Roadmap for the next generation of Children's Oncology Group rhabdomyosarcoma trials.

Clinical trials conducted by the Intergroup Rhabdomyosarcoma (RMS) Study Group and the Children's Oncology Group have been pivotal to establishing current standards for diagnosis and therapy for RMS. Recent advancements in understanding the biology and clinical behavior of RMS have led to more nuanced approaches to diagnosis, risk stratification, and treatment. The complexities introduced by these advancements, coupled with the rarity of RMS, pose challenges to conducting large-scale phase 3 clinical trials to evaluate new treatment strategies for RMS. Given these challenges, systematic planning of future clinical trials in RMS is paramount to address pertinent questions regarding the therapeutic efficacy of drugs, biomarkers of response, treatment-related toxicity, and patient quality of life. Herein, the authors outline the proposed strategic approach of the Children's Oncology Group Soft Tissue Sarcoma Committee to the next generation of RMS clinical trials, focusing on five themes: improved novel agent identification and preclinical to clinical translation, more efficient trial development and implementation, expanded opportunities for knowledge generation during trials, therapeutic toxicity reduction and quality of life, and patient engagement.

A platform for rapid patient-derived cutaneous neurofibroma organoid establishment and screening.

Localized cutaneous neurofibromas (cNFs) are benign tumors that arise in the dermis of patients affected by neurofibromatosis type 1 syndrome. cNFs are benign lesions: they do not undergo malignant transformation or metastasize. Nevertheless, they can cover a significant proportion of the body, with some individuals developing hundreds to thousands of lesions. cNFs can cause pain, itching, and disfigurement resulting in substantial socio-emotional repercussions. Currently, surgery and laser desiccation are the sole treatment options but may result in scarring and potential regrowth from incomplete removal. To identify effective systemic therapies, we introduce an approach to establish and screen cNF organoids. We optimized conditions to support the ex vivo growth of genomically diverse cNFs. Patient-derived cNF organoids closely recapitulate cellular and molecular features of parental tumors as measured by immunohistopathology, methylation, RNA sequencing, and flow cytometry. Our cNF organoid platform enables rapid screening of hundreds of compounds in a patient- and tumor-specific manner.

Membrane damage by MBP-1 is mediated by pore formation and amplified by mtDNA.

Eosinophils play a crucial role in host defense while also contributing to immunopathology through the release of inflammatory mediators. Characterized by distinctive cytoplasmic granules, eosinophils securely store and rapidly release various proteins exhibiting high toxicity upon extracellular release. Among these, major basic protein 1 (MBP-1) emerges as an important mediator in eosinophil function against pathogens and in eosinophil-associated diseases. While MBP-1 targets both microorganisms and host cells, its precise mechanism remains elusive. We demonstrate that formation of small pores by MBP-1 in lipid bilayers induces membrane permeabilization and disrupts potassium balance. Additionally, we reveal that mitochondrial DNA (mtDNA) present in eosinophil extracellular traps (EETs) amplifies MBP-1 toxic effects, underscoring the pivotal role of mtDNA in EETs. Furthermore, we present evidence indicating that absence of CpG methylation in mtDNA contributes to the regulation of MBP-1-mediated toxicity. Taken together, our data suggest that the mtDNA scaffold within extracellular traps promotes MBP-1 toxicity.

Drug screening at single-organoid resolution via bioprinting and interferometry.

High throughput drug screening is an established approach to investigate tumor biology and identify therapeutic leads. Traditional platforms use two-dimensional cultures which do not accurately reflect the biology of human tumors. More clinically relevant model systems such as three-dimensional tumor organoids can be difficult to scale and screen. Manually seeded organoids coupled to destructive endpoint assays allow for the characterization of treatment response, but do not capture transitory changes and intra-sample heterogeneity underlying clinically observed resistance to therapy. We present a pipeline to generate bioprinted tumor organoids linked to label-free, time-resolved imaging via high-speed live cell interferometry (HSLCI) and machine learning-based quantitation of individual organoids. Bioprinting cells gives rise to 3D structures with unaltered tumor histology and gene expression profiles. HSLCI imaging in tandem with machine learning-based segmentation and classification tools enables accurate, label-free parallel mass measurements for thousands of organoids. We demonstrate that this strategy identifies organoids transiently or persistently sensitive or resistant to specific therapies, information that could be used to guide rapid therapy selection.

The landscape of drug sensitivity and resistance in sarcoma.

Sarcomas are a family of rare malignancies composed of over 100 distinct histological subtypes. The rarity of sarcoma poses significant challenges in conducting clinical trials to identify effective therapies, to the point that many rarer subtypes of sarcoma do not have standard-of-care treatment. Even for established regimens, there can be substantial heterogeneity in responses. Overall, novel, personalized approaches for identifying effective treatments are needed to improve patient out-comes. Patient-derived tumor organoids (PDTOs) are clinically relevant models representative of the physiological behavior of tumors across an array of malignancies. Here, we use PDTOs as a tool to better understand the biology of individual tumors and characterize the landscape of drug resistance and sensitivity in sarcoma. We collected n=194 specimens from n=126 sarcoma patients, spanning 24 distinct subtypes. We characterized PDTOs established from over 120 biopsy, resection, and metastasectomy samples. We leveraged our organoid high-throughput drug screening pipeline to test the efficacy of chemotherapeutics, targeted agents, and combination therapies, with results available within a week from tissue collection. Sarcoma PDTOs showed patient-specific growth characteristics and subtype-specific histopathology. Organoid sensitivity correlated with diagnostic subtype, patient age at diagnosis, lesion type, prior treatment history, and disease trajectory for a subset of the compounds screened. We found 90 biological pathways that were implicated in response to treatment of bone and soft tissue sarcoma organoids. By comparing functional responses of organoids and genetic features of the tumors, we show how PDTO drug screening can provide an orthogonal set of information to facilitate optimal drug selection, avoid ineffective therapies, and mirror patient outcomes in sarcoma. In aggregate, we were able to identify at least one effective FDA-approved or NCCN-recommended regimen for 59% of the specimens tested, providing an estimate of the proportion of immediately actionable information identified through our pipeline. Highlights: Standardized organoid culture preserve unique sarcoma histopathological featuresDrug screening on patient-derived sarcoma organoids provides sensitivity information that correlates with clinical features and yields actionable information for treatment guidanceHigh-throughput screenings provide orthogonal information to genetic sequencingSarcoma organoid response to treatment correlates with patient response to therapyLarge scale, functional precision medicine programs for rare cancers are feasible within a single institution.

Personalized chordoma organoids for drug discovery studies.

Chordomas are rare tumors of notochordal origin, most commonly arising in the sacrum or skull base. Chordomas are considered insensitive to conventional chemotherapy, and their rarity complicates running timely and adequately powered trials to identify effective treatments. Therefore, there is a need for discovery of novel therapeutic approaches. Patient-derived organoids can accelerate drug discovery and development studies and predict patient responses to therapy. In this proof-of-concept study, we successfully established organoids from seven chordoma tumor samples obtained from five patients presenting with tumors in different sites and stages of disease. The organoids recapitulated features of the original parent tumors and inter- as well as intrapatient heterogeneity. High-throughput screenings performed on the organoids highlighted targeted agents such as PI3K/mTOR, EGFR, and JAK2/STAT3 inhibitors among the most effective molecules. Pathway analysis underscored how the NF-κB and IGF-1R pathways are sensitive to perturbations and potential targets to pursue for combination therapy of chordoma.

Organoids.

Organoids have attracted increasing attention because they are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration, and repair in human tissues. Organoids can also be used in diagnostics, disease modeling, drug discovery, and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumors. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer serves to highlight the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community is also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and key priorities in organoid engineering for the coming years.

Cervical organoids go viral.

In this issue of Cell Stem Cell, Lõhmussaar et al. (2021) describe a versatile platform for generating long-term patient-derived organoids from hysterectomies and Pap brush cells. They establish malignant and benign cervix tissue organoids from both endo- and ectocervix that have applications ranging from precision medicine to infection modeling.

Patient-Derived Tumor Organoid Rings for Histologic Characterization and High-Throughput Screening.

Tumor organoids are promising tools for cancer biology investigations and preclinical drug screenings because they are often representative of the histology and drug responses of patients. Here, we introduce a facile protocol to overcome technical limitations by generating patient-derived tumor organoids using a simplified ring-like geometry. This facilitates media exchange and drug treatment for histopathology characterization and automated high-throughput drug screenings. For complete details on the use and execution of this protocol, please refer to Phan et al. (2019).

Introductions to the Community: Early-Career Researchers in the Time of COVID-19.

COVID-19 has unfortunately halted lab work, conferences, and in-person networking, which is especially detrimental to researchers just starting their labs. Through social media and our reviewer networks, we met some early-career stem cell investigators impacted by the closures. Here, they introduce themselves and their research to our readers.

A simple high-throughput approach identifies actionable drug sensitivities in patient-derived tumor organoids.

Tumor organoids maintain cell-cell interactions, heterogeneity, microenvironment, and drug response of the sample they originate from. Thus, there is increasing interest in developing tumor organoid models for drug development and personalized medicine applications. Although organoids are in principle amenable to high-throughput screenings, progress has been hampered by technical constraints and extensive manipulations required by current methods. Here we introduce a miniaturized method that uses a simplified geometry by seeding cells around the rim of the wells (mini-rings). This allows high-throughput screenings in a format compatible with automation as shown using four patient-derived tumor organoids established from two ovarian and one peritoneal high-grade serous carcinomas and one carcinosarcoma of the ovary. Using our automated screening platform, we identified personalized responses by measuring viability, number, and size of organoids after exposure to 240 kinase inhibitors. Results are available within a week from surgery, a timeline compatible with therapeutic decision-making.

Journal clubs in the time of preprints.

Early-career researchers can learn about peer review by discussing preprints at journal clubs and sending feedback to the authors.

A Designed Inhibitor of p53 Aggregation Rescues p53 Tumor Suppression in Ovarian Carcinomas.

Half of all human cancers lose p53 function by missense mutations, with an unknown fraction of these containing p53 in a self-aggregated amyloid-like state. Here we show that a cell-penetrating peptide, ReACp53, designed to inhibit p53 amyloid formation, rescues p53 function in cancer cell lines and in organoids derived from high-grade serous ovarian carcinomas (HGSOC), an aggressive cancer characterized by ubiquitous p53 mutations. Rescued p53 behaves similarly to its wild-type counterpart in regulating target genes, reducing cell proliferation and increasing cell death. Intraperitoneal administration decreases tumor proliferation and shrinks xenografts in vivo. Our data show the effectiveness of targeting a specific aggregation defect of p53 and its potential applicability to HGSOCs.

Toxicity of eosinophil MBP is repressed by intracellular crystallization and promoted by extracellular aggregation.

Eosinophils are white blood cells that function in innate immunity and participate in the pathogenesis of various inflammatory and neoplastic disorders. Their secretory granules contain four cytotoxic proteins, including the eosinophil major basic protein (MBP-1). How MBP-1 toxicity is controlled within the eosinophil itself and activated upon extracellular release is unknown. Here we show how intragranular MBP-1 nanocrystals restrain toxicity, enabling its safe storage, and characterize them with an X-ray-free electron laser. Following eosinophil activation, MBP-1 toxicity is triggered by granule acidification, followed by extracellular aggregation, which mediates the damage to pathogens and host cells. Larger non-toxic amyloid plaques are also present in tissues of eosinophilic patients in a feedback mechanism that likely limits tissue damage under pathological conditions of MBP-1 oversecretion. Our results suggest that MBP-1 aggregation is important for innate immunity and immunopathology mediated by eosinophils and clarify how its polymorphic self-association pathways regulate toxicity intra- and extracellularly.

Superresolution imaging of amyloid fibrils with binding-activated probes.

Protein misfolding into amyloid-like aggregates underlies many neurodegenerative diseases. Thus, insights into the structure and function of these amyloids will provide valuable information on the pathological mechanisms involved and aid in the design of improved drugs for treating amyloid-based disorders. However, determining the structure of endogenous amyloids at high resolution has been difficult. Here we employ binding-activated localization microscopy (BALM) to acquire superresolution images of α-synuclein amyloid fibrils with unprecedented optical resolution. We propose that BALM imaging can be extended to study the structure of other amyloids, for differential diagnosis of amyloid-related diseases and for discovery of drugs that perturb amyloid structure for therapy.

On-surface aggregation of α-synuclein at nanomolar concentrations results in two distinct growth mechanisms.

The aggregation of α-synuclein (α-Syn) is believed to be one of the key steps driving the pathology of Parkinson's disease and related neurodegenerative disorders. One of the present hypotheses is that the onset of such pathologies is related to the rise of α-Syn levels above a critical concentration at which toxic oligomers or mature fibrils are formed. In the present study, we find that α-Syn aggregation in vitro is a spontaneous process arising at bulk concentrations as low as 1 nM and below in the presence of both hydrophilic glass surfaces and cell membrane mimicking supported lipid bilayers (SLBs). Using three-dimensional supercritical angle fluorescence (3D-SAF) microscopy, we observed the process of α-Syn aggregation in situ. As soon as α-Syn monomers were exposed to the surface, they started to adsorb and aggregate along the surface plane without a prior lag phase. However, at a later stage of the aggregation process, a second type of aggregate was observed. In contrast to the first type, these aggregates showed an extended structure being tethered with one end to the surface and being mobile at the other end, which protruded into the solution. While both types of α-Syn aggregates were found to contain amyloid structures, their growing mechanisms turned out to be significantly different. Given the clear evidence that surface-induced α-Syn aggregation in vitro can be triggered at bulk concentrations far below physiological concentrations, the concept of a critical concentration initiating aggregation in vivo needs to be reconsidered.

Mechanism of membrane interaction and disruption by α-synuclein.

Parkinson's disease is a common progressive neurodegenerative condition, characterized by the deposition of amyloid fibrils as Lewy bodies in the substantia nigra of affected individuals. These insoluble aggregates predominantly consist of the protein α-synuclein. There is increasing evidence suggesting that the aggregation of α-synuclein is influenced by lipid membranes and, vice versa, the membrane integrity is severely affected by the presence of bound aggregates. Here, using the surface-sensitive imaging technique supercritical angle fluorescence microscopy and Förster resonance energy transfer, we report the direct observation of α-synuclein aggregation on supported lipid bilayers. Both the wild-type and the two mutant forms of α-synuclein studied, namely, the familiar variant A53T and the designed highly toxic variant E57K, were found to follow the same mechanism of polymerization and membrane damage. This mechanism involved the extraction of lipids from the bilayer and their clustering around growing α-synuclein aggregates. Despite all three isoforms following the same pathway, the extent of aggregation and their effect on the bilayers was seen to be variant and concentration dependent. Both A53T and E57K formed cross-ß-sheet aggregates and damaged the membrane at submicromolar concentrations. The wild-type also formed aggregates in this range; however, the extent of membrane disruption was greatly reduced. The process of membrane damage could resemble part of the yet poorly understood cellular toxicity phenomenon in vivo.

In vivo demonstration that alpha-synuclein oligomers are toxic.

The aggregation of proteins into oligomers and amyloid fibrils is characteristic of several neurodegenerative diseases, including Parkinson disease (PD). In PD, the process of aggregation of α-synuclein (α-syn) from monomers, via oligomeric intermediates, into amyloid fibrils is considered the disease-causative toxic mechanism. We developed α-syn mutants that promote oligomer or fibril formation and tested the toxicity of these mutants by using a rat lentivirus system to investigate loss of dopaminergic neurons in the substantia nigra. The most severe dopaminergic loss in the substantia nigra is observed in animals with the α-syn variants that form oligomers (i.e., E57K and E35K), whereas the α-syn variants that form fibrils very quickly are less toxic. We show that α-syn oligomers are toxic in vivo and that α-syn oligomers might interact with and potentially disrupt membranes.