Gene expression down-regulation in CD90+ prostate tumor-associated stromal cells involves potential organ-specific genes

Abstract

Background: The prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. The tumor-associated stroma is marked by increased expression of CD90/THY1. Isolation and characterization of these stromal cells could provide valuable insight into the biology of the tumor microenvironment.

Methods: Prostate CD90+ stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between histologically normal and tumor-associated stromal cells. For comparison, stromal cells were also isolated and analyzed from the urinary bladder.

Results: The tumor-associated stromal cells were found to have decreased expression of genes involved in smooth muscle differentiation, and those detected in prostate but not bladder. Other differential expression between the stromal cell types included that of the CXC-chemokine genes.

Conclusion: CD90+ prostate tumor-associated stromal cells differed from their normal counterpart in expression of multiple genes, some of which are potentially involved in organ development.

Figure 1

Isolation of CD90⁺ stromal cells from cancer tissue. A: Tissue digestion media were probed for TIMP1 and CD90 proteins by Western blot analysis. Sample 00-044NP served as non-cancer control because 08-028NP and 08-032NP were not available. PSA was used as a sample loading control. B: The individual sorted CP stromal transcriptome datasets (second and third columns) contain minimal signals for the epithelial genes that are present in the cancer epithelial cell (CP cancer) transcriptome (first column) in virtual Northern blot format. Affymetrix signal values are represented on a gray scale. C: The expression levels of epithelial genes are below background (≤ 50 RMA) in sorted CP stromal cells.

Figure 2

Sorted cell and whole tissue transcriptome dataset comparison. The most down-regulated (negative) and most up-regulated (positive) genes in sorted tumor-associated stromal cells are also similarly expressed, for the majority, in whole tissue comparison of CP to NP.

Figure 3

Organ-specific stromal genes. A: Up-regulated genes in the bladder (left) and the prostate (right) are identified by brown colored data points. The horizontal axis represent the fraction of spot replicates above (p.up) or below (p.down) the differentially expressed cutoff. The vertical axis represents the mean log-ratios. B: Shown are the qPCR results for prostate SPOCK3, MSMB, CXCL13, PAGE4, and bladder TRPA1, HSD17B2, IL24, SALL1. RPLP0 was used as reaction control. Light blue indicates prostate genes and red bladder genes.

Figure 4

Differential expression of organ-specific stromal genes in cancer. A: Genes found to be differentially expressed in sorted tumor-associated stromal cells of bladder and prostate and prostate tissue. B: CP and NP are matched cancer and non-cancer prostate specimens; bone, liver and LN (lymph node) are prostate cancer metastasis specimens. CNTN1 is detectable in all NP, but it is down-regulated in CP; not detected in metastasis. A similar pattern is shown by MAOB and SPOCK3, but the differential expression is not as pronounced. MAOB is expressed in LN. cDNA quantity of each sample was monitored by ACTA2 (shown for CP1/NP1).

Figure 5

Expression of CXC-chemokines in stromal cells. A: Differential expression of CXC-chemokines in sorted CD90⁺ CP vs. CD49a⁺ NP stromal cells and comparison to whole tissue CP vs. NP and CD26⁺ CP vs. NP. B: Virtual Northern display of expression levels of CXCL1, CXCL3, CXCL5 and CXCL6 in other prostate cell types (darker shading of the boxes indicates higher mRNA levels). C: RT-PCR verification of differentially expressed CXC-chemokines in sorted CD90⁺ CP and CD49a⁺ NP stromal cells.