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3D Tissue-Engineered Model of Ewing's Sarcoma


3D Tissue-Engineered Model of Ewing's Sarcoma

Salah-Eddine Lamhamedi-Cherradi et al. Adv Drug Deliv Rev.


Despite longstanding reliance upon monolayer culture for studying cancer cells, and numerous advantages from both a practical and experimental standpoint, a growing body of evidence suggests that more complex three-dimensional (3D) models are necessary to properly mimic many of the critical hallmarks associated with the oncogenesis, maintenance and spread of Ewing's sarcoma (ES), the second most common pediatric bone tumor. And as clinicians increasingly turn to biologically-targeted therapies that exert their effects not only on the tumor cells themselves, but also on the surrounding extracellular matrix, it is especially important that preclinical models evolve in parallel to reliably measure antineoplastic effects and possible mechanisms of de novo and acquired drug resistance. Herein, we highlight a number of innovative methods used to fabricate biomimetic ES tumors, encompassing both the surrounding cellular milieu and the extracellular matrix (ECM), and suggest potential applications to advance our understanding of ES biology, preclinical drug testing, and personalized medicine.

Keywords: 3D; ECM; Ewing's sarcoma; MCTS; Preclinical testing; Scaffolds; Tissue-engineering; Tumor model.


Figure 1
Figure 1
(A) Principal components of the tumor microenvironment that affect cell behavior. Signaling factors include ECM, GFs, etc. Biomechanical forces include 3D architecture and mechanical loading. Hypoxia, pH, nutrients and stress are affected affecting malignant cell phenotype. (B) Key cell effectors in tumor microenvironment. Abbreviations: GF, growth factor; ECM, extracellular matrix; MMPs, metalloproteinases; IGF-1, insulin growth factor 1; IGF-1R, insulin growth factor 1-receptor; FAD, focal adhesion kinase; PI3K, phosphatidylinositide 3-kinases; ATP, Adenosine triphosphate; mTOR, mammalian target of rapamycin; NHE-Flux, sodium-proton exchanger; HIF-1, hypoxia inducible actor-1; ROS, reactive oxygen species; and ICAM, intercellular adhesion molecule 1.
Figure 2
Figure 2. Multicellular tumor spheroids (MCTS) in vitro production techniques of ES
(A) Spinner flask spheroid cultures. (B) Micro-etched Nano-cultures. (C) Biologically (e.g. Collagen gel) derived 3D matrices cultures. (D) ES Hanging-drop cultures. (D1) ES cell line counting using Beckman Coulter Vi-Cell XR Cell Viability Analyzer. (D2) 20μL of cell suspension (100 ES cells) plated on the lid of a Greiner 96-well plate. (D3) The lid was placed back on the 96-well plate containing 100 mL of RPMI (cell culture complete medium) and carefully placed in the incubator for 72 hours. (D4) The lids were removed and 300 μL of RPMI was added to a Nano-Culture® Plate (Scivax NCP-LS) to allow the drop to come in contact with the media, re-incubate for one hour and remove 100 μL. (D5) Spheroids were imaged using the GE InCell Analyzer 6000. (D6) Images of ES spheroids cells at 2 × 104 cells / mL and 5 × 104 cells /mL.
Figure 3
Figure 3. Chemotherapeutic and biologically targeted drug sensitivity testing ES monolayers and spheroids
ES were cultured as both monolayers and spheroids in the presence of chemotherapeutic and biologically targeted drugs. Cell viability data are shown. (A) Response to doxorubicin (CID: 31703) (B) Response to fully humanized monoclonal antibody R1507 anti-human IGF-1R (Roche).
Figure 4
Figure 4. Scanning electron micrographs of PCL microfiber scaffolds
(A) before cell seeding, (B) after 2 days in static ES cell culture, (C&D) after 24 days in static ES cell culture. After 24 days of static culture, ES exhibit significant 3D cell-cell and cell-matrix contacts.
Figure 5
Figure 5
IHC staining of human ES tumor for VE-Cadherin under low-(A) and high- power (B) magnification confirming the coexistence of EC.
Figure 6
Figure 6
Bioengineered models that mimic ES microenvironment at the primary, hematogenous and secondary sites. (A) Bioengineered preclinical models of ES interacting with osseous-like 3D scaffold (B) or synthetic vasculature (C). (D) Vascular system by which ES disseminate to lung. (E) Lung metastases modeled lung. (F) Lung-on-chip or (G) Decellularized lung. Abbreviations: CTC, circulating tumor cells; EC, endothelial cell; ECM, extracellular matrix; MSC, mesenchymal stem cell; and WBC, white blood cell.

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