PNIPAAm-co-Jeffamine® (PNJ) scaffolds as in vitro models for niche enrichment of glioblastoma stem-like cells

Abstract

Glioblastoma (GBM) is the most common adult primary brain tumor, and the 5-year survival rate is less than 5%. GBM malignancy is driven in part by a population of GBM stem-like cells (GSCs) that exhibit indefinite self-renewal capacity, multipotent differentiation, expression of neural stem cell markers, and resistance to conventional treatments. GSCs are enriched in specialized niche microenvironments that regulate stem phenotypes and support GSC radioresistance. Therefore, identifying GSC-niche interactions that regulate stem phenotypes may present a unique target for disrupting the maintenance and persistence of this treatment resistant population. In this work, we engineered 3D scaffolds from temperature responsive poly(N-isopropylacrylamide-co-Jeffamine M-1000® acrylamide), or PNJ copolymers, as a platform for enriching stem-specific phenotypes in two molecularly distinct human patient-derived GSC cell lines. Notably, we observed that, compared to conventional neurosphere cultures, PNJ cultured GSCs maintained multipotency and exhibited enhanced self-renewal capacity. Concurrent increases in expression of proteins known to regulate self-renewal, invasion, and stem maintenance in GSCs (NESTIN, EGFR, CD44) suggest that PNJ scaffolds effectively enrich the GSC population. We further observed that PNJ cultured GSCs exhibited increased resistance to radiation treatment compared to GSCs cultured in standard neurosphere conditions. GSC radioresistance is supported in vivo by niche microenvironments, and this remains a significant barrier to effectively treating these highly tumorigenic cells. Taken in sum, these data indicate that the microenvironment created by synthetic PNJ scaffolds models niche enrichment of GSCs in patient-derived GBM cell lines, and presents tissue engineering opportunities for studying clinically important behaviors such as radioresistance in vitro.

Three‐dimensional biomimetic hyaluronic acid hydrogels to investigate glioblastoma stem cell behaviors

“…The physical barriers provided by the HA hydrogels caused the GSCs to form smaller but uniform spheroids compared to the tumorspheres in the suspension culture. These observations are consistent with recent studies utilizing PNIPAAm-co-Jeffamine ® (PNJ), PNIPAAm-PEG, and PEGDA-alginate scaffolds (Heffernan et al, 2017;Li et al, 2016;Oh, Cha, Kang, & Kim, 2016) to study GSCs. Overall, our results indicated that HA hydrogels could serve as a valuable tool to study clonally expanded glioblastoma cells.…”

“…This observation is consistent with previous results of more homogenous cell distribution throughout the poly(lactide‐co‐glycolide scaffolds obtained after increasing the initial seeding density of bone‐marrow‐derived cells (Holy, Shoichet, & Davies, ). Glioblastoma cells have been cultured as multicellular tumorspheres to maintain their stemness phenotype (Bez et al, ; Heffernan et al, ). However, the large size of tumorspheres resulted in limited diffusion of nutrients and signaling factors essential for the maintenance of stem characteristics, and thereby, poor stem‐like population (Beier et al, ; Pollard et al, ; Woolard & Fine, ).…”

Design of polymer-based antimicrobial hydrogels through physico-chemical transition

“…For example, Javan and coworkers [41] introduced a jeffamine chemical functionalization in hyaluronic acid-based hydrogels to formulate a prolonged and sustained drug release and reduce the number of hydrogel injections: in vivo studies showed the efficiency of this material thanks to the improved biocompatibility and no signal of cell viability reduction. Similar biocompatibility aims were achieved in other works: the jeffamine was used as monomer to preserve some physico-mechanical properties, blood compatibility and cargo release [42][43][44][45], as copolymer with acrylamides derivatives to design 3D scaffolds able to modulate the lower critical solution temperature (LCST) and internalize cells [46] or as cross-linker to satisfy the no-cytotoxicity criteria [47]. However, the evaluation of the polymer antimicrobial features and the design of a 3D scaffold able to preserve them is not widely investigated.…”

Modeling Microenvironmental Regulation of Glioblastoma Stem Cells: A Biomaterials Perspective

“…,Florczyk et al (2016),and Oh et al (2016) Poly(N-isopropylacrylamide)Heffernan et al (2016Heffernan et al ( , 2017, andLi et al (2016) PolyacrylamideUlrich et al (2009),Pathak and Kumar (2012),Ruiz-Ontañon et al (2013),Fernandez-Fuente et al (2014),,Umesh et al (2014),Wong et al (2015), andGrundy et al (2016) PolycaprolactoneRao et al (2013a),Jain et al (2014),Kievit et al (2014Kievit et al ( , 2016, andCha et al (2016) PolystyreneKievit et al (2014) andMa et al (2016a) Poly(lactic acid)Ma et al (2012) Bioactive peptide/proteinTamaki et al (1997),Cordes et al (2003),Sarkar et al (2006),Ulrich et al (2009), Ananthanarayanan et al (2011), Pathak and Kumar (2012), Ruiz-Ontañon et al (2013), Jain et al (2014), Kim and Kumar (2014), Rape and Kumar (2014), Umesh et al (2014), Wang et al (2014), Rape et al (2015), Wong et al (2015), Heffernan et al (2016), Ma et al (2016a), and Ngo and Harley (2017) Complex three-dimensional (3D) models Ma et al (2012), Pathak and Kumar (2012), Rao et al (2013a), Jain et al (2014), Herrera-Perez et al (2015), Pedron et al (2015), Rape et al (2015), Cha et al (2016), Fan et al (2016), Li et al (2016), and Chonan et al (2017) BiOPHYSiCAL PROPeRTieS Stiffness Kim et al (2008), Ulrich et al (2009, 2010), Yang et al (2010), Ananthanarayanan et al (2011), Pathak and Kumar (2012), Florczyk et al (2013, 2016), Pedron and Harley (2013), Pedron et al (2013, 2015), Rao et al (2013a,b), Fernandez-Fuente et al (2014), Heffernan et al (2014, 2016, 2017), Kim and Kumar (2014), Rape and Kumar (2014), Umesh et al (2014), Wang et al (2014), Herrera-Perez et al (2015), Rape et al (2015), Wong et al (2015), Cha et al (2016), Grundy et al (2016), Chen et al (2017), and Ngo and Harley (2017) Porosity Kim et al (2008), Yang et al (2010, 2014), Ananthanarayanan et al (2011), Ma et al (2012, 2016a), Pathak and Kumar (2012), Florczyk et al (2013, 2016), Pedron and Harley (2013), Rao et al (2013a,b), Kievit et al (2014, 2016), Wang et al (2014, 2016), Herrera-Perez et al (2015), Cha et al (2016), Fan et al (2016), and Oh et al (2016) Microchannels Pathak and Kumar (2012), Pedron et al (2015), Fan et al (2016), and Chonan et al (2017) Fibers/alignment Kim et al (2008), Ulrich et al (2010), Yang et al (2010), Rao et al (2013a,b), Jain et al (2014), Herrera-Perez et al (2015), Cha...…”