Methodology and Applications of Disease Biomarker Identification in Human Serum

Biomarkers are biomolecules that serve as indicators of biological and pathological processes, or physiological and pharmacological responses to a drug treatment. Because of the high abundance of albumin and heterogeneity of plasma lipoproteins and glycoproteins, biomarkers are difficult to identify in human serum. Due to the clinical significance the identification of disease biomarkers in serum holds great promise for personalized medicine, especially for disease diagnosis and prognosis. This review summarizes some common and emerging proteomics techniques utilized in the separation of serum samples and identification of disease signatures. The practical application of each protein separation or identification technique is analyzed using specific examples. Biomarkers of cancers of prostate, breast, ovary, and lung in human serum have been reviewed, as well as those of heart disease, arthritis, asthma, and cystic fibrosis. Despite the advancement of technology few biomarkers have been approved by the Food and Drug Administration for disease diagnosis and prognosis due to the complexity of structure and function of protein biomarkers and lack of high sensitivity, specificity, and reproducibility for those putative biomarkers. The combination of different types of technologies and statistical analysis may provide more effective methods to identify and validate new disease biomarkers in blood

Active Roles of Tumor Stroma in Breast Cancer Metastasis

Metastasis is the major cause of death for breast cancer patients. Tumors are heterogenous cellular entities composed of cancer cells and cells of the microenvironment in which they reside. A reciprocal dynamic interaction occurs between the tumor cells and their surrounding stroma under physiological and pathological conditions. This tumor-host communication interface mediates the escape of tumor cells at the primary site, survival of circulating cancer cells in the vasculature, and growth of metastatic cancer at secondary site. Each step of the metastatic process is accompanied by recruitment of stromal cells from the microenvironment and production of unique array of growth factors and chemokines. Stromal microenvironment may play active roles in breast cancer metastasis. Elucidating the types of cells recruited and signal pathways involved in the crosstalk between tumor cells and stromal cells will help identify novel strategies for cotargeting cancer cells and tumor stromal cells to suppress metastasis and improve patient outcome

Identification of Potential Glycoprotein Biomarkers in Estrogen Receptor Positive (ER+) and Negative (ER-) Human Breast Cancer Tissues by LC-LTQ/FT-ICR Mass Spectrometry

Breast cancer is the second most fatal cancer in American women. To increase the life expectancy of patients with breast cancer new diagnostic and prognostic biomarkers and drug targets must be identified. A change in the glycosylation on a glycoprotein often causes a change in the function of that glycoprotein; such a phenomenon is correlated with cancerous transformation. Thus, glycoproteins in human breast cancer estrogen receptor positive (ER+) tissues and those in the more advanced stage of breast cancer, estrogen receptor negative (ER-) tissues, were compared. Glycoproteins showing differences in glycosylation were examined by 2-dimensional gel electrophoresis with double staining (glyco- and total protein staining) and identified by reversed-phase nano-liquid chromatography coupled with a hybrid linear quadrupole ion trap/ Fourier transform ion cyclotron resonance mass spectrometer. Among the identified glycosylated proteins are alpha 1 acid glycoprotein, alpha-1-antitrypsin, calmodulin, and superoxide dismutase mitochondrial precursor that were further verified by Western blotting for both ER+ and ER- human breast tissues. Results show the presence of a possible glycosylation difference in alpha-1-antitrypsin, a potential tumor-derived biomarker for breast cancer progression, which was expressed highest in the ER- samples

Novel Stromal Biomarkers in Human Breast Cancer Tissues Provide Evidence for the More Malignant Phenotype of Estrogen Receptor-Negative Tumors

Research efforts were focused on genetic alterations in epithelial cancer cells. Epithelial-stromal interactions play a crucial role in cancer initiation, progression, invasion, angiogenesis, and metastasis; however, the active role of stroma in human breast tumorigenesis in relation to estrogen receptor (ER) status of epithelial cells has not been explored. Using proteomics and biochemical approaches, we identified two stromal proteins in ER-positive and ER-negative human breast cancer tissues that may affect malignant transformation in breast cancer. Two putative biomarkers, T-cell receptor alpha (TCR-α) and zinc finger and BRCA1-interacting protein with a KRAB domain (ZBRK1), were detected in leukocytes of ER-positive and endothelial cells of ER-negative tissues, respectively. Our data suggest an immunosuppressive role of leukocytes in invasive breast tumors, propose a multifunctional nature of ZBRK1 in estrogen receptor regulation and angiogenesis, and demonstrate the aggressiveness of ER-negative human breast carcinomas. This research project may identify new stromal drug targets for the treatment of breast cancer patients

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Secretome analysis reveals upregulated granzyme B in human androgen-repressed prostate cancer cells with mesenchymal and invasive phenotype.

Epithelial-mesenchymal transition (EMT) is a critical early step in cancer metastasis and a complex process that involves multiple factors. In this study, we used proteomics approaches to investigate the secreted proteins (secretome) of paired human androgen-repressed prostate cancer (ARCaP) cell lines, representing the epithelial (ARCaP-E) and mesenchymal (ARCaP-M) phenotypes. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed high levels of proteins involved in bone remodeling and extracellular matrix degradation in the ARCaP-M cells, consistent with the bone metastasis phenotype. Furthermore, LC-MS/MS showed a significantly higher level of the serine protease granzyme B (GZMB) in ARCaP-M conditioned media (CM) compared to that of ARCaP-E. Using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) to detect mRNA and Western blot to detect protein expression, we further demonstrated that the GZMB gene was expressed by ARCaP-M and the protein was secreted extracellularly. ARCaP-M cells with GZMB gene knockdown using small interfering RNA (siRNA) have markedly reduced invasiveness as demonstrated by the Matrigel invasion assay in comparison with the scrambled siRNA negative control. This study reports that GZMB secretion by mesenchymal-like androgen-repressed human prostate cancer cells promotes invasion, suggesting a possible extracellular role for GZMB in addition to its classic role in immune cell-mediated cytotoxicity

Subgrouping breast cancer patients based on immune evasion mechanisms unravels a high involvement of transforming growth factor-beta and decoy receptor 3.

In the era of immunotherapy and personalized medicine, there is an urgent need for advancing the knowledge of immune evasion in different cancer types and identifying reliable biomarkers that guide both therapy selection and patient inclusion in clinical trials. Given the differential immune responses and evasion mechanisms in breast cancer, we expect to identify different breast cancer groups based on their expression of immune-related genes. For that, we used the sequential biclustering method on The Cancer Genome Atlas RNA-seq breast cancer data and identified 7 clusters. We found that 77.4% of the clustered tumor specimens evade through transforming growth factor-beta (TGF-β) immunosuppression, 57.7% through decoy receptor 3 (DcR3) counterattack, 48.0% through cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and 34.3% through programmed cell death-1 (PD-1). TGF-β and DcR3 are potential novel drug targets for breast cancer immunotherapy. Targeting TGF-β and DcR3 may provide a powerful approach for treating breast cancer because 57.7% of patients overexpressed these two molecules. Furthermore, triple-negative breast cancer (TNBC) patients clustered equally into two subgroups: one with impaired antigen presentation and another with high leukocyte recruitment but four different evasion mechanisms. Thus, different TNBC patients may be treated with different immunotherapy approaches. We identified biomarkers to cluster patients into subgroups based on immune evasion mechanisms and guide the choice of immunotherapy. These findings provide a better understanding of patients' response to immunotherapies and shed light on the rational design of novel combination therapies

Macrophage-Conditioned Media Promotes Adipocyte Cancer Association, Which in Turn Stimulates Breast Cancer Proliferation and Migration

Background: Breast cancer is the most common cancer in women and the leading cause of female cancer deaths worldwide. Obesity causes chronic inflammation and is a risk factor for post-menopausal breast cancer and poor prognosis. Obesity triggers increased infiltration of macrophages into adipose tissue, yet little research has focused on the effects of macrophages in early stages of breast tumor development in obese patients. In this study, the effects of pro-inflammatory macrophages on breast cancer–adipocyte crosstalk were investigated. Methods: An innovative human cell co-culture system was built and used to model the paracrine interactions among adipocytes, macrophages, and breast cancer cells and how they facilitate tumor progression. The effects on cancer cells were examined using cell counts and migration assays. Quantitative reverse-transcription polymerase chain reaction was used to measure the expression levels of several cytokines and proteases to analyze adipocyte cancer association. Results: Macrophage-conditioned media intensified the effects of breast cancer–adipocyte crosstalk. Adipocytes became delipidated and increased production of pro-inflammatory cytokines, even in the absence of cancer cells, although the expression levels were highest with all three cell components. As a result, co-cultured breast cancer cells became more aggressive, with increased proliferation and migration compared to adipocyte–breast cancer co-cultures treated with unconditioned media. Conclusions: A novel co-culture model was built to evaluate the crosstalk among human macrophages, adipocytes, and breast cancer cells. We found that macrophages may contribute to adipocyte inflammation and cancer association and thus promote breast cancer progression

Metalloproteinase disintegrins ADAM8 and ADAM19 are highly regulated in human primary brain tumors and their expression levels and activities are associated with invasiveness.

Patients with primary brain tumors have bleak prognoses and there is an urgent desire to identify new markers for sensitive diagnosis and new therapeutic targets for effective treatment. A family of proteins, the disintegrin and metalloproteinases (ADAMs or adamalysins), are cell surface and extracellular multidomain proteins implicated in cell-cell signaling, cell adhesion, and cell migration. Their putative biological and pathological roles make them candidates for promoting tumor growth and malignancy. We investigated the expression levels of 12 cerebrally expressed ADAM genes in human primary brain tumors (astrocytoma WHO grade I-III, glioblastoma WHO grade IV, oligoastrocytoma WHO grade II and III, oligodendroglioma WHO grade II and III, ependymoma WHO grade II and III, and primitive neuroectodermal tumor WHO grade IV) using real-time PCR. The mRNAs of the five ADAMs 8, 12, 15, 17, and 19 were significantly upregulated. The ADAM8 and ADAM19 proteins were mainly located in tumor cells and in some tumors in endothelia of blood vessels. In brain tumor tissue, ADAM8 and ADAM19 undergo activation by prodomain removal resulting in active proteases. By using specific peptide substrates for ADAM8 and ADAM19, respectively, we demonstrated that the proteases exert enhanced proteolytic activity in those tumor specimens with the highest expression levels. In addition, expression levels and the protease activities of ADAM8 and ADAM19 correlated with invasive activity of glioma cells, indicating that ADAM8 and ADAM19 may play a significant role in tumor invasion that may be detrimental to patients survival