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.
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.
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.
Groups of five mice maintained in a BL2 facility were infected intranasally with 1 x 105 or 1 x 106 colony-forming units (CFU) of this bioluminescent strain under light anesthesia with 2.5% inhaled ...isoflurane using an established infection model approved by the St. Jude Children's Research Hospital animal care and use committee 11. Visualization of Bioluminescent Bacteria Inside Live, Anesthetized Animals Shows the Induction of Otitis Media (A-B) Six- to eight-week-old female Balb/cJ mice (Jackson Laboratory, http://www.jax.org) were colonized intranasally with 1 x 105 CFU of type 19F S. pneumoniae strain ST16219F suspended in 100 μl of sterile PBS, then seven days later either (A) infected intranasally in a volume of 100 μl of sterile PBS with 100 TCID50 (doses of virus infectious for 50% of tissue culture wells) of the Mount Sinai strain of influenza virus A/Puerto Rico/8/34 (H1N1), or (B) mock-infected with sterile PBS.
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.
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•Pediatric RMSs contain transcriptional states of developing muscle•Treatment of embryonal RMS selects for cells in a progenitor mesoderm-like state•Mesoderm-like cells are sensitive to EGFR inhibition•Targeting myogenic states using both EGFR blockade and chemotherapy improves outcomes
Rhabdomyosarcoma is a pediatric cancer with features of skeletal muscle. Patel et al. show that intratumoral heterogeneity within rhabdomyosarcoma mimics the developmental states of embryonal myogenesis. Furthermore, treatment selects for cells in a mesoderm-like state that are vulnerable to EGFR inhibition. Combining EGFR inhibitors with chemotherapy therefore improves treatment outcomes.
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.
•Few new agents are identified for osteosarcoma due to the paucity of actionable somatic mutations.•Patient-derived orthotopic xenografts can expand tumor cells as short term early-phase cultures.•High-throughput screens identify novel and repurposed drugs with activity against osteosarcoma.•HDAC inhibitors have particular efficacy and are potentiated in combination with anthracyclines.
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 β-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 Tolllike 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.
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