A mouse to human search for plasma proteome changes associated with pancreatic tumor development

Abstract

Background: The complexity and heterogeneity of the human plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. Refined genetically engineered mouse (GEM) models of human cancer have been shown to faithfully recapitulate the molecular, biological, and clinical features of human disease. Here, we sought to exploit the merits of a well-characterized GEM model of pancreatic cancer to determine whether proteomics technologies allow identification of protein changes associated with tumor development and whether such changes are relevant to human pancreatic cancer.

Methods and findings: Plasma was sampled from mice at early and advanced stages of tumor development and from matched controls. Using a proteomic approach based on extensive protein fractionation, we confidently identified 1,442 proteins that were distributed across seven orders of magnitude of abundance in plasma. Analysis of proteins chosen on the basis of increased levels in plasma from tumor-bearing mice and corroborating protein or RNA expression in tissue documented concordance in the blood from 30 newly diagnosed patients with pancreatic cancer relative to 30 control specimens. A panel of five proteins selected on the basis of their increased level at an early stage of tumor development in the mouse was tested in a blinded study in 26 humans from the CARET (Carotene and Retinol Efficacy Trial) cohort. The panel discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 mo prior to the development of symptoms and clinical diagnosis of pancreatic cancer.

Conclusions: Our findings indicate that GEM models of cancer, in combination with in-depth proteomic analysis, provide a useful strategy to identify candidate markers applicable to human cancer with potential utility for early detection.

Figure 1. PanIN and PDAC Mice for Plasma Proteomic Analyses

(A) Pdx1-Cre Ink4a/Arf ^lox/lox and Kras^G12D Ink4a/Arf ^lox/lox mice at average of 5.5 and 7 wk of age representing PanIN and PDAC lesions, respectively, were selected on the basis of histological analysis of tumor for this study. (B and C) show representative histology for PanIN and PDAC stages. Plasma was pooled from each disease group and each corresponding age and sex-matched controls to yield 1 ml per phenotype for proteomic analysis.

Figure 2. Schematic of Mouse Plasma Proteomic Analysis

Pools of plasma from PanIN and PDAC mice along with corresponding controls were similarly processed and combined after differential isotopic labeling. Subsequent protein fractionation involved anion-exchange and reversed-phase chromatography. Individual fractions were analyzed by LC–MS/MS after in-solution digestion. Data were processed using Computational Proteomics Analysis System (CPAS).

Figure 3. Identification of Low Abundance Proteins in Mouse Plasma

(A) Spectral counts (number of MS2-events acquired per protein) in the experiment performed for early stage pancreatic cancer mouse plasma protein (PanIN) were correlated with protein plasma concentration reported by Rules-Based Medicine (http://www.rulesbasedmedicine.com/case3/Table3.htm). The 21 proteins used for this estimation were: Serpina1b, Adipoq, A2m, Apoa1, Apoc3, Apoh, B2m, C3, Ceacam1, Crp, Fabp1, F7, Ftl1, Fgb, Hp, Icam1, Igf1, Mb, Serbp1, Timp1, Vcam1. As an approximation, we estimated the protein concentration with the correlation (log spectral counts = [0.623 × log protein concentration] + 0.0625). (B) Taking into consideration the correlation of spectral counts and protein concentration, we observed an inverse relationship between the total number of proteins identified and their abundance (number of MS2/proteins).

Figure 4. IHC Analysis of Candidate PDAC Biomarkers in Mouse and Human Tissue

Mouse, left photomicrographs: PTPRG expression (A–C). Note islet staining in normal pancreas (A); membranous staining is seen in PanIN and PDAC epithelium (B and C). TNC expression (D–F). Note lack of staining in normal pancreatic tissue (D); strong expression is present in stroma of PanIN (E) and PDAC (F). ALCAM expression (G–I). Note membranous staining of the normal pancreatic acinar and ductal cells (G); increased staining is present in the PanIN epithelium (H) and PDAC cells (I). TIMP1 expression (J–L). Note lack of staining in normal pancreatic tissue (J); staining is observed in association with acinar-ductal metaplasia (K) and both PDAC stromal and tumor cells (L). (A–C, J–L, magnification 200×; D–I, magnification 400×). Human, right photomicrographs: PTPRG expression (A, B). Note membranous staining in PDAC epithelium and absence of staining in normal pancreas. TNC expression (C, D). Note expression in PDAC stroma. TNFRSF1 expression (E, F). Note membranous staining in PDAC epithelium; normal pancreatic tissue is negative. Dashed red lines subdivide different histology of the tissue analysed, and blue boxes indicate the adjacent magnified region. Six independent tumor specimens were stained per antibody for mouse IHC and three for human. PTTGF, TNFRSF1, and ALCAM each showed positive staining in at least 15% of tumor cells. TNC and TIMP1 showed positive staining in at least 50% of stromal cells. A, C, and E, magnification 100×; B, magnification 200×; D and F, magnification 400×.

Figure 5. Validation Study of ALCAM, ICAM1, and TIMP1 in Mouse Plasma by ELISA

(A) Plasma from the same individual mice used for proteomics discovery were utilized for validation by means of ELISA. ALCAM, TIMP1, and ICAM1 were all elevated in plasma of the PDAC mice. (B) TIMP1 was also elevated in plasma of PanIN mice. ALCAM overall (cancer plus controls) concentration in mouse plasma was 19 ng/ml; ICAM1 was 163 ng/ml; and TIMP1was 6.2 ng/ml. The low ng/ml concentrations of these proteins support the substantial depth of analysis achieved with our discovery platform. Normalization of concentration was performed as described in the Materials and Methods.

Figure 6. ROC in Assays of Human Samples for Two Panels of Proteins Identified in Proteomic Analysis of Plasmas from Tumor-Bearing Mice

ROC curves based on ELISA measurements of ALCAM, ICAM1, LCN2, TIMP1, REG1A, REG3, and IGFBP4 as a panel with or without CA19–9 comparing pancreatic cancer versus healthy controls (A) and pancreatic cancer versus pancreatitis (B). This panel of candidates was chosen on the basis of up-regulation in tumor-bearing mice. As expected, CA19–9 performed well in comparisons with healthy individuals as controls; however, the chosen panel was significantly better than CA19–9 alone when pancreatitis patients were used as controls. (C) The panel tested with prediagnostic sera (LCN2, TIMP1, REG1A, REG3, and IGFBP4) was chosen on the basis of up-regulation at the PanIN stage. This panel performed slightly better in comparison to CA19–9 but a combination of CA19–9 with the panel of candidates significantly improved discrimination between early stage (prediagnostic) sera and matched controls. Standardization procedures and composite marker ROCs were generated without fitting, by inclusion of all tested candidate markers. Specimens from controls and pancreatitis patients were obtained from the same institution and with the same protocol for blood collection. For details see Materials and Methods and Tables S1 and S2.