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Title: Validation of Human-Derived Neuroblastoma Organoids and Stem Cells for Fluorescence-Guided Surgery Targeting Neuroblastoma Antigens
Supervisor: Stefano Giuliani, Paolo de Coppi
Project Description:
Background
Neuroblastoma, the most common extracranial solid tumor in children, requires complete surgical resection for optimal outcomes. Accurate tumor margin delineation remains a significant challenge with conventional imaging techniques, leading to incomplete resections and higher recurrence rates. Targeted fluorescence-guided surgery (FGS) is a promising approach that enhances tumor visualization through fluorescent-labeled probes targeting specific cancer antigens. This study will use human-derived neuroblastoma organoids and stem cells to assess the efficacy of FGS targeting various neuroblastoma-specific antigens over a three-year PhD project.
Aim
The study aims to validate patient-derived neuroblastoma organoids and stem cells as models for evaluating FGS targeting neuroblastoma antigens. Specific objectives include:
1-Model Characterization: Establish and characterize neuroblastoma organoids and stem cells for neuroblastoma markers.
2-Target Identification: Identify promising cancer surface antigens and intracellular targets for FGS.
3-Probe Development: Conjugate antibodies with fluorescent dyes and develop nanoparticles for delivering fluorescent molecules. Validate their binding specificity, internalization, and efficacy.
4-Imaging and Testing: Use multispectral near-infrared and short-wave infrared cameras to image neuroblastoma models. Evaluate FGS efficacy in vitro and in vivo using established mouse models.
5-Protocol Development: Develop and finalize protocols for preclinical testing of FGS techniques using these models.
Methods
Organoid and Stem Cell Culture:
-Establish neuroblastoma organoids and stem cells from patient-derived samples using three-dimensional culture methods.
-Characterize these models for neuroblastoma markers such as CD56, GD2, PHOX2B, MYCN, and ALK using flow cytometry, immunostaining, and RNA profiling.
Target Selection:
-Screen and validate cancer surface antigens (e.g., B7H3, GD2) and intracellular targets (e.g., PHOX2B, MYCN, ALK) for FGS.
Quantification and Probe Development:
-Quantify target expression with BD-QuantiBrite beads and flow cytometry.
-Conjugate high-affinity antibodies with fluorescent dyes (e.g., IRDye800) and evaluate their binding specificity and internalization in vitro.
-Develop nanoparticles for fluorescent molecule delivery and assess their efficacy in organoids.
Imaging and In Vivo Testing:
-Image neuroblastoma models using multispectral near-infrared and short-wave infrared cameras to assess FGS effectiveness.
-Test FGS in vivo using neuroblastoma mouse models. Assess the ability to delineate tumor margins with the developed probes and nanoparticles.
Timeline
Year 1:
Months 1-6: Culture and characterize neuroblastoma organoids and stem cells. Initial target identification.
Months 7-12: Screen and validate cancer antigens and intracellular targets.
Year 2:
Months 13-18: Develop and test antibody-fluorophore conjugates. Develop and evaluate nanoparticles.
Months 19-24: In vitro testing of FGS efficacy with organoids and stem cells. Monitor fluorescence emission and probe internalization.
Year 3:
Months 25-30: Image models and assess FGS in neuroblastoma mouse models.
Months 31-36: Data analysis, manuscript preparation, and protocol finalization.
References
- Maris, J. M. (2007). Neuroblastoma. The Lancet, 369(9579), 2106-2120.
- Matthay, K. K. (1999). Treatment of high-risk neuroblastoma with intensive chemotherapy, radiotherapy, autologous bone marrow transplantation, and 13-cis-retinoic acid. New England Journal of Medicine, 341(16), 1165-1173.
- Privitera, L. (2023). Shortwave Infrared Imaging Enables High-Contrast Fluorescence-Guided Surgery in Neuroblastoma. Cancer Research, 83(12), 2077-2089.
Contact Information:
Stefano Giuliani