Up-regulated proteins in the fluid bathing the tumour cell microenvironment as potential serological markers for early detection of cancer of the breast

Abstract

Breast cancer is by far the most common diagnosed form of cancer and the leading cause of cancer death in women today. Clinically useful biomarkers for early detection of breast cancer could lead to a significant reduction in mortality. Here we describe a detailed analysis using gel-based proteomics in combination with mass spectrometry and immunohistochemistry (IHC) of the tumour interstitial fluids (TIF) and normal interstitial fluids (NIF) collected from 69 prospective breast cancer patients. The goal of this study was to identify abundant cancer up-regulated proteins that are externalised by cells in the tumour microenvironment of most if not all these lesions. To this end, we applied a phased biomarker discovery research strategy to the analysis of these samples rather than comparing all samples among each other, with inherent inter and intra-sample variability problems. To this end, we chose to use samples derived from a single tumour/benign tissue pair (patient 46, triple negative tumour), for which we had well-matched samples in terms of epithelial cell numbers, to generate the initial dataset. In this first phase we found 110 proteins that were up-regulated by a factor of 2 or more in the TIF, some of which were confirmed by IHC. In the second phase, we carried out a systematic computer assisted analysis of the 2D gels of the remaining 68 TIF samples in order to identify TIF 46 up-regulated proteins that were deregulated in 90% or more of all the available TIFs, thus representing common breast cancer markers. This second phase singled out a set of 26 breast cancer markers, most of which were also identified by a complementary analysis using LC-MS/MS. The expression of calreticulin, cellular retinoic acid-binding protein II, chloride intracellular channel protein 1, EF-1-beta, galectin 1, peroxiredoxin-2, platelet-derived endothelial cell growth factor, protein disulfide isomerase and ubiquitin carboxyl-terminal hydrolase 5 were further validated using a tissue microarray containing 70 malignant breast carcinomas of various grades of atypia. A significant number of these proteins have already been detected in the blood/plasma/secretome by others. The next steps, which include biomarker prioritization based on the hierarchal evaluation of these markers, antibody and antigen development, assay development, analytical validation, and preliminary testing in the blood of healthy and breast cancer patients, are discussed.

Figure 1

Paraffin‐embedded sections of (A) benign breast tissue 46 and (B) tumour 46 stained with a monoclonal antibody against CK19 (dilution 1:1000).

Figure 2

Silver stained IEF 2D gels of (A) TIF 46 and NIF 46. Proteins up‐regulated by a factor of 2 or more are indicated with arrows.

Figure 3

IHC staining of paraffin‐embedded sections of benign 46 (A, C, E, G and I) and tumour 46 (B, D, F, H and J) stained with antibodies against TIF 46 up‐regulated proteins. (A) and (B) calreticulin, dilution 1:200. (C) and (D) calumenin, dilution 1:1000. (E) and (F) TCTP, dilution 1:2000. (G) and (H) SA100 A9, dilution 1:1000. (I) and (J) p53, dilution 1:250. The arrow in I indicates cells with atypia (compare with J).

Figure 4

PDQuest image analysis of TIF and NIF samples. 2D images were matched and analysed in sets of five to six images each. A master image was established based on the gel of TIF 46. All proteins detected in the set were added to the master image.

Figure 5

Scatter dot plots showing comparisons of the levels of the 26 cancer up‐regulated proteins between TIF (69 sample set) and NIF samples (28 sample set). Data are presented as protein spot intensities measured for each protein in both TIF (blue dots) and NIF (red dots) sample sets. P values were determined using the non‐parametric Mann–Whitney test. Horizontal bars indicate mean with SEM.

Figure 6

IHC staining of paraffin‐embedded sections of normal (A, C, E, G, I, K M, O and Q) and tumour cores (B, D, F, H, J, L, N, P and R) in the Pantomics BRC1503 breast cancer tissue array stained with various antibodies against up‐regulated proteins. (A) and (B) calreticulin, dilution 1:200. (C) and (D) CRABP II, dilution 1:1500. (E) and (F) CL1C 1, dilution 1:250. (G) and (H) EF‐1‐beta, dilution 1:1500. (I) and (J) galectin 1, dilution 1:250. (K) and (L) PRDX 2, dilution 1:3000. (M) and (N) PD‐ECGF 1, dilution 1:300. (O) and (P) PDI, dilution 1:800. (Q) and (R) USTH 5, dilution 1:50.

Figure 7

Representative tumour cores of the Pantomics BRC1503 breast cancer tissue array stained with various antibodies. (A) Calreticulin. (B) Platelet‐derived endothelial cell growth factor. (C) Peroxiredoxin‐2. (D) Protein disulfide isomerase.

Figure 8

Indirect immunofluorescence analysis of fat tissue peripheral to a breast tumour stained with A‐FABP (dilution 1:250; Alexa Fluor 488; green) and keratin 19 (dilution 1:1000: Alexa Fluor 594; red) specific antibodies.

Figure 9

Silver stained 2D gels of (A) FIF 51 and (B) TIF 51. TIF up‐regulated proteins (black arrows) as well as fat tissue specific proteins (blue arrows) are indicated in the figure.