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PMC12852540
Choosing the right animal model for sarcoma research
Accurate animal models must reflect not only the molecular characteristics of these tumours, but also their microenvironment and dynamic interaction with the host immune system.
PMC12852540
Choosing the right animal model for sarcoma research
The diversity of this group of cancers presents different challenges for animal model selection, particularly as sarcoma-specific resources are very limited.
PMC12852540
Choosing the right animal model for sarcoma research
Therefore, we have reviewed in detail several types of models, including: syngeneic (e.g., MCA205 and KRIMS series), chemically induced (e.g., MCA, DMBA), cell-derived xenografts (CDX; e.g., KCS8 and KCS9 osteosarcoma lines), patient-derived xenograft (PDX; e.g., pleomorphic leiomyosarcoma and GIST models), including humanised PDX (huPDX; e.g. HuNOG-EXL), and zebrafish (e.g. tp53M214K PNST and EWS-FLI1 transgenics) - to illustrate their sarcoma-specific use cases and discuss their advantages and limitations.
PMC12852540
Choosing the right animal model for sarcoma research
Genetically engineered models and their development are not a subject of this review, as they represent a very broad subject independently and are discussed elsewhere.
PMC12852540
Choosing the right animal model for sarcoma research
Some sarcomas are driven by simple translocations (e.g. EWS-FLI1 in Ewing’s Sarcoma), others by complex karyotypic instability or recurrent mutations, with corresponding differences translate into variable biology - including immune-microenvironment profiles - across subtypes .
PMC12852540
Choosing the right animal model for sarcoma research
Clinically, this heterogeneity is reflected in generally poor outcomes: The 5-year overall survival (OS) for Soft Tissue Sarcomas (STS) is 90% for stage I, 81% for stage II, and 56% for stage III , with the influence of multiple factors beyond TNM stage, including surgical margins, age, anatomic location, and histological factors, especially grade and subtype .
PMC12852540
Choosing the right animal model for sarcoma research
Studies have shown that median OS after first documentation of metastatic disease is approximately 24 months, with 1-, 2- and 5-year OS rates of 70%, 49.9% and 24.8%, respectively .
PMC12852540
Choosing the right animal model for sarcoma research
Therefore, there is the urgent need for further therapies development.
PMC12852540
Choosing the right animal model for sarcoma research
Adequately-chosen models improve the fidelity of preclinical testing, allowing scientists to distinguish between drugs that are truly promising and those that are unlikely to succeed in the clinic.
PMC12852540
Choosing the right animal model for sarcoma research
Better animal models streamline the drug development pipeline, preventing costly failures and accelerating the introduction of effective therapies to patients in need.
PMC12852540
Choosing the right animal model for sarcoma research
Preclinical sarcoma research encompasses 2D monolayers, 3D spheroids, organoids, and organotypic tissue slices [6–8].
PMC12852540
Choosing the right animal model for sarcoma research
While these platforms enable mechanistic interrogation and medium-to-high-throughput screening, they lack perfused vasculature, intact stroma and an adaptive immune system.
PMC12852540
Choosing the right animal model for sarcoma research
This limits analyses of exposure-response, metastatic tropism and host-tumour crosstalk [9–11].
PMC12852540
Choosing the right animal model for sarcoma research
Animal models restore the pharmacokinetics, vascular transport and myeloid-lymphoid interactions of the whole organism that are required for metastasis, drug delivery and immunotherapy read-outs .
PMC12852540
Choosing the right animal model for sarcoma research
Humanised mouse systems incorporate human-specific immune effectors and have longer timelines, higher costs and constraints relating to xenogeneic graft-versus-host disease (xGvHD) .
PMC12852540
Choosing the right animal model for sarcoma research
Rapid non-mammalian assays, such as zebrafish xenografts and chick CAM, can triage angiogenesis, invasion and short-course drug responses within days.
PMC12852540
Choosing the right animal model for sarcoma research
This reduces the use of mammals, but the results must be confirmed in mammalian models [15–17].
PMC12852540
Choosing the right animal model for sarcoma research
In line with the 3Rs and EU Directive 2010/63/EU, we cite non-animal methods where they credibly replace or reduce the need for animal studies, while reserving the use of mammalian models for questions that demand an intact organism.
PMC12852540
Choosing the right animal model for sarcoma research
The diversity of different models makes translational research in sarcoma particularly challenging.
PMC12852540
Choosing the right animal model for sarcoma research
Because each subtype is rare and biologically distinct, patients are underrepresented in clinical trials and subtype-specific data are scarce.
PMC12852540
Choosing the right animal model for sarcoma research
Similarly, approved treatments show highly variable responses between subtypes .
PMC12852540
Choosing the right animal model for sarcoma research
These factors - and the historically poor correlation between preclinical models and clinical outcomes - mean that many promising sarcoma therapies have stalled or failed in translation.
PMC12852540
Choosing the right animal model for sarcoma research
For example, the PDGFRα antibody olaratumab showed activity in cell line xenograft models and an initial phase II trial , but failed to improve survival in a definitive phase III STS trial .
PMC12852540
Choosing the right animal model for sarcoma research
This and similar discrepancies highlight how model selection can make or break translational success in sarcoma.
PMC12852540
Choosing the right animal model for sarcoma research
Syngeneic tumour models are among the oldest and most widely used preclinical models for testing anticancer therapies.
PMC12852540
Choosing the right animal model for sarcoma research
Syngeneic models involve transplanting tumour cells into immunocompetent mice of the same genetic background, enabling the study of tumour behaviour within a fully functional immune system.
PMC12852540
Choosing the right animal model for sarcoma research
Some of the earliest cancer models in mice were created using cell lines derived from tumours that developed spontaneously in inbred mouse strains .
PMC12852540
Choosing the right animal model for sarcoma research
Tumor cells are derived from the same mouse strain as the host, ensuring consistent tumor engraftment and growth without rejection, enhancing experimental reliability.
PMC12852540
Choosing the right animal model for sarcoma research
The driver mutations in these tumours usually do not match those found in human cancers of the same type.
PMC12852540
Choosing the right animal model for sarcoma research
While some spontaneous tumour-derived models show immune responses, these responses are generally weak or only partially induced.
PMC12852540
Choosing the right animal model for sarcoma research
Additionally, the models vary significantly from one another, making it challenging to determine which secondary models are most suitable for validating results .
PMC12852540
Choosing the right animal model for sarcoma research
This model is particularly relevant for understanding the interactions between tumours and the immune microenvironment, which are vital for the development and progression of sarcoma .
PMC12852540
Choosing the right animal model for sarcoma research
By using inbred mouse strains like C57BL/6, BALB/c, and FVB, researchers can develop tumour cell lines from spontaneous, carcinogen-induced, or transgenic sources, expand them in vitro, and then introduce them into similar wild-type mice to create a tumour-bearing system .
PMC12852540
Choosing the right animal model for sarcoma research
For instance, Nafia et al. used a syngeneic mouse model of sarcoma, specifically the MCA205 fibrosarcoma cell line, to study the immune response to anti-PDL1 immunotherapy.
PMC12852540
Choosing the right animal model for sarcoma research
MCA205 is a methylcholanthrene-induced fibrosarcoma derived on the C57BL/6 background - a canonical syngeneic model used for immuno-oncology studies.
PMC12852540
Choosing the right animal model for sarcoma research
As previously stated, in syngeneic models, tumour cells are derived from the same species and genetic background as the host, allowing for a fully functional immune system.
PMC12852540
Choosing the right animal model for sarcoma research
This makes them particularly valuable for immuno-oncology research as they more accurately reflect immune responses than models lacking immune components.
PMC12852540
Choosing the right animal model for sarcoma research
The MCA205 model allows researchers to study how immune checkpoint inhibitors, such as anti-PDL1, affect tumour growth in an immunocompetent environment.
PMC12852540
Choosing the right animal model for sarcoma research
This model is useful because it reflects specific tumour-immune interactions seen in sarcomas, including immune-related metabolic features.
PMC12852540
Choosing the right animal model for sarcoma research
Within the MCA205 tumour, the researchers observed distinct metabolic features such as kynurenine pathway activation, arginase activity and adenosine production - factors that contribute to the immunosuppressive tumour microenvironment and may influence response to therapies.
PMC12852540
Choosing the right animal model for sarcoma research
The model was further analysed using advanced techniques such as intratumoral microdialysis to assess pathways and flow cytometry and immunohistofluorescence to map immune cell populations within the tumour.
PMC12852540
Choosing the right animal model for sarcoma research
This allowed detailed profiling of immune dynamics, revealing increased CD8 + T cells and shifts in macrophage populations following treatment, highlighting the utility of this syngeneic sarcoma model for dissecting specific immune mechanisms.
PMC12852540
Choosing the right animal model for sarcoma research
In addition, the model confirmed that CD8 + T cells are essential for the anti-PDL1 response, as their depletion abolished the efficacy of the treatment.
PMC12852540
Choosing the right animal model for sarcoma research
Unlike xenograft models that use immunodeficient mice, syngeneic models maintain a functional immune system.
PMC12852540
Choosing the right animal model for sarcoma research
Therefore, it was used in a separate osteosarcoma syngeneic model to evaluate the combination of trabectedin and oncolytic herpes simplex virus (oHSV) therapy .
PMC12852540
Choosing the right animal model for sarcoma research
Osteosarcoma cells were subcutaneously or orthotopically implanted into syngeneic mice to generate tumors.
PMC12852540
Choosing the right animal model for sarcoma research
The site of implantation was chosen to replicate key features of osteosarcoma growth and to allow for effective monitoring of tumor size.
PMC12852540
Choosing the right animal model for sarcoma research
Trabectedin was administered systemically at doses optimized for murine models to evaluate its ability to modulate the tumor microenvironment and reduce immunosuppressive cells.
PMC12852540
Choosing the right animal model for sarcoma research
oHSV was delivered intratumorally to target and lyse tumor cells directly while promoting an immune response against tumor.
PMC12852540
Choosing the right animal model for sarcoma research
Trabectedin significantly reduced the number of immunosuppressive Tregs and TAMs in the tumour microenvironment.
PMC12852540
Choosing the right animal model for sarcoma research
This effect was enhanced by oHSV, which promoted immune recognition of tumour cells.
PMC12852540
Choosing the right animal model for sarcoma research
Increased infiltration of NK cells and CD8 + T cells was observed, indicating a shift towards a more immune-activating tumour microenvironment.
PMC12852540
Choosing the right animal model for sarcoma research
While trabectedin alone demonstrated moderate anti-tumour activity, its combination with oHSV resulted in enhanced tumour regression and prolonged survival in the syngeneic mice.
PMC12852540
Choosing the right animal model for sarcoma research
This synergy was attributed to the dual mechanisms of immune modulation by trabectedin and the oHSV-induced immune response.
PMC12852540
Choosing the right animal model for sarcoma research
As a murine model, it lacked the full genetic diversity and heterogeneity of human osteosarcomas, which may affect the applicability of the results to clinical settings.
PMC12852540
Choosing the right animal model for sarcoma research
This limitation was illustrated by Guttierrez et al. .
PMC12852540
Choosing the right animal model for sarcoma research
investigated syngeneic mouse models specifically derived from the K-Ras Induced Murine Sarcoma (KRIMS) series, which are derived from primary genetically engineered mouse models (GEMMs) designed to study undifferentiated pleomorphic sarcoma (UPS) and rhabdomyosarcoma (RMS).
PMC12852540
Choosing the right animal model for sarcoma research
The KRIMS series was developed by harvesting tumour cells from GEMM-derived sarcomas with Kras activation and p53 loss, and then transplanting these cells into immunocompetent syngeneic mice.
PMC12852540
Choosing the right animal model for sarcoma research
The syngeneic KRIMS models showed a different distribution of CD8 + T cells and Treg (regulatory T cells) compared to primary GEMM tumours.
PMC12852540
Choosing the right animal model for sarcoma research
Specifically, KRIMS tumours contained a higher proportion of CD8 + T cells, which are key players in the antitumor immune response, but with an altered activation state.
PMC12852540
Choosing the right animal model for sarcoma research
This difference in T-cell profiles may indicate an enhanced immune response in KRIMS models, which could influence the outcome of immunotherapy trials.
PMC12852540
Choosing the right animal model for sarcoma research
This model-specific immune variation highlights the complex interactions within the tumour microenvironment and demonstrates that while syngeneic models reflect certain features of the primary tumour, they also have unique immune profiles.
PMC12852540
Choosing the right animal model for sarcoma research
Unlike primary GEMMs, which can take months to develop spontaneous tumours, the KRIMS syngeneic models allow for rapid and consistent tumour formation upon injection, making them more time-efficient and accessible for high-throughput experiments.
PMC12852540
Choosing the right animal model for sarcoma research
The syngeneic model enables repeated, reliable tumour formation under standardised conditions, reducing the variability often seen with spontaneous GEMM tumour models and lowering the cost per experiment .
PMC12852540
Choosing the right animal model for sarcoma research
This reproducibility is particularly useful in preclinical studies where consistent tumour characteristics are critical.
PMC12852540
Choosing the right animal model for sarcoma research
The main advantage of this model is that it allows tumours to be studied in a fully functional, immunocompetent host.
PMC12852540
Choosing the right animal model for sarcoma research
Because the tumor cells and the host animal share the same genetic background, the immune response to the tumor remains intact, closely resembling the complex interplay between cancer cells and the immune system.
PMC12852540
Choosing the right animal model for sarcoma research
This facilitates the evaluation of immunotherapies, tumour-infiltrating immune cells and immune-mediated resistance mechanisms, providing valuable insights that are often not possible in immunodeficient or artificially humanised systems.
PMC12852540
Choosing the right animal model for sarcoma research
In addition, syngeneic models are relatively inexpensive, easy to establish and amenable to genetic manipulation, allowing for straightforward and reproducible experiments.
PMC12852540
Choosing the right animal model for sarcoma research
The main limitation of this model is its low translational relevance to human cancers.
PMC12852540
Choosing the right animal model for sarcoma research
Mouse tumours and their immune environment, including their stromal and vascular structures, often differ significantly from their human counterparts.
PMC12852540
Choosing the right animal model for sarcoma research
This can lead to discrepancies in how therapies, particularly immunomodulatory treatments, perform in the model compared with the clinic.
PMC12852540
Choosing the right animal model for sarcoma research
In addition, the genetic homogeneity, rapid tumor growth and sometimes less complex microenvironment found in syngeneic models may not accurately reflect the heterogeneity, slow progression and treatment-resistant nature of many human cancers.
PMC12852540
Choosing the right animal model for sarcoma research
While genetically engineered mouse (GEM) models are excellent at recapitulating specific genetic drivers of tumorigenesis and providing precise, reproducible insights into cancer biology, they often lack the genetic diversity and mutational complexity found in human cancers.
PMC12852540
Choosing the right animal model for sarcoma research
This limitation makes them less suitable for studying cancers with high mutational burden or exploring novel, unpredictable oncogenic pathways.
PMC12852540
Choosing the right animal model for sarcoma research
To address these gaps, chemically induced models offer an alternative approach, relying on random mutagenesis to generate tumours with significant heterogeneity that better reflect the complexity of human malignancies .
PMC12852540
Choosing the right animal model for sarcoma research
Chemically induced models involve administering carcinogenic compounds, such as MCA and 7,12-dimethylbenz[a]anthracene (DMBA), to induce tumour formation in laboratory animals .
PMC12852540
Choosing the right animal model for sarcoma research
This approach mimics the environmental contributions to cancer development and allows studying tumour biology in a controlled setting .
PMC12852540
Choosing the right animal model for sarcoma research
DMBA primarily induces mutations by forming DNA adducts that cause specific A→T transversions at certain hotspot codons, particularly in the ras gene family.
PMC12852540
Choosing the right animal model for sarcoma research
In addition to genetic mutations, DMBA also induces oxidative stress and cellular damage that contribute to tumourigenesis.
PMC12852540
Choosing the right animal model for sarcoma research
Its effects are well suited to study the carcinogen-induced fibrosarcomas, particularly in rodent models.
PMC12852540
Choosing the right animal model for sarcoma research
DMBA induces fibrosarcomas through its ability to form DNA adducts, leading to mutations in key oncogenes such as the ras gene family.
PMC12852540
Choosing the right animal model for sarcoma research
The researchers aimed to analyse the relationship between DMBA-induced chromosomal abnormalities, in particular gains in rat chromosome 2 (RNO2), and mutations in the Hras, Kras and Nras oncogenes .
PMC12852540
Choosing the right animal model for sarcoma research
Rats of the BN/Han and LE/Mol strains were used and crossed to produce genetically uniform F1 progeny.
PMC12852540
Choosing the right animal model for sarcoma research
DMBA was injected subcutaneously in two doses (1 mg or 2 mg) dissolved in tricapryline, resulting in tumour formation in 62% of the animals over a period of 220 days.
PMC12852540
Choosing the right animal model for sarcoma research
Tumour cells were analysed by karyotyping, PCR and DNA sequencing to identify chromosomal alterations and ras gene mutations.
PMC12852540
Choosing the right animal model for sarcoma research
A high frequency of tetraploid cells was observed in the tumours, with notable numerical increases in RNO2 gene material.
PMC12852540
Choosing the right animal model for sarcoma research
RNO2 gains were consistent across sarcomas, highlighting their importance in DMBA-induced carcinogenesis.
PMC12852540
Choosing the right animal model for sarcoma research
Mutations were found in codon 61 of Kras (CAA→CAT) and Nras (CAA→CTA), with an amino acid substitution to histidine and leucine, respectively.
PMC12852540
Choosing the right animal model for sarcoma research
These mutations were present in 18% of the tumours, suggesting an important but not exclusive role in tumour initiation.
PMC12852540
Choosing the right animal model for sarcoma research
Despite being induced in genetically uniform animals, tumours displayed diverse genetic alterations, reflecting the stochastic nature of DMBA carcinogenesis.
PMC12852540
Choosing the right animal model for sarcoma research
DMBA preferentially induces RAS pathway lesions and broad aneuploidy, and canonical RAS mutations are uncommon in human adult-type fibrosarcoma.
PMC12852540
Choosing the right animal model for sarcoma research
In the initial reports, tumour classification relied on histology and immunophenotype rather than molecular criteria.
PMC12852540
Choosing the right animal model for sarcoma research
Consequently, while the model is informative for carcinogen-driven fibroblastic sarcomagenesis, it should be considered only hypothesis-generating, rather than confirmatory, for adult fibrosarcoma.
PMC12852540
Choosing the right animal model for sarcoma research
Unlike DMBA, which predominantly targets the ras gene family, MCA’s mutagenic effects are less specific, affecting multiple oncogenes and tumor suppressors .
PMC12852540
Choosing the right animal model for sarcoma research
MCA is used to model slowly progressing tumours and to study interactions with the immune system, including immune editing and the role of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs).
PMC12852540
Choosing the right animal model for sarcoma research
It is valuable for studying the immune microenvironment but requires long tumour development times, making it less suitable for rapid studies.
PMC12852540
Choosing the right animal model for sarcoma research
MCA typically produces high-grade spindle-cell sarcomas with UPS- or fibrosarcoma-like histology, as well as polygenic mutation spectra that are shaped by immune editing.
PMC12852540
Choosing the right animal model for sarcoma research
While these tumours recapitulate slow carcinogenesis and immune suppression, they only partially mirror human driver architecture.
PMC12852540
Choosing the right animal model for sarcoma research
Consequently, they are better suited to studying tumour-immune dynamics and carcinogen responses than genotype-matched targeted therapy.