Abstract
IntroductionAs cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs) while keeping other factors unchanged. We have developed a synthetic inert 3D polyethylene glycol diacrylate (PEGDA) gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. We have reported that CSCs formed in the inert PEGDA gel by encapsulation of breast cancer cells maintain their stemness within a certain range of gel stiffness. The objective was to investigate the effect of CD44 binding peptide (CD44BP) conjugated to the gel on the maintenance of breast CSCs.Methods4T1 or MCF7 breast cancer cells were encapsulated in PEGDA gel with CD44BP conjugation. Control groups included dissolved CD44BP and the gel with mutant CD44BP conjugation. Tumorsphere size and density, and expression of CSC markers were determined after 9 days. For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined after 4 weeks. Effect of CD44BP conjugation on breast CSC maintenance was compared with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP).ResultsConjugation of CD44BP to the gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP.ConclusionCD44BP and IBP conjugated to the gel abolished tumorsphere formation by encapsulated 4T1 cells while FHBP enhanced tumorsphere formation compared to cells in the gel without peptide. The PEGDA hydrogel culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors.
Highlights
As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs) while keeping other factors unchanged
CD44 binding peptide (CD44BP) and integrin binding RGD peptide (IBP) conjugated to the gel abolished tumorsphere formation by encapsulated 4T1 cells while fibronectin-derived heparin-binding peptide (FHBP) enhanced tumorsphere formation compared to cells in the gel without peptide
Our results show that conjugation of FHBP to the gel matrix enhanced tumorsphere formation by the encapsulated 4T1 breast cancer cells while CD44BP and IBP abolished sphere formation in vitro
Summary
As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs) while keeping other factors unchanged. We have developed a synthetic inert 3D polyethylene glycol diacrylate (PEGDA) gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. Increasing evidence suggests that the heterogeneity of the tumor tissue is rooted in the existence of cancer stem cells (CSCs) [3]. Consistent with this notion, the triple negative breast cancer, which is one of the most aggressive types of breast cancer, contains a high fraction of CSCs [4,5]. We have developed an inert polyethylene glycol diacrylate (PEGDA) based in vitro 3D cell culture system which does not have cell interaction ligands, providing a unique tool to study tumor microenvironment in vitro. The CSCs of breast cancer can maintain their stemness and proliferate while the growth of nonCSCs is inhibited when encapsulated in the PEGDA gel within a certain range of elastic moduli
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