A Three-Dimensional-Engineered Matrix to Study the Microenvironment of Cancer Stem Cells

Maintenance of cancer stem cells (CSCs) is regulated by their microenvironment. As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on CSCs while keeping other factors unchanged. We developed a synthetic inert 3D Poly Ethylene Glycol Di-Acrylate (PEGDA) gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. Synthetic hydrogels provide the flexibility to design three-dimensional (3D) matrices to isolate and study individual factors in the tumor microenvironment. The first objective of this work was to investigate the effect of physical properties of microenvironment including modulus and geometry on maintenance of CSCs isolated of 4T1 (mouse breast cancer cell line), MCF7 and MDA-MB-231 (human Breast carcinoma cells) HCT116 (Colon), AGS (Gastric) and U-2 OS (Bone) human cancer cell lines in an inert microenvironment. We have shown that the gel elastic modulus had a strong effect on tumorsphere formation. CSCs formed in the inert PEGDA gel keep their stemness within certain ranges of gel stiffness. Briefly, mouse and human breast cancer cells encapsulated in the gel with approximate modulus of 5 kPa formed the largest and highest density of tumorspheres with highest expression of breast CSC markers.The highest number, diameter and CSC marker expression of HCT116 and AGS spheroids were observed in 25 kPa where it occurred at an optimum modulus of 50 kPa for U-2 OS cell line. We have also shown that geometry and spatial properties of ECM can significantly affect the proliferation rate and size of tumorspheres formed by MDA-MB-231 cells in PEGDA micropatternedhydrogesl. Weintroduced a 3D culture system as a model that controls the uniformity, size and proliferation of tumorspheres. As thesecondary objective,we investigated the effect of ECM composition on CSCs maintenance by using ECM-derived cell binding peptides active in malignancy. CD44 binding peptide (CD44bp), Integrin binding peptides (Ibps) and Heparin binding peptides (Hbps) were conjugated to the gel or dissolved in polymer solution to study their effect on the maintenance of CSCs. We have shown that peptides derived from ECM proteins with different mechanisms of action can influence the microenvironment and cancer cells functions by either promotion or abolition of the spheroid forming ability in the inert 3D PEGDA hydrogel cell culture system. Conjugation of CD44bp and Ibps to the gel inhibited tumorsphere formation in vitro and in vivo where Hbps enhanced tumorsphere formation and CSCs marker expression compared to cells in the gel without peptide