RESUMEN
Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.
Asunto(s)
Animales Modificados Genéticamente/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias de los Músculos , Rabdomiosarcoma , Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/inmunología , Femenino , Xenoinjertos , Humanos , Células K562 , Masculino , Neoplasias de los Músculos/tratamiento farmacológico , Neoplasias de los Músculos/inmunología , Neoplasias de los Músculos/metabolismo , Neoplasias de los Músculos/patología , Trasplante de Neoplasias , Ftalazinas/farmacología , Piperazinas/farmacología , Rabdomiosarcoma/tratamiento farmacológico , Rabdomiosarcoma/inmunología , Rabdomiosarcoma/metabolismo , Rabdomiosarcoma/patología , Temozolomida/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/genética , Pez Cebra/inmunologíaRESUMEN
Overall survival rates for pediatric patients with high-risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Desarrollo de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Rabdomiosarcoma , Niño , Humanos , Proyectos de InvestigaciónRESUMEN
High-throughput vertebrate animal model systems for the study of patient-specific biology and new therapeutic approaches for aggressive brain tumors are currently lacking, and new approaches are urgently needed. Therefore, to build a patient-relevant in vivo model of human glioblastoma, we expressed common oncogenic variants including activated human EGFRvIII and PI3KCAH1047R under the control of the radial glial-specific promoter her4.1 in syngeneic tp53 loss-of-function mutant zebrafish. Robust tumor formation was observed prior to 45 days of life, and tumors had a gene expression signature similar to human glioblastoma of the mesenchymal subtype, with a strong inflammatory component. Within early stage tumor lesions, and in an in vivo and endogenous tumor microenvironment, we visualized infiltration of phagocytic cells, as well as internalization of tumor cells by mpeg1.1:EGFP+ microglia/macrophages, suggesting negative regulatory pressure by pro-inflammatory cell types on tumor growth at early stages of glioblastoma initiation. Furthermore, CRISPR/Cas9-mediated gene targeting of master inflammatory transcription factors irf7 or irf8 led to increased tumor formation in the primary context, while suppression of phagocyte activity led to enhanced tumor cell engraftment following transplantation into otherwise immune-competent zebrafish hosts. Altogether, we developed a genetically relevant model of aggressive human glioblastoma and harnessed the unique advantages of zebrafish including live imaging, high-throughput genetic and chemical manipulations to highlight important tumor-suppressive roles for the innate immune system on glioblastoma initiation, with important future opportunities for therapeutic discovery and optimizations.
Glioblastoma is the most common and deadly type of brain cancer in adults. Fewer than 7% of patients survive for more than five years after diagnosis. This poor prognosis for patients with glioblastoma has not significantly improved for decades. The standard treatment for glioblastoma consists of surgery, radiotherapy and the same chemotherapy that has been prescribed for twenty years. This suggests that there is still much to learn about glioblastoma and how better to treat it. Scientists use various laboratory models to mimic human disease. They can study human glioblastoma cells grown in the laboratory or transplanted into mice, and they can also use genetically engineered mice that develop brain tumors from their own tissue. These systems provide valuable information about glioblastoma, but each model has certain drawbacks. For example, glioblastoma cells in a dish do not grow in an environment containing other types of cells found in the body, such as immune cells. And although studying glioblastoma in mice bypasses this problem, such experiments often take years to perform and are very expensive. To address these limitations, Weiss et al. asked whether introducing some of the same genetic mutations that cause glioblastoma in humans could lead to brain tumors in zebrafish. Zebrafish have multiple advantages as models of human disease: they are inexpensive to maintain and have a rapid life cycle, they are relatively easy to manipulate using various genetic tools, and they are transparent so that the growth of tumors can be filmed. Weiss et al. expressed mutant versions of genes found in many patients with glioblastoma in the brains of developing zebrafish. These zebrafish rapidly developed tumor-like growths and detailed analyses confirmed that these tumors highly resembled human glioblastomas. Zebrafish glioblastomas contained active immune cells in addition to the cancer cells and showed signs of being inflamed. Weiss et al. filmed interactions between immune cells and cancer cells in zebrafish brains. They noted that specific immune cells called macrophages (commonly known to destroy certain disease-causing pathogens like bacteria) had pieces of tumors inside them. This and other evidence suggested that these macrophages counteracted the growth of tumors by potentially engulfing (or 'eating') glioblastoma cells during the early stages of tumor development. Altogether, these experiments indicate that zebrafish containing specific genes that cause glioblastoma in humans can mimic disease in many respects. Future studies will build on this work by testing other genes and further studying interactions between immune cells and cancer cells in the animal body.
Asunto(s)
Neoplasias Encefálicas , Modelos Animales de Enfermedad , Receptores ErbB , Glioblastoma , Inflamación , Proteína p53 Supresora de Tumor , Proteínas de Pez Cebra , Pez Cebra , Animales , Glioblastoma/genética , Glioblastoma/patología , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Inflamación/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Humanos , Microambiente Tumoral/genéticaRESUMEN
Survival rates among patients with high-risk neuroblastoma remain low and novel therapies for recurrent neuroblastomas are required. ALK is commonly mutated in primary and relapsed neuroblastoma tumors and ALK tyrosine kinase inhibitors (TKI) are promising treatments for ALK-driven neuroblastoma; however, innate or adaptive resistance to single-agent ALK-TKIs remain a clinical challenge. Recently, SHP2 inhibitors have been shown to overcome ALK-TKI resistance in lung tumors harboring ALK rearrangements. Here, we have assessed the efficacy of the SHP2 inhibitor TNO155 alone and in combination with the ALK-TKIs crizotinib, ceritinib, or lorlatinib for the treatment of ALK-driven neuroblastoma using in vitro and in vivo models. In comparison to wild-type, ALK-mutant neuroblastoma cell lines were more sensitive to SHP2 inhibition with TNO155. Moreover, treatment with TNO155 and ALK-TKIs synergistically reduced cell growth and promoted inactivation of ALK and MAPK signaling in ALK-mutant neuroblastoma cells. ALK-mutant cells engrafted into larval zebrafish and treated with single agents or dual SHP2/ALK inhibitors showed reduced growth and invasion. In murine ALK-mutant xenografts, tumor growth was likewise reduced or delayed, and survival was prolonged upon combinatorial treatment of TNO155 and lorlatinib. Finally, we show that lorlatinib-resistant ALK-F1174L neuroblastoma cells harbor additional RAS-MAPK pathway alterations and can be resensitized to lorlatinib when combined with TNO155 in vitro and in vivo. Our results report the first evaluation of TNO155 in neuroblastoma and suggest that combinatorial inhibition of ALK and SHP2 could be a novel approach to treating ALK-driven neuroblastoma, potentially including the increasingly common tumors that have developed resistance to ALK-TKIs. SIGNIFICANCE: These findings highlight the translatability between zebrafish and murine models, provide evidence of aberrant RAS-MAPK signaling as an adaptive mechanism of resistance to lorlatinib, and demonstrate the clinical potential for SHP2/ALK inhibitor combinations for the treatment of ALK-mutant neuroblastoma, including those with acquired tolerance or potentially resistance to ALK-TKIs.
Asunto(s)
Neuroblastoma , Pez Cebra , Humanos , Ratones , Animales , Pez Cebra/metabolismo , Quinasa de Linfoma Anaplásico , Resistencia a Antineoplásicos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Lactamas Macrocíclicas/farmacología , Neuroblastoma/tratamiento farmacológicoRESUMEN
In embryonal rhabdomyosarcoma (ERMS) and generally in sarcomas, the role of wild-type and loss- or gain-of-function TP53 mutations remains largely undefined. Eliminating mutant or restoring wild-type p53 is challenging; nevertheless, understanding p53 variant effects on tumorigenesis remains central to realizing better treatment outcomes. In ERMS, >70% of patients retain wild-type TP53, yet mutations when present are associated with worse prognosis. Employing a kRASG12D-driven ERMS tumor model and tp53 null (tp53-/-) zebrafish, we define wild-type and patient-specific TP53 mutant effects on tumorigenesis. We demonstrate that tp53 is a major suppressor of tumorigenesis, where tp53 loss expands tumor initiation from <35% to >97% of animals. Characterizing three patient-specific alleles reveals that TP53C176F partially retains wild-type p53 apoptotic activity that can be exploited, whereas TP53P153Δ and TP53Y220C encode two structurally related proteins with gain-of-function effects that predispose to head musculature ERMS. TP53P153Δ unexpectedly also predisposes to hedgehog-expressing medulloblastomas in the kRASG12D-driven ERMS-model.
Asunto(s)
Neoplasias Cerebelosas , Rabdomiosarcoma Embrionario , Animales , Carcinogénesis , Mutación , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Rabdomiosarcoma Embrionario/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.
Asunto(s)
Rabdomiosarcoma Embrionario , Rabdomiosarcoma , Niño , Humanos , Músculo Esquelético/patología , Rabdomiosarcoma/genética , Análisis de la Célula Individual , Células Madre/patologíaRESUMEN
The TP53 tumor-suppressor gene is mutated in >50% of human tumors and Li-Fraumeni patients with germ line inactivation are predisposed to developing cancer. Here, we generated tp53 deleted zebrafish that spontaneously develop malignant peripheral nerve-sheath tumors, angiosarcomas, germ cell tumors, and an aggressive Natural Killer cell-like leukemia for which no animal model has been developed. Because the tp53 deletion was generated in syngeneic zebrafish, engraftment of fluorescent-labeled tumors could be dynamically visualized over time. Importantly, engrafted tumors shared gene expression signatures with predicted cells of origin in human tissue. Finally, we showed that tp53del/del enhanced invasion and metastasis in kRASG12D-induced embryonal rhabdomyosarcoma (ERMS), but did not alter the overall frequency of cancer stem cells, suggesting novel pro-metastatic roles for TP53 loss-of-function in human muscle tumors. In summary, we have developed a Li-Fraumeni zebrafish model that is amenable to large-scale transplantation and direct visualization of tumor growth in live animals.
Asunto(s)
Rabdomiosarcoma Embrionario/metabolismo , Rabdomiosarcoma Embrionario/patología , Proteína p53 Supresora de Tumor/deficiencia , Pez Cebra/metabolismo , Animales , Recuento de Células , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Hemangiosarcoma/metabolismo , Hemangiosarcoma/patología , Homocigoto , Leucemia/metabolismo , Leucemia/patología , Invasividad Neoplásica , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias de Células Germinales y Embrionarias/metabolismo , Neoplasias de Células Germinales y Embrionarias/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Rabdomiosarcoma Embrionario/genética , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/genéticaRESUMEN
Tumor growth and relapse are driven by tumor propagating cells (TPCs). However, mechanisms regulating TPC fate choices, maintenance, and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway, affects TPC self-renewal in rhabdomyosarcoma (RMS)-a pediatric cancer of muscle. VANGL2 is expressed in a majority of human RMS and within early mononuclear progenitor cells. VANGL2 depletion inhibited cell proliferation, reduced TPC numbers, and induced differentiation of human RMS in vitro and in mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS), we determined that Vangl2 expression enriches for TPCs and promotes their self-renewal. Expression of constitutively active and dominant-negative isoforms of RHOA revealed that it acts downstream of VANGL2 to regulate proliferation and maintenance of TPCs in human RMS. Our studies offer insights into pathways that control TPCs and identify new potential therapeutic targets.
Asunto(s)
Autorrenovación de las Células , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Células Madre Neoplásicas/patología , Rabdomiosarcoma/patología , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Células Madre Neoplásicas/metabolismo , Rabdomiosarcoma/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth.
Asunto(s)
Proteína MioD/metabolismo , Factor 5 Regulador Miogénico/metabolismo , Rabdomiosarcoma/fisiopatología , Transcripción Genética , Animales , Animales Modificados Genéticamente , Inmunoprecipitación de Cromatina , Humanos , Análisis de Secuencia de ADN , Pez CebraRESUMEN
Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.
Asunto(s)
Factores de Transcripción MEF2/metabolismo , Receptor Notch1/metabolismo , Rabdomiosarcoma Embrionario/metabolismo , Factores de Transcripción de la Familia Snail/metabolismo , Animales , Diferenciación Celular/fisiología , Humanos , Rabdomiosarcoma Embrionario/patología , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas de Xenopus/metabolismo , Pez CebraRESUMEN
Cancers contain a wide diversity of cell types that are defined by differentiation states, genetic mutations and altered epigenetic programmes that impart functional diversity to individual cells. Elevated tumour cell heterogeneity is linked with progression, therapy resistance and relapse. Yet, imaging of tumour cell heterogeneity and the hallmarks of cancer has been a technical and biological challenge. Here we develop optically clear immune-compromised rag2(E450fs) (casper) zebrafish for optimized cell transplantation and direct visualization of fluorescently labelled cancer cells at single-cell resolution. Tumour engraftment permits dynamic imaging of neovascularization, niche partitioning of tumour-propagating cells in embryonal rhabdomyosarcoma, emergence of clonal dominance in T-cell acute lymphoblastic leukaemia and tumour evolution resulting in elevated growth and metastasis in BRAF(V600E)-driven melanoma. Cell transplantation approaches using optically clear immune-compromised zebrafish provide unique opportunities to uncover biology underlying cancer and to dynamically visualize cancer processes at single-cell resolution in vivo.