Detection of S100A6 in serum over time from mice with OVCA, in association with estimated peritoneal cavity tumor burden.

<p>(A) Photon flux measurements obtained from mice harboring defined numbers of bioluminescent SKOV-3-Luc cells within the peritoneal cavity. Regression analysis was performed on (log₁₀) photon output vs. (log₁₀) numbers of inoculated cells to produce a standard calibration curve using an inverse prediction equation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007670#s4" target="_blank">Materials and Methods</a>). This was used to predict tumor burden from photon flux measurements (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007670#pone-0007670-g006" target="_blank">figure 6C</a>). (B) Comparisons of serum S100A6 in tumor-bearing (T, closed circles) and saline-inoculated control mice (SC, open circles). Serum S100A6 concentrations, tested as individual specimens from multiple mice by ECLISA (plotted as individual values with horizontal line at median value), were significantly different between T vs. SC at each of three times tested post inoculation, 9 days (p = 0.015), 15 days (p = 0.036), and 21 days (p = 0.0031) (Wilcoxon Rank Sum test). (C) Correlation between estimate of tumor burden in OVCA carcinomatosis and concentration of serum S100A6, as measured by ECLISA from tumor-bearing mice (r = 0.79, p<0.0001).</p

S100A6 protein sequence.

<p>(A) Aligned human and mouse S100A6 sequences depicting 3 amino acid differences between the two species (vertical lines). Three peptides were detected by LC-LTQ MS/MS from tumor-bearing mouse sera; sequences are shown in color font. Peptides in red and blue font are homologous for human and mouse, while the peptide sequence in green font is unique to human S100A6, due to one amino acid difference. (B) MS/MS spectrum of the human-specific S100A6 peptide, LMEDLDR. This sequence was not detected in saline-inoculated control mice sera.</p

Human OVCA cell xenograft in mouse peritoneal cavity.

<p>(A) Representative OVCA exhibits predominantly solid tumor growth, with evidence of papillary projections and occasional minute cysts (bar = 25 µm). Inset, higher resolution photomicrograph depicting cuboidal to polygonal pleomorphic microcystic epithelium with anisokaryosis and atypical mitoses of ovarian cancer cell nuclei (bar = 50 µm). (B) OVCA tumor nodule implant on uterine infundibulo-ovarian ligament (arrowhead) adjacent to the ovary (arrow) (bar = 25 µm). Tumor implants occurred throughout the peritoneal cavity, peri-ovarian connective tissues, and invaded surrounding tissues such as intestines, liver and diaphragm. Hematoxylin and eosin (H&E) stained, paraffin-embedded tissue sections.</p

S100A6 immunohistochemical findings in epithelial OVCA and ovary tissues.

<p>*Equivocal  =  weak labeling or rare positive cells. † Reactive hyperplastic ovarian surface epithelium.</p

Differentially abundant proteins in serum from OVCA and control (C) mice selected based upon spectral counting.

<p>*One peptide sequence identified in controls is unconfirmed and differs from those identified in cancers.</p

Reverse phase protein micorarray data obtained from the analysis of diagnostic sera from women with OVCA.

<p>Scatterplot displays relative intensity of serum S100A6 values for women with early stage (open circles, n = 23) and advanced stage (closed circles, n = 43) OVCA. The early stage disease group includes 2 tumors diagnosed as borderline OVCA. The means of the relative intensity values of the analyte concentrations shown (horizontal lines) are significantly different for the two groups (p = 0.031, two-sample t-test), and depict relatively greater mean S100A6 concentration in sera of advanced stage patients.</p

Experimental strategy to use MS discovery proteomics in low molecular weight (LMW) serum fraction from a mouse model of human ovarian cancer.

<p>To determine whether cancer-derived proteins have candidate biomarker potential, (A) a bioluminescent xenograft model, (B) ECLISA, (C) Western blot, and (D) OVCA tissue array immunohistochemistry (IHC) are utilized. This was followed up for S100A6 by analysis of human patient sera.</p

S100A6 protein expression in mouse serum and OVCA tissue.

<p>(A) Immunoblot analysis for the presence of S100A6 in pooled raw sera from cancer-bearing mice (1) and saline control mice (2). The sera were used in LMW protein fraction preparation for MS analysis in biomarker discovery. (B) OVCA xenografts express S100A6 as analyzed by IHC. Tumor xenograft implant on intestinal mesentery reveals S100A6 protein limited to tumor, with lack of immunolabeling in adjacent intestines and mesentery. (C) Serial section from tissue shown in B, used for IHC reaction control, reveals lack of immunoreactivity when primary S100A6 antibody is omitted from IHC. Immunoperoxidase, hematoxylin counterstain (Bars = 50 µm).</p

S100A6 expression in human OVCA cell lines and tissues by Western blot analysis.

<p>(A and B) Paired lysates of OVCA (cancer) and adjacent normal ovary tissue (normal) from 2 OVCA patients were blotted for S100A6 expression. SKOV-3 cell lysate was used for comparison. (C and D) S100A6 expression in OVCA cell lines from NCI60 (SKOV-3, OVCAR-3, 4 and 5) as well as cell lines derived from OVCA patients (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007670#s4" target="_blank">Materials and Methods</a>).</p