Background

Astrocytomas are central nervous system tumors of glial origin. Poor survival rates among patients within 5 years of diagnosis and almost inevitable relapse are the hallmarks of this life-threatening disease (Krex et al., 2007). Our understanding of disease pathogenesis is limited, resulting in the lack of effective intervention strategies. In vitro culture systems could prove useful for studying cell-cell interactions and intracellular signaling events in astrocytoma, and provide platforms for testing potential therapeutic compounds (Reardon et al., 2006; Baker and Chen, 2012). Three-dimensional (3D) in vitro culture platforms have especially gained popularity in recent years due to their biological relevance to in vivo tumors (Li et al., 2007; Whiteside, 2008; Tibbitt and Anseth, 2009; Xu et al., 2014; Jogalekar and Serrano, 2018). 3D cultures established using various tissue engineering scaffolds (e.g., hydrogels) allow for multi-layered growth of cells in a fashion similar to that of native tissue, unlike two-dimensional (2D) cultures, making them better predictors of responses to drug candidates.

Hydrogel culture systems could be exploited in parallel with monolayer cultures to further our understanding of cell-cell and cell-matrix interactions with high-throughput applications that analyze cellular gene expression, such as RNA-sequencing, microarray analysis and cloning. To accomplish this, a pure, intact, high quality RNA needs to be isolated in parallel from separately cultured monolayer and hydrogel cultures so that both cultures are processed under identical conditions and are therefore comparable. Several protocols have been reported for extracting RNA from 3D cultures established using a variety of commonly used tissue engineering scaffolds such as Matrigel and collagen (Lee et al., 2007; Mroue and Bissell, 2013). However, there is a paucity of information available with respect to RNA extraction from 3D cultures maintained in Geltrex^TM, a relatively new basement membrane matrix derived from murine Engelbreth-Holm-Swarm tumors and manufactured by Invitrogen.

When we used existing protocols to carry out RNA isolation from multiple cell lines including astrocytoma, we found that our astrocytoma cell line (CCF-STTG1) was highly sensitive to the reagents that dissolve extracellular matrices used in 3D cultures and could not yield an intact RNA at the end of the procedure, although this procedure worked well with other mammalian and amphibian cell lines we used in our experiment. Therefore, we modified RNA isolation protocols available in the literature to extract total RNA from monolayer and hydrogel cultures of astrocytoma. We used this RNA to carry out gene expression analyses for key autophagy pathway genes, Beclin-1 (BECN1) and microtubule associated protein 1 light chain 3 beta (MAP1LC3B), using quantitative polymerase chain reaction (qPCR) (Jogalekar et al., 2017). Our findings indicate a modest upregulation of these genes in hydrogel cultures of astrocytoma and are consistent with previous reports suggesting their upregulation in correlation with poor prognosis in glioma patients (Pirtoli et al., 2009; Giatromanolaki et al., 2014).