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PMC12852540
Choosing the right animal model for sarcoma research
The primary limitation of this model is the lack of a fully developed immune system in early embryonic stages limits their use for studying immune-related mechanisms of sarcoma progression or responses to immunotherapy.
PMC12852540
Choosing the right animal model for sarcoma research
In addition, differences in drug metabolism and genetic regulation between zebrafish and humans may affect the translational applicability of findings.
PMC12852540
Choosing the right animal model for sarcoma research
However, the development of adult immunodeficient zebrafish strains has extended their utility for xenotransplantation studies, including patient-derived sarcoma cells, thus filling some gaps in immunological studies .
PMC12852540
Choosing the right animal model for sarcoma research
Compared to PDXs, zebrafish models offer distinct advantages in scalability and visualisation, although PDXs retain the upper hand in recapitulating human tumour architecture and immune responses.
PMC12852540
Choosing the right animal model for sarcoma research
PDXs are better suited for long-term studies and assessing treatment response in a context that closely mimics human physiology .
PMC12852540
Choosing the right animal model for sarcoma research
However, their high cost, longer timelines and reliance on immunocompromised mice limit their utility for large-scale studies, where zebrafish excel.
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish models also complement syngeneic and humanised mouse models by providing rapid, cost-effective insights into genetic mutations, tumour dynamics and initial drug screening.
PMC12852540
Choosing the right animal model for sarcoma research
Table 3Summary of sarcoma studies using zebrafish modelsStudySarcoma subtype Model descriptionStudy findingsLeacock et al. Ewing sarcomaMosaic EWSR1-FLI1 expression under heat-shock or β-actin promoters; ± tp53 lossInduced small-round-blue-cell tumours; incidence increased on tp53-deficient background; transplantable lesionsKendal et al. .Alveolar rhabdomyosarcomaβ-actin/ubiquitin-driven PAX3-FOXO1 or PAX7-FOXO1 mosaics with lineage tracingFusion reprogrammed lineage and produced ARMS-like tumours; identified her3/HES3-mediated dependenciesLangenau et al. .Embryonal rhabdomyosarcomarag2:KRAS^G12D transgenic driving myogenic precursorsRobust ERMS with transplantability; established in vivo platform for genetics and drug testingIgnatius et al. .Embryonal rhabdomyosarcomaStage-specific KRAS^G12D driven by rag2, cdh15, mylz2 promotersPromoter and developmental timing dictated ERMS latency, location and differentiation statusStorer et al. .Embryonal rhabdomyosarcomaPatient-relevant TP53 variants modelled in KRAS^G12D ERMSTP53 genotype modulated ERMS initiation, proliferation and apoptosis; enabled functional TP53 classificationWatson et al. .CIC-rearranged round-cell sarcomaUbiquitous CIC-DUX4 expressionGenerated aggressive round-cell tumours; ETV4 activity was required for tumorigenesisBerghmans et al. .Malignant peripheral nerve sheath tumourtp53^M214K germline mutant with nf1a/b lossHigh-penetrance MPNST; nf1 loss cooperated with tp53 to accelerate onset and increase incidenceOppel et al. .Malignant peripheral nerve sheath tumoursuz12a/b CRISPR in tp53/nf1 mutant backgroundsPRC2 loss accelerated MPNST development and broadened tumour spectrum; epigenetic cooperation demonstratedChoorapoikayil et al. .Angiosarcomaptena^+/−; ptenb^+/− germline mutantsSpontaneous vascular tumours consistent with hemangiosarcoma/angiosarcoma predisposition; PI3K-pathway involvement supportedChen et al. .Angiosarcomatp53-null zebrafish linesBroad spontaneous tumour spectrum with high-penetrance angiosarcoma componentOppel et al. .SMARCB1-deficient sarcoma (incl.
PMC12852540
Choosing the right animal model for sarcoma research
epithelioid sarcoma)smarcb1 CRISPR knockout with p53-pathway inactivationGenerated SMARCB1-deficient sarcoma phenotypes including epithelioid sarcoma and angiosarcoma; enabled MDM2-pathway testing Summary of sarcoma studies using zebrafish models Sarcomas are divided into sarcomas with simple karyotypes, driven by specific genetic mutations such as the EWS-FLI1 fusion in Ewing sarcoma, and sarcomas with complex karyotypes, such as osteosarcoma and leiomyosarcoma, which exhibit widespread chromosomal instability and multiple genetic alterations.
PMC12852540
Choosing the right animal model for sarcoma research
These differences require tailored approaches to model development.
PMC12852540
Choosing the right animal model for sarcoma research
To gain more insight into these molecular events, scientists began using patient-derived sarcoma cell lines, which have been instrumental in modelling sarcoma progression, particularly through their application in xenograft experiments [168–170].
PMC12852540
Choosing the right animal model for sarcoma research
Despite these advancements, such models have significant limitations .
PMC12852540
Choosing the right animal model for sarcoma research
For instance, many sarcoma cell lines have been continuously passed over time, leading to changes in mutation rates and genetic stability due to cell culture shock .
PMC12852540
Choosing the right animal model for sarcoma research
Additionally, cell lines from individual patients reflect their unique genetic backgrounds, making it difficult to generalise findings across diverse genetic profiles .
PMC12852540
Choosing the right animal model for sarcoma research
Then, xenograft models using immunocompromised mice have helped advance cancer research by allowing scientists to study tumour growth, but they have limitations.
PMC12852540
Choosing the right animal model for sarcoma research
These models do not fully replicate the tumour’s behaviour in a living organism because they lack a functional immune system crucial for cancer progression and immune response .
PMC12852540
Choosing the right animal model for sarcoma research
Additionally, key interactions between the tumour and surrounding tissues must be included, making it harder to study how tumours behave in their natural environment .
PMC12852540
Choosing the right animal model for sarcoma research
GEMMs have proven useful in recapitulating specific genetic events, such as p53 and Rb deletions, that drive osteosarcoma formation .
PMC12852540
Choosing the right animal model for sarcoma research
Similarly, fusion-driven sarcomas such as Ewing sarcoma have been modelled using conditional expression systems, although challenges such as embryonic lethality and inconsistent tumour formation remain.
PMC12852540
Choosing the right animal model for sarcoma research
CRISPR-Cas9 technology has enabled rapid multi-gene editing, allowing the study of complex sarcoma subtypes, including undifferentiated pleomorphic sarcoma (UPS).
PMC12852540
Choosing the right animal model for sarcoma research
The combination of CRISPR and Cre-loxP systems has accelerated the generation of accurate models, reproducing features such as metastasis and heterogeneity with greater efficiency.
PMC12852540
Choosing the right animal model for sarcoma research
Compared to genetically defined cancer models, carcinogen-induced models exhibit greater genomic instability, creating a tumour microenvironment that more closely resembles real-world physiological conditions.
PMC12852540
Choosing the right animal model for sarcoma research
However, this complexity also presents challenges, including variability in tumour incidence (penetrance), delays in tumour development (latency) and the limited presence of common tumour antigens.
PMC12852540
Choosing the right animal model for sarcoma research
The utilisation of GEMMs is advocated for the elucidation of driver causality, lineage-of-origin and immune-competent metastasis.
PMC12852540
Choosing the right animal model for sarcoma research
Conditional alleles are employed to circumvent embryonic lethality and to synchronise tumour initiation with developmental windows.
PMC12852540
Choosing the right animal model for sarcoma research
The potential applications of somatic CRISPR-Cas9 ± Cre-loxP encompass the expeditious perturbation of multiple genes, facilitating the modelling of intratumoural heterogeneity and the efficient reproduction of metastatic dissemination.
PMC12852540
Choosing the right animal model for sarcoma research
In the field of carcinogen-induced models, the focus is directed towards immunoediting and antigenicity questions, as opposed to driver-matched therapeutics.
PMC12852540
Choosing the right animal model for sarcoma research
To ensure the management of variability, it is essential to specify penetrance and latency in advance .
PMC12852540
Choosing the right animal model for sarcoma research
PDXs and organoid models retain the genetic and phenotypic characteristics of primary tumours, making them valuable for the development of personalised therapies.
PMC12852540
Choosing the right animal model for sarcoma research
However, these models lack a functional immune system, which limits their use in immunotherapy research.
PMC12852540
Choosing the right animal model for sarcoma research
Syngeneic models provide insights into tumour-immune interactions, but are limited by their reduced genetic diversity.
PMC12852540
Choosing the right animal model for sarcoma research
In addition, challenges remain in modelling immunologically ‘cold’ sarcomas, which exhibit low immune activity and evade immune surveillance.
PMC12852540
Choosing the right animal model for sarcoma research
Advances in humanised mouse models and immunomodulatory therapies, such as CSF-1R and TGF-β inhibitors, offer potential solutions to these limitations.
PMC12852540
Choosing the right animal model for sarcoma research
We recommend PDXs for exposure–response, formulation and schedule optimisation with architectural fidelity, limiting passages to ≤ 3 and favouring orthotopic implantation when metastasis is the endpoint.
PMC12852540
Choosing the right animal model for sarcoma research
The potential applications of syngeneic models encompass the interrogation of tumour–immune interactions and immuno-oncology combinations, acknowledging constrained genetic diversity.
PMC12852540
Choosing the right animal model for sarcoma research
In circumstances where human-restricted immunity is deemed paramount, the deployment of humanised strains emerges as a rational approach, exhibiting set-up times of approximately 20–28 weeks and a graft-versus-host risk profile.
PMC12852540
Choosing the right animal model for sarcoma research
The employment of immunomodulatory strategies, such as CSF-1R or TGF-β blockade, has been shown to effect a conversion of ‘cold’ phenotypes into a state amenable to testing.
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish have emerged as a versatile and cost-effective alternative for cancer research.
PMC12852540
Choosing the right animal model for sarcoma research
Zebrafish models enable rapid, high-throughput studies of tumour growth, drug screening and angiogenesis, with the added benefit of real-time observation of tumour behaviour due to their transparent embryos.
PMC12852540
Choosing the right animal model for sarcoma research
Unlike huPDX models, which rely on the complex integration of the immune system, zebrafish offer simplicity and scalability, making them particularly suitable for early-stage drug discovery and functional genomics.
PMC12852540
Choosing the right animal model for sarcoma research
It is recommended that zebrafish xenografts be used as a preliminary screening method to determine the most effective treatment regimens and to quantify invasion/dissemination within 48–72 h and drug response within approximately 3–7 days.
PMC12852540
Choosing the right animal model for sarcoma research
This approach is intended to reduce the number of downstream mammalian cells required.
PMC12852540
Choosing the right animal model for sarcoma research
The potential applications of genetically defined zebrafish lines are manifold.
PMC12852540
Choosing the right animal model for sarcoma research
These include driver-specific hypothesis testing and scalable in vivo perturbation.
PMC12852540
Choosing the right animal model for sarcoma research
Furthermore, adaptive immunity matures at approximately 14 days post-fertilisation, which limits direct immunotherapy readouts but permits early assays without rejection.
PMC12852540
Choosing the right animal model for sarcoma research
Notably, Directive 2010/63/EU is predicated on the principles of replacement, reduction and refinement (3Rs) and protects fish from the independently feeding larval stage (in zebrafish ≈ 5 days post-fertilisation).
PMC12852540
Choosing the right animal model for sarcoma research
In several EU jurisdictions, CAM assays are often classified outside animal experimentation.
PMC12852540
Choosing the right animal model for sarcoma research
In 2021, the European Parliament issued a call to accelerate the transition to innovation that does not involve the use of animals, thereby signalling stronger expectations to justify the continued use of mammals.
PMC12852540
Choosing the right animal model for sarcoma research
Consequently, we recommend: (i) replacement where feasible with PDOs, tumour slices and organ-on-chip systems; (ii) reduction by using zebrafish/CAM as front-end screens and statistically powered “mouse clinical trials” with fewer passages; and (iii) refinement via improved analgesia, endpoints and longitudinal imaging.
PMC12852540
Choosing the right animal model for sarcoma research
Improving functional immunity across preclinical sarcoma models is likely to require coordinated host-, tumour- and microenvironment-level engineering.
PMC12852540
Choosing the right animal model for sarcoma research
In murine models, the integration of cytokine knock-in backbones that facilitate the organisation of myeloid, natural killer (NK), and T-cell compartments, along with the expression of human SIRPα to regulate CD47-SIRPα signalling, and the incorporation of HLA class I/II transgenes with autologous or HLA-matched reconstitution, has been shown to attenuate alloreactivity and facilitate antigen-restricted interrogation.
PMC12852540
Choosing the right animal model for sarcoma research
In order to preserve stromal crosstalk and antigenicity, it is recommended that co-engraftment of human fibroblasts and endothelium, orthotopic implantation, and low-passage material be utilised.
PMC12852540
Choosing the right animal model for sarcoma research
In GEMMs, the presence of switchable or lineage-restricted alleles, as well as human HLA or FcγR knock-ins, has been observed to enhance immune readouts while preserving the capacity for spontaneous metastasis.
PMC12852540
Choosing the right animal model for sarcoma research
In zebrafish models and CAM assays, immune add-backs, temperature-adapted protocols, and immune reporter lines could permit quantitative cytotoxic and trafficking readouts within short assay windows.
PMC12852540
Choosing the right animal model for sarcoma research
In zebrafish, adaptive immunity matures at approximately 14 days post-fertilisation, which constricts direct immunotherapy testing but enables scalable xenografts without rejection.
PMC12852540
Choosing the right animal model for sarcoma research
Across diverse platforms, the integration of barcoded perturbations, in conjunction with single-cell and spatial profiling, microbiome standardisation or humanisation, and predefined criteria for penetrance, latency and passage number, has the potential to enhance translational fidelity while concurrently minimising animal usage (Fig. 3).Fig.
PMC12852540
Choosing the right animal model for sarcoma research
3Preclinical xenograft and in vivo tumour models mapped to key translational applications Preclinical xenograft and in vivo tumour models mapped to key translational applications
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Gastrointestinal stromal tumors (GISTs) frequently show KIT mutations, accompanied by overexpression and aberrant localization of mutant KIT (MT-KIT).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
As previously established by multiple studies, including ours, we confirmed that MT-KIT initiates downstream signaling in the Golgi complex.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Basic leucine zipper nuclear factor 1 (BLZF1) was identified as a novel MT-KIT-binding partner that tethers MT-KIT to the Golgi complex.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Sustained activation of activated transcription factor 6 (ATF6), which belongs to the unfolded protein response (UPR) family, alleviates endoplasmic reticulum (ER) stress by upregulating chaperone expression, including heat shock protein 90 (HSP90), which assists in MT-KIT folding.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
BLZF1 knockdown and ATF6 inhibition suppressed both imatinib-sensitive and -resistant GIST in vitro.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
ATF6 inhibitors further showed potent antitumor effects in GIST xenografts, and the effect was enhanced with ER stress-inducing drugs.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
ATF6 activation was frequently observed in 67% of patients with GIST (n = 42), and was significantly associated with poorer relapse-free survival (P = 0.033).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Overall, GIST bypasses ER quality control (QC) and ER stress-mediated cell death via UPR activation and uses the QC-free Golgi to initiate signaling.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor in the stomach and small intestine, is a well-known model of oncogene addiction.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Approximately 75% of patients with GIST show primary gain-of-function mutations in KIT, and secondary mutations additionally occur in KIT during imatinib treatment .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
The primary mutations are mostly detected in exon 11 of KIT, and patients with these mutations show more than 80% of the overall response rate (ORR) to imatinib .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
The major challenge of GIST treatment is that there are insufficient regimens for patients showing poor response or developing resistance to imatinib .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Other kinase inhibitors, such as sunitinib and regorafenib, and more recently ripretinib, have been applied to patients who are refractory to imatinib.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
However, these drugs have shown limited therapeutic efficacy, especially in a metastatic setting .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Therefore, breakthroughs beyond KIT, such as identification of novel druggable targets and tumorigenic mechanisms, are needed for GIST treatment.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Mutant KIT (MT-KIT) was previously reported to be aberrantly localized in the Golgi complex and to exhibit sustained activation .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
However, it remains unclear how MT-KIT escapes the endoplasmic reticulum quality control (ERQC) system to reach the Golgi complex, and how the Golgi-retained MT-KIT contributes to GIST pathogenesis.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Therefore, identifying the mechanisms underlying these processes could provide important therapeutic insights into the challenges faced by conventional GIST treatment.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Cancer cells are constantly exposed to extrinsic and intrinsic stresses, such as hypoxia, nutrition insufficiency, and increased folding burden of proteins, which collectively cause ER stress and cell death.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
To survive unfavorable conditions, cancer cells activate the unfolded protein response (UPR) pathway, which ameliorates ER stresses by regulating protein degradation, chaperone expression, and adaptive pathways related to cell proliferation and survival .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
GIST is a relatively large solid tumor and most solid tumors develop regions of low oxygen tension.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
In addition, GIST exhibits MT-KIT folding stress .
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Therefore, GIST is subjected to chronic ER stress during tumorigenesis, which suggests the potential role of UPR in overcoming ER stress.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Moreover, UPR is involved in mutant protein folding, which also indicates that UPR might be involved in ERQC bypass and Golgi retention of MT-KIT.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
In this study, we aimed to elucidate the pathological mechanisms of Golgi-retained MT-KIT in GISTs to better understand its biological and clinical significance, and to investigate the unknown roles of ER stress and UPR to discover potential non-KIT drug targets.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Here, we show the underlying mechanism for the tumorigenic roles of Golgi-localized MT-KIT, with the assistance of BLZF1 and a novel pro-survival mechanism, based on constant activation of the ATF6-dependent UPR pathway in GIST.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
To detect the cellular localization of MT-KIT (GIST cells: GIST430 and GIST882) and wild-type KIT (colon cancer cells: DLD-1 and Colo320DM), immunocytochemistry (ICC) analysis was performed.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
In GIST cells, fluorescence signal of MT-KIT was barely observed at the plasma membrane (PM) without permeabilization, whereas a strong perinuclear fluorescence signal of MT-KIT was detected following permeabilization (Fig. 1A).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
In colon cancer (CC) cells, wild-type KIT (WT-KIT) was detected only in the PM, regardless of permeabilization (Fig. 1B).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
The perinuclear MT-KIT did not colocalize with the ER marker calnexin, but clearly colocalized with the cis- (GM130), medial- (mannosidase ll), and trans-Golgi (Golgin-97) markers (Fig. 1C, D).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Quantification of immunofluorescence intensity further demonstrated that MT-KIT evidently colocalized to the Golgi complex (Supplementary Fig. S1).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Deglycosylation analysis of MT- and WT-KIT further revealed that KIT localized in the ER (endoglycosidase H-sensitive) was barely detected in GIST and CC cells, which supports the hypothesis that MT-KIT in GISTs is primarily localized in post-ER organelles (Fig. 1E).
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Golgi localization of MT-KIT was validated in tissues from 42 patients with GIST.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Immunohistochemistry (IHC) analysis showed that perinuclear MT-KIT expression was detected in 28 patients (67%) (Supplementary Fig. S2 and Supplementary Table S1).Fig.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
1Mutant KIT (MT-KIT) and wild type KIT (WT-KIT) show perinuclear Golgi and membranous expression patterns, respectively.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
A, B Immunocytochemistry (ICC) analysis of KIT expression in gastrointestinal stromal tumor (GIST) (GIST430 and GIST882) and colon cancer (CC) cells (DLD-1 and Colo320DM) with or without permeabilization using triton X-100.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
C ICC analysis of KIT and calnexin (endoplasmic reticulum (ER) marker) in GIST cells.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
D ICC analysis of KIT, GM130 (cis-Golgi), mannosidase ll (medial-Golgi), and Golgin-97 (trans-Golgi) in GIST cells.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
E Lysates from GIST and CC cells were deglycosylated using endoglycosidase H (Endo-H) or PNGase-F, and analyzed by western blotting.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
F Biotinylation, streptavidin pull-down, and western blotting were performed using GIST and CC cells.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Because the surface expression of MT-KIT was low, twice the amount of biotin-labeled lysates was used for GIST cells than in CC cell lysates.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
G Quantification of the band intensities for the western blots as shown in F. H Stem cell factor (SCF)-treatment-mediated KIT degradation was measured by western blot lysates in GIST and CC cells.
PMC10589262
Identification of novel pathogenic roles of BLZF1/ATF6 in tumorigenesis of gastrointestinal stromal tumor showing Golgi-localized mutant KIT
Band intensities of the western blots were quantified.