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PMC12852540
Choosing the right animal model for sarcoma research
The accompanying illustration depicts the creation of a humanised patient-derived xenograft (PDX) model to sarcoma research.
PMC12852540
Choosing the right animal model for sarcoma research
The illustration depicts the methodology employed for the engraftment of human immune cells into an immunodeficient mouse, followed by the implantation of sarcoma tissue derived from a patient.
PMC12852540
Choosing the right animal model for sarcoma research
Researchers are exploring embryonic systems like the chorioallantoic membrane (CAM) and zebrafish embryos as alternatives to mouse PDX models (Fig. 2) .
PMC12852540
Choosing the right animal model for sarcoma research
These systems offer a faster and more affordable approach for tumor and patient-derived cells engraftment.
PMC12852540
Choosing the right animal model for sarcoma research
These models are largely used to study invasion, early metastatic behaviour and angiogenesis, and to perform short-term drug screening in a time- and resource-efficient manner that complements slower mammalian models .
PMC12852540
Choosing the right animal model for sarcoma research
Also, the transposition of the EU Directive 2010/63/EU on protecting animals used for scientific purposes under national laws has resulted in strict regulation, reflecting the growing ethical concerns around the use of adult animal experimentation .
PMC12852540
Choosing the right animal model for sarcoma research
As a result, researchers are focusing on non-mammalian embryonic models, including zebrafish larvae and chick embryos .
PMC12852540
Choosing the right animal model for sarcoma research
Embryonic models are subject to less stringent regulation until they can feed independently (chick embryo: hatching; zebrafish: 5 days post-fertilisation) and, crucially, until they develop the capacity to experience pain (chick embryo: >13 egg development day; zebrafish: 5 days post-fertilisation) .
PMC12852540
Choosing the right animal model for sarcoma research
Mice are kept in environments that meet animal welfare standards, but these conditions do not accurately reflect their natural habitats.
PMC12852540
Choosing the right animal model for sarcoma research
In contrast, embryonic systems are not exposed to environmental stressors.
PMC12852540
Choosing the right animal model for sarcoma research
Embryonic systems offer improved manageability, eliminating the need for animal facilities, while providing increased throughput and reduced costs [135–137].Fig.
PMC12852540
Choosing the right animal model for sarcoma research
2Steps to Develop a Zebrafish Model for Sarcoma Steps to Develop a Zebrafish Model for Sarcoma CAM (chorionic allantoic membrane) assays are increasingly being used in sarcoma research, particularly for rapid angiogenesis readouts, short-window xenograft growth and pilot drug response testing.
PMC12852540
Choosing the right animal model for sarcoma research
However, they remain supplementary to murine PDX/CDX/zebrafish pipelines rather than being the dominant preclinical standard, and therefore we only discuss their use briefly.
PMC12852540
Choosing the right animal model for sarcoma research
There are protocol papers and examples for fresh sarcoma tissue and cell-line grafts, including osteosarcoma and Ewing sarcoma, with extensions to rarer entities such as CIC-DUX4 and fibrosarcoma [138–142].
PMC12852540
Choosing the right animal model for sarcoma research
Contemporary reviews characterise CAM as a low-cost and rapid procedure that is well-suited to the study of vascular and invasion biology.
PMC12852540
Choosing the right animal model for sarcoma research
However, these reviews also underscore the limitations that hinder its complete replacement of mouse models.
PMC12852540
Choosing the right animal model for sarcoma research
These limitations encompass a brief observation window, the presence of non-mammalian stroma, and an immature immune system.
PMC12852540
Choosing the right animal model for sarcoma research
Collectively, these factors elucidate its niche and complementary utilisation in sarcoma programmes .
PMC12852540
Choosing the right animal model for sarcoma research
In practice, the majority of translational sarcoma groups continue to anchor efficacy packages in murine PDX/CDX for durability, PK/PD, and immuno-oncology, using CAM selectively for early ranking or mechanism-specific assays .
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish (Danio rerio) have emerged as an alternative to traditional mammalian models due to their rapid developmental cycles, optical transparency and low cost of maintenance (Table 3).
PMC12852540
Choosing the right animal model for sarcoma research
Their use is more frequent in sarcoma studies than CAM.
PMC12852540
Choosing the right animal model for sarcoma research
These characteristics allow researchers to study sarcoma initiation and progression in vivo, providing real-time insights into cellular dynamics.
PMC12852540
Choosing the right animal model for sarcoma research
Disease modelling in zebrafish is adaptable and can be carried out using a variety of methods, including the generation of gene-targeted mutations and stable transgenes, as well as the generation of fish with transient overexpression or downregulation of specific genes.
PMC12852540
Choosing the right animal model for sarcoma research
Initial forward genetic screens in zebrafish have shown that common mutagens, including ethylnitrosourea (ENU) and N-methylnitrosoguanidine (MNNG), induce a variety of neoplasms, including adenomas and rhabdomyosarcomas (RMS) .
PMC12852540
Choosing the right animal model for sarcoma research
One of the first zebrafish cancer models identified by an ENU screen was the fish with a tumour suppressor gene tp53 (tp53M214K) mutation.
PMC12852540
Choosing the right animal model for sarcoma research
TP53 is the most commonly mutated tumour suppressor gene in human malignancies.
PMC12852540
Choosing the right animal model for sarcoma research
Mutant tp53-/- animals develop malignant peripheral nerve sheath tumours (PNST), which are classified as a subtype of sarcoma.
PMC12852540
Choosing the right animal model for sarcoma research
PNST were rarely observed in wild-type (WT) fish.
PMC12852540
Choosing the right animal model for sarcoma research
The zebrafish phenotype partially mirrors the conditions seen in TP53-inactivated human patients.
PMC12852540
Choosing the right animal model for sarcoma research
They develop a variety of cancers in addition to sarcomas, including breast cancer, brain tumours and leukaemia .
PMC12852540
Choosing the right animal model for sarcoma research
A recent description describes a novel zebrafish model with a tp53del/del loss-of-function deletion allele developed in the CG1 syngeneic zebrafish strain.
PMC12852540
Choosing the right animal model for sarcoma research
These zebrafish exhibit a range of tumour types in addition to PNST, including leukaemia and germ cell tumours, paralleling conditions observed in human patients .
PMC12852540
Choosing the right animal model for sarcoma research
For instance, in rhabdomyosarcoma and liposarcoma can be induced in transgenic zebrafish by targeting the Ras-Raf-MEK-ERK and PI(3)K-Akt pathways, respectively, whereas hemangiosarcoma in zebrafish is associated with pten haploinsufficiency .
PMC12852540
Choosing the right animal model for sarcoma research
TP53 mutant zebrafish spontaneously develop PNST after around 8.5 months, which establishes Schwann-lineage sarcomagenesis in vivo Furthermore, engineered lines recapitulate driver-defined sarcomas.
PMC12852540
Choosing the right animal model for sarcoma research
RAS pathway activation is sufficient to induce embryonal rhabdomyosarcoma (RMS) in 49 out of 105 fish by 80 days post-fertilisation (dpf) Pten haploinsufficiency predisposes to haemangiosarcoma , and mosaic or Cre-inducible expression of EWSR1-FLI1 initiates Ewing sarcoma (ES)-like tumours with canonical markers, including CD99 .
PMC12852540
Choosing the right animal model for sarcoma research
The optical transparency of zebrafish embryos allows unparalleled visualisation of processes such as angiogenesis and metastasis .
PMC12852540
Choosing the right animal model for sarcoma research
In sarcoma studies, zebrafish models have been used to understand how tumours recruit blood vessels to support their growth and how cancer cells spread from primary tumours to distant sites.
PMC12852540
Choosing the right animal model for sarcoma research
Fluorescent imaging techniques allow cancer cells and their interactions with the tumour microenvironment to be tracked in real-time.
PMC12852540
Choosing the right animal model for sarcoma research
One of the studies using zebrafish to study metastasis in primary bone tumour including sarcomas obtained from surgical resections and prepared for implantation .
PMC12852540
Choosing the right animal model for sarcoma research
Tumour fragments were excised, cut into small pieces (~ 1/5 to 1/2 the size of zebrafish yolk) and stained with fluorescent dyes such as CM-DIL for visualisation.
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish embryos at two days post-fertilisation (dpf) were dechorionated and anaesthetised with tricaine to facilitate manipulation.
PMC12852540
Choosing the right animal model for sarcoma research
Transgenic zebrafish lines such as Tg(fli1:eGFP) with fluorescent vasculature were used to improve imaging of tumour interactions with blood vessels.
PMC12852540
Choosing the right animal model for sarcoma research
Tumor fragments or dissociated cells were injected via fine needles into the yolk sac or zebrafish embryos liver.
PMC12852540
Choosing the right animal model for sarcoma research
The yolk sac serves as a nutrient-rich site to support tumour growth, while liver implantation provides an organotypic environment to assess metastasis.
PMC12852540
Choosing the right animal model for sarcoma research
Confocal laser scanning microscopy was used to live track tumour cell invasion, migration and micrometastasis formation.
PMC12852540
Choosing the right animal model for sarcoma research
Tumour behaviour, including intravasation into blood vessels, circulation and colonisation of distant sites, was observed as early as 24 h post-transplantation.
PMC12852540
Choosing the right animal model for sarcoma research
Tumour cells infiltrated zebrafish tissues within hours of transplantation, indicating a high metastatic potential.
PMC12852540
Choosing the right animal model for sarcoma research
The cells also spread through the vasculature and colonised distant tissues such as the liver, intestine and caudal fin.
PMC12852540
Choosing the right animal model for sarcoma research
These behaviours mimicked metastatic patterns observed in human cancers, including sarcomas.
PMC12852540
Choosing the right animal model for sarcoma research
The implanted tumour cells exhibited migration and invasion distinct from non-malignant control tissues, which remained confined to the injection site.
PMC12852540
Choosing the right animal model for sarcoma research
The metastatic behaviour was critically dependent on the zebrafish vasculature.
PMC12852540
Choosing the right animal model for sarcoma research
In cloche mutant zebrafish lacking functional blood vessels, metastatic spread was significantly impaired, confirming that intravasation and vascular dissemination are essential for metastasis.
PMC12852540
Choosing the right animal model for sarcoma research
Also, Vasileva et al. .
PMC12852540
Choosing the right animal model for sarcoma research
developed an innovative zebrafish model of Ewing sarcoma (ES) to address critical gaps in understanding the role of the tumour microenvironment in ES initiation and progression.
PMC12852540
Choosing the right animal model for sarcoma research
ES is a highly aggressive bone and soft tissue tumour driven by the fusion oncogene EWSR1-FLI1 (EF1), a key driver of tumourigenesis.
PMC12852540
Choosing the right animal model for sarcoma research
The researchers created a Cre-inducible zebrafish model that allowed mosaic expression of the human EF1 fusion oncogene in a subset of zebrafish cells.
PMC12852540
Choosing the right animal model for sarcoma research
This broad mosaic expression resulted in the rapid onset of ES with high penetrance, closely mimicking the aggressive behaviour of human ES.
PMC12852540
Choosing the right animal model for sarcoma research
The model incorporated GFP-tagged cancer cells, allowing real-time imaging of tumour initiation and progression in the living organism.
PMC12852540
Choosing the right animal model for sarcoma research
Tumours generated in the zebrafish expressed canonical EF1 target genes and known ES markers such as CD99, validating the relevance of the model.
PMC12852540
Choosing the right animal model for sarcoma research
The ability of the zebrafish model to faithfully reproduce these molecular features confirmed its utility in studying ES biology.
PMC12852540
Choosing the right animal model for sarcoma research
Using live imaging, the researchers tracked the invasive behaviour of GFP-tagged cancer cells and analysed their interactions with the surrounding microenvironment during tumour initiation and progression.
PMC12852540
Choosing the right animal model for sarcoma research
Proteomic analysis revealed that EF1 expression dysregulates heparan sulphate proteoglycan (HSPG) metabolism and activates HSPG-mediated ERK signalling, pathways that promote cancer cell survival and proliferation.
PMC12852540
Choosing the right animal model for sarcoma research
The study also showed that the specific heparan sulphate antagonist Surfen effectively reduced ERK1/2 signalling and impaired the tumourigenicity of ES cells both in vitro and in vivo in the zebrafish model.
PMC12852540
Choosing the right animal model for sarcoma research
This Cre-inducible system overcame the limitations of low tumour initiation rates commonly seen in mammalian models by ensuring efficient and rapid tumour formation.
PMC12852540
Choosing the right animal model for sarcoma research
GFP labelling allowed direct visualisation of cancer cell behaviour, including invasion, proliferation and interactions with the extracellular matrix, in real time.
PMC12852540
Choosing the right animal model for sarcoma research
In addition, the zebrafish model allowed ES to be studied in a complex developmental and microenvironmental context, providing insights that are difficult to achieve in traditional mouse models.
PMC12852540
Choosing the right animal model for sarcoma research
The main advantage of zebrafish models in sarcoma research is their unique combination of cost effectiveness, rapid generation times and transparency, making them an exceptional tool for studying tumour development, angiogenesis and metastasis in real time.
PMC12852540
Choosing the right animal model for sarcoma research
The model’s pipeline is renowned for its short cycle times.
PMC12852540
Choosing the right animal model for sarcoma research
Injection at two days post-fertilisation yields invasion, dissemination and early treatment response readouts within 48–72 h post-engraftment.
PMC12852540
Choosing the right animal model for sarcoma research
This is enabled by the absence of a mature adaptive immune system until around 14 days post-fertilisation Across multiple series, concordance between drug sensitivity and dissemination and clinical behaviour has been demonstrated within a 3–5 day assay window, including patient-derived samples.
PMC12852540
Choosing the right animal model for sarcoma research
This supports the rapid execution of experiments that enable informed decision-making .
PMC12852540
Choosing the right animal model for sarcoma research
Practically, this compresses the end-to-end timeline for zebrafish xenografts to ≤ 1 week from sample preparation to the first pharmacodynamic readout under standard conditions .
PMC12852540
Choosing the right animal model for sarcoma research
The model’s pipeline is renowned for its short cycle times.
PMC12852540
Choosing the right animal model for sarcoma research
Injection at two days post-fertilisation yields invasion, dissemination and early treatment response readouts within 48–72 h post-engraftment.
PMC12852540
Choosing the right animal model for sarcoma research
This is enabled by the absence of a mature adaptive immune system until around 14 days post-fertilisation .
PMC12852540
Choosing the right animal model for sarcoma research
Across multiple series, concordance between drug sensitivity and dissemination and clinical behaviour has been demonstrated within a 3–5 day assay window, including patient-derived samples.
PMC12852540
Choosing the right animal model for sarcoma research
This supports the rapid execution of experiments that enable informed decision-making .
PMC12852540
Choosing the right animal model for sarcoma research
Practically, this compresses the end-to-end timeline for zebrafish xenografts to ≤ 1 week from sample preparation to the first pharmacodynamic readout under standard conditions .
PMC12852540
Choosing the right animal model for sarcoma research
Optical transparency of this model allows sarcoma cell behaviour visualisation, such as metastasis and dissemination, at high resolution without invasive procedures.
PMC12852540
Choosing the right animal model for sarcoma research
This is particularly useful for studying metastatic sarcomas such as osteosarcoma, which often spread to the lungs and other tissues.
PMC12852540
Choosing the right animal model for sarcoma research
Using fluorescently labelled tumour cells, zebrafish models allow direct observation of cellular dynamics, including interactions with the tumour microenvironment (TME) and blood vessels.
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish models are excellent at recapitulating key aspects of the TME, such as vascular development and immune interactions, which are critical for sarcoma progression.
PMC12852540
Choosing the right animal model for sarcoma research
For example, studies of soft tissue sarcomas have shown how tumour cells exploit angiogenesis to sustain growth and facilitate metastasis.
PMC12852540
Choosing the right animal model for sarcoma research
The conserved vascular architecture between zebrafish and humans allows researchers to study the effects of anti-angiogenic therapies, such as VEGF inhibitors like bevacizumab, in a physiologically relevant context .
PMC12852540
Choosing the right animal model for sarcoma research
The rapid development of zebrafish embryos also allows high-throughput screening of drug candidates, enabling the identification of novel therapies for sarcomas with limited treatment options, such as synovial sarcoma or alveolar rhabdomyosarcoma .
PMC12852540
Choosing the right animal model for sarcoma research
Another major advantage of the zebrafish model is that it is easily amenable to genetic manipulation.
PMC12852540
Choosing the right animal model for sarcoma research
Techniques such as CRISPR-Cas9 genome editing or morpholino oligonucleotide knockdowns can be used to rapidly generate zebrafish with genetic alterations that mimic human sarcoma drivers, such as TP53 mutations or fusion oncogenes such as EWS-FLI1 .
PMC12852540
Choosing the right animal model for sarcoma research
For example, zebrafish models of Ewing sarcoma have been generated by expressing the EWS-FLI1 fusion gene in mesenchymal progenitor cells, resulting in tumours with histological and molecular features similar to the human disease.
PMC12852540
Choosing the right animal model for sarcoma research
These models are invaluable for understanding how specific genetic alterations contribute to sarcomagenesis and for testing targeted therapies that target these genetic drivers.
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish also provide an efficient platform for studying sarcoma metastasis.
PMC12852540
Choosing the right animal model for sarcoma research
By injecting sarcoma cells into the bloodstream of zebrafish embryos, researchers can follow metastatic spread to secondary sites, such as the caudal fin or liver, in real time.
PMC12852540
Choosing the right animal model for sarcoma research
This ability has been particularly useful for investigating the mechanisms underlying lung metastasis of osteosarcoma, a major cause of patient mortality.
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish models have revealed how osteosarcoma cells interact with endothelial cells and the extracellular matrix to establish metastatic niches, providing insights into potential therapeutic targets to prevent metastasis .
PMC12852540
Choosing the right animal model for sarcoma research
The cost-effectiveness and scalability of zebrafish models further enhance their utility in sarcoma research.
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish are inexpensive to maintain compared to mouse models, and their high fecundity allows researchers to generate large cohorts for experiments in a short period of time.
PMC12852540
Choosing the right animal model for sarcoma research
This scalability makes zebrafish ideal for high-throughput drug screening, allowing thousands of compounds to be screened for anti-tumour activity.
PMC12852540
Choosing the right animal model for sarcoma research
Finally, zebrafish models provide valuable insights into sarcoma-immune interactions.
PMC12852540
Choosing the right animal model for sarcoma research
Although zebrafish lack an adaptive immune system during early stages of development, their innate immune system is functional and shares similarities with humans.
PMC12852540
Choosing the right animal model for sarcoma research
This makes them a useful model for studying innate immune responses to sarcoma, such as the recruitment of macrophages or neutrophils to the tumour site.
PMC12852540
Choosing the right animal model for sarcoma research
Recent advances in humanised zebrafish models, in which human immune cells are introduced, have further extended their applicability to immunotherapy studies, including the evaluation of immune checkpoint inhibitors or CAR-T cell therapies.