Primary cancer cell culture: mammary-optimized vs conditional reprogramming

Abstract

The impact of different culture conditions on biology of primary cancer cells is not always addressed. Here conditional reprogramming (CRC) was compared with mammary-optimized EpiCult™-B (EpiC) for primary mammary epithelial cell isolation and propagation, allograft generation and genome-wide transcriptional consequences using cancer and non-cancer mammary tissue from mice with different dosages of Brca1 and p53. Selective comparison to DMEM was included. Primary cultures were established with all three media but CRC was most efficient for initial isolation (p<0.05). Allograft development was faster using cells grown in EpiC as compared to CRC (p<0.05). Transcriptome comparison of paired CRC and EpiC cultures revealed 1700 differentially expressed genes by passage 20. CRC promoted Trp53 gene family up-regulation and increased expression of epithelial differentiation genes while EpiC elevated expression of epithelial-mesenchymal-transition genes. Differences did not persist in allografts where both methods yielded allografts with relatively similar transcriptomes. Restricting passage (<7) reduced numbers of differentially expressed genes below 50. In conclusion CRC was most efficient for initial cell isolation but EpiC was quicker for allograft generation. The extensive culture-specific gene expression patterns that emerged with longer passage could be limited by reducing passage number when both culture transcriptomes were equally similar to that of the primary tissue. Defining impact of culture condition and passage on the transcriptome of primary cells could assist experimental design and interpretation. For example differences that appear with passage and culture condition are potentially exploitable for comparative studies targeting specific biological networks in different transcriptional environments.

Responsiveness of Brca1 and Trp53 Deficiency–Induced Mammary Preneoplasia to Selective Estrogen Modulators versus an Aromatase Inhibitor in Mus musculus

“…Regions demonstrating deviation from the reference genome were identified and compared to regions that were modified in the Brca1 and Trp53 loci in the GEMM (30,31). For these analyses DNA was extracted directly from noncancerous mammary gland (MG) or cancer (CA) tissue or primary cultures of noncancerous or cancer MEC under conditional reprogramming culture without feeder cells (CRC) or EpiCult ™ B Mouse Medium (EpiC) (Stemcell Technologies, Vancouver, BC, Canada) (38). Primary mammary epithelial cell cultures were obtained from mammary glands of Brca1 fl11/fl11/Cre/p53−/+ and Brca1 WT/fl11/Cre/p53−/+ mice aged 8–11m (2011–2012), stored viably at −80°c when not passaged, authenticated (2015) for presence of Brca1 exon 11 deletion and Cre transgene by RT-PCR and PCR (38), and verified as having predicted genetically engineered deviations from the reference genome in Brca1 and Trp53 genes in this study (2016).…”

Expanding primary cells from mucoepidermoid and other salivary gland neoplasms for genetic and chemosensitivity testing

“…Conditionally reprogrammed cell (CRC) technology can generate primary epithelial cell cultures from normal, benign and malignant tissue from different species (30)(31)(32)(33)(34)(35). Here we explored its utility for establishing primary cell cultures from different types of human salivary gland neoplasms, examined if sufficient material for next generation sequencing could be obtained from the initial cultures, and tested if the cells could be expanded to evaluate growth and chemosensitivity under different culture conditions.…”

“…Here we explored its utility for establishing primary cell cultures from different types of human salivary gland neoplasms, examined if sufficient material for next generation sequencing could be obtained from the initial cultures, and tested if the cells could be expanded to evaluate growth and chemosensitivity under different culture conditions. In human normal keratinocytes the Rho kinase inhibitor (ROCK) Y-27632 induces down-regulation of genes involved in keratinization and differentiation (36) while in malignant mouse mammary epithelial cells use of CRC technology favors up-regulation of keratin (KRT) differentiation genes when compared to mammary optimized media (34). In mammary cells this is associated with up-regulation of Tumor Protein (TP)p53 family genes (34,37).…”

“…In human normal keratinocytes the Rho kinase inhibitor (ROCK) Y-27632 induces down-regulation of genes involved in keratinization and differentiation (36) while in malignant mouse mammary epithelial cells use of CRC technology favors up-regulation of keratin (KRT) differentiation genes when compared to mammary optimized media (34). In mammary cells this is associated with up-regulation of Tumor Protein (TP)p53 family genes (34,37). Therefore, the study compared expression of TP53 and salivary epithelial cell KRT differentiation-linked proteins (38,39) in original tissue and cells cultured under CRC and non-CRC conditions.…”

“…Whole exome sequencing is a means to evaluate DNA for known driver mutations, single nucleotide polymorphisms (SNPs), and insertions and deletions (indels) (42). RNAseq results from CRC cultured cells and original tissue show high concordance when passage number is limited (34). Here we tested if combining CRC technology with next generation sequencing (NGS) could be used to correctly identify candidate therapeutic pathways present in the parenchyma of the original tissue.…”