Developing zebrafish avatars for pediatric sarcomas to support functional precision oncology

Pediatric sarcomas, including rhabdomyosarcoma (RMS) and osteosarcoma, remain associated with poor outcomes in relapsed or high-risk disease, highlighting the need for rapid functional models to guide patient-specific therapeutic decisions. Zebrafish patient-derived xenografts (zPDX “avatars”) provide a low-cost, scalable in vivo platform to test drug responses within days. As a foundation for pediatric sarcoma avatars, we optimized zebrafish xenografts using human RMS cell lines (RD, embryonal; RH30, alveolar) prior to introducing primary patient samples. GFP-labeled RD cells injected into the dorsal perivitelline space (PVS) of 48-hours-post-fertilization larvae produced approximately 60% larval survival, nearly 100% engraftment at 1 day postinjection (dpi), and stable tumors through 4 dpi, enabling reproducible conditions for standardized tumor size and short-term drug testing. CellTiter-Glo assays at 34 °C, the incubation temperature required for zPDX experiments, confirmed that RD cells retain drug sensitivity under these conditions: dactinomycin (DAC) significantly reduced RD viability at low nanomolar concentrations (≥10 nM), whereas vincristine (VIN) was effective at 2-10 nM. Parallel larval toxicity assays indicated that both drugs were generally well tolerated by zebrafish larvae at concentrations ≤200 nM for 72 h, establishing a practical exposure window for in vivo testing. As a proof-of-concept for drug response assessment, GFP+ RD cells were injected into 48 hpf larvae and tumor burden (fluorescence intensity) and dissemination were evaluated following treatment with VIN and DAC at 15 nM. At this concentration, no statistically significant differences were observed versus control, although a trend was noted toward reduced tumor size and dissemination. In contrast, RH30 cells did not persist beyond 1-2 dpi when injected in simple carriers (PBS or HBSS + 1-2% FBS), prompting systematic optimization of the injection medium. Guided by media described for zPDX, we developed an enriched medium containing HEPES, glutamine, MEM-NEAA, B27, nicotinamide, ITS, Y-27632, SB202190, N-acetylcysteine, and EGF/FGF with reduced serum. Using this formulation, we achieved approximately 100% tumor-positive larvae up to 4 dpi, indicating that medium composition, particularly exogenous growth factors, are critical for xenograft retention. Together, these preliminary data define robust survival and engraftment parameters for RMS xenografts in zebrafish, validate the feasibility of this platform for rapid chemotherapy response readouts, and establish an optimized injection medium that enables even challenging sarcoma subtypes to persist in vivo. These advances provide the foundation for extending this platform to primary pediatric sarcomas, including osteosarcoma, to generate zebrafish avatars for functional precision oncology.