Secretome analysis using a hollow fiber culture system for cancer biomarker discovery

Secreted proteins, collectively referred to as the secretome, were suggested as valuable biomarkers in disease diagnosis and prognosis. However, some secreted proteins from cell cultures are difficult to detect because of their intrinsically low abundance; they are frequently masked by the released proteins from lysed cells and the substantial amounts of serum proteins used in culture medium. The hollow fiber culture (HFC) system is a commercially available system composed of small fibers sealed in a cartridge shell; cells grow on the outside of the fiber. Recently, because this system can help cells grow at a high density, it has been developed and applied in a novel analytical platform for cell secretome collection in cancer biomarker discovery. This article focuses on the advantages of the HFC system, including the effectiveness of the system for collection of secretomes, and reviews the process of cell secretome collection by the HFC system and proteomic approaches to discover cancer biomarkers. The HFC system not only provides a high-density three-dimensional (3D) cell culture system to mimic tumor growth conditions in vivo but can also accommodate numerous cells in a small volume, allowing secreted proteins to be accumulated and concentrated. In addition, cell lysis rates can be greatly reduced, decreasing the amount of contamination by abundant cytosolic proteins from lysed cells. Therefore, the HFC system is useful for preparing a wide range of proteins from cell secretomes and provides an effective method for collecting higher amounts of secreted proteins from cancer cells. This article is part of a Special Issue entitled: An Updated Secretome.     

The cancer secretome,current status and opportunities in the lung,breast and colorectal cancer context

Despite major improvements on the knowledge and clinical management, cancer is still a deadly disease. Novel biomarkers for better cancer detection, diagnosis and treatment prediction are urgently needed. Proteins secreted, shed or leaking from the cancer cell, collectively termed the cancer secretome, are promising biomarkers since they might be detectable in blood or other biofluids. Furthermore, the cancer secretome in part represents the tumor microenvironment that plays a key role in tumor promoting processes such as angiogenesis and invasion. The cancer secretome, sampled as conditioned medium from cell lines, tumor/tissue interstitial fluid or tumor proximal body fluids, can be studied comprehensively by nanoLC-MS/MS-based approaches. Here, we outline the importance of current cancer secretome research and describe the mass spectrometry-based analysis of the secretome. Further, we provide an overview of cancer secretome research with a focus on the three most common cancer types: lung, breast and colorectal cancer. We conclude that the cancer secretome research field is a young, but rapidly evolving research field. Up to now, the focus has mainly been on the discovery of novel promising secreted cancer biomarker proteins. An interesting finding that merits attention is that in cancer unconventional secretion, e.g. via vesicles, seems increased. Refinement of current approaches and methods and progress in clinical validation of the current findings are vital in order to move towards applications in cancer management. This article is part of a Special Issue entitled: An Updated Secretome.     

The human secretome atlas initiative: Implications in health and disease conditions

Proteomic analysis of human body fluids is highly challenging, therefore many researchers are redirecting efforts toward secretome profiling. The goal is to define potential biomarkers and therapeutic targets in the secretome that can be traced back in accessible human body fluids. However, currently there is a lack of secretome profiles of normal human primary cells making it difficult to assess the biological meaning of current findings. In this study we sought to establish secretome profiles of human primary cells obtained from healthy donors with the goal of building a human secretome atlas. Such an atlas can be used as a reference for discovery of potential disease associated biomarkers and eventually novel therapeutic targets. As a preliminary study, secretome profiles were established for six different types of human primary cell cultures and checked for overlaps with the three major human body fluids including plasma, cerebrospinal fluid and urine. About 67% of the 1054 identified proteins in the secretome of these primary cells occurred in at least one body fluid. Furthermore, comparison of the secretome profiles of two human glioblastoma cell lines to this new human secretome atlas enabled unambiguous identification of potential brain tumor biomarkers. These biomarkers can be easily monitored in different body fluids using stable isotope labeled standard proteins. The long term goal of this study is to establish a comprehensive online human secretome atlas for future use as a reference for any disease related secretome study. This article is part of a Special Issue entitled: An Updated Secretome.     

Secretome proteomics for discovery of cancer biomarkers

“Secretome” is referred to as the rich, complex set of molecules secreted from living cells. In a less strict definition frequently followed in “secretome” studies, the term also includes molecules shed from the surface of living cells. Proteins of secretome (will be referred to as secreted) play a key role in cell signaling, communication and migration. The need for developing more effective cancer biomarkers and therapeutic modalities has led to the study of cancer cell secretome as a means to identify and characterize diagnostic and prognostic markers and potential drug and therapeutic targets. Significant technological advances in the field of proteomics during the last two decades have greatly facilitated research towards this direction. Nevertheless, secretome analysis still faces some difficulties mainly related to sample collection and preparation. The goal of this article is to provide an overview of the main findings from the analysis of cancer cell secretome. Specifically, we focus on the presentation of main methodological approaches that have been developed for the study of secreted proteins and the results thereof from the analysis of secretome in different types of malignancies; special emphasis is given on correlation of findings with protein expression in body fluids.     

Secretome compartment is a valuable source of biomarkers for cancer-relevant pathways

In principle, targeted therapies have optimal activity against a specific subset of tumors that depend upon the targeted molecule or pathway for growth, survival, or metastasis. Consequently, it is important in drug development and clinical practice to have predictive biomarkers that can reliably identify patients who will benefit from a given therapy. We analyzed tumor cell-line secretomes (conditioned cell media) to look for predictive biomarkers; secretomes represent a potential source for potential biomarkers that are expressed in intracellular signaling and therefore may reflect changes induced by targeted therapy. Using Gene Ontology, we classified by function the secretome proteins of 12 tumor cell lines of different histotypes. Representations and hierarchical relationships among the functional groups differed among the cell lines. Using bioinformatics tools, we identified proteins involved in intracellular signaling pathways. For example, we found that secretome proteins related to TGF-beta signaling in thyroid cancer cells, such as vasorin, CD109, and βIG-H3 (TGFBI), were sensitive to RPI-1 and dasatinib treatments, which have been previously demonstrated to be effective in blocking cell proliferation. The secretome may be a valuable source of potential biomarkers for detecting cancer and measuring the effectiveness of cancer therapies.     

Agrin and Perlecan Mediate Tumorigenic Processes in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma is the most common type of cancer in the oral cavity, representing more than 90% of all oral cancers. The characterization of altered molecules in oral cancer is essential to understand molecular mechanisms underlying tumor progression as well as to contribute to cancer biomarker and therapeutic target discovery. Proteoglycans are key molecular effectors of cell surface and pericellular microenvironments, performing multiple functions in cancer. Two of the major basement membrane proteoglycans, agrin and perlecan, were investigated in this study regarding their role in oral cancer. Using real time quantitative PCR (qRT-PCR), we showed that agrin and perlecan are highly expressed in oral squamous cell carcinoma. Interestingly, cell lines originated from distinct sites showed different expression of agrin and perlecan. Enzymatically targeting chondroitin sulfate modification by chondroitinase, oral squamous carcinoma cell line had a reduced ability to adhere to extracellular matrix proteins and increased sensibility to cisplatin. Additionally, knockdown of agrin and perlecan promoted a decrease on cell migration and adhesion, and on resistance of cells to cisplatin. Our study showed, for the first time, a negative regulation on oral cancer-associated events by either targeting chondroitin sulfate content or agrin and perlecan levels.     

A high-quality secretome of A549 cells aided the discovery of C4b-binding protein as a novel serum biomarker for non-small cell lung cancer

Cancer secretomes are a promising source for biomarker discovery. The analysis of cancer secretomes still faces some difficulties mainly related to the intracellular contamination, which hinders the qualification and follow-up validations. This study aimed to establish a high-quality secretome of A549 cells by using the cellular proteome as a reference and to test the merits of this refined secretome for biomarker discovery for non-small cell lung cancer (NSCLC). Using one-dimensional gel electrophoresis followed by liquid-chromatography tandem mass spectrometry, we comprehensively investigated the secretome and the concurrent cellular proteome of A549 cells. A high-quality secretome consisting of 382 proteins was refined from 889 initial secretory proteins. More than 85.3% of proteins were annotated as secreted and 76.8% as extracellular or membrane-bound. The discriminative power of the lung-cancer associated secretome was confirmed by gene expression and serum proteomic data. The elevated level of C4b-binding Protein (C4BP) in NSCLC blood was verified by enzyme-linked immunosorbent assays (ELISA, p = 6.07e-6). Moreover, the serum C4BP level in 89 patients showed a strong association with the clinical staging of NSCLC. Our reference-experiment-driven strategy is simple and widely applicable, and may facilitate the identification of novel promising biomarkers of lung cancer.     

N-glycoproteome Analysis of the Secretome of Human Metastatic Hepatocellular Carcinoma Cell Lines Combining Hydrazide Chemistry,HILIC Enrichment and Mass Spectrometry

Cancer cell metastasis is a major cause of cancer death. Unfortunately, the underlying molecular mechanisms remain unknown, which results in the lack of efficient diagnosis, therapy and prevention approaches. Nevertheless, the dysregulation of the cancer cell secretome is known to play key roles in tumor transformation and progression. The majority of proteins in the secretome are secretory proteins and membrane-released proteins, and, mostly, the glycosylated proteins. Until recently, few studies have explored protein N-glycosylation changes in the secretome, although protein glycosylation has received increasing attention in the study of tumor development processes. Here, the N-glycoproteins in the secretome of two human hepatocellular carcinoma (HCC) cell lines with low (MHCC97L) or high (HCCLM3) metastatic potential were investigated with a in-depth characterization of the N-glycosites by combining two general glycopeptide enrichment approaches, hydrazide chemistry and zwitterionic hydrophilic interaction chromatography (zic-HILIC), with mass spectrometry analysis. A total of 1,213 unique N-glycosites from 611 N-glycoproteins were confidently identified. These N-glycoproteins were primarily localized to the extracellular space and plasma membrane, supporting the important role of N-glycosylation in the secretory pathway. Coupling label-free quantification with a hierarchical clustering strategy, we determined the differential regulation of several N-glycoproteins that are related to metastasis, among which AFP, DKK1, FN1, CD151 and TGFβ2 were up-regulated in HCCLM3 cells. The inclusion of the well-known metastasis-related proteins AFP and DKK1 in this list provides solid supports for our study. Further western blotting experiments detecting FN1 and FAT1 confirmed our discovery. The glycoproteome strategy in this study provides an effective means to explore potential cancer biomarkers.     

Gaining insights into cancer biology through exploration of the cancer secretome using proteomic and bioinformatic tools

Introduction: Tumor-associated proteins released by cancer cells and by tumor stroma cells, referred as ‘cancer secretome’, represent a valuable resource for discovery of potential cancer biomarkers. The last decade was marked by a great increase in number of studies focused on various aspects of cancer secretome including, composition and identification of components externalized by malignant cells and by the components of tumor microenvironment.

Areas covered: Here, we provide an overview of achievements in the proteomic analysis of the cancer secretome, elicited through the tumor-associated interstitial fluid recovered from malignant tissues ex vivo or the protein component of conditioned media obtained from cultured cancer cells in vitro. We summarize various bioinformatic tools and approaches and critically appraise their outcomes, focusing on problems and challenges that arise when applied for the analysis of cancer secretomic databases.

Expert commentary: Recent achievements in the omics- analysis of structural and metabolic aspects of altered cancer secretome contribute greatly to the various hallmarks of cancer including the identification of clinically significant biomarkers and potential targets for therapeutic intervention.     

Identification of proteins secreted by head and neck cancer cell lines using LC-MS/MS: Strategy for discovery of candidate serological biomarkers

In search of blood-based biomarkers that would enhance the ability to diagnose head and neck/oral squamous cell carcinoma (HNOSCC) in early stages or predict its prognosis, we analyzed the HNOSCC secretome (ensemble of proteins secreted and/or shed from the tumor cells) for potential biomarkers using proteomic technologies. LC-MS/MS was used to identify proteins in the conditioned media of four HNOSCC cell lines (SCC4, HSC2, SCC38, and AMOSIII); 140 unique proteins were identified on the basis of 5% global false discovery rate, 122 of which were secretory proteins, with 29 being previously reported to be overexpressed in HNOSCC in comparison to normal head and neck tissues. Of these, five proteins including α-enolase, peptidyl prolyl isomerase A/cyclophilin A, 14-3-3 ζ, heterogeneous ribonucleoprotein K, and 14-3-3 σ were detected in the sera of HNOSCC patients by Western blot analysis. Our study provides the evidence that analysis of head and neck cancer cells' secretome is a viable strategy for identifying candidate serological biomarkers for HNOSCC. In future, these biomarkers may be useful in predicting the likelihood of transformation of oral pre-malignant lesions, prognosis of HNOSCC patients and evaluate response to therapy using minimally invasive tests.     

Identification of collapsin response mediator protein-2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes

The cancer cell secretome may contain many potentially useful biomarkers. We therefore sought to identify proteins in the conditioned media of colorectal carcinoma (CRC) cell lines but not in those from other cancer cell lines. The secretomes of 21 cancer cell lines derived from 12 cancer types were analyzed by SDS-PAGE combined with MALDI-TOF MS. Among the 325 proteins identified, collapsin response mediator protein-2 (CRMP-2) was chosen for evaluation as a potential CRC biomarker, since it was selectively detected in the CRC cell line secretome and has never been reported as a cancer biomarker. Immunohistochemical analysis of 169 CRC specimens showed that CRMP-2 was positively detected in 58.6% of the tumors, but weakly or not detected in >90% of the adjacent nontumor epithelial cells. Moreover, the CRMP-2-positive rate was significantly increased in earlier stage tumors and lymph node metastasis. Plasma CRMP-2 levels were significantly higher in CRC patients (N = 201) versus healthy controls (N = 201) (61.3 +/- 34.6 vs. 40.2 +/- 24.3 ng/mL, p = 0.001). Our results indicate that comparative analysis of cancer cell secretome is a feasible strategy for identifying potential cancer biomarkers, and that CRMP-2 may be a novel CRC biomarker.     

Proteomic profiling of the cancer cell secretome: informing clinical research

Introduction: Cancer represents one of the major causes of human deaths. Identification of proteins as biomarkers for early detection of cancer and therapeutic targets for cancer treatment are important issues in precision medicine. Secretome of cancer cells represents the collection of proteins secreted or shed from cancer cells. Proteomic profiling of the cancer cell secretome has been proven to be a convenient and efficient way to discover cancer biomarker and/or therapeutic targets.

Areas covered: There have been numerous reviews describing the history and application of secretome analysis in cancer biomarker/therapeutic target research. The present review focuses on the technological advancement for profiling low-molecular-mass proteins in secretome, the latest information regarding the new candidate biomarkers and molecular mechanisms discovered on the basis of cancer cell secretome analysis, as well as the previously discovered candidate biomarkers that enter into clinical trials.

Expert commentary: Current technologies for protein sample preparation/separation and MS-based protein identification have allowed in-depth analysis of cancer cell secretome. Future efforts should focus on the comprehensiveness of cancer cell secretome, meta-analysis of different secretome datasets and integrated analysis via combining other omics datasets, as well as the incorporation of MS-based biomarker verification pipeline into both preclinical studies and clinical trials.     

Identification of head and neck squamous cell carcinoma biomarker candidates through proteomic analysis of cancer cell secretome

Protein biomarker discovery for early detection of head and neck squamous cell carcinoma (HNSCC) is a crucial unmet need to improve patient outcomes. Mass spectrometry-based proteomics has emerged as a promising tool for identification of biomarkers in different cancer types. Proteins secreted from cancer cells can serve as potential biomarkers for early diagnosis. In the current study, we have used isobaric tag for relative and absolute quantitation (iTRAQ) labeling methodology coupled with high resolution mass spectrometry to identify and quantitate secreted proteins from a panel of head and neck carcinoma cell lines. In all, we identified 2,472 proteins, of which 225 proteins were secreted at higher or lower abundance in HNSCC-derived cell lines. Of these, 148 were present in higher abundance and 77 were present in lower abundance in the cancer-cell derived secretome. We detected a higher abundance of some previously known markers for HNSCC including insulin like growth factor binding protein 3, IGFBP3 (11-fold) and opioid growth factor receptor, OGFR (10-fold) demonstrating the validity of our approach. We also identified several novel secreted proteins in HNSCC including olfactomedin-4, OLFM4 (12-fold) and hepatocyte growth factor activator, HGFA (5-fold). IHC-based validation was conducted in HNSCC using tissue microarrays which revealed overexpression of IGFBP3 and OLFM4 in 70% and 75% of the tested cases, respectively. Our study illustrates quantitative proteomics of secretome as a robust approach for identification of potential HNSCC biomarkers. This article is part of a Special Issue entitled: An Updated Secretome.     

Sieving through the cancer secretome

Cancer is among the most prevalent and serious health problems worldwide. Therefore, there is an urgent need for novel cancer biomarkers with high sensitivity and specificity for early detection and management of the disease. The cancer secretome, encompassing all the proteins that are secreted by cancer cells, is a promising source of biomarkers as the secreted proteins are most likely to enter the blood circulation. Moreover, since secreted proteins are responsible for signaling and communication with the tumor microenvironment, studying the cancer secretome would further the understanding of cancer biology. Latest developments in proteomics technologies have significantly advanced the study of the cancer secretome. In this review, we will present an overview of the secretome sample preparation process and summarize the data from recent secretome studies of six common cancers with high mortality (breast, colorectal, gastric, liver, lung and prostate cancers). In particular, we will focus on the various platforms that were employed and discuss the clinical applicability of the key findings in these studies. This article is part of a Special Issue entitled: An Updated Secretome.     

The cancer cell secretome: A good source for discovering biomarkers?

Cancer is a leading cause of death. Early detection is usually associated with better clinical outcomes. Recent advances in genomics and proteomics raised hopes that new biomarkers for diagnosis, prognosis or monitoring therapeutic response will soon be discovered. Proteins secreted by cancer cells, referred also as “the cancer cell secretome”, is a promising source for biomarker discovery. In this review we will summarize recent advances in cancer cell secretome analysis, focusing on the five most fatal cancers (lung, breast, prostate, colorectal, and pancreatic). For each cancer type we will describe the proteomic approaches utilized for the identification of novel biomarkers. Despite progress, identification of markers that are superior to those currently used has proven to be a difficult task and very few, if any, newly discovered biomarker has entered the clinic the last 10 years.     

A mouse model repository for cancer biomarker discovery

Early detection of cancer using biomarkers obtained from blood or other easily accessible tissues would have a significant impact on reducing cancer mortality. However, identifying new blood-based biomarkers has been hindered by the dynamic complexity of the human plasma proteome, confounded by genetic and environmental variability, and the scarcity of high quality controlled samples. In this report, we discuss a new paradigm for biomarker discovery through the use of mouse models. Inbred mouse models of cancer recapitulate many critical features of human cancer, while eliminating sources of environmental and genetic variability. The ability to collect samples from highly matched cases and controls under identical conditions further reduces variability which is critical for successful biomarker discovery. We describe the establishment of a repository containing tumor, plasma, urine, and other tissues from 10 different mouse models of human cancer, including two breast, two lung, two prostate, two gastrointestinal, one ovarian, and one skin tumor model. We present the overall design of this resource and its potential use by the research community for biomarker discovery.     

Negative regulators of cell death pathways in cancer: perspective on biomarkers and targeted therapies

Cancer is a primary cause of human fatality and conventional cancer therapies, e.g., chemotherapy, are often associated with adverse side-effects, tumor drug-resistance, and recurrence. Molecularly targeted therapy, composed of small-molecule inhibitors and immunotherapy (e.g., monoclonal antibody and cancer vaccines), is a less harmful alternative being more effective against cancer cells whilst preserving healthy tissues. Drug-resistance, however, caused by negative regulation of cell death signaling pathways, is still a challenge. Circumvention of negative regulators of cell death pathways or development of predictive and response biomarkers is, therefore, quintessential. This review critically discusses the current state of knowledge on targeting negative regulators of cell death signaling pathways including apoptosis, ferroptosis, necroptosis, autophagy, and anoikis and evaluates the recent advances in clinical and preclinical research on biomarkers of negative regulators. It aims to provide a comprehensive platform for designing efficacious polytherapies including novel agents for restoring cell death signaling pathways or targeting alternative resistance pathways to improve the chances for antitumor responses. Overall, it is concluded that nonapoptotic cell death pathways are a potential research arena for drug discovery, development of novel biomarkers and targeted therapies.     

Discovery of biomarkers for gastric cancer: a proteomics approach

Gastric cancer is the second leading cause of cancer-related deaths worldwide. Although many treatment options exist for patients with gastric tumors, the incidence and mortality rate of gastric cancer are on the rise. The early stages of gastric cancer are non-symptomatic, and the treatment response is unpredictable. This situation is further aggravated by a lack of diagnostic biomarkers that can aid in the early detection and prognosis of gastric cancer and in the prediction of chemoresistance. Moreover, clinical surgical specimens are rarely obtained, and traditional biomarkers of gastric cancer are not very effective. Many studies in the field of proteomics have contributed to the discovery and establishment of powerful diagnostic tools (e.g., ProteinChip array) in the management of cancer. The evolution in proteomic technologies has not only enabled the screening of a large number of samples but also enabled the identification of pathologically significant proteins, such as phosphoproteins, and the quantitation of difference in protein expression under different conditions. Multiplexed assays are used widely to accurately fractionate various complex samples such as blood, tissue, cells, and Helicobacter pylori-infected specimens to identify differentially expressed proteins. Biomarker detection studies have substantially contributed to the areas of secretome, metabolome, and phosphoproteome. Here, we review the development of potential biomarkers in the natural history of gastric cancer, with specific emphasis on the characteristics of target protein convergence.     

Extracellular heat shock proteins and cancer: New perspectives

Heat shock proteins (HSPs) are a large family of molecular chaperones aberrantly expressed in cancer. The expression of HSPs in tumor cells has been shown to be implicated in the regulation of apoptosis, immune responses, angiogenesis and metastasis. Given that extracellular vesicles (EVs) can serve as potential source for the discovery of clinically useful biomarkers and therapeutic targets, it is of particular interest to study proteomic profiling of HSPs in EVs derived from various biological fluids of cancer patients. Furthermore, a divergent expression of circulating microRNAs (miRNAs) in patient samples has opened new opportunities in exploiting miRNAs as diagnostic tools. Herein, we address the current literature on the expression of extracellular HSPs with particular interest in HSPs in EVs derived from various biological fluids of cancer patients and different types of immune cells as promising targets for identification of clinical biomarkers of cancer. We also discuss the emerging role of miRNAs in HSP regulation for the discovery of blood-based biomarkers of cancer. We outline the importance of understanding relationships between various HSP networks and co-chaperones and propose the model for identification of HSP signatures in cancer. Elucidating the role of HSPs in EVs from the proteomic and miRNAs perspectives may provide new opportunities for the discovery of novel biomarkers of cancer.     

Biomarker research with prospective study designs for the early detection of cancer

This article describes the principles of marker research with prospective studies along with examples for diagnostic tumor markers. A plethora of biomarkers have been claimed as useful for the early detection of cancer. However, disappointingly few biomarkers were approved for the detection of unrecognized disease, and even approved markers may lack a sound validation phase. Prospective studies aimed at the early detection of cancer are costly and long-lasting and therefore the bottleneck in marker research. They enroll a large number of clinically asymptomatic subjects and follow-up on incident cases. As invasive procedures cannot be applied to collect tissue samples from the target organ, biomarkers can only be determined in easily accessible body fluids. Marker levels increase during cancer development, with samples collected closer to the occurrence of symptoms or a clinical diagnosis being more informative than earlier samples. Only prospective designs allow the serial collection of pre-diagnostic samples. Their storage in a biobank upgrades cohort studies to serve for both, marker discovery and validation. Population-based cohort studies, which may collect a wealth of data, are commonly conducted with just one baseline investigation lacking serial samples. However, they can provide valuable information about factors that influence the marker level. Screening programs can be employed to archive serial samples but require significant efforts to collect samples and auxiliary data for marker research. Randomized controlled trials have the highest level of evidence in assessing a biomarker's benefit against usual care and present the most stringent design for the validation of promising markers as well as for the discovery of new markers. In summary, all kinds of prospective studies can benefit from a biobank as they can serve as a platform for biomarker research. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.