CD90/THY1 is overexpressed in prostate cancer-associated fibroblasts and could serve as a cancer biomarker

Abstract

A by-product in the processing of prostate tissue for cell sorting by collagenase digestion is the media supernatant that remains after the cells are harvested. These supernatants contain proteins made by the cells within the tissue. Quantitative proteomic analysis of N-glycosylated proteins detected an increased amount of CD90/THY1 in cancer supernatants compared with non-cancer supernatants. Immunohistochemistry showed that in all carcinomas, regardless of Gleason grade, a layer of CD90-positive stromal fibroblastic cells, ∼5 to 10 cells deep, was localized to tumor glands. In contrast, a no more than 1-cell wide girth of CD90-positive stromal cells was found around benign glands. The increased number of CD90-positive stromal cells in cancer correlated with overexpression of CD90 mRNA detected by gene expression analysis of stromal cells obtained by laser-capture microdissection. There is increasing evidence that cancer-associated stroma has a function in both tumor progression and carcinogenesis. Most experiments to identify cancer biomarkers have focused on the cancer cells. CD90, being a marker for prostate cancer-associated stroma, might be a potential biomarker for this cancer. A non-invasive test could be provided by a urine test. Proteomic analysis of urine from patients with prostate cancer identified CD90; conversely, CD90 was not detected in the urine of post-prostatectomy patients. Furthermore, this urinary CD90 protein was a variant CD90 protein not known to be expressed by such cells as lymphocytes that express CD90. These CD90 results were obtained from ∼90 cases consisting of proteomic analysis of tissue and urine, immunohistochemistry, western blot analysis of tissue media, flow cytometry of cells from digested tissue, and reverse transcriptase polymerase chain reaction analysis of isolated stromal cells.

Fig. 1

CD90 glycoprotein in cancer tissue. Western blot analysis of tissue digestion media detected a diffuse CD90 band at ∼27 kDa, which is larger than the calculated molecular weight of CD90 (∼16 kDa) based on the amino acid sequence. Cancer is characterized as CD90^hi and non-cancer as CD90^lo. Both peripheral zone and transition zone cancer show increased CD90 expression compared to non-cancer. Expression level does not appear to vary with Gleason scores. Note that the Gleason score applies to the case not necessarily to the specimens obtained for collagenase digestion. The 30-kDa TIMP1 can be used to characterize non-cancer specimens since TIMP1 is synthesized by luminal cells and not cancer cells.

Fig. 2

CD90 staining of cancer-associated stromal cells. A. CD90 staining of tissue specimen 99-068D shows increased reactivity localized to the tumor foci. Staining in non-cancer is restricted to a cell layer adjacent to the basal epithelium. Blood vessels are also positive for CD90. Original magnifications are indicated in the individual photomicrographs. B. The transition zone cancer, 04-023, contained both tissue without benign glands (top panels) and cancer-rich tissue with scattered benign glands (bottom left). The tumor cells in 04-023C are stained by CD26 (top left). 01-180C contained a large tumor focus with crowded glands. Original magnification 40×.

Fig. 2

CD90 staining of cancer-associated stromal cells. A. CD90 staining of tissue specimen 99-068D shows increased reactivity localized to the tumor foci. Staining in non-cancer is restricted to a cell layer adjacent to the basal epithelium. Blood vessels are also positive for CD90. Original magnifications are indicated in the individual photomicrographs. B. The transition zone cancer, 04-023, contained both tissue without benign glands (top panels) and cancer-rich tissue with scattered benign glands (bottom left). The tumor cells in 04-023C are stained by CD26 (top left). 01-180C contained a large tumor focus with crowded glands. Original magnification 40×.

Fig. 3

CD90 expression analysis in laser-captured stromal cells. Matched cancer and non-cancer specimens were prepared from cases listed on the x-axis. The fold differences in CD90 transcript expression for the laser-captured stromal cell samples are indicated on the y-axis in the range of 0.1- to 10-fold cancer over non-cancer. Increased transcript was seen in 8 of 10 cancer-associated stromal cell samples.

Fig. 4

Major human urinary proteins. Polyacrylamide gel-resolved urinary proteins from 4 healthy donor specimens are shown. Identification was achieved by in-gel digestion and mass spectrometry. The band intensities show that there is no significant variation between samples.

Fig. 5

CD90 in urine. Single ion traces of the mass from heavy (d9, pre-op urine) and light (d0, post-op urine) ICAT-labeled peptides indicate that PSA and CD90 are detectable in prostate cancer patient urine. The elution profiles (single ion traces of the peptide mass over time) of heavy labeled peptide masses and those of the corresponding light labeled peptide masses are highlighted. The detected peptide sequences are shown below the traces. The weight difference of 18 in PSA is due to the two labeled Cys residues. Both PSA (more abundant than CD90, y-axis) and CD90 show a similar trend wherein both proteins are present in the pre-op sample but below detection limit in the post-op sample.

Fig. 6

CD90 variant. The panel shows LC-SRM-MS detection of 7 MS/MS fragments of a peptide specific to the CD90 variant protein (BAD92446) encoded by a CD90 cDNA clone (AB209209) in a pre-op urine sample. In the same sample, a peptide of the cancer upregulated BCMP11 was also detected.

Fig. 7

CD90 gene structure. The top panel shows the amino acid sequence difference (in red) between the T-cell CD90 (AAA61180) and CD90 variant (BAD92446). In the AAA61180 sequence, MNLAISIALLLTVLQVSR constitutes the signal sequence. The bottom panel shows the gene arrangements of these two CD90 coding regions: nucleotides in red are for the T-cell CD90, and nucleotides in green are for variant CD90; common coding sequence is in blue. The start codon of AAA61180 and the stop codons of the two coding regions are underlined. A stop codon (TAG) precedes the CD90 variant coding sequence. Sequences of the CD90 primers in RT-PCR analysis are highlighted in gray.