A physiologically relevant 3D collagen-based scaffold–neuroblastoma cell system exhibits chemosensitivity similar to orthotopic xenograft models
3D scaffold-based in vitro cell culture is a recent technological advancement in cancer research bridging the gap between conventional 2D culture and in vivo tumors. The main challenge in treating neuroblastoma, a pediatric cancer of the sympathetic nervous system, is to combat tumor metastasis and resistance to multiple chemotherapeutic drugs. The aim of this study was to establish a physiologically relevant 3D neuroblastoma tissue-engineered system and explore its therapeutic relevance. Two neuroblastoma cell lines, chemotherapeutic sensitive Kelly and chemotherapeutic resistant KellyCis83 were cultured in a 3D in vitro model on two collagen-based scaffolds containing either glycosaminoglycan (CollGAG) or nanohydroxyapatite (Coll-nHA) and compared to 2D cell culture and an orthotopic murine model. Both neuroblastoma cell lines actively infiltrated the scaffolds and proliferated displaying >100-fold increased resistance to cisplatin treatment when compared to 2D cultures, exhibiting chemosensitivity similar to orthotopic xenograft in vivo models. This model demonstrated its applicability to validate miRNA-based gene delivery. The efficacy of liposomes bearing miRNA mimics uptake and gene knockdown was similar in both 2D and 3D in vitro culturing models highlighting the proof-of-principle for the applicability of 3D collagen-based scaffolds cell system for validation of miRNA function. Collectively, this data shows the successful development and characterisation of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies and investigation of neuroblastoma pathogenesis. While neuroblastoma is the specific disease being focused upon, the platform may have multifunctionality beyond this tumor type. Statement of Significance Traditional 2D cell cultures do not completely capture the 3D architecture of cells and extracellular matrix contributing to a gap in their understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results.
Here, they demonstrated the successful development and characterization of a physiologically relevant, scaffold-based 3D tissue-engineered neuroblastoma cell model, strongly supporting its value in the evaluation of chemotherapeutics, targeted therapies, and investigation of neuroblastoma pathogenesis1.
The proliferation of KellyLuc sensitive and KellyCis83Luc resistant neuroblastoma cell lines on two different collagen-based scaffolds compared to traditional 2D monolayer cell culture. (A) Schematic of 3D tissue-engineering model for neuroblastoma using collagen-based scaffolds. (B) Representative confocal imaging of the collagen-based scaffold colonized by neuroblastoma cells on day 14. Bar, 200 lm.
The ability to test drugs in this reproducible and controllable tissue-engineered model system will help reduce the attrition rate of the drug development process and lead to more effective and tailored therapies. Importantly, such 3D cell models help to reduce and replace animals for pre-clinical research addressing the principles of the 3Rs.
In the native microenvironment, tumor cells are surrounded by three-dimensional (3D) essential physical scaffolding called extracellular matrix (ECM). The ECM composition is shaped by proteoglycans and fibrous proteins that are secreted locally by cells and remain closely connected. The ECM shapes cellular architecture and maintains tissue homeostasis. The tumor ECM can influence disease progression, patient prognosis, and response to treatment. A major challenge today is to distinguish the relative contributions of structural, molecular, and microenvironmental changes to cancer progression. Traditional 2D cultures do not completely capture the 3D architecture of cells and ECM leading to a gap in their understanding of mammalian biology at the tissue level and may explain some of the discrepancies between in vitro and in vivo results leading to only 1 in 10 drugs reaching clinical trials and approval by the FDA. 3D scaffold-based in vitro cell culture is a new innovative approach in cancer research to bridge the gap between conventional 2D culture and in vivo tumors. The use of scaffold-based cell culturing would help to reduce and/or replace animals for pre-clinical research aligning with the guiding principles for the care and use of animals in biomedical research known as the 3Rs (Replacement, Reduction, and Refinement ) and increase the potential for a strong economic impact and patient benefit. The use of such 3D culture systems allows for the precise manipulation of cell and ECM components of the microenvironment. The analysis of their contribution to the structure and function of a cell or tissue is vital in their understanding of disease progression and the discovery of new effective drugs. Scaffolds provide a 3D structural matrix that offers the necessary support for cells to proliferate, migrate, differentiate, deposit ECM and respond to stimuli, similar to in vivo biological systems.
Bioluminescent assessment of KellyLuc (A) and KellyCis83Luc cell lines (B). Representative bioluminescent imaging of KellyLuc (C) and KellyCis83Luc (D) tumors in orthotopic xenograft models of neuroblastoma obtained at Day 7, Day 14, and Day 21 post-implantation. Tumor weight analysis following 18 days of 3.5 mg/kg cisplatin treatment (E). Typical KellyLuc and KellyCis83Luc tumors after extraction without cisplatin treatment (F). Asterisks indicate statistical significance obtained using a paired Student’s t-test.
Collagen is a very attractive material for tissue-engineering and regenerative medicine applications because of its natural occurrence in the human body. Collagen triggers the driving force underlying the cell adhesion, migration, chemotaxis, and tissue development as well as contributes to its mechanical properties. 3D collagen-based scaffolds can be specifically engineered to mimic the intrinsic physiological conditions and have controllable and adaptable properties that facilitate the infiltration of cells and nutrients while being biocompatible, biodegradable, and non-toxic. Originally developed for regenerative medicine applications with a particular focus on bone repair, they are increasingly being used for disease modeling and drug screening. The use of such scaffolds to culture cells in a 3D environment in vitro is gaining greater recognition as a technologically progressive platform for further investigation of disease mechanisms and pathology in an environment that partially mimics a solid tumor microenvironment. Neuroblastoma is a highly aggressive pediatric solid tumor arising from the sympathetic nervous system, which accounts for approximately 15% of all childhood cancer deaths. The disease displays certain clinical, genomic, and transcriptomic alterations that contribute to a highly heterogeneous clinical behavior and are associated with very unfavorable outcomes in pediatric patients. Results from analyzing the survival of almost 60,000 children with cancer identified no improvements in survival for children with aggressive disease (e.g. high-risk neuroblastoma) despite an intensive induction treatment. Almost 20% of children with the aggressive disease do not respond at all, and up to 50% of children that do respond experience disease recurrence with metastatic foci resistant to multiple drugs. The main challenge in treating high-risk neuroblastoma is to combat tumor metastasis and the development of resistance to multiple chemotherapeutic drugs thus emphasizing an imminent need for new treatment options. Current neuroblastoma studies employ either 2D cell culture systems, murine models, or alternatively a mix of both, increasing the risk of inconsistencies between these two research models, and thus highlighting the limited translational efficacy of the results obtained in 2D models and requiring new pre-clinical models. In order to establish and characterize physiologically relevant tissue-engineered 3D neuroblastoma in vitro models capable of recapitulating elements of a native tumor tissue microenvironment, we aimed to examine cisplatin sensitive and resistant neuroblastoma cells on different collagen-based scaffolds, collagen glycosaminoglycan (Coll-GAG) and collagen-nanohydroxyapatite (Coll-nHA).
CgA expression by KellyLuc and KellyCis83Luc cells grown in 2D monolayer as determined using ELISA (A). Levels of human CgA in the blood of mice harboring KellyLuc and KellyLucCis83 tumours directly correlates with tumour weight. KellyLuc = blue dots and KellyLucCis83 = red squares. Pearson correlation, p = 0.0059 (B). Expression of human CgA is higher in KellyCis83Luc tumours than in KellyLuc tumours as demonstrated by Western blot and densitometry analysis (C). Secretion of CgA by KellyLuc and KellyCis83Luc cells over 21 days on Coll-GAG (D) and Coll-nHA scaffolds (E). Asterisks indicate statistical significance obtained using an unpaired Student’s t-test.
If successful, this model could be used further to gain insights into mechanisms of disease pathogenesis and improve the translational efficacy between results obtained in vitro, in vivo, and in the clinic. Both Coll-GAG and Coll-nHA scaffolds have been successfully used to study the primary tumor microenvironment in breast cancer and metastasis to bone in prostate cancer, respectively. Therefore, they may represent attractive matrices for modeling metastatic neuroblastoma as bone marrow (70.5%) and bone (55.7%) are the most common sites for metastases. Both types of scaffolds have controllable physical and biological properties and consist of a porous, collagen-based layer fabricated using freeze-drying techniques that were originally developed and extensively studied for bone tissue engineering applications. GAGs, negatively charged carbohydrates, are commonly found in the ECM involved in cell attachment, migration, proliferation, and differentiation. Nano-hydroxyapatites (nHAs), a calcium phosphate, are common elements of the mineral composition of the human bone tissues and extensively used as a biocompatible material for bone replacement and regeneration. Coll-nHA scaffolds have been extensively characterized for their biocompatibility, toxicity, and osteoconductive and osteoinductive features. Thus GAGs and nHA are attractive composites for reconstructing primary and metastatic bone/bone marrow tumor microenvironment. Having characterized the cell response to chemotherapeutic in the proposed 3D in vitro cell model, they then used this model to evaluate miRNA-mediated gene regulation. MiRNAs are a class of small, noncoding RNAs that regulate gene expression at the translational level. These molecules control the expression of a great variety of genes driving cell cycle, migration, differentiation, development, apoptosis, and metabolism. Numerous studies describe dysregulation of miRNA expression in tumors, including neuroblastoma emphasizing their potential in the generation of new drugs for therapeutic intervention. In neuroblastoma, some miRNA was found to be over-or under-expressed demonstrating their complex role as either ‘‘oncomirs” or tumor-suppressors, respectively. These functions can be exploited to repair signaling pathways essential for normal cellular function and block those upregulated in pathological conditions. Unsurprisingly, the development of miRNA therapeutics is under extensive investigation by several companies for a variety of health conditions, including cancer.
Here, they explored the 3D scaffold-based in vitro cell culturing platform for neuroblastoma, however, it may have multi-functionality beyond this tumor type1.
(A) Ectopic overexpression of miR-324-5p mimics transfection into Kelly cells grown in 2D significantly reduced cell viability (p = 0.016), indicating a potential tumor-suppressive function. Expression of miRNA (B) and VDAC1 mRNA (C) 48hr after transfection with miR-324-5p mimic or scrambled negative control in neuroblastoma Kelly cell line in 2D as determined by RT-qPCR. (D) Effect of miR-324-5p mimics transfection on cell DNA content 7 days after transfection on Coll-nHA scaffold. Expression of miRNA (E) and VDAC1 mRNA (F) 7 days after transfection with miR-324-5p mimic or scrambled negative control in neuroblastoma Kelly cell line in 3D as determined by RT-qPCR. (G) Assessment of viability of neuroblastoma cells grown on collagen-based scaffolds 7 days after transfection with miR-324-5p mimic or scrambled negative control using DAPI labeled cell nuclei, Calcein AM (green fluorescence) labeled live cells and EthD-1 (red fluorescence) labeled dead cells. Labeling miR-324-5p mimic, miR-324-5p, scrambled negative control, NC, positive control siKinesin, KIFF. Asterisks indicate statistical significance obtained using a paired Student’s t-test.
1. C. Curtin, J.C. Nolan, R. Conlon, L. Deneweth, C. Gallagher, Y.J. Tan, B.L. Cavanagh, A.Z. Asraf, H. Harvey, S. Miller-Delaney, J. Shohet, I. Bray, F.J. O'Brien, R.L. Stallings, O. Piskareva,
A physiologically relevant 3D collagen-based scaffold–neuroblastoma cell system exhibits chemosensitivity similar to orthotopic xenograft models,
Acta Biomaterialia, Volume 70, 2018, Pages 84-97, ISSN 1742-7061,