Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Integrative analysis of neuroblastoma by single-cell RNA sequencing identifies the NECTIN2-TIGIT axis as a target for immunotherapy.

Pediatric patients with high-risk neuroblastoma have poor survival rates and urgently need more effective treatment options with less side effects. Since novel and improved immunotherapies may fill this need, we dissect the immunoregulatory interactions in neuroblastoma by single-cell RNA-sequencing of 24 tumors (10 pre- and 14 post-chemotherapy, including 5 pairs) to identify strategies for optimizing immunotherapy efficacy. Neuroblastomas are infiltrated by natural killer (NK), T and B cells, and immunosuppressive myeloid populations. NK cells show reduced cytotoxicity and T cells have a dysfunctional profile. Interaction analysis reveals a vast immunoregulatory network and identifies NECTIN2-TIGIT as a crucial immune checkpoint. Combined blockade of TIGIT and PD-L1 significantly reduces neuroblastoma growth, with complete responses (CR) in vivo. Moreover, addition of TIGIT+PD-L1 blockade to standard relapse treatment in a chemotherapy-resistant Th-ALKF1174L/MYCN 129/SvJ syngeneic model induces CR. In conclusion, our integrative analysis provides promising targets and a rationale for immunotherapeutic combination strategies.

Ewing Sarcoma Single-cell Transcriptome Analysis Reveals Functionally Impaired Antigen-presenting Cells.

Novel therapeutic strategies are urgently needed for patients with high-risk Ewing sarcoma and for the reduction of severe side effects for all patients. Immunotherapy may fill this need, but its successful application has been hampered by a lack of knowledge on the composition and function of the Ewing sarcoma immune microenvironment. Here, we explore the immune microenvironment of Ewing sarcoma, by single-cell RNA sequencing of 18 Ewing sarcoma primary tissue samples. Ewing sarcoma is infiltrated by natural killer, T, and B cells, dendritic cells, and immunosuppressive macrophages. Ewing sarcoma-associated T cells show various degrees of dysfunction. The antigen-presenting cells found in Ewing sarcoma lack costimulatory gene expression, implying functional impairment. Interaction analysis reveals a clear role for Ewing sarcoma tumor cells in turning the Ewing sarcoma immune microenvironment into an immunosuppressive niche. These results provide novel insights into the functional state of immune cells in the Ewing sarcoma tumor microenvironment and suggest mechanisms by which Ewing sarcoma tumor cells interact with, and shape, the immune microenvironment. SIGNIFICANCE: This study is the first presenting a detailed analysis of the Ewing sarcoma microenvironment using single-cell RNA sequencing. We provide novel insight into the functional state of immune cells and suggests mechanisms by which Ewing tumor cells interact with, and shape, their immune microenvironment. These insights provide help in understanding the failures and successes of immunotherapy in Ewing sarcoma and may guide novel targeted (immuno) therapeutic approaches.

ErbB2 (HER2)-CAR-NK-92 cells for enhanced immunotherapy of metastatic fusion-driven alveolar rhabdomyosarcoma.

Introduction: Metastatic rhabdomyosarcoma (RMS) is a challenging tumor entity that evades conventional treatments and endogenous antitumor immune responses, highlighting the need for novel therapeutic strategies. Applying chimeric antigen receptor (CAR) technology to natural killer (NK) cells may offer safe, effective, and affordable therapies that enhance cancer immune surveillance. Methods: Here, we assess the efficacy of clinically usable CAR-engineered NK cell line NK-92/5.28.z against ErbB2-positive RMS in vitro and in a metastatic xenograft mouse model. Results: Our results show that NK-92/5.28.z cells effectively kill RMS cells in vitro and significantly prolong survival and inhibit tumor progression in mice. The persistence of NK-92/5.28.z cells at tumor sites demonstrates efficient antitumor response, which could help overcome current obstacles in the treatment of solid tumors. Discussion: These findings encourage further development of NK-92/5.28.z cells as off-the-shelf immunotherapy for the treatment of metastatic RMS.

Systematic discovery of gene fusions in pediatric cancer by integrating RNA-seq and WGS.

BACKGROUND: Gene fusions are important cancer drivers in pediatric cancer and their accurate detection is essential for diagnosis and treatment. Clinical decision-making requires high confidence and precision of detection. Recent developments show RNA sequencing (RNA-seq) is promising for genome-wide detection of fusion products but hindered by many false positives that require extensive manual curation and impede discovery of pathogenic fusions. METHODS: We developed Fusion-sq to overcome existing disadvantages of detecting gene fusions. Fusion-sq integrates and "fuses" evidence from RNA-seq and whole genome sequencing (WGS) using intron-exon gene structure to identify tumor-specific protein coding gene fusions. Fusion-sq was then applied to the data generated from a pediatric pan-cancer cohort of 128 patients by WGS and RNA sequencing. RESULTS: In a pediatric pan-cancer cohort of 128 patients, we identified 155 high confidence tumor-specific gene fusions and their underlying structural variants (SVs). This includes all clinically relevant fusions known to be present in this cohort (30 patients). Fusion-sq distinguishes healthy-occurring from tumor-specific fusions and resolves fusions in amplified regions and copy number unstable genomes. A high gene fusion burden is associated with copy number instability. We identified 27 potentially pathogenic fusions involving oncogenes or tumor-suppressor genes characterized by underlying SVs, in some cases leading to expression changes indicative of activating or disruptive effects. CONCLUSIONS: Our results indicate how clinically relevant and potentially pathogenic gene fusions can be identified and their functional effects investigated by combining WGS and RNA-seq. Integrating RNA fusion predictions with underlying SVs advances fusion detection beyond extensive manual filtering. Taken together, we developed a method for identifying candidate gene fusions that is suitable for precision oncology applications. Our method provides multi-omics evidence for assessing the pathogenicity of tumor-specific gene fusions for future clinical decision making.

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors.

Background: Liquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors. Materials and methods: Regions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed. Results: TLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse. Conclusion: We demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.

Single-cell transcriptomics reveals immune suppression and cell states predictive of patient outcomes in rhabdomyosarcoma.

Paediatric rhabdomyosarcoma (RMS) is a soft tissue malignancy of mesenchymal origin that is thought to arise as a consequence of derailed myogenic differentiation. Despite intensive treatment regimens, the prognosis for high-risk patients remains dismal. The cellular differentiation states underlying RMS and how these relate to patient outcomes remain largely elusive. Here, we use single-cell mRNA sequencing to generate a transcriptomic atlas of RMS. Analysis of the RMS tumour niche reveals evidence of an immunosuppressive microenvironment. We also identify a putative interaction between NECTIN3 and TIGIT, specific to the more aggressive fusion-positive (FP) RMS subtype, as a potential cause of tumour-induced T-cell dysfunction. In malignant RMS cells, we define transcriptional programs reflective of normal myogenic differentiation and show that these cellular differentiation states are predictive of patient outcomes in both FP RMS and the less aggressive fusion-negative subtype. Our study reveals the potential of therapies targeting the immune microenvironment of RMS and suggests that assessing tumour differentiation states may enable a more refined risk stratification.

Molecular Characterization Reveals Subclasses of 1q Gain in Intermediate Risk Wilms Tumors.

Chromosomal alterations have recurrently been identified in Wilms tumors (WTs) and some are associated with poor prognosis. Gain of 1q (1q+) is of special interest given its high prevalence and is currently actively studied for its prognostic value. However, the underlying mutational mechanisms and functional effects remain unknown. In a national unbiased cohort of 30 primary WTs, we integrated somatic SNVs, CNs and SVs with expression data and distinguished four clusters characterized by affected biological processes: muscle differentiation, immune system, kidney development and proliferation. Combined genome-wide CN and SV profiles showed that tumors profoundly differ in both their types of 1q+ and genomic stability and can be grouped into WTs with co-occurring 1p-/1q+, multiple chromosomal gains or CN neutral tumors. We identified 1q+ in eight tumors that differ in mutational mechanisms, subsequent rearrangements and genomic contexts. Moreover, 1q+ tumors were present in all four expression clusters reflecting activation of various biological processes, and individual tumors overexpress different genes on 1q. In conclusion, by integrating CNs, SVs and gene expression, we identified subgroups of 1q+ tumors reflecting differences in the functional effect of 1q gain, indicating that expression data is likely needed for further risk stratification of 1q+ WTs.

Implementation of paediatric precision oncology into clinical practice: The Individualized Therapies for Children with cancer program 'iTHER'.

iTHER is a Dutch prospective national precision oncology program aiming to define tumour molecular profiles in children and adolescents with primary very high-risk, relapsed, or refractory paediatric tumours. Between April 2017 and April 2021, 302 samples from 253 patients were included. Comprehensive molecular profiling including low-coverage whole genome sequencing (lcWGS), whole exome sequencing (WES), RNA sequencing (RNA-seq), Affymetrix, and/or 850k methylation profiling was successfully performed for 226 samples with at least 20% tumour content. Germline pathogenic variants were identified in 16% of patients (35/219), of which 22 variants were judged causative for a cancer predisposition syndrome. At least one somatic alteration was detected in 204 (90.3%), and 185 (81.9%) were considered druggable, with clinical priority very high (6.1%), high (21.3%), moderate (26.0%), intermediate (36.1%), and borderline (10.5%) priority. iTHER led to revision or refinement of diagnosis in 8 patients (3.5%). Temporal heterogeneity was observed in paired samples of 15 patients, indicating the value of sequential analyses. Of 137 patients with follow-up beyond twelve months, 21 molecularly matched treatments were applied in 19 patients (13.9%), with clinical benefit in few. Most relevant barriers to not applying targeted therapies included poor performance status, as well as limited access to drugs within clinical trial. iTHER demonstrates the feasibility of comprehensive molecular profiling across all ages, tumour types and stages in paediatric cancers, informing of diagnostic, prognostic, and targetable alterations as well as reportable germline variants. Therefore, WES and RNA-seq is nowadays standard clinical care at the Princess Máxima Center for all children with cancer, including patients at primary diagnosis. Improved access to innovative treatments within biology-driven combination trials is required to ultimately improve survival.

Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes.

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions.

Hypoxic, glycolytic metabolism is a vulnerability of B-acute lymphoblastic leukemia-initiating cells.

High-risk forms of B-acute lymphoblastic leukemia (B-ALL) remain a therapeutic challenge. Leukemia-initiating cells (LICs) self-renew and spark relapse and therefore have been the subject of intensive investigation; however, the properties of LICs in high-risk B-ALL are not well understood. Here, we use single-cell transcriptomics and quantitative xenotransplantation to understand LICs in MLL-rearranged (MLL-r) B-ALL. Compared with reported LIC frequencies in acute myeloid leukemia (AML), engraftable LICs in MLL-r B-ALL are abundant. Although we find that multipotent, self-renewing LICs are enriched among phenotypically undifferentiated B-ALL cells, LICs with the capacity to replenish the leukemic cellular diversity can emerge from more mature fractions. While inhibiting oxidative phosphorylation blunts blast proliferation, this intervention promotes LIC emergence. Conversely, inhibiting hypoxia and glycolysis impairs MLL-r B-ALL LICs, providing a therapeutic benefit in xenotransplantation systems. These findings provide insight into the aggressive nature of MLL-r B-ALL and provide a rationale for therapeutic targeting of hypoxia and glycolysis.

Improved Gene Fusion Detection in Childhood Cancer Diagnostics Using RNA Sequencing.

PURPOSE: Gene fusions play a significant role in cancer etiology, making their detection crucial for accurate diagnosis, prognosis, and determining therapeutic targets. Current diagnostic methods largely focus on either targeted or low-resolution genome-wide techniques, which may be unable to capture rare events or both fusion partners. We investigate if RNA sequencing can overcome current limitations with traditional diagnostic techniques to identify gene fusion events. METHODS: We first performed RNA sequencing on a validation cohort of 24 samples with a known gene fusion event, after which a prospective pan-pediatric cancer cohort (n = 244) was tested by RNA sequencing in parallel to existing diagnostic procedures. This cohort included hematologic malignancies, tumors of the CNS, solid tumors, and suspected neoplastic samples. All samples were processed in the routine diagnostic workflow and analyzed for gene fusions using standard-of-care methods and RNA sequencing. RESULTS: We identified a clinically relevant gene fusion in 83 of 244 cases in the prospective cohort. Sixty fusions were detected by both routine diagnostic techniques and RNA sequencing, and one fusion was detected only in routine diagnostics, but an additional 24 fusions were detected solely by RNA sequencing. RNA sequencing, therefore, increased the diagnostic yield by 38%-39%. In addition, RNA sequencing identified both gene partners involved in the gene fusion, in contrast to most routine techniques. For two patients, the newly identified fusion by RNA sequencing resulted in treatment with targeted agents. CONCLUSION: We show that RNA sequencing is sufficiently robust for gene fusion detection in routine diagnostics of childhood cancers and can make a difference in treatment decisions.

Identification and characterization of relapse-initiating cells in MLL-rearranged infant ALL by single-cell transcriptomics.

Infants with MLL-rearranged infant acute lymphoblastic leukemia (MLL-r iALL) undergo intense therapy to counter a highly aggressive malignancy with survival rates of only 30-40%. The majority of patients initially show therapy response, but in two-thirds of cases the leukemia returns, typically during treatment. The glucocorticoid drug prednisone is established as a major player in the treatment of leukemia and the in vivo response to prednisone monotreatment is currently the best indicator of risk for MLL-r iALL. We used two different single-cell RNA sequencing technologies to analyze the expression of a prednisone-dependent signature, derived from an independent study, in diagnostic bone marrow and peripheral blood biopsies. This allowed us to classify individual leukemic cells as either resistant or sensitive to treatment and show that quantification of these two groups can be used to better predict the occurrence of future relapse in individual patients. This work also sheds light on the nature of the therapy-resistant subpopulation of relapse-initiating cells. Leukemic cells associated with high relapse risk are characterized by basal activation of glucocorticoid response, smaller size, and a quiescent gene expression program with cell stemness properties. These results improve current risk stratification and elucidate leukemic therapy-resistant subpopulations at diagnosis.

A systematic analysis of genetic interactions and their underlying biology in childhood cancer.

Childhood cancer is a major cause of child death in developed countries. Genetic interactions between mutated genes play an important role in cancer development. They can be detected by searching for pairs of mutated genes that co-occur more (or less) often than expected. Co-occurrence suggests a cooperative role in cancer development, while mutual exclusivity points to synthetic lethality, a phenomenon of interest in cancer treatment research. Little is known about genetic interactions in childhood cancer. We apply a statistical pipeline to detect genetic interactions in a combined dataset comprising over 2,500 tumors from 23 cancer types. The resulting genetic interaction map of childhood cancers comprises 15 co-occurring and 27 mutually exclusive candidates. The biological explanation of most candidates points to either tumor subtype, pathway epistasis or cooperation while synthetic lethality plays a much smaller role. Thus, other explanations beyond synthetic lethality should be considered when interpreting genetic interaction test results.

Single-cell atlas reveals meningeal leukocyte heterogeneity in the developing mouse brain.

The meninges are important for brain development and pathology. Using single-cell RNA sequencing, we have generated the first comprehensive transcriptional atlas of neonatal mouse meningeal leukocytes under normal conditions and after perinatal brain injury. We identified almost all known leukocyte subtypes and found differences between neonatal and adult border-associated macrophages, thus highlighting that neonatal border-associated macrophages are functionally immature with regards to immune responses compared with their adult counterparts. We also identified novel meningeal microglia-like cell populations that may participate in white matter development. Early after the hypoxic-ischemic insult, neutrophil numbers increased and they exhibited increased granulopoiesis, suggesting that the meninges are an important site of immune cell expansion with implications for the initiation of inflammatory cascades after neonatal brain injury. Our study provides a single-cell resolution view of the importance of meningeal leukocytes at the early stage of development in health and disease.

Tumor to normal single-cell mRNA comparisons reveal a pan-neuroblastoma cancer cell.

Neuroblastoma is a childhood cancer that resembles developmental stages of the neural crest. It is not established what developmental processes neuroblastoma cancer cells represent. Here, we sought to reveal the phenotype of neuroblastoma cancer cells by comparing cancer (n = 19,723) with normal fetal adrenal single-cell transcriptomes (n = 57,972). Our principal finding was that the neuroblastoma cancer cell resembled fetal sympathoblasts, but no other fetal adrenal cell type. The sympathoblastic state was a universal feature of neuroblastoma cells, transcending cell cluster diversity, individual patients, and clinical phenotypes. We substantiated our findings in 650 neuroblastoma bulk transcriptomes and by integrating canonical features of the neuroblastoma genome with transcriptional signals. Overall, our observations indicate that a pan-neuroblastoma cancer cell state exists, which may be attractive for novel immunotherapeutic and targeted avenues.

Single-cell atlas of developing murine adrenal gland reveals relation of Schwann cell precursor signature to neuroblastoma phenotype.

Neuroblastoma is the most common extracranial solid tumor and accounts for ∼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor ("SCP") and the "neuroblast" cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.

An organoid biobank for childhood kidney cancers that captures disease and tissue heterogeneity.

Kidney tumours are among the most common solid tumours in children, comprising distinct subtypes differing in many aspects, including cell-of-origin, genetics, and pathology. Pre-clinical cell models capturing the disease heterogeneity are currently lacking. Here, we describe the first paediatric cancer organoid biobank. It contains tumour and matching normal kidney organoids from over 50 children with different subtypes of kidney cancer, including Wilms tumours, malignant rhabdoid tumours, renal cell carcinomas, and congenital mesoblastic nephromas. Paediatric kidney tumour organoids retain key properties of native tumours, useful for revealing patient-specific drug sensitivities. Using single cell RNA-sequencing and high resolution 3D imaging, we further demonstrate that organoid cultures derived from Wilms tumours consist of multiple different cell types, including epithelial, stromal and blastemal-like cells. Our organoid biobank captures the heterogeneity of paediatric kidney tumours, providing a representative collection of well-characterised models for basic cancer research, drug-screening and personalised medicine.

CHETAH: a selective, hierarchical cell type identification method for single-cell RNA sequencing.

Cell type identification is essential for single-cell RNA sequencing (scRNA-seq) studies, currently transforming the life sciences. CHETAH (CHaracterization of cEll Types Aided by Hierarchical classification) is an accurate cell type identification algorithm that is rapid and selective, including the possibility of intermediate or unassigned categories. Evidence for assignment is based on a classification tree of previously available scRNA-seq reference data and includes a confidence score based on the variance in gene expression per cell type. For cell types represented in the reference data, CHETAH's accuracy is as good as existing methods. Its specificity is superior when cells of an unknown type are encountered, such as malignant cells in tumor samples which it pinpoints as intermediate or unassigned. Although designed for tumor samples in particular, the use of unassigned and intermediate types is also valuable in other exploratory studies. This is exemplified in pancreas datasets where CHETAH highlights cell populations not well represented in the reference dataset, including cells with profiles that lie on a continuum between that of acinar and ductal cell types. Having the possibility of unassigned and intermediate cell types is pivotal for preventing misclassification and can yield important biological information for previously unexplored tissues.

FOXO Transcription Factors Both Suppress and Support Breast Cancer Progression.

FOXO transcription factors are regulators of cellular homeostasis and putative tumor suppressors, yet the role of FOXO in cancer progression remains to be determined. The data on FOXO function, particularly for epithelial cancers, are fragmentary and come from studies that focused on isolated aspects of cancer. To clarify the role of FOXO in epithelial cancer progression, we characterized the effects of inducible FOXO activation and loss in a mouse model of metastatic invasive lobular carcinoma. Strikingly, either activation or loss of FOXO function suppressed tumor growth and metastasis. We show that the multitude of cellular processes critically affected by FOXO function include proliferation, survival, redox homeostasis, and PI3K signaling, all of which must be carefully balanced for tumor cells to thrive.Significance: FOXO proteins are not solely tumor suppressors, but also support tumor growth and metastasis by regulating a multitude of cellular processes essential for tumorigenesis. Cancer Res; 78(9); 2356-69. ©2018 AACR.