Embryonal carcinoma cell induction of miRNA and mRNA changes in co-cultured prostate stromal fibromuscular cells

Abstract

The prostate stromal mesenchyme controls organ-specific development. In cancer, the stromal compartment shows altered gene expression compared to non-cancer. The lineage relationship between cancer-associated stromal cells and normal tissue stromal cells is not known. Nor is the cause underlying the expression difference. Previously, the embryonal carcinoma (EC) cell line, NCCIT, was used by us to study the stromal induction property. In the current study, stromal cells from non-cancer (NP) and cancer (CP) were isolated from tissue specimens and co-cultured with NCCIT cells in a trans-well format to preclude heterotypic cell contact. After 3 days, the stromal cells were analyzed by gene arrays for microRNA (miRNA) and mRNA expression. In co-culture, NCCIT cells were found to alter the miRNA and mRNA expression of NP stromal cells to one like that of CP stromal cells. In contrast, NCCIT had no significant effect on the gene expression of CP stromal cells. We conclude that the gene expression changes in stromal cells can be induced by diffusible factors synthesized by EC cells, and suggest that cancer-associated stromal cells represent a more primitive or less differentiated stromal cell type.

Fig. 1

CP stromal cells. A: In tissue section 99-068D, the tumor in the right half of the field is outlined in red and stained strongly by CD90. The small cancer glands are surrounded by CD90⁺ stromal cells (magnification 40X). In normal glands, stromal CD90 staining is confined to cells adjacent to the basal epithelium (black arrows). B: The principal components analysis plot shows the separation between sorted NP stromal (labeled as NP strom) and CP stromal cells (labeled as CP strom 1 and CP strom 2 from two cases), luminal and G3 cancer cells based on their transcriptome datasets. This analytic space is defined by the transcriptomes of prostate luminal, stromal, basal, and endothelial cells, and can be rotated for different aspects of view.

Fig. 1

CP stromal cells. A: In tissue section 99-068D, the tumor in the right half of the field is outlined in red and stained strongly by CD90. The small cancer glands are surrounded by CD90⁺ stromal cells (magnification 40X). In normal glands, stromal CD90 staining is confined to cells adjacent to the basal epithelium (black arrows). B: The principal components analysis plot shows the separation between sorted NP stromal (labeled as NP strom) and CP stromal cells (labeled as CP strom 1 and CP strom 2 from two cases), luminal and G3 cancer cells based on their transcriptome datasets. This analytic space is defined by the transcriptomes of prostate luminal, stromal, basal, and endothelial cells, and can be rotated for different aspects of view.

Fig. 2

Cultured cell morphology. The micrographs show phase contrast images of NP strom, CP strom and NCCIT cells in culture (magnification 200X). Note the increase in space between the NCCIT colonies (dark lacunae) as an indication of lower cell density compared to untreated NCCIT (see main text for an explanation). In contrast, the stromal morphology was not appreciably altered by co-culture with NCCIT.

Fig. 3

MicroRNA expression profiles of stromal/NCCIT interaction. Shown are the hierarchical clustering and tree-view analysis of relative expression levels for miRNA in the different cell cultures arranged in the order written on the right. Array signal intensities are represented in color, blue hues for low and yellow for high. Arrow indicates miRNA shared between CP strom and NCCIT.

Fig. 4

MicroRNA-21 expression pattern. The histogram shows array signal values, which are listed in the table below, scored by miR-21 probesets in the different cultures. Array signal intensity values are indicated on the y-axis. The display gives a visual representation of the data. The fold-change between NP strom and NP strom + NCCIT is relatively constant for the two probesets. MicroRNA-21* was only detected (>300) in NP strom.

Fig. 5

MicroRNA-210 expression analysis by PCR. The relative product levels are indicated on the y-axis. Array signal values for each condition are indicated next to the culture identifiers.

Fig. 6

Stromal mRNA expression in co-culture with NCCIT. A: Array signal values are shown for the genes listed on the x-axis with array signal intensity values on the y-axis. The first 5 examples show increased expression in NP strom after NCCIT signaling while the next 2 examples show decreased expression. Expression levels of these genes in CP strom were not much altered by NCCIT. THY1 is CD90. B: A principal components analysis plot shows the “migration” of NP strom + NCCIT transcriptome towards the CP strom transcriptome, and little change of CP strom + NCCIT compared to CP strom. The NCCIT transcriptome is distinct from the others. Note the difference shown by sorted stromal cells (labeled stromal) and cultured stromal cells (labeled NP strom).

Fig. 6

Stromal mRNA expression in co-culture with NCCIT. A: Array signal values are shown for the genes listed on the x-axis with array signal intensity values on the y-axis. The first 5 examples show increased expression in NP strom after NCCIT signaling while the next 2 examples show decreased expression. Expression levels of these genes in CP strom were not much altered by NCCIT. THY1 is CD90. B: A principal components analysis plot shows the “migration” of NP strom + NCCIT transcriptome towards the CP strom transcriptome, and little change of CP strom + NCCIT compared to CP strom. The NCCIT transcriptome is distinct from the others. Note the difference shown by sorted stromal cells (labeled stromal) and cultured stromal cells (labeled NP strom).

Fig. 7

MicroRNA in media of co-culture. Expression patterns of the different cultures for cytoplasmic (left half) and culture media (right half) are compared. Three overall patterns are seen in the cytoplasmic data: [NP + NCCIT, CP, CP + NCCIT], [NCCIT, NCCIT + NP, NCCIT + CP], and [NP strom]. Two general patterns are seen in the culture media data: [NCCIT] and [all others]. Cell types underlined are the ones analyzed by arrays. Red hues represent high signal values and green low signal values.