A spatial cell atlas of neuroblastoma reveals developmental, epigenetic and spatial axis of tumor heterogeneity.

Neuroblastoma is a pediatric cancer arising from the developing sympathoadrenal lineage with complex inter- and intra-tumoral heterogeneity. To chart this complexity, we generated a comprehensive cell atlas of 55 neuroblastoma patient tumors, collected from two pediatric cancer institutions, spanning a range of clinical, genetic, and histologic features. Our atlas combines single-cell/nucleus RNA-seq (sc/scRNA-seq), bulk RNA-seq, whole exome sequencing, DNA methylation profiling, spatial transcriptomics, and two spatial proteomic methods. Sc/snRNA-seq revealed three malignant cell states with features of sympathoadrenal lineage development. All of the neuroblastomas had malignant cells that resembled sympathoblasts and the more differentiated adrenergic cells. A subset of tumors had malignant cells in a mesenchymal cell state with molecular features of Schwann cell precursors. DNA methylation profiles defined four groupings of patients, which differ in the degree of malignant cell heterogeneity and clinical outcomes. Using spatial proteomics, we found that neuroblastomas are spatially compartmentalized, with malignant tumor cells sequestered away from immune cells. Finally, we identify spatially restricted signaling patterns in immune cells from spatial transcriptomics. To facilitate the visualization and analysis of our atlas as a resource for further research in neuroblastoma, single cell, and spatial-omics, all data are shared through the Human Tumor Atlas Network Data Commons at www.humantumoratlas.org.

The myogenesis program drives clonal selection and drug resistance in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a pediatric cancer with features of skeletal muscle; patients with unresectable or metastatic RMS fare poorly due to high rates of disease recurrence. Here, we use single-cell and single-nucleus RNA sequencing to show that RMS tumors recapitulate the spectrum of embryonal myogenesis. Using matched patient samples from a clinical trial and orthotopic patient-derived xenografts (O-PDXs), we show that chemotherapy eliminates the most proliferative component with features of myoblasts within embryonal RMS; after treatment, the immature population with features of paraxial mesoderm expands to reconstitute the developmental hierarchy of the original tumor. We discovered that this paraxial mesoderm population is dependent on EGFR signaling and is sensitive to EGFR inhibitors. Taken together, these data serve as a proof of concept that targeting each developmental state in embryonal RMS is an effective strategy for improving outcomes by preventing disease recurrence.

Author Correction: A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

Combinatorial screening using orthotopic patient derived xenograft-expanded early phase cultures of osteosarcoma identify novel therapeutic drug combinations.

Lead discovery in osteosarcoma has been hampered by the lack of new agents, limited representative clinical samples and paucity of accurate preclinical models. We developed orthotopic patient-derived xenografts (PDXs) that recapitulated the molecular, cellular and histologic features of primary tumors, and screened PDX-expanded short-term cultures and commercial cell lines of osteosarcoma against focused drug libraries. Osteosarcoma cells were most sensitive to HDAC, proteasome, and combination PI3K/MEK and PI3K/mTOR inhibitors, and least sensitive to PARP, RAF, ERK and MEK inhibitors. Correspondingly, PI3K signaling pathway genes were up-regulated in metastatic tumors compared to primary tumors. In combinatorial screens, as a class, HDAC inhibitors showed additive effects when combined with standard-of-care agents gemcitabine and doxorubicin. This lead discovery strategy afforded a means to perform high-throughput drug screens of tumor cells that accurately recapitulated those from original human tumors, and identified classes of novel and repurposed drugs with activity against osteosarcoma.

Orthotopic patient-derived xenografts of paediatric solid tumours.

Paediatric solid tumours arise from endodermal, ectodermal, or mesodermal lineages. Although the overall survival of children with solid tumours is 75%, that of children with recurrent disease is below 30%. To capture the complexity and diversity of paediatric solid tumours and establish new models of recurrent disease, here we develop a protocol to produce orthotopic patient-derived xenografts at diagnosis, recurrence, and autopsy. Tumour specimens were received from 168 patients, and 67 orthotopic patient-derived xenografts were established for 12 types of cancer. The origins of the patient-derived xenograft tumours were reflected in their gene-expression profiles and epigenomes. Genomic profiling of the tumours, including detailed clonal analysis, was performed to determine whether the clonal population in the xenograft recapitulated the patient's tumour. We identified several drug vulnerabilities and showed that the combination of a WEE1 inhibitor (AZD1775), irinotecan, and vincristine can lead to complete response in multiple rhabdomyosarcoma orthotopic patient-derived xenografts tumours in vivo.

The Childhood Solid Tumor Network: A new resource for the developmental biology and oncology research communities.

Significant advances have been made over the past 25 years in our understanding of the most common adult solid tumors such as breast, colon, lung and prostate cancer. Much less is known about childhood solid tumors because they are rare and because they originate in developing organs during fetal development, childhood and adolescence. It can be very difficult to study the cellular origins of pediatric solid tumors in developing organs characterized by rapid proliferative expansion, growth factor signaling, developmental angiogenesis, programmed cell death, tissue reorganization and cell migration. Not only has the etiology of pediatric cancer remained elusive because of their developmental origins, but it also makes it more difficult to treat. Molecular targeted therapeutics that alter developmental pathway signaling may have devastating effects on normal organ development. Therefore, basic research focused on the mechanisms of development provides an essential foundation for pediatric solid tumor translational research. In this article, we describe new resources available for the developmental biology and oncology research communities. In a companion paper, we present the detailed characterization of an orthotopic xenograft of a pediatric solid tumor derived from sympathoadrenal lineage during development.

Systematic Screening Identifies Dual PI3K and mTOR Inhibition as a Conserved Therapeutic Vulnerability in Osteosarcoma.

PURPOSE: Osteosarcoma is the most common cancer of bone occurring mostly in teenagers. Despite rapid advances in our knowledge of the genetics and cell biology of osteosarcoma, significant improvements in patient survival have not been observed. The identification of effective therapeutics has been largely empirically based. The identification of new therapies and therapeutic targets are urgently needed to enable improved outcomes for osteosarcoma patients. EXPERIMENTAL DESIGN: We have used genetically engineered murine models of human osteosarcoma in a systematic, genome-wide screen to identify new candidate therapeutic targets. We performed a genome-wide siRNA screen, with or without doxorubicin. In parallel, a screen of therapeutically relevant small molecules was conducted on primary murine- and primary human osteosarcoma-derived cell cultures. All results were validated across independent cell cultures and across human and mouse osteosarcoma. RESULTS: The results from the genetic and chemical screens significantly overlapped, with a profound enrichment of pathways regulated by PI3K and mTOR pathways. Drugs that concurrently target both PI3K and mTOR were effective at inducing apoptosis in primary osteosarcoma cell cultures in vitro in both human and mouse osteosarcoma, whereas specific PI3K or mTOR inhibitors were not effective. The results were confirmed with siRNA and small molecule approaches. Rationale combinations of specific PI3K and mTOR inhibitors could recapitulate the effect on osteosarcoma cell cultures. CONCLUSIONS: The approaches described here have identified dual inhibition of the PI3K-mTOR pathway as a sensitive, druggable target in osteosarcoma, and provide rationale for translational studies with these agents.

Targeting the DNA repair pathway in Ewing sarcoma.

Ewing sarcoma (EWS) is a tumor of the bone and soft tissue that primarily affects adolescents and young adults. With current therapies, 70% of patients with localized disease survive, but patients with metastatic or recurrent disease have a poor outcome. We found that EWS cell lines are defective in DNA break repair and are sensitive to PARP inhibitors (PARPis). PARPi-induced cytotoxicity in EWS cells was 10- to 1,000-fold higher after administration of the DNA-damaging agents irinotecan or temozolomide. We developed an orthotopic EWS mouse model and performed pharmacokinetic and pharmacodynamic studies using three different PARPis that are in clinical development for pediatric cancer. Irinotecan administered on a low-dose, protracted schedule previously optimized for pediatric patients was an effective DNA-damaging agent when combined with PARPis; it was also better tolerated than combinations with temozolomide. Combining PARPis with irinotecan and temozolomide gave complete and durable responses in more than 80% of the mice.

Toll-like receptor 2 mediates fatal immunopathology in mice during treatment of secondary pneumococcal pneumonia following influenza.

Host inflammatory responses contribute to the significant immunopathology that occurs during treatment of secondary bacterial pneumonia following influenza. We undertook the present study to determine the mechanisms underlying disparate outcomes in a mouse model with ß-lactam and macrolide antibiotics. Lysis of superinfecting bacteria by ampicillin caused an extensive influx of neutrophils into the lungs resulting in a consolidative pneumonia, necrotic lung damage, and significant mortality. This was mediated through Toll-like receptor (TLR) 2 and was independent of TLR4 and the Streptococcus pneumoniae cytotoxin pneumolysin. Treatment with azithromycin prevented neutrophil accumulation and rescued mice from subsequent mortality. This effect was independent of the antibacterial activity of this macrolide since dual therapy with ampicillin and azithromycin against an azithromycin-resistant strain also was able to cure secondary pneumonia. These data suggest that strategies for eliminating bacteria without lysis coupled with immunomodulation of inflammation should be pursued clinically.

Treatment with protein synthesis inhibitors improves outcomes of secondary bacterial pneumonia after influenza.

Pneumonia occurring as a secondary infection after influenza is a major cause of excess morbidity and mortality, despite the availability and use of antibiotics active against Streptococcus pneumoniae. We hypothesized that the use of a bacteriostatic protein synthesis inhibitor would improve outcomes by reducing the inflammatory response. BALB/cJ mice infected with influenza virus and superinfected with S. pneumoniae were treated with either the cell-wall-active antibiotic ampicillin or the protein synthesis inhibitor clindamycin or azithromycin. In the model, ampicillin therapy performed significantly worse (survival rate, 56%) than (1) clindamycin therapy used either alone (82%) or in combination with ampicillin (80%) and (2) azithromycin (92%). Improved survival appeared to be mediated by decreased inflammation manifested as lower levels of inflammatory cells and proinflammatory cytokines in the lungs and by observation of less-severe histopathologic findings. These data suggest that beta-lactam therapy may not be optimal as a first-line treatment for community-acquired pneumonia when it follows influenza.

Vaccination of horses against strangles using recombinant antigens from Streptococcus equi.

Strangles is an upper respiratory tract infection in horses, which is highly contagious and one of the more costly diseases of the horse. Three recombinant antigens were used to vaccinate horses, which were then experimentally challenged with Streptococcus equi, the causative agent for strangles. The vaccinated horses showed significantly reduced bacterial growth (p=0.02) and nasal discharge (p=0.0004), a typical symptom of strangles. Other clinical signs of strangles were also reduced and at post mortem examination, lower rate of empyaema or scarring of the guttural pouches was found in the vaccinated group (p=0.01). The antigens used were EAG (alpha2-macroglobulin, albumin, and IgG-binding protein), CNE (a collagen-binding protein), and SclC (a collagen-like protein). The adjuvant used was Abisco, a saponin derived matrix. No adverse effects were observed following vaccination with the antigens and adjuvant.

A fibronectin-binding protein from Streptococcus equi binds collagen and modulates cell-mediated collagen gel contraction.

The N-terminal fragment (FNZN) of the fibronectin-binding protein FNZ from Streptococcus equi subspecies zooepidemicus was investigated as to effects on murine cell interactions with extracellular matrix proteins. FNZN bound to immobilized fibronectin (FN) and native, but not denatured, collagen type I. FNZN had no effect on primary adhesion of cells from the murine myoblastic C2C12 cell line to immobilized fibronectin. C2C12 cells adhered to immobilized FNZN, a process that was not inhibited by anti-human FN IgG or by an inhibitor of integrin alphaVbeta3. C2C12 cells lack collagen-binding beta1 integrins and neither adhere to native collagen nor mediate contraction of three-dimensional collagen gels. FNZN stimulated collagen gel contraction by C2C12 cells but not adhesion of C2C12 cells to collagen. Experiments with an alphaVbeta3-inhibitor suggested that FNZN promoted contraction by a process requiring alphaVbeta3. Our data suggest that FNZN by binding to cells, collagen, and FN modulate complex adhesive processes mediated by the alphaVbeta3 integrin. Since alphaVbeta3-mediated contractile events function to counteract edema formation during inflammation, it is possible that FNZN and its secreted homologue FNE modulate edema responses in infected tissues.

SclC is a member of a novel family of collagen-like proteins in Streptococcus equi subspecies equi that are recognised by antibodies against SclC.

Previously we have reported on a cell surface collagen-like protein, called SclC, from Streptococcus equi subspecies equi. In the present study we show that this protein is a member of a family of seven collagen-like proteins, called SclC-SclI in this subspecies. All proteins contain an N-terminal signal sequence, followed by a unique non-repetitive region called A, a highly repetitive collagen-like region (CL) consisting of Glycine-Xaa-Yaa-triplet repeats. Following the CL-region a C-terminal proline-rich putative wall spanning region (W) preceding an LPXTG-motif and a hydrophobic transmembrane region (M) are found, typical features of cell surface exposed proteins in Gram-positive bacteria. The nucleotide and amino acid sequences, were analysed to investigate the similarities between them, and recombinant proteins encoding different domains (A- and CL-regions) were expressed and purified. Although the novel collagen-like proteins display differences in amino acid sequences, affinity purified antibodies against SclC were found to cross react with the other members of the novel collagen-like proteins. Furthermore, in sera from horses previously diagnosed having strangles, antibodies against these proteins were detected suggesting that these proteins are expressed during the infection.

Identification of a novel collagen-like protein, SclC, in Streptococcus equi using signal sequence phage display.

Strangles is a serious disease in horses caused by Streptococcus equi subspecies equi. In this study, genes encoding putative extracellular proteins in this subspecies have been identified using signal sequence phage display. Among these, one showed similarities to the SclB protein, a member of the collagen-like proteins of Streptococcus pyogenes. The novel gene denoted sclC encodes a protein, SclC, of 302 amino acids, containing typical features found in cell wall-anchored proteins in Gram-positive bacteria. Based on similarities to the S. pyogenes collagen-like proteins the mature SclC protein can be divided into various domains: an N-terminal non-repetitive region (A), a highly repetitive collagen-like region (CL), and a C-terminal proline-rich wall-associated region (W). Using PCR, the sclC gene was detected in all studied strains of S. equi subsp. equi and S. equi subsp. zooepidemicus. Further, antibodies against recombinant SclC were detected in a collection of sera from horses with no history of strangles as well as horses previously infected with S. equi subsp. equi. Interestingly, the sera from convalescence horses were found to have significantly increased antibody titers against the SclC protein indicating that this protein is expressed during infection of S. equi subsp. equi.