back Flow Injection needle did not penetrate tibial growth plate. Need to make sure tibial plate has been penetrated, resistance will be much less once that’s accomplished.
No Growth Cells did not grow, unhealthy cells, or not properly injected. Check for cell viability prior to injection.
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ACKNOWLEDGEMENTS: (mandatory for NIH, optional for all others)

TDP, RSK, and CC were partially supported by The Cancer Prevention Institute of Texas (CPRIT) grants RP170005, RP210227, RP200504, NIH P30 shared resource grant CA125123, NIEHS grants P30 ES030285 and P42 ES027725, and NIMHD grant P50 MD015496. TDP and JTY are supported by NIH grants R21 CA267914, R01 EB026453 and CA215452. AD and JTY are supported by NIH 1R21CA260331 and NIH 1R21CA255894. JTY is also supported by NIH 1R21HL157810.

Footnotes

CONFLICT OF INTEREST STATEMENT:

No conflict of interest to declare.

DATA AVAILABILITY STATEMENT:

The data, tools, and material (or their source) that support the protocol are available from the corresponding author upon reasonable request.

LITERATURE CITED:

  1. Abboud A, Masrouha K, Saliba M, Haidar R, Saab R, Khoury N, … Saghieh S (2021). Extraskeletal Ewing sarcoma: Diagnosis, management and prognosis. Oncol Lett, 21(5), 354. doi: 10.3892/ol.2021.12615 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bakir B, Chiarella AM, Pitarresi JR, & Rustgi AK (2020). EMT, MET, Plasticity, and Tumor Metastasis. Trends Cell Biol, 30(10), 764–776. doi: 10.1016/j.tcb.2020.07.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bielenberg DR, & Zetter BR (2015). The Contribution of Angiogenesis to the Process of Metastasis. Cancer J, 21(4), 267–273. doi: 10.1097/PPO.0000000000000138 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bos PD, Zhang XH, Nadal C, Shu W, Gomis RR, Nguyen DX, … Massague J (2009). Genes that mediate breast cancer metastasis to the brain. Nature, 459(7249), 1005–1009. doi: 10.1038/nature08021 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carrabotta M, Laginestra MA, Durante G, Mancarella C, Landuzzi L, Parra A, … Scotlandi K (2022). Integrated Molecular Characterization of Patient-Derived Models Reveals Therapeutic Strategies for Treating CIC-DUX4 Sarcoma. Cancer Res, 82(4), 708–720. doi: 10.1158/0008-5472.CAN-21-1222 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chaffer CL, & Weinberg RA (2011). A perspective on cancer cell metastasis. Science, 331(6024), 1559–1564. doi: 10.1126/science.1203543 [DOI] [PubMed] [Google Scholar]
  7. Cocchiararo I, Cornut M, Soldati H, Bonavoglia A, & Castets P (2022). Back to basics: Optimization of DNA and RNA transfer in muscle cells using recent transfection reagents. Exp Cell Res, 421(2), 113392. doi: 10.1016/j.yexcr.2022.113392 [DOI] [PubMed] [Google Scholar]
  8. Cotterill SJ, Ahrens S, Paulussen M, Jurgens HF, Voute PA, Gadner H, & Craft AW (2000). Prognostic factors in Ewing’s tumor of bone: analysis of 975 patients from the European Intergroup Cooperative Ewing’s Sarcoma Study Group. J Clin Oncol, 18(17), 3108–3114. doi: 10.1200/JCO.2000.18.17.3108 [DOI] [PubMed] [Google Scholar]
  9. Fan Z, Huang G, Zhao J, Li W, Lin T, Su Q, … Shen J (2021). Establishment and characterization of a highly metastatic human osteosarcoma cell line from osteosarcoma lung metastases. J Bone Oncol, 29, 100378. doi: 10.1016/j.jbo.2021.100378 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hanahan D, & Weinberg RA (2011). Hallmarks of cancer: the next generation. Cell, 144(5), 646–674. doi: 10.1016/j.cell.2011.02.013 [DOI] [PubMed] [Google Scholar]
  11. Hu G, Kang Y, & Wang XF (2009). From breast to the brain: unraveling the puzzle of metastasis organotropism. J Mol Cell Biol, 1(1), 3–5. doi: 10.1093/jmcb/mjp005 [DOI] [PubMed] [Google Scholar]
  12. Lambert AW, & Weinberg RA (2021). Linking EMT programmes to normal and neoplastic epithelial stem cells. Nat Rev Cancer, 21(5), 325–338. doi: 10.1038/s41568-021-00332-6 [DOI] [PubMed] [Google Scholar]
  13. Leacock SW, Basse AN, Chandler GL, Kirk AM, Rakheja D, & Amatruda JF (2012). A zebrafish transgenic model of Ewing’s sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis. Dis Model Mech, 5(1), 95–106. doi: 10.1242/dmm.007401 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Minas TZ, Surdez D, Javaheri T, Tanaka M, Howarth M, Kang HJ, … Uren A (2017). Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model. Oncotarget, 8(21), 34141–34163. doi: 10.18632/oncotarget.9388 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Molina ER, Chim LK, Salazar MC, Koons GL, Menegaz BA, Ruiz-Velasco A, … Mikos AG (2020). 3D Tissue-Engineered Tumor Model for Ewing’s Sarcoma That Incorporates Bone-like ECM and Mineralization. ACS Biomater Sci Eng, 6(1), 539–552. doi: 10.1021/acsbiomaterials.9b01068 [DOI] [PubMed] [Google Scholar]
  16. Molnar C, Reina J, Herrero A, Heinen JP, Méndiz V, Bonnal S, … Gonzalez C (2022). Human EWS-FLI protein recapitulates in Drosophila the neomorphic functions that induce Ewing sarcoma tumorigenesis. PNAS Nexus, 1(4). doi: 10.1093/pnasnexus/pgac222 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Nwabo Kamdje AH, Takam Kamga P, Tagne Simo R, Vecchio L, Seke Etet PF, Muller JM, … Krampera M (2017). Developmental pathways associated with cancer metastasis: Notch, Wnt, and Hedgehog. Cancer Biol Med, 14(2), 109–120. doi: 10.20892/j.issn.2095-3941.2016.0032 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Riggi N, & Stamenkovic I (2007). The Biology of Ewing sarcoma. Cancer Lett, 254(1), 1–10. doi: 10.1016/j.canlet.2006.12.009 [DOI] [PubMed] [Google Scholar]
  19. Shulman DS, Whittle SB, Surdez D, Bailey KM, de Alava E, Yustein JT, … Grohar PJ (2022). An international working group consensus report for the prioritization of molecular biomarkers for Ewing sarcoma. NPJ Precis Oncol, 6(1), 65. doi: 10.1038/s41698-022-00307-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Surdez D, Landuzzi L, Scotlandi K, & Manara MC (2021). Ewing Sarcoma PDX Models. Methods Mol Biol, 2226, 223–242. doi: 10.1007/978-1-0716-1020-6_18 [DOI] [PubMed] [Google Scholar]
  21. Vergara D, Simeone P, Franck J, Trerotola M, Giudetti A, Capobianco L, … Maffia M (2016). Translating epithelial mesenchymal transition markers into the clinic: Novel insights from proteomics. EuPA Open Proteom, 10, 31–41. doi: 10.1016/j.euprot.2016.01.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Winkler J, Abisoye-Ogunniyan A, Metcalf KJ, & Werb Z (2020). Concepts of extracellular matrix remodelling in tumour progression and metastasis. Nat Commun, 11(1), 5120. doi: 10.1038/s41467-020-18794-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Womer RB, West DC, Krailo MD, Dickman PS, Pawel BR, Grier HE, … Weiss AR (2012). Randomized controlled trial of interval-compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children’s Oncology Group. J Clin Oncol, 30(33), 4148–4154. doi: 10.1200/JCO.2011.41.5703 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data, tools, and material (or their source) that support the protocol are available from the corresponding author upon reasonable request.

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