DIPG-50. A NOVEL ORTHOTOPIC PATIENT-DERIVED XENOGRAFT MODEL OF RADIATION-INDUCED GLIOMA

Abstract Diffuse midline glioma (DMG) can arise as a primary tumour but also as a consequence of radiation therapy (RT) in survivors of other paediatric brain tumours. Radiation-associated gliomas are molecularly distinct from primary gliomas and have poorer overall survival. We report a case of radiation-associated DMG following treatment for medulloblastoma, and the development of a matched patient-derived xenograft (PDX) model. A four-year-old boy diagnosed with medulloblastoma was treated with surgical resection, RT and chemotherapy (COG:CCG-99701-Arm B). Eleven years post-diagnosis, the patient relapsed with radiation-associated DMG, participated in a Phase I clinical trial (COG:ACNS0927), and passed away eight months later. Tumour tissue collected at autopsy was intracranially implanted into immunodeficient mice and serially transplanted in vivo. Immunohistochemistry demonstrated both the primary DMG and PDXs expressed PDGFR-alpha and PTEN, were H3K27me3-positive, and had undetectable levels of p53. Sequencing revealed an activating mutation in PI3-kinase (H1047L) and variants of unknown significance in GRK4, FLG, BAZ2A, and CRTC3. DNA methylation array of the PDX demonstrated 1p loss, which is not typically associated with primary DMG, and broad deletion within 9p including CDKN2A/B, MTAP and multiple interferon genes. The methylation profile did not significantly classify with other tumours in the Molecular Neuropathology database (molecularneuropathology.org/mnp). We describe the first reported PDX model of radiation-associated DMG following medulloblastoma, which recapitulates the patient disease and is molecularly distinct from primary DMG. Interrogation of this model through RNA and whole genome sequencing presents a valuable opportunity to better understand and identify novel therapeutic vulnerabilities against this currently incurable disease.

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Secondary gliosarcoma: the clinicopathological features and the development of a patient-derived xenograft model of gliosarcoma

BMC Cancer ◽

10.1186/s12885-021-08008-y ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Karrie Mei-Yee Kiang ◽

Andrian A. Chan ◽

Gilberto Ka-Kit Leung

Keyword(s):

Tumor Resection ◽

Xenograft Model ◽

Treatment Modalities ◽

Future Research ◽

Significant Implication ◽

Therapeutic Benefits ◽

Patient Derived Xenograft ◽

Pdx Model ◽

Immunocompromised Mice

Abstract Background Gliosarcoma (GSM) is a distinct and aggressive variant of glioblastoma multiforme (GBM) with worse prognosis and few treatment options. It is often managed with the same treatment modalities with temozolomide (TMZ) as in GBM. However, the therapeutic benefits on GSM from such treatment regimen is largely unknown. Patient-derived xenograft (PDX) models have been used widely to model tumor progression, and subsequently to validate biomarkers and inform potential therapeutic regimens. Here, we report for the first time the successful development of a PDX model of secondary GSM. Methods Tissue obtained from a tumor resection revealed a secondary GSM arising from GBM. The clinical, radiological, and histopathological records of the patient were retrospectively reviewed. Samples obtained from surgery were cultured ex vivo and/or implanted subcutaneously in immunocompromised mice. Histopathological features between the primary GBM, secondary GSM, and GSM PDX are compared. Results In explant culture, the cells displayed a spindle-shaped morphology under phase contrast microscopy, consistent with the sarcomatous component. GSM samples were subcutaneously engrafted into immunocompromised mice after single-cell suspension. Xenografts of serial passages showed enhanced growth rate with increased in vivo passage. We did not observe any histopathological differences between the secondary GSM and its serial in vivo passages of PDX tumors. Conclusions Our PDX model for GSM retained the histopathological characteristics of the engrafted tumor from the patient. It may provide valuable information to facilitate molecular and histopathological modelling of GSM and be of significant implication in future research to establish precise cancer medicine for this highly malignant tumor.

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Combination therapy of the EZH2 inhibitor GSK126 and the PARP inhibitor Olaparib shows in vivo synergy in a patient-derived xenograft model of BRCA1-deficient breast cancer

Geburtshilfe und Frauenheilkunde ◽

10.1055/s-0034-1388546 ◽

2014 ◽

Vol 74 (S 01) ◽

Author(s):

J Puppe ◽

P ter Brugge ◽

M Seressi ◽

O van Tellingen ◽

E van der Burg ◽

...

Keyword(s):

Breast Cancer ◽

Combination Therapy ◽

Parp Inhibitor ◽

Xenograft Model ◽

Patient Derived Xenograft

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TMOD-15. EFFICACY OF THE MDM2 INHIBITOR KRT-232 IN GLIOBLASTOMA PATIENT-DERIVED XENOGRAFT MODELS

Neuro-Oncology ◽

10.1093/neuonc/noaa215.966 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii231-ii231

Author(s):

Rachael Vaubel ◽

Ann Mladek ◽

Yu Zhao ◽

Shiv K Gupta ◽

Minjee Kim ◽

...

Keyword(s):

Target Genes ◽

Xenograft Model ◽

Culture Conditions ◽

Patient Derived Xenograft ◽

Fold Induction ◽

Extended Survival ◽

Mdm2 Amplification ◽

Mdm2 Inhibitor

Abstract Non-genotoxic reactivation of p53 by MDM2 inhibitors represents a promising therapeutic strategy for tumors with wild-type TP53, particularly tumors harboring MDM2 amplification. MDM2 controls p53 levels by targeting it for degradation, while disruption of the MDM2-p53 interaction causes rapid accumulation of p53 and activation of the p53 pathway. We examined the efficacy of the small molecule MDM2 inhibitor KRT-232, alone and in combination with radiation therapy (RT), in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of glioblastoma in vitro and in vivo. In vitro, glioblastoma PDX explant cultures showed sensitivity to KRT-232, both tumors with MDM2 amplification (GBM108 and G148) and non-amplified but TP53-wildtype lines (GBM10, GBM14, and GBM39), with IC50s ranging from 300-800 nM in FBS culture conditions. A TP53 p.F270C mutant PDX (GBM43) was inherently resistant, with IC50 >3000 nM. In the MDM2-amplified GBM108 line, KRT-232 led to a robust (5-6 fold) induction of p53-target genes p21, PUMA, and NOXA, with initiation of both apoptosis and senescence. Expression of p21 and PUMA was greater with KRT-232 in combination with RT (25-35 fold induction), while stable knock-down of p53 in GBM108 led to complete resistance to KRT-232. In contrast, GBM10 showed lower induction of p21 and PUMA (2-3 fold) and was more resistant to KRT-232. In an orthotopic GBM108 xenograft model, treatment with KRT-232 +/- RT for one week extended survival from 22 days (placebo) to 46 days (KRT-232 alone); combination KRT-232 + RT further extended survival (77 days) over RT alone (31 days). KRT-232 is an effective treatment in a subset of glioblastoma pre-clinical models alone and in combination with RT. Further studies are underway to understand the mechanisms conferring innate sensitivity or resistance to KRT-232.

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Induction of Acquired Resistance towards EGFR Inhibitor Gefitinib in a Patient-Derived Xenograft Model of Non-Small Cell Lung Cancer and Subsequent Molecular Characterization

Cells ◽

10.3390/cells8070740 ◽

2019 ◽

Vol 8 (7) ◽

pp. 740 ◽

Cited By ~ 5

Author(s):

Julia Schueler ◽

Cordula Tschuch ◽

Kerstin Klingner ◽

Daniel Bug ◽

Anne-Lise Peille ◽

...

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Cell Lung Cancer ◽

Xenograft Model ◽

Small Cell ◽

Small Cell Lung ◽

Protein Ratio ◽

Patient Derived Xenograft ◽

Resistant Lines

In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.

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Conditionally Reprogrammed Cells from Patient-Derived Xenograft to Model Neuroendocrine Prostate Cancer Development

Cells ◽

10.3390/cells9061398 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1398 ◽

Cited By ~ 2

Author(s):

Xinpei Ci ◽

Jun Hao ◽

Xin Dong ◽

Hui Xue ◽

Rebecca Wu ◽

...

Keyword(s):

Prostate Cancer ◽

Ex Vivo ◽

Cancer Cell Line ◽

Marker Genes ◽

Patient Derived Xenograft ◽

Pdx Model ◽

Neuroendocrine Prostate Cancer ◽

Parental Tumor

Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It develops mainly via NE transdifferentiation of prostate adenocarcinoma in response to androgen receptor (AR)-inhibition therapy. The study of NEPC development has been hampered by a lack of clinically relevant models. We previously established a unique and first-in-field patient-derived xenograft (PDX) model of adenocarcinoma (LTL331)-to-NEPC (LTL331R) transdifferentiation. In this study, we applied conditional reprogramming (CR) culture to establish a LTL331 PDX-derived cancer cell line named LTL331_CR_Cell. These cells retain the same genomic mutations as the LTL331 parental tumor. They can be continuously propagated in vitro and can be genetically manipulated. Androgen deprivation treatment on LTL331_CR_Cells had no effect on cell proliferation. Transcriptomic analyses comparing the LTL331_CR_Cell to its parental tumor revealed a profound downregulation of the androgen response pathway and an upregulation of stem and basal cell marker genes. The transcriptome of LTL331_CR_Cells partially resembles that of post-castrated LTL331 xenografts in mice. Notably, when grafted under the renal capsules of male NOD/SCID mice, LTL331_CR_Cells spontaneously gave rise to NEPC tumors. This is evidenced by the histological expression of the NE marker CD56 and the loss of adenocarcinoma markers such as PSA. Transcriptomic analyses of the newly developed NEPC tumors further demonstrate marked enrichment of NEPC signature genes and loss of AR signaling genes. This study provides a novel research tool derived from a unique PDX model. It allows for the investigation of mechanisms underlying NEPC development by enabling gene manipulations ex vivo and subsequent functional evaluations in vivo.

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37. IN VIVO FUNCTIONAL GENOMIC SCREEN TO IDENTIFY NOVEL DRIVERS OF LUNG-TO-BRAIN METASTASIS

Neuro-Oncology Advances ◽

10.1093/noajnl/vdaa073.025 ◽

2020 ◽

Vol 2 (Supplement_2) ◽

pp. ii6-ii7

Author(s):

Nikoo Aghaei ◽

Fred Lam ◽

Chitra Venugopal ◽

Sheila Singh

Keyword(s):

Brain Metastasis ◽

Brain Metastases ◽

Primary Tumour ◽

Unmet Need ◽

Xenograft Model ◽

Lung Tumour ◽

Loss Of Function ◽

Mri Imaging ◽

Genome Wide

Abstract Brain metastasis, the most common tumour of the central nervous system, occurs in 20–36% of primary cancers. In particular, 40% of patients with non-small cell lung cancer (NSCLC) develop brain metastases, with a dismal survival of approximately 4–11 weeks without treatment, and 16 months with treatment. This highlights a large unmet need to develop novel targeted therapies for the treatment of lung-to-brain metastases (LBM). Genomic interrogation of LBM using CRISPR technology can inform preventative therapies targeting genetic vulnerabilities in both primary and metastatic tumours. Loss-of-function studies present limitations in metastasis research, as knocking out genes essential for survival in the primary tumour cells can thwart the metastatic cascade prematurely. However, gene overexpression using CRISPR activation (CRISPRa) has the potential for overcoming dependencies of gene essentiality. We theorize that an in vivo genome-wide CRISPRa screen will identify novel genes that, when overexpressed, drive LBM. We have developed a patient-derived orthotopic murine xenograft model of LBM using primary patient-derived NSCLC cell lines (termed LTX cells) from the Swanton Lab TRACERx study. We are now poised to transduce LTX cells with a human genome-wide CRISPRa single guide RNA (sgRNA) library, and to subsequently inject the cells into the lungs of immunocompromised mice. We will then track the process of LBM using bioluminescent and MRI imaging until mice reach endpoint. Sequencing of primary lung tumours and subsequent brain metastases promises to uncover enriched sgRNAs, which may represent novel drivers of primary lung tumour formation and LBM. To the best of our knowledge, this study is the first in vivo genome-wide CRISPRa screen focused on identifying novel drivers of LBM, and can inform future preventative therapies to improve survival outcomes for NSCLC patients.

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Zebrafish research in Greece: swimming against the current

The International Journal of Developmental Biology ◽

10.1387/ijdb.210129db ◽

2021 ◽

Author(s):

Dimitris Beis

Keyword(s):

Disease Modeling ◽

Association Studies ◽

Genome Wide Association Studies ◽

Variants Of Unknown Significance ◽

Organ Regeneration ◽

Unknown Significance ◽

Vertebrate Model ◽

The Cost ◽

Mutant Lines

Zebrafish is a vertebrate model extensively used in Developmental Biology and Human Disease modeling as it shares high genetic and physiological similarities with humans. It has become the second most popular animal model, following mice, with several advantages: zebrafish are easily housed and cared for, the cost of installing and maintaining a zebrafish facility is significantly lower than mice, they reproduce often and develop quickly. Using zebrafish complies with the 3Rs principles of laboratory animal use. Zebrafish embryos develop externally and are transparent, allowing for in vivo non-invasive imaging. There are many transgenic and mutant lines available that mimic most Human Diseases, including reporter lines for most signaling pathways. There are also several reverse genetic tools to functionally verify genes or variants of unknown significance, identified in Genome-Wide Association Studies (GWAS) or using Next Generation Sequencing (NGS) approaches. In addition, the model emerges as an invaluable whole animal platform for various stages of drug discovery efforts by exploring the ability to do high-throughput phenotypic-driven screens. These include phenotypic screenings, determinations of general and/or specific toxicity (cardiac, renal, hepatotoxicity etc.), and mechanism of action studies. Finally, zebrafish are able to retain their capacity to regenerate most organs during their entire life span, making them a well-established model to study organ regeneration. The European Zebrafish Society consists of more than 180 research labs throughout Europe. In Greece however, zebrafish use remains rather limited. I present here a brief historical overview of zebrafish research in Greece.

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The Effects of PI3K-δ/γ Inhibitor, Duvelisib, in Mantle Cell Lymphoma in Vitro and in Patient-Derived Xenograft Studies

Blood ◽

10.1182/blood.v128.22.3016.3016 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3016-3016 ◽

Cited By ~ 4

Author(s):

Jack Wang ◽

Victoria Zhang ◽

Taylor Bell ◽

Yang Liu ◽

Hui Guo ◽

...

Keyword(s):

Growth Inhibition ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Cell Lymphoma ◽

Oral Gavage ◽

Patient Derived Xenograft ◽

Pdx Model ◽

Mantle Cell

Abstract Background: Mantle cell lymphoma (MCL) is an incurable subtype of B-cell lymphoma. Ibrutinib, a first-in-class, once-daily, oral covalent inhibitor of Bruton's tyrosine kinase (BTK) was approved by the FDA for the treatment of MCL in patients previously treated. In our prior multicenter Phase 2 clinical trial, the overall response rate in relapsed/refractory MCL was 68%, with a median progression-free survival (PFS) of 13.9 months. However, the majority of MCL patients treated with ibrutinib relapsed; in these relapsed patients, the one-year survival rate was only 22%. Therefore, there exists an urgent need for additional novel targeted therapies to improve the mortality rate in these patients. In this study, we assessed the in vitro and in vivo effects of duvelisib, a PI3K-δ,-γ inhibitor, in MCL. Methods: The PI3K/AKT/mTOR and other cell survival signaling pathways were investigated by RNASeq and reverse phase protein array (RPPA) in ibrutinib-sensitive and -resistant MCL samples. The expression of PI3K isoforms, α, β, γ, and δ was tested in 11 MCL cell lines, patient and patient-derived xenograft (PDX) MCL cells by western blot analysis. We then investigated the growth inhibition and apoptosis of duvelisib (IPI-145, Infinity Pharmaceuticals, Inc.) in MCL cells by CellTiter-Glo® Luminescent Cell Viability Assay (Promega) and Annexin V-binding assay (BD Biosciences). We established a primary MCL-bearing PDX model and passaged the primary MCL tumor to next generations. Mice were administrated with 50 mg/kg duvelisib daily by oral gavage. Tumor burden and survival time were investigated in the MCL-PDX model. Results: We found that the PI3K/AKT/mTOR signaling pathway was activated in both primary and acquired ibrutinib-resistant MCL cell lines and PDX MCL cells. We immunoblotted PI3K isoforms, α, β, γ, and δ in 11 MCL cell lines and the result demonstrated that both ibrutinib-sensitive and ibrutinib-resistant MCL cells dominantly expressed PI3K-δ and -γ. Next, we tested the effects of duvelisib on these MCL cells. Duvelisib had effects on the growth inhibition and apoptosis in both ibrutinib-sensitive and ibrutinib-resistant MCL cells as good as the PI3K-δ inhibitor, idelalisib (Cal-101, GS-1101). The PI3K-δ isoform could play a very important role in PI3K-mediated signals in MCL. We then investigated the effects of duvelisib in vivo through our established MCL-bearing PDX mouse models. These models are created by inoculating the primary tumor cells from MCL patients into a human fetal bone chip implanted into NSG mice to provide a microenvironment that reconstitutes the human environment. MCL tumor mass was then passaged to next generations for therapeutic investigation of duvelisib. Mice were treated with 50 mg/kg duvelisib daily by oral gavage. Our data demonstrated that duvelisib significantly inhibited tumor growth and prolonged survival of MCL-PDX mice. Conclusion: Duvelisib, an oral dual inhibitor of PI3K-δ,-γ, inhibits MCL growth both in vitro and in PDX mice. These preclinical results suggests duvelisib may be effective in the treatment of patients with relapsed/refractory MCL. Disclosures No relevant conflicts of interest to declare.

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Covalent 18F-Radiotracers for SNAPTag: a New Toolbox for Reporter Gene Imaging

10.20944/preprints202108.0260.v1 ◽

2021 ◽

Author(s):

Sophie Stotz ◽

Gregory D. Bowden ◽

Jonathan M. Cotton ◽

Bernd J. Pichler ◽

Andreas Maurer

Keyword(s):

Reporter Gene ◽

Xenograft Model ◽

Nuclear Imaging ◽

Hek293 Cells ◽

In Vivo Studies ◽

Reporter Gene Imaging ◽

Immunodeficient Mice ◽

Biodistribution Studies ◽

Sds Page

There is a need for versatile in vivo nuclear imaging reporter systems to foster preclinical and clinical research. We explore the applicability of the SNAPTag and novel radiolabeled small-molecule ligands as a versatile reporter gene system for in vivo nuclear imaging. SNAPTag is a high-affinity protein tag used in a variety of biochemical research areas and based on the suicide DNA repair enzyme O6-methylguanine methyl transferase (MGMT). Its ligands are well suited for reporter gene imaging as the benzyl guanine core scaffold can be derivatized with fluorescent or radiolabeled moieties for various applications. Three guanine-based SNAPTag ligands ([18F]FBBG, [18F]pFBG and [18F]mFBG) were synthesized in high yields and were (radio)chemically characterized. HEK293 cells were engineered to express the SNAPTag on the cell surface and served as cell model to assess target affinity by radiotracer uptake assays, Western blotting and SDS-PAGE autoradiography. A subcutaneous HEK293-SNAPTag xenograft model in immunodeficient mice was used for in vivo evaluation of [18F]FBBG amd [18F]pFBG while the biodistribution of [18F]mFBG was characterized in naïve animals. The results were validated by ex vivo biodistribution studies and immunofluorescence staining of the xenografts. All three radiotracers were produced in high radiochemical purity, molar activity and good yields. Western blot analysis revealed successful SNAPTag expression by the transfected HEK293 cells. In vitro testing revealed high target affinity of all three tracers with an up to 191-fold higher signal in the HEK293-SNAPTag cells compared to untransfected cells. This was further supported by a prominent radioactive protein band at the expected size in the SDS-PAGE autoradiograph of cells incubated with [18F]FBBG or [18F]pFBG. The in vivo studies demonstrated high uptake in HEK293-SNAP xenografts compared to HEK293 xenografts with excellent tumor-to-muscle ratios (7.5 ± 4.2 for [18F]FBBG and 10.6 ± 6.2 for [18F]pFBG). In contrast to [18F]pFBG and its chemical analogue [18F]mFBG, [18F]FBBG showed no signs of unspecific bone uptake and defluorination in vivo. Radiolabeled SNAPTag ligands bear great potential for clinical applications such as in vivo tracking of cell populations, antibody fragments and targeted radiotherapy. With excellent target affinity, good stability, and low non-specific binding, [18F]FBBG is a highly promising candidate for further preclinical evaluation.

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