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.

Two-dimensional gel electrophoresis analysis of the leiomyoma interstitial fluid reveals altered protein expression with a possible involvement in pathogenesis

“…The mean age of the patients was 45.6 years with a minimum of 40 years and a maximum of 53 years. Clean fresh leiomyoma and myometrium (500 µg each) were washed with sterile PBS at 4˚C as previously described (16). Tissues were cut into sections of ~1-3 mm 3 , rinsed in sterile PBS at 4˚C and placed in sterile Petri dishes with 5 ml sterile PBS.…”

“…The tumor interstitial fluid (TIF) consists of a liquid phase that accumulates inside the tumor interstitium (15). In addition to the group of blood soluble proteins, it contains a large subset of abnormal proteins released by tumor cells through classical secretion, non-classical secretion, membrane protein shedding and secretion via exosomes (16). The proteomic approach has been described for leiomyoma tissue (17) and plasma (18).…”

Identification of proteins with different abundance associated with cell migration and proliferation in leiomyoma interstitial fluid by proteomics

“…The IF was obtained as previously described (14,15). Two samples (300 mg each) were collected from each patient: One from the unaffected myometrium and one from the central area of the leiomyoma.…”

Proteomics of Triple Negative Breast Cancers in Asian Population- The Need for Further Studies

“…Schulz et al [23] compared the protein expression pattern of triple-negative breast carcinomas versus those being positive for HER2 and negative for the hormone receptors (HER2+, ER-, PR-) by 2-D DIGE and mass spectrometry. They obtained differential expression patterns for several glycolytic enzymes (MDH2, PGK1, TKT, Aldolase1), cytokeratins (CK7, 8,9,14,17,19), further structure proteins (vimentin, fibronectin, L-plastin), for NME1-NME2, lactoferrin, and members of the Annexin family. They suggest that the identified marker proteins may advance a more detailed characterization of triple-negative breast cancers and may contribute to the development of better treatment strategies.…”

“…Over expression of Cyclin E1 [5], metastasis-related proteins [6] as well as glycolytic enzymes, cytokeratins, and structural proteins [7] were seen in triple negative breast cancers. Gromov et al [8] identified 46 proteins that were upregulated in triple negative tumors. Zhang et al [9] have studied proteomic signature in 3 breast cancer cell lines of inflammatory breast cancer (SKBR3, SUM149 and SUM190).…”