Clinical cancer proteomics aims at the identification of markers for early detection and predictive purposes, as well as to provide novel targets for drug discovery and therapeutic intervention. Proteomics-based analysis of traditional sources of biomarkers, such as serum, plasma, or tissue lyzates, has resulted in a wealth of information and the finding of several potential tumor biomarkers. However, many of these markers have shown limited usefulness in a clinical setting, underscoring the need for new clinically relevant sources. Here we present a novel and highly promising source of biomarkers, the tumor interstitial fluid (TIF) that perfuses the breast tumor microenvironment. We collected TIFs from small pieces of freshly dissected invasive breast carcinomas and analyzed them by two-dimensional polyacrylamide gel electrophoresis in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Western immunoblotting, as well as by cytokine-specific antibody arrays. This approach provided for the first time a snapshot of the protein components of the TIF, which we show consists of more than one thousand proteins—either secreted, shed by membrane vesicles, or externalized due to cell death—produced by the complex network of cell types that make up the tumor microenvironment. So far, we have identified 267 primary translation products including, but not limited to, proteins involved in cell proliferation, invasion, angiogenesis, metastasis, inflammation, protein synthesis, energy metabolism, oxidative stress, the actin cytoskeleton assembly, protein folding, and transport. As expected, the TIF contained several classical serum proteins. Considering that the protein composition of the TIF reflects the physiological and pathological state of the tissue, it should provide a new and potentially rich resource for diagnostic biomarker discovery and for identifying more selective targets for therapeutic intervention.

Formalin fixation and paraffin‐embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)–based proteomics has so far proven challenging. Herein we describe a MS‐based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low‐input cancer tissue isolated by laser microdissection. Using state‐of‐the‐art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single‐run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long‐term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

It has become clear that growth and progression of breast tumor cells not only depend on their malignant potential but also on factors present in the tumor microenvironment. Of the cell types that constitute the mammary stroma, the adipocytes are perhaps the least well studied despite the fact that they represent one of the most prominent cell types surrounding the breast tumor cells. There is compelling evidence demonstrating a role for the mammary fat pad in mammary gland development, and some studies have revealed the ability of fat tissue to augment the growth and ability to metastasize of mammary carcinoma cells. Very little is known, however, about which factors adipocytes produce that may orchestrate these actions and how this may come about. In an effort to shed some light on these questions, we present here a detailed proteomic analysis, using two-dimensional gel-based technology, mass spectrometry, immunoblotting, and antibody arrays, of adipose cells and interstitial fluid of fresh fat tissue samples collected from sites topologically distant from the tumors of high risk breast cancer patients that underwent mastectomy and that were not treated prior to surgery. A total of 359 unique proteins were identified, including numerous signaling molecules, hormones, cytokines, and growth factors, involved in a variety of biological processes such as signal transduction and cell communication; energy metabolism; protein metabolism; cell growth and/or maintenance; immune response; transport; regulation of nucleobase, nucleoside, and nucleic acid metabolism; and apoptosis. Apart from providing a comprehensive overview of the mammary fat proteome and its interstitial fluid, the results offer some insight as to the role of adipocytes in the breast tumor microenvironment and provide a first glance of their molecular cellular circuitry. In addition, the results open new possibilities to the study of obesity, which has a strong association with type 2 diabetes, hypertension, and coronary heart disease.

Histone deacetylase inhibitors (HDACIs) induce hyperacetylation of core histones modulating chromatin structure and affecting gene expression. These compounds are also able to induce growth arrest, cell differentiation, and apoptotic cell death of tumor cells in vitro as well as in vivo. Even though several genes modulated by HDAC inhibition have been identified, those genes clearly responsible for the biological effects of these drugs have remained elusive. We investigated the pharmacological effect of the HDACI and potential anti-cancer agent Trichostatin A (TSA) on primary T cells. To ascertain the effect of TSA on resting and activated T cells we used a model system where an enriched cell population consisting of primary T-cells was stimulated in vitro with immobilized anti-CD3/anti-CD28 antibodies whilst exposed to pharmacological concentrations of Trichostatin A. We found that this drug causes a rapid decline in cytokine expression, accumulation of cells in the G1 phase of the cell cycle, and induces apoptotic cell death. The mitochondrial respiratory chain (MRC) plays a critical role in the apoptotic response to TSA, as dissipation of mitochondrial membrane potential and reactive oxygen species (ROS) scavengers block TSA-induced T-cell death. Treatment of T cells with TSA results in the altered expression of a subset of genes involved in T cell responses, as assessed by microarray gene expression profiling. We also observed up- as well as down-regulation of various costimulatory/adhesion molecules, such as CD28 and CD154, important for T-cell function. Taken together, our findings indicate that HDAC inhibitors have an immunomodulatory potential that may contribute to the potency and specificity of these antineoplastic compounds and might be useful in the treatment of autoimmune disorders.

Current standard treatments for metastatic colorectal cancer (CRC) are based on combination regimens with one of the two chemotherapeutic drugs, irinotecan or oxaliplatin. However, drug resistance frequently limits the clinical efficacy of these therapies. In order to gain new insights into mechanisms associated with chemoresistance, and departing from three distinct CRC cell models, we generated a panel of human colorectal cancer cell lines with acquired resistance to either oxaliplatin or irinotecan. We characterized the resistant cell line variants with regards to their drug resistance profile and transcriptome, and matched our results with datasets generated from relevant clinical material to derive putative resistance biomarkers. We found that the chemoresistant cell line variants had distinctive irinotecan‐ or oxaliplatin‐specific resistance profiles, with non‐reciprocal cross‐resistance. Furthermore, we could identify several new, as well as some previously described, drug resistance‐associated genes for each resistant cell line variant. Each chemoresistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of chemotherapy resistance. In addition, and given the potential implications for selection of subsequent treatment, we also performed an exploratory analysis, in relevant patient cohorts, of the predictive value of each of the specific genes identified in our cellular models.

Genome‐wide association studies recently linked the locus for Na⁺,HCO₃⁻‐cotransporter NBCn1 (SLC4A7) to breast cancer susceptibility, yet functional insights have been lacking. To determine whether NBCn1, by transporting HCO₃⁻ into cells, may dispose of acid produced during high metabolic activity, we studied the expression of NBCn1 and the functional impact of Na⁺,HCO₃⁻‐cotransport in human breast cancer. We found that the plasmalemmal density of NBCn1 was 20–30% higher in primary breast carcinomas and metastases compared to matched normal breast tissue. The increase in NBCn1 density was similar in magnitude to that observed for Na⁺/H⁺‐exchanger NHE1 (SLC9A1), a transporter previously implicated in cell migration, proliferation and malignancy. In primary breast carcinomas, the apparent molecular weight for NBCn1 was increased compared to normal tissue. Using pH‐sensitive fluorophores, we showed that Na⁺,HCO₃⁻‐cotransport is the predominant mechanism of acid extrusion and is inhibited 34 ± 9% by 200 μM 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid in human primary breast carcinomas. At intracellular pH (pH_i) levels >6.6, CO₂/HCO₃⁻‐dependent mechanisms accounted for >90% of total net acid extrusion. Na⁺/H⁺‐exchange activity was prominent only at lower pH_i‐values. Furthermore, steady‐state pH_i was 0.35 ± 0.06 units lower in the absence than in the presence of CO₂/HCO₃⁻. In conclusion, expression of NBCn1 is upregulated in human primary breast carcinomas and metastases compared to normal breast tissue. Na⁺,HCO₃⁻‐cotransport is a major determinant of pH_i in breast cancer and the modest DIDS‐sensitivity is consistent with NBCn1 being predominantly responsible. Hence, our results suggest a major pathophysiological role for NBCn1 that may be clinically relevant.