Three-Dimensional-Engineered Matrix to Study Cancer Stem Cells and Tumorsphere Formation: Effect of Matrix Modulus

Abstract

Maintenance of cancer stem cells (CSCs) is regulated by the tumor microenvironment. Synthetic hydrogels provide the flexibility to design three-dimensional (3D) matrices to isolate and study individual factors in the tumor microenvironment. The objective of this work was to investigate the effect of matrix modulus on tumorsphere formation by breast cancer cells and maintenance of CSCs in an inert microenvironment without the interference of other factors. In that regard, 4T1 mouse breast cancer cells were encapsulated in inert polyethylene glycol diacrylate hydrogels and the effect of matrix modulus on tumorsphere formation and expression of CSC markers was investigated. The gel modulus had a strong effect on tumorsphere formation and the effect was bimodal. Tumorsphere formation and expression of CSC markers peaked after 8 days of culture. At day 8, as the matrix modulus was increased from 2.5 kPa to 5.3, 26.1, and 47.1 kPa, the average tumorsphere size changed from 37 -6 mm to 57 -6, 20 -4, and 12 -2 mm, respectively; cell number density in the gel changed from 0.8 -0.1 · 10 5 cells/mL to 1.7 -0.2 · 10 5 , 0.4 -0.1 · 10 5 , and 0.2 -0.1 · 10 5 cells/mL after initial encapsulation of 0.14 · 10 5 cells/mL; and the expression of CD44 breast CSC marker changed from 17 -4-fold to 38 -9-, 3 -1-, and 2 -1-fold increase compared with the initial level. Similar results were obtained with MCF7 human breast carcinoma cells. Mouse 4T1 and human MCF7 cells encapsulated in the gel with 5.3 kPa modulus formed the largest tumorspheres and highest density of tumorspheres, and had highest expression of breast CSC markers CD44 and ABCG2. The inert polyethylene glycol hydrogel can be used as a model-engineered 3D matrix to study the role of individual factors in the tumor microenvironment on tumorigenesis and maintenance of CSCs without the interference of other factors.

The Role of Biomaterials on Cancer Stem Cell Enrichment and Behavior

“…19 Similar to results from other studies discussed earlier, 17 CSC marker expression was time-dependent, peaking at 8 days of culture. 19 In another study, alginate beads with soft (21 kPa), moderate (70 kPa) and hard (105 kPa) stiffness were generated by changing alginate concentration and used to study the effect of 3D matrix stiffness on the biological characteristics of CSCs from human head and neck squamous cell carcinoma (HNSCC). 62 The authors found that significant CSC enrichment as well as gene expression of stemness markers was achieved in the alginate beads with moderate matrix stiffness.…”

“…For example, Yang et al used an inert polyethylene glycol with a range of modulus of 2.5-70 kPa to study the effect of stiffness on CSC marker expression and tumor spheroid formation. 19 Results showed that the gel modulus had a strong bimodal effect on the tumor spheroid formation and the expression of CSC markers: cells encapsulated in the gel with modulus of 5.3 kPa had the highest growth rate and formed the largest and highest number of tumor spheroids, further exhibiting the highest expression of CSC markers. 19 Similar to results from other studies discussed earlier, 17 CSC marker expression was time-dependent, peaking at 8 days of culture.…”

“…19 Results showed that the gel modulus had a strong bimodal effect on the tumor spheroid formation and the expression of CSC markers: cells encapsulated in the gel with modulus of 5.3 kPa had the highest growth rate and formed the largest and highest number of tumor spheroids, further exhibiting the highest expression of CSC markers. 19 Similar to results from other studies discussed earlier, 17 CSC marker expression was time-dependent, peaking at 8 days of culture. 19 In another study, alginate beads with soft (21 kPa), moderate (70 kPa) and hard (105 kPa) stiffness were generated by changing alginate concentration and used to study the effect of 3D matrix stiffness on the biological characteristics of CSCs from human head and neck squamous cell carcinoma (HNSCC).…”

“…18 For example, biomaterials afford the ability to fine tune the matrix stiffness that better mimics the stem cell niche. [19][20][21] These studies show that stiffness and mechanical matching could direct the fate of stem cells, instructing the stem cells to produce differentiated progeny cells rather than self-renew. Insights into the striking similarities between stem cells and CSCs have surfaced;…”

“…An added benefit of synthetic hydrogels over natural materials is the improved flexibility to design 3D matrices with a wide range of mechanical, physical, and biological properties that are crucial to the maintenance, differentiation, maturation, and fate of the encapsulated cells. 19 For example, an enzymatically degradable polyethylene glycol hydrogels with orthogonally controlled biophysical and biochemical properties have been developed for the study of pancreatic ductal adenocarcinoma. 58 When collagen was added to the matrix (i.e.…”