Engineering a multicellular metastatic bone marrow niche using foamed beta tricalcium phosphate scaffolds for advanced neuroblastoma in vitro modeling and drug testing

Next Materials, vol. 13, pp. 103575

Abstract

Bone marrow metastasis is a hallmark of high-risk neuroblastoma (NB) and is associated with poor prognosis, therapy resistance, and disease recurrence. Despite significant advances in cancer biology, current in vitro models inadequately reproduce the complexity of the metastatic bone marrow microenvironment, limiting the predictive value of preclinical studies. Here, we developed versatile three-dimensional (3D) foamed beta tricalcium phosphate (βTCP) scaffolds to engineer an in vitro multicellular NB metastatic bone marrow niche. We fabricated spongy βTCP scaffolds using a polyurethane mold and an organic slurry, and then sintered them to obtain mechanically stable constructs with interconnected porosity. We used thermal and spectroscopic analyses to assess scaffold structure, and advanced microscopy for morphological and functional evaluations of the metastatic in vitro model. Human mesenchymal stromal cells efficiently colonized the scaffolds, maintained viability for 28 days, and remodeled the acellular microenvironment through extracellular matrix protein deposition. Moreover, modulation of RUNX2 and Collagen I gene levels confirmed their pro-osteogenic commitment. Sequential introduction of endothelial cells, hematopoietic stem cells, and NB cells enabled generation of a multicellular metastatic niche that recapitulates key cellular components of the bone marrow. Chemokine profiling revealed modulation of inflammatory, angiogenic, and stromal-remodeling pathways, including NF-κB, STAT3, IL-2 signaling, epithelial-to-mesenchymal transition, and pro-angiogenic signatures. Functional validation demonstrated that therapeutic responses differed substantially between non-malignant and metastatic multicellular systems, highlighting the critical contribution of the complex cell-to-cell interactions to treatment outcomes. Collectively, these findings establish a biomimetic, versatile 3D in vitro model that reproduces essential structural and biological features of the metastatic bone marrow niche, providing a valuable tool for further NB research and preclinical drug screening.

Authors 20

1. Diana Corallo

   Città della Speranza Foundation

   Affiliation as printed

   Laboratory of Target Discovery and Biology of Neuroblastoma, Pediatric Research Institute Città della Speranza Foundation, 35127 Padua, Italy

2. Sara Menegazzo

   Città della Speranza Foundation

   Affiliation as printed

   Laboratory of Target Discovery and Biology of Neuroblastoma, Pediatric Research Institute Città della Speranza Foundation, 35127 Padua, Italy

3. Alessandro Davini

   Città della Speranza Foundation

   Affiliation as printed

   Laboratory of Target Discovery and Biology of Neuroblastoma, Pediatric Research Institute Città della Speranza Foundation, 35127 Padua, Italy

4. Lucio Litti

   University of Padua

   Affiliation as printed

   Department of Chemical Sciences, University of Padova, 35131 Padua, Italy

5. Chiara Frasson

   Città della Speranza Foundation

   Affiliation as printed

   Flow Cytometry Core Service, Pediatric Research Institute Città della Speranza Foundation, 35127 Padua, Italy

6. Elena Mariotto

   University of Padua

   Affiliation as printed

   Department of Women's and Children's Health, University of Padova, 35128 Padua, Italy

7. Stefania Benazzato

   University of Padua · National Interuniversity Consortium of Materials Science and Technology

   Affiliation as printed

   Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali (INSTM), 50121 Florence, Italy

   Department of Chemical Sciences, University of Padova, 35131 Padua, Italy

8. Ester Marotta

   University of Padua · National Interuniversity Consortium of Materials Science and Technology

   Affiliation as printed

   Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali (INSTM), 50121 Florence, Italy

   Department of Chemical Sciences, University of Padova, 35131 Padua, Italy

9. Marcella Pantile

   Città della Speranza Foundation

   Affiliation as printed

   Laboratory of Target Discovery and Biology of Neuroblastoma, Pediatric Research Institute Città della Speranza Foundation, 35127 Padua, Italy

10. Marta Rizk Aachen Department of Orthodontics

    Universitätsklinikum Aachen · RWTH Aachen University

    Affiliation as printed

    Department of Orthodontics, RWTH Aachen University Hospital, 52074 Aachen, Germany

11. Michela Pozzobon

    University of Padua · Città della Speranza Foundation

    Affiliation as printed

    Department of Women's and Children's Health, University of Padova, 35128 Padua, Italy

    Stem Cells and Regenerative Medicine Lab, Pediatric Research Institute Città della Speranza Foundation, 35127, Padua, Italy

12. Stephan Rütten Aachen Electron Microscopy Facility Institute of Pathology

    Universitätsklinikum Aachen · RWTH Aachen University

    Affiliation as printed

    Electron Microscopy Facility, Institute of Pathology, RWTH Aachen University Hospital, 52074 Aachen, Germany

13. Michael Vogt Aachen Two-Photon Imaging Facility Interdisciplinary Center for Clinical Research (IZKF)

    Universitätsklinikum Aachen · RWTH Aachen University

    Affiliation as printed

    Two-Photon Imaging Facility, Interdisciplinary Center for Clinical Research, RWTH Aachen University Hospital, 52074 Aachen, Germany

14. Ronny Schmidt
    Affiliation as printed

    Sciomics GmbH, 69151 Neckargemünd, Germany

15. Ramy Girgis
    Affiliation as printed

    Sciomics GmbH, 69151 Neckargemünd, Germany

16. Florian Skwirblis

    Teamtechnik (Germany)

    Affiliation as printed

    Sciomics GmbH , Neckargemünd , 69151 , Germany

17. Horst Fischer Aachen Department of Dental Materials and Biomaterials Research

    Universitätsklinikum Aachen · RWTH Aachen University

    Affiliation as printed

    Department of Dental Materials and Biomaterials Research, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074 Aachen, Germany

18. Moreno Meneghetti

    University of Padua

    Affiliation as printed

    Department of Chemical Sciences, University of Padova, 35131 Padua, Italy

19. Enzo Menna

    University of Padua · National Interuniversity Consortium of Materials Science and Technology

    Affiliation as printed

    CNR Institute on Membrane Technology “Enrico Drioli”, Unit of Padova, 35131 Padua, Italy

    Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali (INSTM), 50121 Florence, Italy

    Department of Chemical Sciences, University of Padova, 35131 Padua, Italy

20. Sanja Aveic corresponding Aachen Department of Dental Materials and Biomaterials Research

    Universitätsklinikum Aachen · Città della Speranza Foundation · RWTH Aachen University

    Affiliation as printed

    Department of Dental Materials and Biomaterials Research, RWTH Aachen University Hospital, Pauwelsstrasse 30, 52074 Aachen, Germany

    Laboratory of Target Discovery and Biology of Neuroblastoma, Pediatric Research Institute Città della Speranza Foundation, 35127 Padua, Italy