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Supplementary Table S3. Related to Figure 3. Gene ontology for genes with higher expression in patient tumors vs. PDOXs. Supplementary Table S4. Related to Figure 3. List of the gene clusters defined across PDOXs #1-5 obtained from serial orthotopic passaging. Supplementary Table S5. Related to Figure 3. List of enriched Gene Ontology terms by cluster defined in PDOXs #1-5.

Supplementary Table S8. Related to Figure 5. List of top varying peptides based on SD across all samples derived from Patient/PDOX #5. Supplementary Table S9. Related to Figure 5. List of top varying phospho-peptides based on SD across all samples derived from Patient/PDOX #5. Supplementary Table S10. Related to Figure 5. List of enriched Gene Ontology terms by cluster derived from the protein clusters defined in Suppl. Table S8. Supplementary Table S11. Related to Figure 5. List of enriched Gene Ontology terms by cluster derived from the phospho-protein clusters defined in Suppl. Table S9. Supplementary Table S12. Related to Figure 5. List of differentially expressed proteins from group 1-3 of PDOX #5. Supplementary Table S13. Related to Figure 5. List of differentially expressed phosphorylated proteins from group 1-3 of PDOX #5. Supplementary Table S14. Related to Figure 5. List of peptides measured in the targeted PRM assay.

Supplementary Figure S2. Related to Figure 4. Intratumor heterogeneity is less prominent than intertumor heterogeneity.

Supplementary Table S6. Related to Figure 4. Top 1000 varying genes based on SD across all samples derived from Patient/PDOX #5. Supplementary Table S7. Related to Figure 4. List of enriched Gene Ontology terms by cluster derived from the gene clusters defined in Suppl. Table S6.

Supplementary Table S1. Related to Figure 1. List of all somatic mutations detected in patient tumors, and in PDOXs from low and high in vivo generations. Supplementary Table S2. Related to Figure 2. List of all chromosomal copy number changes in patient tumors, and in PDOXs from low and high in vivo generations.

Supplementary Figure S3. Related to Figure 5. Proteomic analysis of multiple samples derived from Patient #5.

Supplementary Figure S1. Related to Figure 3. Correlation of PDOX gene signatures to overall survival.

Patient-derived xenografts (PDX) and the Avatar, a single PDX mirroring an individual patient, are emerging tools in preclinical cancer research. However, the consequences of intratumor heterogeneity for PDX modeling of biomarkers, target identification, and treatment decisions remain underexplored. In this study, we undertook serial passaging and comprehensive molecular analysis of neuroblastoma orthotopic PDXs, which revealed strong intrinsic genetic, transcriptional, and phenotypic stability for more than 2 years. The PDXs showed preserved neuroblastoma-associated gene signatures that correlated with poor clinical outcome in a large cohort of patients with neuroblastoma. Furthermore, we captured spatial intratumor heterogeneity using ten PDXs from a single high-risk patient tumor. We observed diverse growth rates, transcriptional, proteomic, and phosphoproteomic profiles. PDX-derived transcriptional profiles were associated with diverse clinical characteristics in patients with high-risk neuroblastoma. These data suggest that high-risk neuroblastoma contains elements of both temporal stability and spatial intratumor heterogeneity, the latter of which complicates clinical translation of personalized PDX–Avatar studies into preclinical cancer research.Significance: These findings underpin the complexity of PDX modeling as a means to advance translational applications against neuroblastoma. Cancer Res; 78(20); 5958–69. ©2018 AACR.

Supplementary methods and legends to supplementary figures and tables

The prognosis for children with high-risk neuroblastoma is often poor and survivors can suffer from severe side effects. Predictive preclinical models and novel therapeutic strategies for high-risk disease are therefore a clinical imperative. However, conventional cancer cell line-derived xenografts can deviate substantially from patient tumors in terms of their molecular and phenotypic features. Patient-derived xenografts (PDXs) recapitulate many biologically and clinically relevant features of human cancers. Importantly, PDXs can closely parallel clinical features and outcome and serve as excellent models for biomarker and preclinical drug development. Here, we review progress in and applications of neuroblastoma PDX models. Neuroblastoma orthotopic PDXs share the molecular characteristics, neuroblastoma markers, invasive properties and tumor stroma of aggressive patient tumors and retain spontaneous metastatic capacity to distant organs including bone marrow. The recent identification of genomic changes in relapsed neuroblastomas opens up opportunities to target treatment-resistant tumors in well-characterized neuroblastoma PDXs. We highlight and discuss the features and various sources of neuroblastoma PDXs, methodological considerations when establishing neuroblastoma PDXs, in vitro 3D models, current limitations of PDX models and their application to preclinical drug testing.

Patient-derived xenografts (PDX) and the Avatar, a single PDX mirroring an individual patient, are emerging tools in preclinical cancer research. However, the consequences of intratumor heterogeneity for PDX modeling of biomarkers, target identification, and treatment decisions remain under-explored. In this study, we undertook serial passaging and comprehensive molecular analysis of neuroblastoma orthotopic PDXs, which revealed strong intrinsic genetic, transcriptional, and phenotypic stability for more than 2 years. The PDXs showed preserved neuroblastoma-associated gene signatures that correlated with poor clinical outcome in a large cohort of patients with neuroblastoma. Furthermore, we captured spatial intratumor heterogeneity using ten PDXs from a single high-risk patient tumor. We observed diverse growth rates, transcriptional, proteomic, and phosphoproteomic profiles. PDX-derived transcriptional profiles were associated with diverse clinical characteristics in patients with high-risk neuroblastoma. These data suggest that high-risk neuroblastoma contains elements of both temporal stability and spatial intratumor heterogeneity, the latter of which complicates clinical translation of personalized PDX-Avatar studies into preclinical cancer research. Significance: These findings underpin the complexity of PDX modeling as a means to advance translational applications against neuroblastoma. (C) 2018 AACR.

Cultured cancer cells serve as important models for preclinical testing of anti-cancer compounds. However, the optimal conditions for retaining original tumor features during in vitro culturing of cancer cells have not been investigated in detail. Here we show that serum-free conditions are critical for maintaining an immature phenotype of neuroblastoma cells isolated from orthotopic patient-derived xenografts (PDXs). PDX cells could be grown either as spheres or adherent on laminin in serum-free conditions with retained patient-specific genomic aberrations as well as tumorigenic and metastatic capabilities. However, addition of serum led to morphological changes, neuronal differentiation and reduced cell proliferation. The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were central for PDX cell proliferation and MYCN expression, and also hindered the serum-induced differentiation. Although serum induced a robust expression of neurotrophin receptors, stimulation with their cognate ligands did not induce further sympathetic differentiation, which likely reflects a block in PDX cell differentiation capacity coupled to their tumor genotype. Finally, PDX cells cultured as spheres or adherent on laminin responded similarly to various cytotoxic drugs, suggesting that both conditions are suitable in vitro screening models for neuroblastoma-targeting compounds.

Treatment of high-risk childhood neuroblastoma is a clinical challenge which has been hampered by a lack of reliable neuroblastoma mouse models for preclinical drug testing. We have previously established invasive and metastasising patient-derived orthotopic xenografts (PDXs) from high-risk neuroblastomas that retained the genotypes and phenotypes of patient tumours. Given the important role of the tumour microenvironment in tumour progression, metastasis, and treatment responses, here we analysed the tumour microenvironment of five neuroblastoma PDXs in detail. The PDXs resembled their parent tumours and retained important stromal hallmarks of aggressive lesions including rich blood and lymphatic vascularisation, pericyte coverage, high numbers of cancer-associated fibroblasts, tumour-associated macrophages, and extracellular matrix components. Patient-derived tumour endothelial cells occasionally formed blood vessels in PDXs; however, tumour stroma was, overall, of murine origin. Lymphoid cells and lymphatic endothelial cells were found in athymic nude mice but not in NSG mice; thus, the choice of mouse strain dictates tumour microenvironmental components. The murine tumour microenvironment of orthotopic neuroblastoma PDXs reflects important hallmarks of aggressive and metastatic clinical neuroblastomas. Neuroblastoma PDXs are clinically relevant models for preclinical drug testing.

Neuroblastoma is a tumor derived from immature neuroblasts predestined to form the sympathetic nervous system. Children with aggressive neuroblastoma are at high-risk of disease relapse despite their initial response to treatment. The molecular mechanisms underlying these relapses are poorly understood, although clonal expansion and de novo mutations have been suggested as potential mechanisms. An alternative hypothesis is that neuroblastoma initiating capacity and resistance to chemotherapy reside in a subpopulation of tumor initiating cells or cancer stem cells, which are not genetically different from most tumor bulk cells but through unknown mechanisms differ phenotypically and functionally. Here we examine aldehyde dehydrogenase (ALDH) expression and activity in neuroblastoma patient-derived xenograft (PDX) cells, cultured under stem cell-promoting conditions, as a potential marker for neuroblastoma stem cells. We found that neuroblastoma PDX cells in general expressed higher levels of various ALDH isoforms as compared to an aggressive classical serum-grown neuroblastoma cell line. Also, a distinct subgroup with high ALDH activity was observed in PDX cells but not in the serum-grown cell line. Growing the PDX cells in serum induced neuronal differentiation accompanied with lower expression of ALDH1 isoforms and reduced ALDH activity. The change in ALDH expression and activity was reversible, implying phenotypic plasticity. Re-analyzing cells initially sorted on ALDH activity also confirmed an inter-conversion of cells with low and high ALDH activity. Furthermore, in vitro clonogenic capacity was significantly higher in cells with high ALDH activity. Serial in vivo passaging of PDXs allowed us to compare PDX cells isolated from different in vivo generations. Interestingly, cells derived from later in vivo generations contained a higher frequency of cells with high ALDH activity. Finally, orthotopic injection of cells with either low or high ALDH activity showed that both subgroups led to tumor formation, however a higher tumor take was observed when injecting cells with high ALDH activity. In conclusion, our results suggest that ALDH activity is associated with an immature phenotype, which is of clinical importance since less differentiated tumors are more aggressive and associated with poor prognosis. Also, cells with high ALDH activity showed higher in vivo tumor initiating capacity. We are currently pursuing a more extensive characterization of neuroblastoma cells with high ALDH activity. Citation Format: Camilla Persson, Kristoffer von Stedingk, Daniel Bexell, My Merselius, Noémie Braekeveldt, Sven Påhlman, Caroline Wigerup. Assessment of ALDH activity in neuroblastoma patient-derived xenograft cells as a potential marker of neuroblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3880. doi:10.1158/1538-7445.AM2017-3880

Background Recently, we established orthotopic neuroblastoma patient-derived xenografts (PDXs) which maintain the phenotypic, genomic, and stromal hallmarks of patient tumors. Here we examined how PDXs evolve following years of in vivo growth. Materials and Methods We established up to eight in vivo generations of neuroblastoma orthotopic PDXs through serial passaging in NSG mice. RNA sequencing, exome sequencing and SNP array analysis were used to analyze patient tumors and PDXs from different in vivo generations. Results Using SNP analysis, we found mostly a remarkable genomic stability at chromosomal level between patient tumors, early and late PDX generations. RNA-seq revealed that patient tumors expressed higher levels of genes involved in immune responses and ECM metabolism compared to PDXs. Different PDX samples clustered correctly into their respective tumor type. PDXs from all early generations did not separate from PDXs from late generations. Thus, gene expression levels are surprisingly often quite stable despite years of in vivo growth. To shed light on neuroblastoma intratumor heterogeneity, we implanted 10 different tumor fragments from a single patient tumor into mice. We classified the 10 mice into three groups based on the time periods required for tumor growth. RNA-seq showed that each of these groups had a distinct gene expression profile and pathways involved in neuroblastoma progression have been identified. Conclusions Neuroblastoma orthotopic PDXs are often very stable at chromosomal and gene expression levels despite years of in vivo growth. We utilized multiple PDXs to show functional intratumor heterogeneity coupled to distinct gene expression profile. Citation Format: Noémie Braekeveldt, Susanne Fransson, Kristoffer von Stedingk, Ingrid Öra, Rosa Noguera, Tommy Martinsson, David Gisselsson Nord, Sven Påhlman, Daniel Bexell. Evolution of neuroblastoma patient-derived orthotopic xenografts through space and time [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1937. doi:10.1158/1538-7445.AM2017-1937

Genetic differences among neoplastic cells within the same tumour have been proposed to drive cancer progression and treatment failure. Whether data on intratumoral diversity can be used to predict clinical outcome remains unclear. We here address this issue by quantifying genetic intratumoral diversity in a set of chemotherapy-treated childhood tumours. By analysis of multiple tumour samples from seven patients we demonstrate intratumoral diversity in all patients analysed after chemotherapy, typically presenting as multiple clones within a single millimetre-sized tumour sample (microdiversity). We show that microdiversity often acts as the foundation for further genome evolution in metastases. In addition, we find that microdiversity predicts poor cancer-specific survival (60%; P=0.009), independent of other risk factors, in a cohort of 44 patients with chemotherapy-treated childhood kidney cancer. Survival was 100% for patients lacking microdiversity. Thus, intratumoral genetic diversity is common in childhood cancers after chemotherapy and may be an important factor behind treatment failure.

Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease often have a poor outcome. Here we describe the establishment of neuroblastoma patient-derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high risk neuroblastoma into immunodeficient mice. In vivo tumour growth was monitored by magnetic resonance imaging and fluorodeoxyglucose–positron emission tomography. Neuroblastoma PDXs retained the undifferentiated histology and proliferative capacity of their corresponding patient tumours. The PDXs expressed neuroblastoma markers neural cell adhesion molecule, chromogranin A, synaptophysin and tyrosine hydroxylase. Whole genome genotyping array analyses demonstrated that PDXs retained patient-specific chromosomal aberrations such as MYCN amplification, deletion of 1p and gain of chromosome 17q. Thus, neuroblastoma PDXs recapitulate the hallmarks of high-risk neuroblastoma in patients. PDX-derived cells were cultured in serum-free medium where they formed free-floating neurospheres, expressed neuroblastoma gene markers MYCN, CHGA, TH, SYP and NPY, and retained tumour-initiating and metastatic capacity in vivo. PDXs showed much higher degree of infiltrative growth and distant metastasis as compared to neuroblastoma SK-N-BE(2)c cell line-derived orthotopic tumours. Importantly, the PDXs presented with bone marrow involvement, a clinical feature of aggressive neuroblastoma. Thus, neuroblastoma PDXs serve as clinically relevant models for studying and targeting high-risk metastatic neuroblastoma. What's new? Neuroblastoma is a childhood tumour with heterogeneous characteristics and children with metastatic disease have a poor outcome. Here, the authors established neuroblastoma patient-derived xenografts (PDXs) by orthotopic implantation of viably cryopreserved or fresh tumour explants of patients with high-risk neuroblastoma into immunodeficient mice. The PDXs retained the genotype and phenotype of patient tumours and exhibited substantial infiltrative growth and metastasis to distant organs including bone marrow. PDX-derived neuroblastoma cells were expanded in vitro and retained tumourigenic and metastatic capacity in vivo. The PDXs may thus represent an important tool for investigating neuroblastoma growth and metastasis as well as drug targeting.

Background: We previously established neuroblastoma patient-derived orthotopic xenografts (PDXs) by implanting patient neuroblastoma fragments into immunodeficient NSG mice. SNP array analysis confirmed that PDXs maintain patient-specific chromosomal aberrations 1p del, MYCN amp and 17q gain. Immunohistochemistry showed that PDXs retain neuroblastoma markers and a highly infiltrative growth pattern. Importantly, we found spontaneous distant metastasis to lungs, liver and bone marrow. In vitro cultures established from the PDXs express neuroblastoma markers and retain their tumorigenic and metastatic ability in vivo after orthotopic injection. Methods and Results: Given the important role of the tumor stroma for tumor progression and treatment response, we examined PDX stroma by immunohistochemistry. PDXs were highly vascularized with mouse endothelial cells and two PDX models also formed tumor vasculature by co-engrafted human tumor endothelial cells. Tumors contained pericytes, cancer-associated fibroblasts, macrophages and extracellular components resembling the patient disease. PDXs established in athymic nude mice additionally developed intratumoral lymphatic vessels and contained mouse-derived CD45+ lymphoid cells. Thus, PDXs reflect important tumor microenvironment hallmarks for high-stage neuroblastoma. Furthermore, we demonstrate the feasibility of using short-term in vitro cultured PDX-derived cells as a drug-testing model, and show that the HIF2A oncogene is transcriptionally regulated at hypoxia via the PI3K/mTORC2 pathway in these cells. The results establish the PI3K and mTORC2 pathways as potential therapeutic targets of aggressive neuroblastoma. Conclusion: Neuroblastoma orthotopic PDXs reflect clinical aspects of high-risk disease including spontaneous metastasis, copy number changes and microenvironmental hallmarks. Neuroblastoma PDXs are relevant models for in vitro and in vivo preclinical drug testing. Citation Format: Daniel Bexell, Noémie Braekeveldt, Sofie Mohlin, Caroline Wigerup, David Gisselsson, Irene Tadeo, Ana P. Berbegall, Sam Navarro, Rosa Noguera, Sven Påhlman. Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B23.

Widespread metastasis is a major problem for the treatment of high-risk neuroblastoma. Relevant neuroblastoma animal models are hence needed to study and target high-risk metastatic neuroblastoma. We developed neuroblastoma patient-derived orthotopic xenografts (PDXs) using viably cryopreserved or fresh patient neuroblastoma fragments which were implanted orthotopically into immunodeficient NSG mice. Immunohistochemistry showed that PDXs retain neuroblastoma markers and a highly infiltrative growth pattern. Importantly, we found distant metastasis to lungs, liver and bone marrow. Single nucleotide polymorphism array analysis confirmed that PDXs maintain patient-specific chromosomal aberrations 1p del, MYCN amp and 17q gain. In vitro cultures established from the PDXs express neuroblastoma markers and retain their tumorigenic and metastatic ability in vivo after orthotopic injection. PDXs were highly vascularized with mouse endothelial cells and one PDX model formed tumor vasculature through co-engrafted human tumor endothelial cells. Tumors also contained pericytes, cancer-associated fibroblasts, macrophages and extracellular components resembling the patient disease. PDXs established in athymic nude mice additionally developed intratumoral lymphatic vessels and contained mouse-derived CD45+ lymphoid cells. Thus, PDXs maintain important tumor microenvironment hallmarks for high stage neuroblastoma. Furthermore, we demonstrate the feasibility of using short-term in vitro cultured PDX-derived cells as a drug-testing model, and show that the HIF2A oncogene is transcriptionally regulated at hypoxia via the PI3K/mTORC2 pathway in these cells. The results establish the PI3K and mTORC2 pathways as potential therapeutic targets of aggressive neuroblastoma. In conclusion, neuroblastoma orthotopic PDXs resemble many clinical aspects of the patient disease, e.g., distant metastases, and PDXs are valuable models for preclinical drug testing. Citation Format: Noémie Braekeveldt, Caroline Wigerup, David Gisselsson, Sofie Mohlin, My Merselius, Siv Beckman, Irene Tadeo, Anna P. Berbegall, Ingrid Öra, Samuel Navarro, Rosa Noguera, Sven Påhlman, Daniel Bexell. Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A02.

Neuroblastoma is a childhood tumor located in the sympathetic nervous system and most often found in the adrenal gland. Half of the patients have metastases already at diagnosis. There is a need for improved preclinical in vivo models of human high-stage neuroblastoma. Aims: 1) To establish relevant xenograft in vivo models for studying neuroblastoma orthotopic growth and metastatic growth in bone marrow, 2) to detect tumor growth by the two imaging modalities utilized in the clinic, i.e. magnetic resonance imaging (MRI) and I-123-metaiodobenzylguanidine (MIBG)-Single photon emission computed tomography (SPECT), and 3) to characterize orthotopic and bone marrow tumor growth at a pathohistological level. Methods: SK-N-Be2c and KCN-69 neuroblastoma cells were injected into adrenal gland of immunodeficient NSG mice to establish orthotopic tumors. SK-N-Be2c cells were injected directly into femur bone marrow of NSG mice to model bone marrow metastatic growth in a reproducible way. Tumor growth was monitored by MRI or by MIBG-SPECT. Histopathological assessment was performed on formalin-fixed paraffin-embedded tissue sections. Conclusion: We have established a model for orthotopic neuroblastoma growth and characterized the model by MRI and MIBG-SPECT. Orthotopic tumors grow infiltratively into surrounding tissues and resemble the histopathological hallmarks of patient neuroblastoma. We also describe a reproducible in vivo model for neuroblastoma bone marrow metastatic growth. The in vivo models will be important tools for studying mechanisms of neuroblastoma growth and for preclinical drug testing. Citation Format: Daniel Bexell, Noémie Braekeveldt, Siv Beckman, Sofie Mohlin, David Gisselsson, Sven Påhlman. Modeling human metastatic neuroblastoma in immunodeficient mice. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B57.