Secretome analysis using Affinity Proteomics and Immunoassays: a focus on Tumor Biology.

The study of the cellular secretome using proteomic techniques continues to capture the attention of the research community across a broad range of topics in biomedical research. Due to their untargeted nature, independence from the model system employed, historically superior depth of analysis, as well as comparative affordability, mass spectrometry-based approaches traditionally dominate such analyses. More recently, however, affinity-based proteomic assays have massively gained in analytical depth, which together with their high sensitivity, dynamic range coverage as well as high throughput capabilities render them exquisitely suited to secretome analysis. In this review, we revisit the analytical challenges implied by secretomics and provide an overview of affinity-based proteomic platforms currently available for such analyses, using the study of the tumor secretome as an example for basic and translational research.

Sample-Treatment with the Virucidal ß-Propiolactone Does Not Preclude Analysis by Large Panel Affinity Proteomics, Including the Discovery of Biomarker Candidates.

Virus inactivation is a prerequisite for safe handling of high-risk infectious samples. ß-Propiolactone (BPL) is an established reagent with proven virucidal efficacy. BPL primarily reacts with DNA, RNA, and amino acids. The latter may modify antigenic protein epitopes interfering with binding properties of affinity reagents such as antibodies and aptamers used in affinity proteomic screens. We investigated (i) the impact of BPL treatment on the analysis of protein levels in plasma samples using the aptamer-based affinity proteomic platform SomaScan and (ii) effects on protein detection in conditioned medium samples using the proximity extension assay-based Olink Target platform. In the former setup, BPL-treated and native plasma samples from patients with ovarian cancer (n = 12) and benign diseases (n = 12) were analyzed using the SomaScan platform. In the latter, conditioned media samples collected from cultured T cells with (n = 3) or without (n = 3) anti-CD3 antibody stimulation were analyzed using the Olink Target platform. BPL-related changes in protein detection were evaluated comparing native and BPL-treated states, simulating virus inactivation, and impact on measurable group differences was assessed. While approximately one-third of SomaScan measurements were significantly changed by the BPL treatment, a majority of antigen/aptamer interactions remained unaffected. Interaction effects of BPL treatment and disease state, potentially altering detectability of group differences, were observable for less than one percent of targets (0.6%). BPL effects on protein detection with Olink Target were also limited, affecting 3.6% of detected proteins with no observable interaction effects. Thus, effects of BPL treatment only moderately interfere with affinity proteomic detectability of differential protein expression between different experimental groups. Overall, the results prove high-throughput affinity proteomics well suited for the analysis of high-risk samples inactivated using BPL.

Advances in aqueous humor proteomics for biomarker discovery and disease mechanisms exploration: a spotlight on primary open angle glaucoma.

Altered protein levels in the aqueous humor (AH) may be a valuable source of novel biomarkers in neurodegenerative retinal disease. The proximity of this body fluid to the disease focus, and its corresponding enrichment for tissue specific proteins, renders it an excellent matrix to study underlying molecular mechanisms. Novel proteomic methods accordingly hold large potential for insight into pathologies based on the composition of the AH proteome, including primary open angle glaucoma (POAG). Recent mass spectrometry-based studies use novel approaches to tackle the challenges arising from the combination of low available sample volume and protein concentration, thereby increasing proteome coverage. But despite significant improvements in mass spectrometry (MS), a different class of proteomic technologies is poised to majorly impact the analysis of ocular biofluids. Affinity proteomic workflows, having become available commercially recently, have started to complement data obtained by MS and likely will grow into a crucial tool for ophthalmological biomarker research. This review highlights corresponding approaches in proteome analysis of aqueous humor and discusses recent findings on alterations of the AH proteome in POAG.

Reciprocal crosstalk between Th17 and mesothelial cells promotes metastasis-associated adhesion of ovarian cancer cells.

BACKGROUND: IL-17A and TNF synergistically promote inflammation and tumorigenesis. Their interplay and impact on ovarian carcinoma (OC) progression are, however, poorly understood. We addressed this question focusing on mesothelial cells, whose interaction with tumor cells is known to play a pivotal role in transcoelomic metastasis formation. METHODS: Flow-cytometry and immunohistochemistry experiments were employed to identify cellular sources of IL-17A and TNF. Changes in transcriptomes and secretomes were determined by bulk and single cell RNA sequencing as well as affinity proteomics. Functional consequences were investigated by microscopic analyses and tumor cell adhesion assays. Potential clinical implications were assessed by immunohistochemistry and survival analyses. RESULTS: We identified Th17 cells as the main population of IL-17A- and TNF producers in ascites and detected their accumulation in early omental metastases. Both IL-17A and its receptor subunit IL-17RC were associated with short survival of OC patients, pointing to a role in clinical progression. IL-17A and TNF synergistically induced the reprogramming of mesothelial cells towards a pro-inflammatory mesenchymal phenotype, concomitantly with a loss of tight junctions and an impairment of mesothelial monolayer integrity, thereby promoting cancer cell adhesion. IL-17A and TNF synergistically induced the Th17-promoting cytokines IL-6 and IL-1ß as well as the Th17-attracting chemokine CCL20 in mesothelial cells, indicating a reciprocal crosstalk that potentiates the tumor-promoting role of Th17 cells in OC. CONCLUSIONS: Our findings reveal a novel function for Th17 cells in the OC microenvironment, which entails the IL-17A/TNF-mediated induction of mesothelial-mesenchymal transition, disruption of mesothelial layer integrity and consequently promotion of OC cell adhesion. These effects are potentiated by a positive feedback loop between mesothelial and Th17 cells. Together with the observed clinical associations and accumulation of Th17 cells in omental micrometastases, our observations point to a potential role in early metastases formation and thus to new therapeutic options.

BAG6 restricts pancreatic cancer progression by suppressing the release of IL33-presenting extracellular vesicles and the activation of mast cells.

Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration. EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.