This project aims to develop a targeted therapy for pediatric ependymoma, an aggressive brain tumor frequently driven by the ZFTA-RELA oncogenic fusion. Unlike adult cancers, which are often defined by recurrent mutations, childhood cancers are commonly driven by gene fusions that remain difficult to treat. Current therapies for fusion-positive CNS tumors, chemotherapy and radiation, have limited success and cause devastating long-term toxicities, underscoring the urgent need for new approaches. This project proposes a nucleic acid–based therapy designed to selectively bind and degrade the ZFTA-RELA fusion transcript, thereby suppressing tumor growth and survival while minimizing off-target effects.

The project has two main aims: (1) identify and optimize oligonucleotides in patient-derived tumor cell lines and organoids, and (2) rigorously evaluate safety and toxicity in human brain organoids, eliminating reliance on animal models. By directly mimicking CNS drug delivery through microinjection into organoid ventricle-like structures, this approach will generate a human-relevant safety profile that de-risks clinical translation. Ultimately, this work seeks to deliver a clinical-grade therapeutic candidate and lay the groundwork for first-in-human trials within two years, offering a new paradigm for treating ependymoma and other fusion-driven childhood cancers.

Childhood hemispheric pediatric high-grade glioma (PHGG) is uniformly lethal. Progress is limited by the lack of predictive human models. This project will build a fully human, non-animal platform that integrates patient-derived tumoroids, human forebrain organoids, and MHC-matched human macrophages. This system models invasion, brain tumor interactions, and the myeloid microenvironment, and supports testing of BBB-penetrant therapies. The project meets the program's dual goals by advancing mechanism-based treatment for childhood cancer and by replacing animal screens with a modular, scalable human platform. The long-term objective is a validated precision system that predicts response, identifies biomarkers of sensitivity and resistance, and accelerates effective regimens to early-phase trials in PHGG.

Aim 1: Develop and validate the tumoroid-organoid-macrophage model of hemispheric PHGG. The team will establish tumoroids from banked and fresh patient samples, confirm fidelity by transcriptomic and pathway similarity to tumors, and quantify cell-state diversity. They will also generate forebrain organoids from iPSCs that recapitulate expected cortical lineages. They will then engraft PHGG cells to model invasion and early tumorigenesis and incorporate macrophages to enable immune readouts with single-cell RNA sequencing, spatial profiling, and phospho-flow cytometry.

Aim 2: Evaluate therapeutic responses and mechanistic biomarkers using multimodality treatments relevant to hemispheric PHGG. They will use radiation, cytotoxic and targeted chemotherapies, and immunotherapies in the models. They will quantify direct cytotoxicity, immune modulation, and macrophage state transitions in tumoroids and in the full co-culture. They will also integrate single-cell and spatial readouts to define resistance programs and combinatorial opportunities.

Medulloblastoma, the most common malignant pediatric brain tumor, lacks United States Food and Drug Administration–approved, subgroup-specific drugs for non-WNT/non-SHH disease, and outcomes for Group 3 and Group 4 remain poor. This project proposes a fully human, animal-free platform to de-risk a radiotherapy-synchronized pro-digoxin strategy that aims to achieve tumor-selective efficacy beyond the blood–brain barrier while widening the cardiac safety margin.

Aim 1 builds and validates a medulloblastoma-blood-brain barrier “assembloid” by integrating induced-pluripotent-stem-cell-derived cerebellar organoids, patient-derived medulloblastoma cells (including paired primary/recurrent lines), and induced-pluripotent-stem-cell-derived vascular organoids; success is defined by physiologic barrier integrity, active efflux, and preserved tumor biology with a shareable standard operating procedure.

Aim 2 quantifies barrier transport and efflux, maps radiotherapy-triggered unmasking of the pro-drug, measures intratumoral exposure and tumor-to-normal selectivity, and fits an interpretable human pharmacokinetic–pharmacodynamic–efficacy model to guide dose and schedule for fractionated radiotherapy. Predictions will be benchmarked to an ongoing multicenter Phase II digoxin trial, enabling immediate translational use without new animal studies. If successful, this work will replace exploratory animal experiments for brain delivery, pharmacodynamic effect, and cardiotoxicity in medulloblastoma and provide a generalizable human-only blueprint for other pediatric brain tumors.

Fusion-positive rhabdomyosarcoma (FPRMS) is an aggressive childhood cancer with poor clinical outcomes, characterized by collective cell invasion driven by the cooperation between “leader” and “follower” cells. This project investigates the spatial molecular heterogeneity that establishes this leader-follower coordination and sustains invasive growth. Preliminary data from patient tissues and spheroid models suggest that leader-like cells at the tumor margin exhibit lower PAX3-FOXO1 fusion gene expression, a key driver of this cancer. Our non-animal FPRMS spheroid model, which successfully recapitulates this spatial molecular heterogeneity, will be leveraged to better understand these mechanisms. This approach addresses the dual goals of the program by improving the understanding of a devastating childhood cancer while demonstrating the value of innovative, non-animal research methods for biomedical research. The long-term objective is to develop new therapeutic strategies that target the mechanisms of collective cell invasion to suppress tumor growth and improve patient outcomes.

The specific aims are to: (1) Determine if the marginal regions of FPRMS masses and the FPRMS spheroid models, which exhibit lower PAX3-FOXO1 levels, demonstrate leader cell molecular features; and (2) Elucidate whether interaction with the stromal extracellular matrix defines marginal cells as leaders by reducing PAX3-FOXO1 expression and conferring their resistance to anti-proliferative drugs.

Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor and a leading cause of childhood cancer death. Despite multimodal therapy, relapse is frequent and toxicities substantial. CAR-T cells are promising, yet early trials show limited efficacy. Converging data implicate the NB tumor microenvironment (TME), rich in M2-like macrophages and a stiff extracellular matrix, as a key barrier that restricts anti-tumor response. Matrix stiffening activates mechanosensory pathways (e.g., CaMKK2-AKT), reprogramming macrophages toward immunosuppressive states.

Dual-goal focus: (1) Advance understanding and treatment of childhood cancer by defining actionable mechanisms of CAR-T resistance in NB and testing macrophage-targeted therapies; (2) demonstrate the value of non-animal methods via a human-relevant organ-on-chip that replaces/reduces studies in mice.

Approach: The NB Tumor Microenvironment Chip (NB-TEMC) will integrate the MIVO perfusion system with stiffness-tunable hydrogels to embed NB cells and suppressive macrophages, thereby delivering CAR-T under physiologic conditions. Real-time readouts (infiltration, cytotoxicity, exhaustion, cytokines) will be integrated into a Composite CAR-T Efficacy on-Chip Score (CARECS) for standardized comparisons. Specific Aims: (1) Determine whether ECM stiffness–induced macrophage reprogramming blunts CAR-T efficacy in NBTEMC; (2) test whether inhibition of the CaMKK2-AKT axis blocks M2 macrophages, enhancing CAR-T function; down-select top regimen(s) for preclinical follow-up.

The long-term objectives are to prioritize macrophage-targeted drug-CAR-T combinations for translation; establish NB-TEMC as a reproducible, immune-competent NAM for NB; extend the modular platform (e.g., marrow, vasculature) to support efficacy and safety across pediatric solid tumors. The impact is coupling mechanistic discovery with a scalable non-animal platform that delivers clinically relevant insights and accelerates translational decision-making for high-risk NB.