Jonas Bossart

Tumor-associated macrophages (TAMs) comprise functionally diverse states that can suppress anti-tumor immunity and promote tumor progression, yet the tumor microenvironmental cues and signaling programs that generate these states remain incompletely defined. Here, we systematically stimulate primary human monocyte-derived macrophages with a panel of cytokines and metabolites abundant in the tumor microenvironment (TME), and profile their transcriptomic and phosphoproteomic responses to resolve stimulus-specific molecular programs. We observe that potassium (K ⁺ ) and adenosine (Ado) stimulation, which accumulate in necrotic tumor cores, downregulate antigen-presentation genes and their master regulator CIITA . K ⁺ stimulation results in the upregulated fibronectin 1 expression, associated with immunosuppressive, metastasis-promoting TAM subsets. Ado induces upregulated expression of tryptophan (Trp) catabolism genes, myeloid checkpoints and metallothioneins (MTs). Although MT-high TAM states have been recurrently observed across tumor single cell RNA sequencing studies, their function remains poorly defined. We show that elevated MT expression in tumor tissue is associated with shorter overall survival. By aligning in vitro transcriptomes with single-cell RNA sequencing (scRNA-seq) signatures from a pan-cancer TAM atlas, we identify significant similarities between several in vitro states and clinically observed TAM populations, with Ado-stimulated macrophages closely resembling a MT-expressing TAM cluster. Overall, this work provides a systematic molecular context linking tumor microenvironmental cues to clinically relevant TAM states and offers a framework for recapitulating their functions in vitro . STATEMENT OF SIGNIFICANCE: This study explores how cytokines and metabolites from the tumor microenvironment shape macrophage molecular phenotypes and lead to the upregulation of clinically relevant marker genes and recapitulation of functional states of interest.

For the successful commercial development of emerging 2D materials, it is crucial to understand their potential biological effects on healthy and diseased individuals. The present study demonstrates that a repeated low‐dose (1 µg cm ⁻² for 5 weeks) exposure of primary human broncho‐epithelial (HBE) cell cultures to hexagonal boron nitride nanosheets ( h ‐BN) and boron nitride nanotubes (BNNTs) increases the phospholipid, sphingolipid, and diglyceride content in cell membranes. Global lipidomics profiling further shows the induction of lipid mediator biosynthesis, especially after exposure to BNNTs in asthmatic cell cultures. The significant increase in leukotriene biosynthesis including its extracellular release is also confirmed in vivo in exposed mouse lungs. Mechanistically, extracellular release of lipid mediators prompts the recruitment and activation of immune cells. Mass cytometry‐based single‐cell profiling of human peripheral blood mononuclear cells reveals the activation of distinct lymphocyte populations expressing cytotoxic granzyme B and perforin mainly after exposure to conditioned medium from BNNT‐exposed asthmatic HBE cultures. These findings unveil the sub‐cytotoxic impact of BN nanomaterials on cellular lipid homeostasis and associated immunomodulation from repeated‐dose exposures, which may pose a potential health hazard, particularly for immune‐compromised individuals, and therefore, needs to be considered for the responsible and sustainable production and use of BN‐based products.

Thyroid nodules are a widespread phenomenon, with follicular cell-derived thyroid tumors being the most prevalent type of endocrine tumor, spanning from benign through low grade malignant to aggressive neoplasms with dismal prognosis. In clinical practice, histopathological criteria are primarily used to determine malignancy and aggressiveness. Therefore, accurate classification may result in surgical procedures for diagnostic reasons, associated with an imbalanced risk/benefit ratio. In recent years, the use of integrated proteomic approaches has proven valuable in expanding the molecular understanding of thyroid neoplasms, with implications in classification, yet remains understudied in divergent thyroid nodules. Here we show the delineation of subtype-specific and malignancy-dependent molecular characteristics through integrative proteomic and phosphoproteomic analysis of 53 human thyroid tissues, encompassing five frequent benign and malignant tumors. We found that the (phospho)-proteomic profiles enable a clear stratification of malignant and benign thyroid tissues. The method also performs well in delineating follicular adenoma (FA) and follicular thyroid carcinoma (FTC) samples. Beside the dysregulation of cell cycle control, apoptosis, and metabolic reprogramming associated with tumor development and malignancy, we further report increased alterations within the well-established oncogenic RAS/BRAF/MAPK and AKT/MTOR signaling pathways, which, contrary to the prevailing paradigm, did not clearly differentiate between FTC and papillary thyroid carcinoma (PTC). In addition, activities of ATM, PLK2-3, and GRK5-6 kinases were predicted to be strongly upregulated in malignant subtypes. Together, this study provides an in-depth insight into molecular changes in different thyroid tumor subtypes. These findings highlight the potential of integrated proteomic approaches to refine our understanding of complex diseases like cancer. As such, they offer a pathway to more precise diagnostic and personalized treatment strategies.

Exposure to nanoparticles (NPs) in pregnancy is increasingly linked to adverse effects on embryo‐fetal development and health later in life. However, the developmental toxicity mechanisms of NPs are largely unknown, in particular potential effects on the placental secretome, which orchestrates many developmental processes pivotal for pregnancy success. This study demonstrates extensive material‐ and pregnancy stage‐specific deregulation of placental signaling from a single exposure of human placental explants to physiologically relevant concentrations of engineered (silica (SiO₂) and titanium dioxide (TiO₂) NPs) and environmental NPs (diesel exhaust particles, DEPs). This includes a multitude of secreted inflammatory, vascular, and endocrine placental factors as well as extracellular vesicle (EV)‐associated proteins. Moreover, conditioned media (CM) from NP‐exposed explants induce pronounced anti‐angiogenic and anti‐vasculogenic effects, while early neurodevelopmental processes are only marginally affected. These findings underscore the potential of metal oxide NPs and DEPs for widespread interference with the placental secretome and identify vascular morphogenesis as a sensitive outcome for the indirect developmental toxicity of different NPs. Overall, this work has profound implications for the future safety assessment of NPs for industrial, commercial, or medical applications in pregnancy, which should consider placenta‐mediated toxicity by holistic secretomics approaches to ensure the development of safe nanotechnologies.

Macrophages represent a major immune cell type in tumor microenvironments, they exist in multiple functional states and are of a strong interest for therapeutic reprogramming. While signaling cascades defining pro-inflammatory macrophages are better characterized, pathways that drive polarization in immunosuppressive macrophages are incompletely mapped. Here, we performed an in-depth characterization of signaling events in primary human macrophages in different functional states using mass spectrometry-based proteomic and phosphoproteomic profiling. Analysis of direct and indirect footprints of kinase activities has suggested PAK2 and PKCα kinases as important regulators of in vitro immunosuppressive macrophages (IL-4/IL-13 or IL-10 stimulated). Network integration of these data with the corresesponding transcriptome profiles has further highlighted FOS and NCOR2 as central transcription regulators in immunosuppressive states. Furthermore, we retrieved single cell sequencing datasets for tumors from cancer patients and found that the unbiased signatures identified here through proteomic analysis were able to successfully separate pro-inflammatory macrophage populations in a clinical setting and could thus be used to expand state-specific markers. This study contributes to in-depth multi-omics characterizations of macrophage phenotypic landscapes, which could be valuable for assisting future interventions that therapeutically alter immune cell compartments. Abstract Figure: Highlights: Global proteomic characterization of primary human macrophages in different states Mapping of main signaling events through in-depth data analysis PKCα and PAK2 kinases are important regulators of immunosuppressive macrophages Proteomic signatures enable accurate detection of pro-inflammatory macrophages in patient tumors