Gene expression profiling of tumor-initiating stem cells from mouse Krebs-2 carcinoma using a novel marker of poorly differentiated cells

Abstract

Using the ability of poorly differentiated cells to natively internalize fragments of extracellular double-stranded DNA as a marker, we isolated a tumorigenic subpopulation present in Krebs-2 ascites that demonstrated the features of tumor-inducing cancer stem cells. Having combined TAMRA-labeled DNA probe and the power of RNA-seq technology, we identified a set of 168 genes specifically expressed in TAMRA-positive cells (tumor-initiating stem cells), these genes remaining silent in TAMRA-negative cancer cells. TAMRA+ cells displayed gene expression signatures characteristic of both stem cells and cancer cells. The observed expression differences between TAMRA+ and TAMRA- cells were validated by Real Time PCR. The results obtained corroborated the biological data that TAMRA+ murine Krebs-2 tumor cells are tumor-initiating stem cells. The approach developed can be applied to profile any poorly differentiated cell types that are capable of immanent internalization of double-stranded DNA.

Keywords: DNA internalization; RNAseq; Real Time PCR; TAMRA; tumor-initiating stem cells.

Figure 1. Analysis of internalization of a TAMRA-labeled DNA into the Krebs-2 cells (Axioskop 2 Plus, Zeiss)

(A) Internalization of a TAMRA-labeled human Alu-fragment into the ascites Krebs-2 cells. (B) Internalization of TAMRA-labeled total fragmented human DNA by Krebs-2 ascites. (C) Analysis of enrichment of the population TAMRA– and TAMRA+ Krebs-2 cells by the sorting procedure (BD FACSAria, Becton Dickinson).

Figure 2

(A) The general view of the expression profiles for TAMRA+ (blue profile) and TAMRA– (green profile) cells overlaid to melanoregulin gene (Mreg, uc007bkg.2, chromosome 1, – strand) marking. The gene consists of 5 exons visualized as yellow and dark purple filled rectangles. The yellow color corresponds to non-coding (untranslatable) sequences, the dark purple one – to coding sequences. Both profiles contain peaks that lay inside the boundaries of the gene, but in the case of TAMRA– cells, most of them are located over introns and therefore present no interest for us, while the TAMRA+ profile contains the peaks exactly corresponding to exons. (B) The detailed view of expression profiles for TAMRA+ (blue profile) and TAMRA– (green profile) cells overlaid to Cd5l gene (Cd5l, uc008psa.2, chromosome 3, + strand) marking. 1 – Gene layout. The gene consists of 6 exons that are drawn as filled rectangles over the black line, which in turn represents the gene's location over the chromosome. The yellow color corresponds to non-coding sequences, the dark purple– to coding sequences. Exons are counted from left to right. 2– Expression profiles for TAMRA+ (the upper blue profile) and TAMRA – (the lower green profile). The peaks height represents the number of reads dropped to a certain chromosome region. 3 – Detailed view of exon 3. It is demonstrative that despite the gap inside the exon's boundaries (marked by vertical blue lines), the short tails of the reads were aligned precisely to the exon layout. TAMRA– profile also contains a single read that dropped to this exon. We cannot determine whether this event is accidental or not, since it cannot be discriminated by nucleotide mismatching or supported by intronic evidence. 4 – Detailed view of exon 4 to exon 5 junction. It is demonstrative that “after splicing” the expression profile would be smooth and continuous. (C) The detailed view of expression profiles for TAMRA+ (the blue profile) and TAMRA– (the green profile) cells overlaid to Claudin 1 gene (Cldn1, uc007yuz.2, chromosome 16, – strand) marking. 1 – Gene layout. 2 – Expression profiles. 3 – “Contrasting bar”. This “profile”, which consists of red bars representing the difference between two expression profiles exactly inside exons boundaries, is used just for greater convenience in the visual analysis. The higher the bar, the more significant the difference is. The absolute difference (one of profiles contains no reads dropped to a certain exon) is represented by a full bar and corresponds to 100%. The direction of a bar (up or down) displays the profile with a lower expression level. It can be seen that TAMRA – cells profile demonstrates almost absolute lack of expression. 4 – Detailed view of exon 3. The TAMRA– profile contains a single read dropped to exon but its right tail lays outside the exon's boundary, which means the accidental nature of this event. In fact, we have an absolute difference between TAMRA+ and TAMRA– profiles, but the viewer had drawn a limited difference bar due to “formal hit” in TAMRA– profile.

Figure 3. The cAMP-dependent signaling network

Genes forming cAMP signaling core are shown in yellow; blue denotes upstream genes that regulate cAMP metabolism and activity of the signaling core; green – downstream genes that are affected by cAMP-signaling system; pink – genes which have an inhibitory effect on cAMP signaling pathway; genes that can form a positive feedback are shown in red.

Figure 4. Real Time PCR validation of gene expression data of select genes identified in RNAseq

The genes are split into main GO groups: stemness, cancer, metastasis, control of the metabolism.

Figure 5

(A) Distribution of all gene expression of TAMRA+ Krebs-2 cells in qPCR. (B) List of 22 genes whose expression in TAMRA+ cells relative TAMRA– cells was maximal in qPCR.

Figure 6. Hypothetical interaction networks of 168 genes expressed in TAMRA+ Krebs-2 TISCs as assessed by IPA

(A) Cell functions in which genes overexpressed in TAMRA+ cells (RNAseq) participate. (B) 4 gene networks and functional molecules uniting them (a schematic view). (C) zoom-in of the 4 main gene networks.