Tumoral glucocorticoids induce a phagocytic CD68+/CD163+/C1Q+ macrophage phenotype primed for IFNγ-driven CXCL9 secretion

Tumoral glucocorticoids induce a phagocytic CD68+/CD163+/C1Q+ macrophage phenotype primed for IFNγ-driven CXCL9 secretion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Tumoral glucocorticoids induce a phagocytic CD68+/CD163+/C1Q+ macrophage phenotype primed for IFNγ-driven CXCL9 secretion Matthias Kroiss, Alexandra Triebig, Tanja Maier, Paul Schwarzlmueller, and 17 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6213228/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Glucocorticoids (GCs) play a multifaceted role in modulating immune responses in cancer. Adrenocortical carcinoma (ACC) is a rare endocrine malignancy that produces excessive glucocorticoids in ~ 60%, providing a unique model to study intra-tumoral GC activity. Here, we report that ACC tumors are strongly infiltrated by CD68+/CD163+ 'M2-like' macrophages, independent of cortisol overproduction. In vitro , GC exposure drives the polarization of macrophages towards a C1Q + subtype with enhanced phagocytic activity, mediated by upregulated expression and secretion of the complement component C1q. IFNγ stimulation of C1Q + macrophages significantly enhanced the production and secretion of the T cell chemoattractant CXCL9, surpassing the concentrations produced by classical pro-inflammatory 'M1-like' macrophages. Notably, the presence of intra-tumoral macrophages correlated with increased T cell infiltration, improved patient survival and response to ICI therapy in ACC. Collectively, this study identifies a GC-driven CD68+/CD163+/C1Q + macrophage phenotype with high phagocytic capacity and IFNγ-induced cytokine secretion, suggesting a potential role in T cell recruitment and the enhancement of immunotherapy efficacy in ACC and other solid tumors. Biological sciences/Immunology/Tumour immunology/Immunosurveillance/Immunoediting Biological sciences/Cancer/Cancer microenvironment Health sciences/Oncology/Cancer/Endocrine cancer/Adrenal tumours Health sciences/Biomarkers/Prognostic markers Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages/Phagocytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Glucocorticoids (GCs) are a class of steroid hormones predominantly synthesized and secreted by the zona fasciculata of the adrenal cortex in response to hypothalamic-pituitary-adrenal (HPA) axis activation. Within cancer tissues, GCs have emerged as an important component of the tumor microenvironment (TME), originating from three main sources: endogenous production 1 , 2 , recycling of inactive metabolites 3 or as a consequence of therapeutic administration. Notably, GCs in the context of cancer are believed to promote tumor cell proliferation, survival and migration while concurrently suppressing antitumor immunity through functional modulation of immune cells 1 , 4 , 5 . Adrenocortical carcinoma (ACC) is a rare, yet highly aggressive tumor of the adrenal cortex, with an estimated annual incidence ranging from approximately 0.5 to 2.0 cases per million individuals 6 , 7 . While non-functional ACCs are commonly diagnosed incidentally or due to mass effects, the majority of ACCs is hormonally functional leading to distinct clinical presentations caused by hormonal hypersecretion. These clinical manifestations include Cushing’s syndrome in GC-producing tumors or virilization in androgen-secreting ACCs. Immune checkpoint inhibitors (ICI) that have profoundly changed the management of many malignancies have demonstrated limited efficacy in ACC. So far, a combined analysis of twenty studies with a total of 250 patients reported an objective response in only 14% of patients with an overall survival of 13.9 months 8 . However, a clinically meaningful overall response rate of 52% and an overall survival (OS) of almost 21 months in one recent study suggest that a subset of patients may benefit from ICI therapy 9 . Tumor associated macrophages (TAMs) exhibit a remarkable plasticity and can assume a dual role in the progression of cancer. Pro-inflammatory 'M1-like' (classically activated) macrophages (MΦ) are known for their antitumoral functions including cytotoxicity and immune stimulation, e.g. by the release of pro-inflammatory cytokines. Meanwhile, anti-inflammatory 'M2-like' (alternatively activated) MΦ often promote tumor growth fostering a profoundly immune-suppressed TME that negatively impacts on cancer outcome 10 , 11 or the responsiveness to ICI therapy 12 . Even though this simplified classification proposed by Mills et al. broadly describes the inflammatory capacity of MΦ, this terminology is considered outdated as the current understanding suggests a spectrum of diverse MΦ phenotypes, each with a unique contribution to inflammation 13 , 14 . Therapeutic modulation of MΦ differentiation such as inhibition of colony stimulating factor 1 (CSF-1) or its receptor (CSF1R), has emerged as a promising approach to increase the utility of ICI in unresponsive or refractory tumors. However, clinical trials investigating CSF1R blockade, either as monotherapy or in combination with ICIs, have largely failed to produce significant clinical responses 15 . This highlights the need for a more sophisticated approach that goes beyond broad depletion of TAMs. Specifically in ACC, therapeutic approaches targeting MΦ remain completely unexplored, primarily due to the limited understanding of MΦ infiltration and function in this rare endocrine malignancy. Current in vitro studies propose the activation of the glucocorticoid receptor (GR) or reprogramming of the mitochondrial metabolism by GCs to profoundly suppress pro-inflammatory (M1) MΦ polarization and promote anti-inflammatory (M2) polarization 16 , 17 . However, in the context of cancer, the impact of tumor-derived GCs on the function of TAMs remains largely unknown. The utilization of ACC as a model to study the role of GC activity in cancer may shed light on the relevance of endogenously produced or administered GCs across several malignancies. We here set out to identify the contribution of tumoral GCs on MΦ polarization in cancer and describe how therapeutic targeting of the underlying mechanisms may be relevant for clinical treatment concepts in regard to ACC and other solid tumors. Materials and Methods Tissue samples Tissue samples from patients with ACC were collected as part of the European Network for the Study of Adrenal Tumors (ENSAT) registry study or the MASTER (Molecularly Aided Stratification for Tumor Eradication Research) program. The ENSAT study has been approved by the ethics committees of the Ludwig-Maximilians-University; Munich; Germany (RRID:SCR_011358) (approval number: 379/10) and the Julius-Maximilians-University Würzburg (approval number: 88/11). Written informed consent was received from each patient before surgery and the study was conducted in accordance with the ethical principles of the declaration of Helsinki. The MASTER program (ClinicalTrials.gov: NCT05852522), a prospective observational study conducted by the German Cancer Research Center (DKFZ), the National Center for Tumor Diseases (NCT), and the German Cancer Consortium (DKTK), leverages whole-genome/exome sequencing (WGS/WES), RNA sequencing (RNA-seq), DNA methylation profiling, proteomics, and phosphoproteomics to guide treatment in young adults with advanced malignancies and patients with incurable rare cancers 18 . Anonymized non-small cell lung cancer (NSCLC) and colorectal cancer samples (CRC) were obtained from the Human Tissue and Cell Research (HTCR) foundation. Immunohistochemistry Immunohistochemistry (IHC) was conducted as described 19 . In short, formalin-fixed, paraffin-embedded (FFPE) tumor sections were deparaffinized in xylene and dehydrated with increasing dilutions of ethanol. Antigen retrieval was performed in 10 mM citric acid monohydrate buffer at pH 6.5. Endogenous peroxidase activity was blocked with methanol containing 3% H 2 O 2 for 10 min at RT. Non-specific binding was blocked using 20% human AB serum for 1 h at RT. Primary antibodies ( suppl. table 1 ) were applied overnight at 4°C. Signals were developed using the HiDef Detection Polymer System Detection Amplifier (Medac, Germany) and HiDef Detection Polymer System HRP Polymer Detector (Medac, Germany). DAB (DAB Liquid Kit; Dako) was applied and the sections were counterstained with hematoxylin. Stained slides were dehydrated in 100% EtOH and dried at 60°C for 1 h. Slides were scanned using the Microscopes International uScopeMXII-20 Digital Microscope (RRID:SCR_026399) with 20x magnification and positive cell detection was performed using the software QuPath (RRID:SCR_018257). Cell culture and drug treatment NCI-H295R cells (RRID:CVCL 0458) were obtained from Cytion and cultured in DMEM/F12 supplemented with 1x Insulin-Transferrin-Selenium (Gibco) and 2.5% Nu-Serum (Corning). JIL-2266 cells were established and characterized in our laboratory and cultured as previously described 20 . All cell lines were cultured at 37°C and 5% CO2. Cells were checked regularly for mycoplasma contamination using the Venor® GeM Classic Mycoplasma Detection Kit (Minerva Biolabs). Ferroptosis was induced using RSL3 (Selleckchem). Inhibitors (liproxstatin-1, metyrapone or celecoxib (all Sigma)) were added 1 h prior cell death initiation or the start point of cell culture supernatant collection. Dexamethasone (Jenapharm®) and hydrocortisone (Pfizer) were used for exogenous glucocorticoid treatment. The glucocorticoid receptor antagonist relacorilant was obtained from Corcept Therapeutics (Menlo Park, CA, USA). PGE 2 was obtained from Selleckchem. Cas9 expressing THP-1 (RRID:CVCL_0006) monocytes were obtained from Prof. Jörg Wischhusen, University Hospital Würzburg and cultured in RPMI 10% FBS, 1% P/S and 50 µM ß-mercaptoethanol. For differentiation, THP-1 cells were plated into 12-well plates and exposed to 10 ng/ml PMA for 48 h before further polarizing factors were added for a duration of 48 h. For NR3C1 KO cells, THP-1 cells were first differentiated for 2 days using 10 ng/ml PMA, followed by detachment of cells using accutase. Cells were counted and 2.5*10^6 cells were transfected with 1.6 µg sgRNA (IDT) using the LONZA™ Nucleofector 2b Device (RRID:SCR_022262) (program V-001). After 5 h medium was changed to THP-1 medium containing 2.5 ng/ml PMA and 50 µM β-mercaptoethanol. 48 h after transfection, cytokines or CM was added for 48 h. Macrophage isolation and polarization Human PBMCs from healthy blood donors were isolated by density gradient using Lymphoprep™ (StemCell) and transferred to 12-well plates at a density of 5*10^6 cells/ml in macrophage medium (RPMI-1640 (Gibco) supplemented with 10% FBS (Sigma), 1% penicillin-streptomycin (Sigma) and 1% L-glutamine (Gibco)). Monocytes attached to the culture vessel after 24 h were incubated in macrophage medium supplemented with either GM-CSF (Gibco; 100 ng/ml) or M-CSF (Gibco; 40 ng/ml) for 'M1-like' or 'M2-like' MΦ differentiation, respectively. On day 7, further polarizing factors IFNγ (Gibco; 10 ng/ml), IL-4 (Gibco; 20 ng/ml), LPS (Invitrogen; 100 ng/ml) or IL-10 (Gibco; 150 ng/ml) were added for a total of 6 days with medium change and PBS washing every other day. A schematic representation of the macrophage polarization protocol and their phenotype characteristics is depicted in the supplementary Figure S1 . For the generation of conditioned media (CM), ACC cell lines were cultured for 48 h at 3.5*10^5 cells/well of a 6 well plate. Supernatant was collected, sterile-filtered and mixed with macrophage medium (30% v/v). This medium was added to the monocytes from day 1 onwards. Medium was changed on day 6 and then every other day. Immuno-Cytochemistry PBMCs were isolated and macrophages were differentiated and polarized according to the protocol stated above. For immunostaining, cells were fixed with 4% PFA for 10 min at 4°C. Subsequently, cells were blocked with PBS/4% BSA for 30 min and incubated with the primary antibodies ( suppl. table S1 ) overnight at 4°C. Secondary antibodies ( suppl. table S1 ) were added for 1 h RT in the dark. Pictures were taken using the EVOS M7000 Imaging System (RRID:SCR_025070). Immunofluorescence FFPE tumor sections were deparaffinized in xylene and dehydrated with increasing dilutions of ethanol. Antigen retrieval was performed in 10 mM citric acid monohydrate buffer at pH 6.5. Non- specific binding was blocked using 20% human AB serum for 30 min at RT. Primary antibodies ( suppl. table S1 ) were applied overnight at 4°C. Secondary antibodies ( suppl. table S1 ) were incubated for 1 h at RT followed by Hoechst staining (1:2000 in PBS) for 8 min. Slides were mounted with Prolong Gold Antifade reagent (ThermoFisher). Phagocytosis assay ACC cells were stained with CSFE (BioTracker 488 Green CSFE Cell Proliferation Kit, Sigma) and seeded in 12 well plates at a density of 3,5*10^5 cells/well. After 24 h incubation at 37°C, cells were treated with RSL3 for 2 h. Medium was discarded and the wells were washed with PBS twice. MФ were added to the cultured early ferroptotic ACC cells at a ratio of 10:1 tumor cell:MФ overnight. Flow cytometry was used to quantify percentages of CSFE + CD64 + macrophages. For inhibition studies, MФ were pre-incubated with the selective MerTK inhibitor UNC1062 (100 nM; MCE: HY-117548), anti-Gas6 antibody (20 µg/ml; R and D Systems Cat# AB885, RRID:AB_354376), a goat isotype IgG control (20 µg/ml; Thermo Fisher Scientific Cat# 02-6202, RRID:AB_2532946) or purified C1q (Merck; C1740) for 30 minutes before they were added to ferroptotic tumor cells. Flow Cytometry Ferroptotic cell death was monitored with Annexin V/propidium iodide (PI) staining (BioLegend). For phagocytosis assays, the co-cultured ferroptotic NCI-H295R cells and macrophages were trypsinized and washed with PBS. Anti-CD64 antibody was added at a dilution of 1:100 in PBS and cells were incubated for 30 min at RT. Cells were washed with PBS and incubated with secondary antibody for 30 min at RT protected from light. Cells were washed and resuspended in 200 µl FACS buffer. The percentage of CSFE + CD64 + cells was determined using FACS-Fortessa at the Ludwig Maximilian University Hospital Munich Flow Cytometry Core Facility (RRID:SCR_026395). CSFE + cells were detected in the FITC channel, CD64 + cells in the APC channel and cell populations quantified with FlowJo (RRID:SCR_008520). Western Blot Cells were lysed in RIPA buffer (Sigma) supplemented with 1% Protease Inhibitor (Sigma), 1% Phosphatase B Inhibitor and 2% Phosphatase C Inhibitor (Santa Cruz). Protein concentrations were quantified using the Pierce™ BCA Protein Assay Kit. Absorbance values were measured at the BMG Labtech FLUOstar Omega (RRID:SCR_025024) microplate reader at 562 nm. Protein was loaded onto a 4–20% Mini-PROTEAN® TGX™ Precast Protein Gel (BioRad) and separated by SDS-PAGE. Proteins were transferred by semi-dry blot onto a nitrocellulose membrane (Cytiva) that was subsequently blocked in 5% skimmed milk in TBS-Tween at RT for 1 h. Primary antibodies (suppl. table 1) were diluted in 5% skimmed milk in TBS-Tween and incubated over night at 4°C. Membranes were washed and incubated at RT for 1 h with horseradish-peroxidase (HRP)-labeled secondary antibodies. Immunoblots were developed and visualized using Clarity™ Western ECL Substrate at the Bio-Rad Chemidoc XRS Gel Imaging System (RRID:SCR_019690) with Image Lab Software (RRID:SCR_014210). GAPDH expression was used as loading control. qPCR Total RNA was extracted from cells using Maxwell® RSC simplyRNA tissue Kit (Promega) and reversetranscribed into cDNA using GoScript™ Reverse Transcription Mix (Promega). qPCR was performed using TaqMan Real-Time-PCR Assays (PTGS2: Hs00153133m1; ACTB: Hs99999903_m1) and TaqMan Gene Expression Master Mix (both ThermoFisher) in a final volume of 25 µl. qPCR was carried out at the Agilent Stratagene Mx3000P qPCR cycler (RRID:SCR_026398). qPCR conditions consisted of an initial denaturation step of 3 min at 95˚C, followed by 39 cycles of 30 sec denaturation at 95˚C, 30 sec annealing at 60˚C and 30 sec elongation at 72°C. mRNA was quantified using the 2ΔΔCq method and βactin was used as internal control. Nanostring nCounter gene expression analysis Gene expression analysis was performed on RNA isolated either from in vitro differentiated macrophages or from FFPE ACC tissue sections. RNA was extracted using the AllPrep DNA/RNA FFPE Kit (Qiagen). RNA quantity and quality were assessed with a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific) and samples meeting predefined quality criteria were further analyzed for gene expression quantification with the NanoString nCounter Analysis System (RRID:SCR_021712). A panel (NanoString Technologies (RRID:SCR_023912)) consisting of 473 immune related genes plus eleven housekeeping genes was used according to the manufacturer´s instructions. Analysis was performed by nSolver Analysis Software (RRID:SCR_003420). Samples passing imaging quality control thresholds (fields of view read > 75%, binding density of 0.05–2.25, positive control linearity > 0.95, and positive control detection limit > 2) were normalized using technical controls and housekeeping genes, based on positive control normalization with the geometric mean as the normalization factor. For comparisons, a one-tailed Student’s t-test p-value and a false discovery rate (FDR) was calculated by the Benjamini–Yekutieli method. Genes were ranked by fold change (FC) and FDR, with thresholds set at FC ≥ 2 and FDR < 0.1. ELISA Prostaglandin E 2 (PGE 2 ), IL-10 and CXCL9 concentrations in cell supernatants were measured by ELISA (PGE 2 : Cayman Chemical; 514010; Gas6; Invitrogen; BMS2291; IL-10: R&D Systems; D1000B; CXCL9/MIG: Invitrogen, Item No 900-K87K). If not stated otherwise, concentrations were normalized to total protein levels isolated as stated above. LC-MS/MS Steroid profiles of ACC tissues were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS) as previously described 21 . Oxylipin measurements in supernatants of ACC cells and ACC tissues were performed as previously described 22 and outlined in the Supplementary Methods. Survival Analysis To assess the prognostic significance of gene expression levels, we performed survival analysis using clinical and RNA seq data from The Cancer Genome Atlas (TCGA) project and a local ACC biobank (NCT MASTER). Patients were stratified into high- and low-expression groups based on the median expression level of each gene of interest. Survival curves were generated using Kaplan-Meier estimates, exported from the either the cBioPortal platform (TCGA) or generated using R Project for Statistical Computing (RRID:SCR_001905) version R/4.0.0 (NCT MASTER). Differences in survival between groups were assessed using the log-rank test. For the use of data from a public database, an approval exemption has been obtained from the LMUs´ ethics committee. Gene Set Enrichment Analysis For pathway and functional enrichment analysis, we employed Gene Set Enrichment Analysis (GSEA) using the WebGestalt: WEB-based GEne SeT AnaLysis Toolkit (RRID:SCR_006786). Differentially expressed genes identified from our RNA dataset were inputted into WebGestalt to assess their association with known biological pathways, gene ontology (GO) categories, and functional gene sets. Statistical analysis If not stated otherwise, three independent replicates were performed per experiment. For experiments using human MΦ each biological replicate equals a different biological blood donor. Statistical analyses were performed using GraphPad Prism (RRID:SCR_002798) version 9.0. If not stated otherwise, biological replicates are shown in Mean +/- SD and the statistical analyses were performed using a two-tailed Student’s t test for paired comparisons or one way analysis of variance (ANOVA) for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns = not significant. Results Macrophages in ACC exhibit a CD68 + CD163 + phenotype irrespective of tumoral glucocorticoid excess Using chromogenic immunohistochemistry (IHC), we investigated the proportion of TAMs in ACC tissue samples from a total of 25 patients. We observed a heterogenous but overall abundant presence of CD68+ (Fig. 1 A) and CD163 + cells (Fig. 1 B). Intra-tumoral TAM counts varied from 3.1–40.1% (median = 15.0%) for CD68 + cells and 3.0–37.6% (median = 16.0%) for CD163 + cells (Fig. 1 C). Moreover, immunofluorescence microscopy revealed the near-total co-localization of CD68 and CD163 in ACC tissue samples (Fig. 1 D). Tissue cortisol levels measured using LC-MS/MS did not correlate with the total TAM population assessed by IHC staining (Spearman: p = 0.95; Fig. 1 E). Likewise, we did not observe a significant correlation between intra-tumoral cortisol concentrations and the total tumoral expression of the 'M2-like' macrophage marker CD163 and CD206, as determined by immunoblotting of respective tumor lysates (Spearman: p = 0.58; Fig. 1 F, G). In a separate cohort of 56 ACC patients stratified by clinical and biochemical hormone status, tumoral RNA expression of CD68 , CD163 , and CD206 showed no significant difference between patients with or without clinical cortisol excess (Fig. 1 H-J). Moreover, when tumors were categorized as inactive, androgen-overproducing, GC-overproducing, or combined androgen and GC excess, no differential expression of these markers was observed across the four groups ( Fig. S2A-C ). Altogether these findings suggest that MΦ are a prevalent immune cell population in ACC heavily skewed towards a CD68 + CD163 + phenotype independently of tumoral GC overproduction. In vitro , both cortisol-secreting and non-cortisol-secreting ACC cells induce a distinct CD163 + macrophage phenotype To directly study the impact of ACC tumor cells on MΦ polarization in vitro , we next isolated monocytes from healthy donor blood samples and exposed them to conditioned medium (CM; 33% v/v) of two different ACC cell lines as outlined in the experimental setup shown in Fig. 2 A. CM from the cortisol-secreting ACC cell line NCI-H295R and the non-cortisol-secreting ACC cell line JIL-2266 (Fig. 2 B) was collected after 48 h of culture and added to human monocytes from day one onwards. Given that the MΦ differentiation factors GM-CSF and M-CSF are known early drivers of M1 and M2 MΦ differentiation, respectively 16 , exposure of MΦ to CM was investigated in the absence of exogenous growth factors. Additionally, CM of ACC cells was added to MΦ exposed to GM-CSF + IFNγ ('M1-like' MΦ polarization; mimicking T cell activation, e.g. induced by immune checkpoint inhibition) to examine potential inhibitory effects in the context of ICI therapy. Irrespective of whether it was derived from cortisol-secreting or non-cortisol-secreting ACC cells, CM treatment induced an upregulation of CD163 expression in MΦ (Fig. 2 C, D; Fig. S3A ). Remarkably, IL-10 secretion was pronouncedly higher in MΦ polarized with non-secreting JIL-2266 CM (Fig. 2 E), suggesting a difference in the MΦ phenotype induced by the two cell lines. Even though CSF-1 mRNA (encoding M-CSF) levels and M-CSF secretion were higher in JIL-2266 cells ( Fig. S3B, C ), they were negligibly low. To test the hypothesis that MΦ polarization driven by steroidogenic NCI-H295R cells was predominantly caused by tumor cell secreted cortisol, we next cultured this ACC cell line in the presence of the steroidogenesis inhibitor metyrapone (cortisol concentrations in supernatants shown in Fig. S3D ), before CM was collected and applied to isolated monocytes. Accordingly, blockade of cortisol synthesis in tumor cells decreased the 'M2-like' polarization of MΦ, as shown by morphological changes ( Fig. S3E ) and CD163 expression (Fig. 2 F, G). In THP-1 MΦ, knockout of the glucocorticoid receptor (GR; gene: NR3C1 ) efficiently attenuated CD163 expression upon treatment with dexamethasone or NCI-H295R CM, further supporting a GC-GR mediated mechanism (Fig. 2 H, I). Taken together, these experiments show that GC secretion by ACC tumor cells induces a CD163 + MΦ phenotype that is functionally distinct from CD163 + MΦ polarized by non-cortisol-secreting ACC cell lines. Glucocorticoids induce MΦ polarization towards a C1Q + CD163 + phenotype To more precisely define the phenotype of MΦ induced by GCs in the context of ICI therapy, we next analyzed the RNA profile of in vitro differentiated and polarized MΦ via a predesigned Nanostring nCounter panel (Fig. 3 A). MΦ were treated with a combination of GM-CSF (day 1), IFNγ (day 7) and the synthetic glucocorticoid dexamethasone (day 1). Co-exposure to dexamethasone and GM-CSF + IFNγ resulted in a strong upregulation of the 'M2-like' marker CD163 and MARCO compared to GM-CSF + IFNγ monotreatment ( Fig. 3 A-C). Moreover, genes encoding subunits of the complement component C1Q, in particular C1QA and C1QB were among the most discriminatively upregulated transcripts in comparison to 'M1-like' MΦ (Fig. 3 A, D, E). Remarkably, we found significantly elevated expression of C1QA and C1QB also in MΦ cultured in the presence of steroidogenic NCI-H295R CM, but not JIL-2266 CM (Fig. S4A, B) . Additionally, upregulation of C1QA and C1QB in MΦ exposed to CM of NCI-H295R was abrogated when ACC cells were cultured in the presence of the steroidogenesis inhibitor metyrapone (Fig. 3 F-H, Fig. S4C ). Co-immunofluorescence analysis revealed a positive cytosolic staining of C1QA in cells that were also positive for CD163, supporting the presence of a C1Q + CD163 + MΦ phenotype in adrenocortical tissue (Fig. 3 I). In ACC patients, we found C1QA gene expression most upregulated in tumors with high CD163 expression levels ( Fig. S4D ). A pan-cancer analysis of TCGA data demonstrated that the C1QA/CD68 gene expression ratio was notably elevated in ACC samples compared to other tumor types, further supporting the hypothesis of a C1Q-dominated MΦ phenotype in GC-producing tumors, such as ACC (Fig. 3 J). In summary, our findings indicate that local GC synthesis induces a CD163 + MΦ type characterized by high expression of the complement component C1Q. GC-induced MΦ are highly efficient in C1q mediated phagocytosis To evaluate the functional relevance of the GC-induced C1Q + MΦ phenotype, we next conducted a gene set enrichment analysis (GSEA) of RNA data obtained from the Nanostring nCounter analysis. Interestingly, GSEA showed a statistically significant enrichment (p < 2.2e-16; FDR: 0.01) of gene sets associated with phagocytosis in MΦ polarized with CM of the steroidogenic ACC cell line NCI-H295R (Fig. 4 A, B). Following this finding, we next co-cultured GC-induced MΦ with dying ACC cells and assessed their phagocytic capacity (experimental setup shown in Fig. S5A ). As we have previously shown that ferroptosis is a prominent mode of cell death in adrenocortical cells 23 , tumor cell death was initiated by treatment with the ferroptosis inducer RSL3, which led to a strong increase in phosphatidylserine (PS) exposure, as detected by Annexin V positivity ( Fig. S5B, C). Consistent with the GSEA results, MΦ treated with NCI-H295R CM exhibited an enhanced phagocytic index compared to control treatment when co-cultured with ferroptotic ACC cells (Fig. 4 C, D). Similarly, MΦ polarized with the synthetic GC dexamethasone demonstrated a significantly increased phagocytic index relative to 'M1-like' MΦ (Fig. 4 E). Furthermore, these MΦ displayed a notable, albeit non-significant, upregulation of genes encoding MHC class II molecules, pointing towards an improved capacity for antigen presentation. ( Fig. S5D, E) . Given that secreted C1q has been shown to target dying cells for phagocytosis by recognizing phosphatidylserine (PS) on their surface 24 , 25 we next investigated whether adding serum-derived C1q to 'M1-like' MΦ enhances their phagocytic capacity. We observed that exposure of the ACC cell/MΦ co-culture to C1q increased the percentage of CSFE + MΦ in a dose-dependent manner, supporting the hypothesis of C1q-mediated phagocytosis by GC-polarized MΦ (Fig. 4 F; Fig. S5F ). We also observed that GCs induced the expression of MerTK on MΦ (Fig. 4 G), an alternative mediator of phagocytosis via the MerTK/Gas6/PS axis. Intriguingly, phagocytosis by C1Q + MΦ was neither impaired by treatment with the selective MerTK antagonist UNC1062 nor by administration of a neutralizing antibody against its ligand Gas6 (Fig. 4 H). In conclusion, these experiments demonstrate that GCs induce a highly phagocytic MΦ phenotype, primarily mediated by increased expression and secretion of the complement component C1q, rather than through the MerTK pathway. Prostaglandin E 2 (PGE 2 ) is an alternative inducer of CD163 + MΦ polarization derived from non-cortisol-secreting ACC cells To decipher the component in the CM of the non-cs ACC cell line (JIL-2266) that was likewise capable of inducing CD163 + MΦ polarization, we next performed a targeted LC/MS-based analysis of oxylipids of both CM. In comparison to the supernatant of steroidogenic ACC cells, we detected exceptionally elevated levels of the eicosanoid prostaglandin E 2 (PGE 2 ) in the CM of the non-cortisol-secreting JIL-2266 cells (Fig. 5 A). This observation was confirmed by PGE 2 ELISA (Fig. 5 B). Treatment of MΦ with PGE 2 in the absence of exogenous growth factors led to a concentration-independent upregulation of the MΦ marker CD163 (Fig. 5 C). Accordingly, exposure to PGE 2 inhibited IFNγ-induced 'M1-like' polarization, while addition of the COX-2 inhibitor celecoxib prior CM collection blocked PGE 2 production ( Fig. S6A ) and restored 'M1-like' polarization of macrophages (Fig. 5 D-F; Fig. S6B ). In contrast to dexamethasone, however, PGE 2 or JIL-2266 CM treated MΦ showed lesser expression of CD163 and C1Q complement components, while the expression of CXCL1,2 and 8 was most prominently increased in comparison to 'M1-like' MΦ, albeit without reaching statistical significance ( Fig. S6C, D ). Previous reports suggest GC secretion and conversion of arachidonic acid into PGE 2 via COX-2 (gene: PTGS2 ) to be interconnected 26 – 28 . In line, we found high expression of COX-2 in the zona glomerulosa and zona reticularis of the normal adrenal gland but not in the cortisol producing zona fasciculata ( Fig. S6E ). In vitro , exposure of non-steroidogenic ACC cells to increasing concentrations of dexamethasone decreased PTGS2 mRNA and COX-2 protein expression and led to a substantial reduction in PGE 2 secretion (Fig. 5 G-I). Conversely, treatment of ACC cells with the highly specific GR antagonist relacorilant induced an increase in intracellular PTGS2 and COX-2 levels and a consequent increase in secreted PGE 2 (Fig. 5 J-L). These results indicate that MΦ polarization by PGE 2 may be predominantly associated to hormonally inactive tumors but could gain significance upon therapeutic inhibition of steroidogenesis. Supporting this idea, simultaneous treatment of steroidogenic ACC cells with metyrapone and celecoxib most effectively reduced 'M2-like' polarization following steroidogenic CM exposure (Fig. 5 M). Taken together, these results demonstrate that in the absence of GCs or upon GR blockade, PGE 2 can induce the polarization of CD163 + MΦ. Blockade of both, tumor cell steroidogenesis and PGE 2 synthesis, might most efficiently reduce 'M2-like' polarization of tumoral macrophages. IFNγ treatment of C1Q + MΦ strongly induces CXCL9 expression which positively correlates with T cell infiltration and better survival in ACC Upon IFNγ stimulation, we found the T cell chemoattractants CXCL9 and CXCl 10 among the most up-regulated genes in MΦ pre-exposed to dexamethasone in comparison to 'M1-like' MΦ (Fig. 3 A, Fig. 6 A). This finding was confirmed by CXCL9 ELISA (Fig. 6 B). Likewise, exposure to hydrocortisone (HC; 500 ng/ml) strongly increased CXCL9 secretion of MΦ upon treatment with IFNγ (Fig. 6 C). Yet, full establishment of the GC-induced C1Q + MΦ phenotype appeared to be critical in this context, as application of dexamethasone at a later timepoint reduced the expression of C1QA and the increase in CXCL9 production ( Fig. S7A, B ). Accordingly, MΦ that were exposed to IFNγ and dexamethasone at the same timepoint did not produce higher amounts of CXCL9 in comparison to 'M1-like' MΦ ( Fig. S7C ). As CXCL9 and CXCL10 are highly potent T cell attractants by binding to its receptor CXCR3, mainly expressed on T cells 29 , we next analyzed the connection between this MΦ phenotype and T cell infiltration in ACC. In two independent datasets, the complement component C1QA strongly correlated with CD3D , CD8A and most significantly with CD4 gene expression (Fig. 6 D, E; Fig. S7D-G ). A pan-cancer analysis of TCGA data revealed this correlation to be true across several cancer entities (Fig. 6 F). Consistently, CD68 expression was positively correlated with better OS within both datasets ( Fig. S7H, I ) and better PFS within the TCGA dataset (Fig. 6 G). C1QA gene expression was correlated with better OS and PFS in the TCGA dataset (Fig. 6 H, Fig. S7J ), however this correlation was not observed in the MASTER dataset ( Fig. S7K ). Finally, in ACC patients undergoing immune checkpoint inhibition therapy (pembrolizumab or nivolumab), plasma CXCL9 concentrations were significantly elevated after the initiation of immunotherapy compared to baseline, with a more pronounced increase in CXCL9 levels observed in plasma of ACC patients with high intra-tumoral macrophage numbers (> 10%) than in those with low TAM counts (< 10%) (Fig. 6 I). Moreover, in this small cohort, a higher percentage of CD163 + cells within respective tumor tissue samples was associated with response to ICI therapy, yet this trend was non-significant. (Fig. 6 J). Together, these data suggest that the presence of C1Q+/CD163 + macrophages in GC-producing tissues, such as ACC, may increase immunotherapy outcomes through enhancing CXCL9-dependent T cell chemoattraction in response to immunotherapy-induced IFNγ release. Discussion With the success of immunotherapies in many cancer entities, mechanisms that confer resistance and prevent treatment success have come into focus in molecular oncology. The potency of GCs to suppress T cell activity has been proposed to profoundly impact immunotherapy outcomes but the actual consequences of GC action in the tumoral immune microenvironment are poorly understood 30 . To better characterize these local effects of glucocorticoids we here exploited the cell-autonomous secretion of GCs present in the majority of ACC, an immunologically cold tumor responding poorly to immunotherapy 8 . We show that tumor associated macrophages dominate the tumor immune microenvironment in ACC irrespective of the amount of tumoral steroid hormone secretion. We find that GCs lead to a specific 'M2-like' MΦ phenotype characterized by expression of the complement component C1Q and a profoundly increased phagocytic capacity. In the absence of GCs or upon GR blockade, the eicosanoid PGE 2 might promote polarization of 'M2-like' MΦ that however lack C1Q expression. Lastly, C1Q + MΦ efficiently produce the chemokine CXCL9 upon stimulation with IFNγ and are strongly associated with T cell infiltration in ACC and – more importantly - across cancer entities. Tumoral T cell infiltration is generally considered the most crucial factor determining prognosis in cancer patients and the efficacy of immunotherapy 12 , 31 – 33 . In ACC however, T cell infiltration has been shown to be scarce with only a median of 7.7 cells/HPF for CD3 + T cells 34 . Intriguingly, in a recent publication addressing the efficacy of the PD-1 inhibitor camrelizumab in combination with the tyrosine kinase inhibitor apatinib, tumoral infiltration with CD8 T cells prior therapy initiation was not correlated with the hormonal status of ACC patients 9 . TAMs have been shown to play a critical role in determining the tumoral immune control 35 . In most cancers, MΦ infiltration is linked to worse clinical outcome, supporting the notion of a tumor-promoting role of TAMs 36 . Surprisingly, in ACC we observed a higher abundance of MΦ, as specified by high expression of CD68 , to significantly correlate with improved OS and PFS. As TAMs predominantly originate from circulating monocytes that undergo differentiation into MΦ within the TME, they dynamically respond to cues from their microenvironment 35 . As a consequence, the particular polarization of TAMs ultimately determines tumoral T cell infiltration 12 , 37 , 38 . By utilizing both, clinical data and tissue mass spectrometry of fresh frozen ACC tissue specimens, we did not find any significant correlation between tumoral cortisol levels and MΦ numbers. Furthermore, TAMs exhibited a CD68+/CD163+ 'M2-like' phenotype in both, GC secreting and hormonally inactive tumors. In contrast to our results, a deconvolution analysis of TCGA data by Baechle et al. observed a small, yet significantly increased presence of M1 MΦ and a non-significant trend towards more M2 MΦ in non-cortisol-secreting ACCs 39 . This small discrepancy with our study may be due to the fact that the clinical annotation of GC secretion in TCGA was used for classification by Baechle et al. while we also had the opportunity to measure intra-tumoral GCs and to quantify MΦ in corresponding tissues directly. Furthermore, the lack of suitable 'M1-like' MΦ markers for IHC analysis (e.g. CD80 or CD86 which are also strongly expressed on 'M2-like' MΦ and other immune cells) did not allow us to directly address 'M1-like' MΦ in our ACC tumor cohorts. Detailed RNA expression analysis from our in vitro polarized MΦ revealed a significant upregulation of genes encoding the complement component C1Q in MΦ treated with dexamethasone or CM of steroidogenic ACC cells. Fluorescence microscopy confirmed the co-expression of C1QA and CD163 in adrenal macrophages, suggesting that C1Q + MΦ are induced by active steroidogenesis of adrenocortical cells, rather than by other tumor cell-derived factors. However, we did not observe higher C1QA or C1QB RNA expression in tumors with clinical and biochemical GC excess, indicating that MΦ may adopt this phenotype independently of the amount of tumoral GC production. Notably, C1Q + MΦ have previously been identified in other solid cancers, including melanoma, breast cancer and hepatocellular carcinoma 40 – 42 . Interestingly, a recent study also detected GC production in several non-adrenal tumor types. In these entities, GCs were found to be produced by recycling of inactive metabolites via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by HSD11B1) 3 . By utilizing TCGA data, we found a significantly increased C1QA/CD68 gene expression ratio in tumor samples with high HSD11B1 expression across these cancer types ( Fig. S8A ), further supporting the link between local GC production and C1Q + macrophages in diverse tumor microenvironments. Current knowledge on the impact of C1Q + MΦ on T cell function and patient prognosis is ambiguous, highlighting their complex and context-specific roles. A recent multi-omics study identified a PD-L1+/MHCII+/C1Q + macrophage phenotype as immunostimulatory to T cells and associated with favorable clinical outcomes in breast cancer patients 43 . Additionally, a FOLR2 + TAM population (identified by high levels of APOE , APOC1 , C1QA and C1QC ) was recently identified to be associated with CD8 + T cell infiltration in human breast cancer 44 . Conversely, the presence of tumoral (secreted) C1q has also been linked to the promotion of tumor growth in two distinct mouse models 41 , 45 . In contrast, we did not observe C1q treatment of ACC cells to affect their proliferation over 48 hours ( Fig. S8DB ). Moreover, the proliferation of CD8 + T cell in the presence or absence of CD3/CD28 activation remained unaltered when exposed to either C1Q + MΦ or 'M1-like' MΦ supernatant ( Fig. S8C ). Furthermore, we demonstrate that C1Q + macrophages are capable of producing high amounts of the T cell chemoattractant CXCL9 in response to IFNγ, a hallmark of ICI therapy 46 . As GC excess in ACC patients was not associated with altered PFS or OS following ICI therapy 47 , the immunosuppressive effects of GCs on T cells may not be the primary obstacle for ICI efficacy in ACC, pointing to a more complex interplay of factors influencing treatment outcomes. In addition, a recently published study on the efficacy of the PD-1 inhibitor camrelizumab in combination with apatinib in pre-treated ACC patients revealed a strong connection between the response to ICI therapy and the presence of baseline CXCR3 + CD8 + T cells 9 , underlining the relevance of this macrophage-produced cytokine on the CXCL9/CXCR3-dependent T cell chemoattraction in ACC. Our data suggest that C1q-mediated phagocytosis plays a central role in the tumoricidal activity of MΦ in ACC. In line, Wilmouth et al. recently demonstrated a highly phagocytic MΦ phenotype to be present in a spontaneous ACC model in male mice 48 . In this study, the androgen dependent polarization of tumoricidal phagocytic MΦ completely reversed the development of adrenal tumors. Bulk RNA sequencing data from their mouse tumors identified a strong up-regulation of C1QA/B/C in ACC bearing mice, which is in strong agreement with the results observed in our study. However, in the context of cancer, phagocytosis of tumor cells may be a double-edged sword. On the one hand, the clearance of tumor cells and presentation of tumoral antigens can increase the adaptive antitumoral immune response. On the other hand, clearance of cancer cells may prevent the release of damage-associated molecular patterns, thus contributing to immune evasion and tumor progression. Undoubtedly, unraveling the consequences of tumor cell clearance by MΦ in this context requires further investigation. Finally, we here identified PGE 2 as a possible mediator of MΦ polarization in ACC. We hypothesize that the release of PGE 2 is confined to hormonally inactive ACCs while in GC-secreting tumors PGE 2 synthesis is transcriptionally suppressed in a GR-dependent manner. Our findings add to recent reports that describe PGE 2 as a negative regulator of immune responses which contributes to tumoral immune evasion in several tumor models 26 , 49 , 50 . However, even though in vitro PGE 2 appeared as a promising alternative mechanism of 'M2-like' polarization induced by non-cortisol-secreting ACC cells, in ACC tumors we found PGE 2 levels to be generally low compared to other solid tumor entities ( Fig. S8D ), underscoring that even marginal concentrations of GCs may be sufficient to suppress COX-2 expression and subsequent PGE 2 production, but induce the polarization of a C1Q + CD163 + macrophage phenotype. Undeniably, our study has certain limitations. One of the main restrictions is the small number of clinically annotated patient samples. This is primarily due to the rarity of this endocrine malignancy. Additionally, the well-established negative impact of GC excess on patient outcomes 34 , 51 may seem contradictory to the favorable impact of GC-induced C1Q + MΦ observed in this study. However, our data also underlines that C1Q macrophage polarization occurs under physiological cortisol concentrations within the adrenal gland, suggesting that the detrimental impact of GC excess on the immune cell compartment in ACC patients may outweigh the beneficial impact of C1Q macrophages on tumor cell clearance. Furthermore, under conditions of ICI therapy, there is currently no association between response to therapy and tumoral GC overproduction. Besides, in the NCT MASTER dataset, C1QA expression was not correlated with improved OS, likely reflecting the dual effects of GCs: enhanced C1QA expression alongside their adverse systemic impact. Finally, the primary limitation of this work is the lack of in vivo studies. Those would be critical for confirming our results within a physiological context and assessing their potential translational impact. Yet, immunocompetent animal models of both cortisol producing and hormonally inactive ACC are scarce. Despite these open questions, the results obtained in this study contribute to a better understanding and characterization of the GC-induced MΦ phenotype in ACC and other cancers. Our results highlight two important points. First, therapies that inhibit MΦ (such as CSF1R taregting therapies) have to be used very cautiously in the future especially when combined with T cell-based treatments (e.g. CAR-T or ICI). Depletion of MΦ may eradicate key mediators of tumor cell clearance e.g. by efficient phagocytosis and crucial facilitators of T cell infiltration, a major determinant of immunotherapy efficacy. Second, the presence of MΦ within ACC tissues combined with a high percentage of CXCR3 + T cells in the blood, may serve as a potential indicator for predicting the response to ICI therapy. Abbreviations ACC: adrenocortical carcinoma CHX: cycloheximide COX-2: cyclooxygenase 2 CXCL9: CXC motif chemokine ligand 9 FDR: false discovery rate GC(s): glucocorticoid(s) GM-CSF: Granulocyte macrophage-colony stimulating factor GR: glucocorticoid receptor HC: hydrocortisone ICI: immune checkpoint inhibition IF: immunofluorescence IHC: immunohistochemistry Lip-1: Liproxstatin 1 MΦ: macrophage M-CSF: macrophage-colony stimulating factor OS: overall survival PBMC: peripheral blood mononuclear cell PBS: phosphate buffered saline PFA: paraformaldehyde PFS: progression-free survival PGE 2 : prostaglandin E 2 PMA: phorbol-12-myristate-13-acetate PS: phosphatidylserine RSL3: (1S, 3R) Ras-selective lethal small molecule 3 TAM: tumor associated macrophage TIME: tumor immune microenvironment TME: tumor microenvironment WB: western blot Declarations Acknowledgments We thank Prof. Jörg Wischhusen, University Hospital Würzburg, for generating and sharing the Cas9 expressing THP-1 cells. Funding Work in the author’s laboratories was supported by the German Research Organization (Deutsche Forschungsgemeinschaft, DFG) within the collaborative research centers (SFB Transregio) no. TRR205 (project number 314061271) to LSL, NB, MP, MR, JPFA, MF, IW and MKr. Author contributions Conceptualization: AT, IW, MKr Methodology: AT, TM, AG, FR, MH, MKi, NB, MP, NP Investigation: AT, TM, FR, MKi, NB, MP, NP, SF Visualization: AT, TM, MKi, NP, MH Supervision: IW, MKr, Writing—original draft: AT, MKr Writing—review & editing: AT, TM, PS, SA, MKi, AG, FR, LSL, NB, MP, NP, MH, ST, SF, HG, DH, MF, MR, JPFA, IW, MKr All authors have read and approved the final version of the manuscript Competing interests The authors declare that they have no competing interests. Data and materials availability All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The complete RNA datasets generated during the current study can be provided on reasonable request. Requests should be submitted to the corresponding author. The use of the THP-1 monocytic line expressing Cas9 is covered by a material transfer agreement (MTA) between Prof. Dr. Jörg Wischhusen and Dr. Isabel Weigand. References Acharya N, Madi A, Zhang H, Klapholz M, Escobar G, Dulberg S et al. Endogenous Glucocorticoid Signaling Regulates CD8 + T Cell Differentiation and Development of Dysfunction in the Tumor Microenvironment. Immunity 2020; 53: 658–671.e6. Sidler D, Renzulli P, Schnoz C, Berger B, Schneider-Jakob S, Flück C et al. Colon cancer cells produce immunoregulatory glucocorticoids. Oncogene 2011; 30: 2411–2419. Taves MD, Otsuka S, Taylor MA, Donahue KM, Meyer TJ, Cam MC et al. Tumors produce glucocorticoids by metabolite recycling, not synthesis, and activate Tregs to promote growth. J Clin Invest 2023; 133: e164599. Cohen N, Mouly E, Hamdi H, Maillot M-C, Pallardy M, Godot V et al. GILZ expression in human dendritic cells redirects their maturation and prevents antigen-specific T lymphocyte response. Blood 2006; 107: 2037–2044. Hamdi H, Godot V, Maillot M-C, Prejean MV, Cohen N, Krzysiek R et al. Induction of antigen-specific regulatory T lymphocytes by human dendritic cells expressing the glucocorticoid-induced leucine zipper. Blood 2007; 110: 211–219. Kerkhofs TMA, Verhoeven RHA, Van der Zwan JM, Dieleman J, Kerstens MN, Links TP et al. Adrenocortical carcinoma: A population-based study on incidence and survival in the Netherlands since 1993. Eur J Cancer 2013; 49: 2579–2586. Libé R. Adrenocortical carcinoma (ACC): diagnosis, prognosis, and treatment. Front Cell Dev Biol 2015; 3: 45. Ababneh O, Ghazou A, Alawajneh M, Alhaj Mohammad S, Bani-Hani A, Alrabadi N et al. The Efficacy and Safety of Immune Checkpoint Inhibitors in Adrenocortical Carcinoma: A Systematic Review and Meta-Analysis. Cancers 2024; 16: 900. Zhu Y, Cai L, Peng X. Camrelizumab plus apatinib for previously treated advanced adrenocortical carcinoma: A single-arm, open-label, phase 2 trial. J Clin Oncol 2024. doi: 10.1200/JCO.2024.42.16_suppl.4511 . Mills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol Baltim Md 1950 2000; 164: 6166–6173. Liu J, Geng X, Hou J, Wu G. New insights into M1/M2 macrophages: key modulators in cancer progression. Cancer Cell Int 2021; 21: 389. van Elsas MJ, Middelburg J, Labrie C, Roelands J, Schaap G, Sluijter M et al. Immunotherapy-activated T cells recruit and skew late-stage activated M1-like macrophages that are critical for therapeutic efficacy. Cancer Cell 2024; 42: 1032–1050.e10. Sreejit G, Fleetwood AJ, Murphy AJ, Nagareddy PR. Origins and diversity of macrophages in health and disease. Clin Transl Immunol 2020; 9: e1222. Anders CB, Lawton TMW, Smith HL, Garret J, Doucette MM, Ammons MCB. Use of integrated metabolomics, transcriptomics, and signal protein profile to characterize the effector function and associated metabotype of polarized macrophage phenotypes. J Leukoc Biol 2022; 111: 667–693. Mantovani A, Allavena P, Marchesi F, Garlanda C. Macrophages as tools and targets in cancer therapy. Nat Rev Drug Discov 2022; 21: 799–820. Zizzo G, Hilliard BA, Monestier M, Cohen PL. Efficient clearance of early apoptotic cells by human macrophages requires “M2c” polarization and MerTK induction. J Immunol Baltim Md 1950 2012; 189: 3508–3520. Auger J-P, Zimmermann M, Faas M, Stifel U, Chambers D, Krishnacoumar B et al. Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids. Nature 2024; 629: 184–192. Horak P, Heining C, Kreutzfeldt S, Hutter B, Mock A, Hüllein J et al. Comprehensive Genomic and Transcriptomic Analysis for Guiding Therapeutic Decisions in Patients with Rare Cancers. Cancer Discov 2021; 11: 2780–2795. Weigand I, Altieri B, Lacombe AMF, Basile V, Kircher S, Landwehr L-S et al. Expression of SOAT1 in Adrenocortical Carcinoma and Response to Mitotane Monotherapy: An ENSAT Multicenter Study. J Clin Endocrinol Metab 2020; 105: 2642–2653. Landwehr L-S, Schreiner J, Appenzeller S, Kircher S, Herterich S, Sbiera S et al. A novel patient-derived cell line of adrenocortical carcinoma shows a pathogenic role of germline MUTYH mutation and high tumour mutational burden. Eur J Endocrinol 2021; 184: 823–835. Bechmann N, Watts D, Steenblock C, Wallace PW, Schürmann A, Bornstein SR et al. Adrenal Hormone Interactions and Metabolism: A Single Sample Multi-Omics Approach. Horm Metab Res Horm Stoffwechselforschung Horm Metab 2021; 53: 326–334. Meng Y, Bilyal A, Chen L, Schnitzler MM y, Kocabiyik J, Gudermann T et al. Endothelial epoxyeicosatrienoic acid release is intact in aldosterone excess. Atherosclerosis 2024; 398. doi: 10.1016/j.atherosclerosis.2024.118591 . Weigand I, Schreiner J, Röhrig F, Sun N, Landwehr L-S, Urlaub H et al. Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction. Cell Death Dis 2020; 11: 1–12. Païdassi H, Tacnet-Delorme P, Garlatti V, Darnault C, Ghebrehiwet B, Gaboriaud C et al. C1q Binds Phosphatidylserine and Likely Acts as a Multiligand-Bridging Molecule in Apoptotic Cell Recognition. J Immunol Baltim Md 1950 2008; 180: 2329. Liang YY, Arnold T, Michlmayr A, Rainprecht D, Perticevic B, Spittler A et al. Serum-dependent processing of late apoptotic cells for enhanced efferocytosis. Cell Death Dis 2014; 5: e1264–e1264. Park A, Lee Y, Kim MS, Kang YJ, Park Y-J, Jung H et al. Prostaglandin E2 Secreted by Thyroid Cancer Cells Contributes to Immune Escape Through the Suppression of Natural Killer (NK) Cell Cytotoxicity and NK Cell Differentiation. Front Immunol 2018; 9: 1859. Lim W, Park C, Shim MK, Lee YH, Lee YM, Lee Y. Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoidinduced leucine zipper. Br J Pharmacol 2014; 171: 735–745. Carolina E, Kato T, Khanh VC, Moriguchi K, Yamashita T, Takeuchi K et al. Glucocorticoid Impaired the Wound Healing Ability of Endothelial Progenitor Cells by Reducing the Expression of CXCR4 in the PGE2 Pathway. Front Med 2018; 5. doi: 10.3389/fmed.2018.00276 . Tokunaga R, Zhang W, Naseem M, Puccini A, Berger MD, Soni S et al. CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation - A target for novel cancer therapy. Cancer Treat Rev 2018; 63: 40–47. Khadka S, Druffner SR, Duncan BC, Busada JT. Glucocorticoid regulation of cancer development and progression. Front Endocrinol 2023; 14: 1161768. Galon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pagès C et al. Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006; 313: 1960–1964. Fridman WH, Pagès F, Sautès-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 2012; 12: 298–306. Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G et al. Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 2003; 348: 203–213. Landwehr L-S, Altieri B, Schreiner J, Sbiera I, Weigand I, Kroiss M et al. Interplay between glucocorticoids and tumor-infiltrating lymphocytes on the prognosis of adrenocortical carcinoma. J Immunother Cancer 2020; 8: e000469. Wu K, Lin K, Li X, Yuan X, Xu P, Ni P et al. Redefining Tumor-Associated Macrophage Subpopulations and Functions in the Tumor Microenvironment. Front Immunol 2020; 11. doi: 10.3389/fimmu.2020.01731 . Medrek C, Pontén F, Jirström K, Leandersson K. The presence of tumor associated macrophages in tumor stroma as a prognostic marker for breast cancer patients. BMC Cancer 2012; 12: 306. House IG, Savas P, Lai J, Chen AXY, Oliver AJ, Teo ZL et al. Macrophage-Derived CXCL9 and CXCL10 Are Required for Antitumor Immune Responses Following Immune Checkpoint Blockade. Clin Cancer Res Off J Am Assoc Cancer Res 2020; 26: 487–504. Vermare A, Guérin MV, Peranzoni E, Bercovici N. Dynamic CD8 + T Cell Cooperation with Macrophages and Monocytes for Successful Cancer Immunotherapy. Cancers 2022; 14: 3546. Baechle JJ, Hanna DN, Sekhar KR, Rathmell JC, Rathmell WK, Baregamian N. Integrative computational immunogenomic profiling of cortisol-secreting adrenocortical carcinoma. J Cell Mol Med 2021; 25: 10061–10072. Yang Y, Sun L, Chen Z, Liu W, Xu Q, Liu F et al. The immune-metabolic crosstalk between CD3 + C1q + TAM and CD8 + T cells associated with relapse-free survival in HCC. Front Immunol 2023; 14: 1033497. Bulla R, Tripodo C, Rami D, Ling GS, Agostinis C, Guarnotta C et al. C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation. Nat Commun 2016; 7: 10346. Revel M, Sautès-Fridman C, Fridman W-H, Roumenina LT. C1q + macrophages: passengers or drivers of cancer progression. Trends Cancer 2022; 8: 517–526. Wang L, Guo W, Guo Z, Yu J, Tan J, Simons DL et al. PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer. Cell Rep Med 2024; 5. doi: 10.1016/j.xcrm.2024.101420 . Nalio Ramos R, Missolo-Koussou Y, Gerber-Ferder Y, Bromley CP, Bugatti M, Núñez NG et al. Tissue-resident FOLR2 + macrophages associate with CD8 + T cell infiltration in human breast cancer. Cell 2022; 185: 1189–1207.e25. Roumenina LT, Daugan MV, Noé R, Petitprez F, Vano YA, Sanchez-Salas R et al. Tumor Cells Hijack Macrophage-Produced Complement C1q to Promote Tumor Growth. Cancer Immunol Res 2019; 7: 1091–1105. Wong CW, Huang YY, Hurlstone A. The role of IFN-γ-signalling in response to immune checkpoint blockade therapy. Essays Biochem 2023; 67: 991–1002. Remde H, Schmidt-Pennington L, Reuter M, Landwehr L-S, Jensen M, Lahner H et al. Outcome of immunotherapy in adrenocortical carcinoma: a retrospective cohort study. Eur J Endocrinol 2023; 188: 485–493. Wilmouth JJ, Olabe J, Garcia-Garcia D, Lucas C, Guiton R, Roucher-Boulez F et al. Sexually dimorphic activation of innate antitumor immunity prevents adrenocortical carcinoma development. Sci Adv 2022; 8: eadd0422. Mao Y, Sarhan D, Steven A, Seliger B, Kiessling R, Lundqvist A. Inhibition of tumor-derived prostaglandin-e2 blocks the induction of myeloid-derived suppressor cells and recovers natural killer cell activity. Clin Cancer Res Off J Am Assoc Cancer Res 2014; 20: 4096–4106. Thumkeo D, Punyawatthananukool S, Prasongtanakij S, Matsuura R, Arima K, Nie H et al. PGE2-EP2/EP4 signaling elicits immunosuppression by driving the mregDC-Treg axis in inflammatory tumor microenvironment. Cell Rep 2022; 39: 110914. Nastos C, Papaconstantinou D, Paspala A, Pararas N, Vryonidou A, Pikouli A et al. The impact of adrenocortical carcinoma hormone secreting status as a predictor of poor survival: a systematic review and meta-analysis. Langenbecks Arch Surg 2024; 409: 316. Additional Declarations (Not answered) Supplementary Files TableS1.xlsx Table S1 Graphicalabstract.tif Graphical abstract Supplementarymaterial.pdf Supplementary material Westernblotsuncropped.pdf western blots_uncropped_merged Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6213228","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":431106973,"identity":"70a04be3-731c-42bc-aeac-9b9d90249980","order_by":0,"name":"Matthias Kroiss","email":"data:image/png;base64,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","orcid":"","institution":"LMU University Hospital, LMU Munich","correspondingAuthor":true,"prefix":"","firstName":"Matthias","middleName":"","lastName":"Kroiss","suffix":""},{"id":431106974,"identity":"7b1740f9-5f17-4f27-9f81-1a748496599d","order_by":1,"name":"Alexandra Triebig","email":"","orcid":"https://orcid.org/0009-0002-4728-0014","institution":"LMU Klinikum","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Triebig","suffix":""},{"id":431106975,"identity":"ccd27462-b10c-4f00-af3f-e201aa101a84","order_by":2,"name":"Tanja Maier","email":"","orcid":"","institution":"LMU University Hospital, LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Tanja","middleName":"","lastName":"Maier","suffix":""},{"id":431106976,"identity":"8ffe7168-1f4c-4190-a121-7b57a96c4a5f","order_by":3,"name":"Paul Schwarzlmueller","email":"","orcid":"","institution":"LMU University Hospital, LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Paul","middleName":"","lastName":"Schwarzlmueller","suffix":""},{"id":431106977,"identity":"27449ab2-6352-4627-883d-2bb3a0481ca7","order_by":4,"name":"Sena Atici","email":"","orcid":"","institution":"University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Sena","middleName":"","lastName":"Atici","suffix":""},{"id":431106978,"identity":"e7b8fdfa-decf-4eca-92fb-8645088867ac","order_by":5,"name":"Martin Kirmaier","email":"","orcid":"","institution":"Klinikumm der Universität München","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Kirmaier","suffix":""},{"id":431106979,"identity":"5cea6ffb-b9bb-4fc9-88d6-65f05eedf5d1","order_by":6,"name":"Adrian Gottschlich","email":"","orcid":"","institution":"LMU University Hospital, LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Adrian","middleName":"","lastName":"Gottschlich","suffix":""},{"id":431106980,"identity":"59bedbfb-ac26-4b1b-bd93-6d282ac9dc98","order_by":7,"name":"Fabien Riols","email":"","orcid":"","institution":"Helmholtz Zentrum München","correspondingAuthor":false,"prefix":"","firstName":"Fabien","middleName":"","lastName":"Riols","suffix":""},{"id":431106981,"identity":"e61be292-0d38-4c0a-a732-548f170559f4","order_by":8,"name":"Laura-Sophie Landwehr","email":"","orcid":"","institution":"University Hospital, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Laura-Sophie","middleName":"","lastName":"Landwehr","suffix":""},{"id":431106982,"identity":"fb43d0b2-0a15-4263-9d0c-18e6781d5ee6","order_by":9,"name":"Nicole Bechmann","email":"","orcid":"","institution":"University Hospital Carl Gustav Carus, TU Dresden","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Bechmann","suffix":""},{"id":431106983,"identity":"5045cc38-04a7-4398-a7b4-355b69662e85","order_by":10,"name":"Mirko Peitzsch","email":"","orcid":"","institution":"University Hospital Carl Gustav Carus, TU Dresden","correspondingAuthor":false,"prefix":"","firstName":"Mirko","middleName":"","lastName":"Peitzsch","suffix":""},{"id":431106984,"identity":"0c48ca2a-cc0d-47ef-a5a5-afbc1caa026e","order_by":11,"name":"Nagarajan Paramasivam","email":"","orcid":"","institution":"National Center for Tumor Diseases (NCT), NCT Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Nagarajan","middleName":"","lastName":"Paramasivam","suffix":""},{"id":431106985,"identity":"85885c98-c266-4ee3-a1b7-e01cbfd94baa","order_by":12,"name":"Mark Haid","email":"","orcid":"https://orcid.org/0000-0001-6118-1333","institution":"Helmholtz Zentrum München GmbH, German Research Center for Environmental Health","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"Haid","suffix":""},{"id":431106986,"identity":"ccd030a7-05ee-4734-945f-dcd1f0cd4bf8","order_by":13,"name":"Sebastian Theurich","email":"","orcid":"","institution":"Klinikum der Universität München","correspondingAuthor":false,"prefix":"","firstName":"Sebastian","middleName":"","lastName":"Theurich","suffix":""},{"id":431106987,"identity":"8f93fe90-722a-4af5-9a64-c597e394aff4","order_by":14,"name":"Stefan Fröhling","email":"","orcid":"","institution":"German Cancer Research Center (DKFZ), Heidelberg","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Fröhling","suffix":""},{"id":431106988,"identity":"33000408-6a1a-4558-80fe-722d840b0223","order_by":15,"name":"Hanno Glimm","email":"","orcid":"","institution":"University Hospital Carl Gustav Carus, TU Dresden","correspondingAuthor":false,"prefix":"","firstName":"Hanno","middleName":"","lastName":"Glimm","suffix":""},{"id":431106989,"identity":"97ff3e24-0138-495b-9cd7-f9f14bdd7d82","order_by":16,"name":"Daniel Hübschmann","email":"","orcid":"","institution":"Heidelberg Institute for Stem Cell Technology and Experimental Medicine","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Hübschmann","suffix":""},{"id":431106990,"identity":"f9813229-9249-4797-b280-a77b28394d1e","order_by":17,"name":"Martin Fassnacht","email":"","orcid":"","institution":"University Hospital, University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Fassnacht","suffix":""},{"id":431106991,"identity":"4d464d39-93a6-4440-9e86-62ea4d582c0c","order_by":18,"name":"Martin Reincke","email":"","orcid":"","institution":"LMU University Hospital, LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Reincke","suffix":""},{"id":431106992,"identity":"424fee1f-9c98-44de-a49b-cb66c636d0fe","order_by":19,"name":"Jose Pedro Friedmann-Angeli","email":"","orcid":"","institution":"University of Würzburg","correspondingAuthor":false,"prefix":"","firstName":"Jose","middleName":"Pedro","lastName":"Friedmann-Angeli","suffix":""},{"id":431106993,"identity":"4fe9e532-2d90-4998-bd61-99a7861ee8a8","order_by":20,"name":"Isabel Weigand","email":"","orcid":"","institution":"LMU University Hospital, LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Weigand","suffix":""}],"badges":[],"createdAt":"2025-03-12 15:06:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6213228/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6213228/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79573602,"identity":"5471d35c-925d-4cf1-9cd4-47ddb35535e0","added_by":"auto","created_at":"2025-03-31 11:07:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":352016,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacrophages (MΦ) in ACC exhibit a CD163+ “M2-like” phenotype irrespective of tumoral glucocorticoid excess\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA, B\u003c/strong\u003e) Immunohistochemistry (IHC) for macrophage markers was performed in tumor tissues from 25 ACC patients. Representative images of (A) CD68 staining and (B) CD163 staining are shown. Scale bar: 200 µm. (\u003cstrong\u003eC\u003c/strong\u003e) The percentage of IHC-positive cells was quantified using positive cell detection (QuPath). Median percentage of positive cells: 15% (CD68+) and 16% (CD163+), dashed lines represent medians, dotted lines represent upper and lower quartiles. \u003cstrong\u003eN\u003c/strong\u003e=25\u003cstrong\u003e.\u003c/strong\u003e(\u003cstrong\u003eD\u003c/strong\u003e) Co-Immunofluorescence of CD68 and CD163 was performed in tumor samples from ACC patients. A representative image is shown. Scale bar: 100 µm. (\u003cstrong\u003eE\u003c/strong\u003e) The percentage of CD68+ cells was plotted against intra-tumoral cortisol levels. Cortisol was quantified by LC-MS/MS. \u003cstrong\u003eN\u003c/strong\u003e=18. (\u003cstrong\u003eF\u003c/strong\u003e) Immunoblots showing CD163 and CD206 expression in protein lysates of ACC tumors. Tissue cortisol concentrations were determined by LC-MS/MS. BLD=below limit of detection. N=14. (\u003cstrong\u003eG\u003c/strong\u003e) Relative CD163 protein expression in whole tumor lysates of ACC tumors was quantified using ImageJ and correlated with intra-tumoral cortisol levels. (\u003cstrong\u003eH-J\u003c/strong\u003e) Violin plots displaying the total tumoral expression of (H) \u003cem\u003eCD68\u003c/em\u003e, (I) \u003cem\u003eCD163\u003c/em\u003e and (J) \u003cem\u003eCD206\u003c/em\u003e in a total of 56 ACC tumors, stratified by clinical cortisol excess; Red dots represent medians, blue lines connect lower adjusted value and lower quartile and upper adjusted value and upper quartile, respectively. FDR adjusted p-values were calculated using the Benjamin\u0026amp;Yekutieli correction and the Nanostring nSolver software.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/7eed3f0f7a897d444233dee3.png"},{"id":79575553,"identity":"5d07f3d0-059b-44c5-baf9-8b5d5060886a","added_by":"auto","created_at":"2025-03-31 11:15:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163498,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, both cortisol-secreting and non-cortisol-secreting ACC cells induce a distinct CD163+ MΦ phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Experimental setup of the differentiation and polarization protocol of primary human monocytes by conditioned media (CM) of ACC cells. (\u003cstrong\u003eB\u003c/strong\u003e) Cortisol in CM of steroidogenic NCI-H295R and non-steroidogenic JIL-2266 cell lines after 48 h of culture. Cortisol was measured by a chemiluminescence immunoassay (DiaSorin; REF:313261) and normalized to total protein content. N=3 biological replicates are shown in Mean +/- SD. (\u003cstrong\u003eC\u003c/strong\u003e) Relative \u003cem\u003eCD163\u003c/em\u003e mRNA and (\u003cstrong\u003eD\u003c/strong\u003e) CD163 protein expression in MΦ exposed to CM of NCI-H295R and JIL-2266 cells. N=3 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eE\u003c/strong\u003e) IL-10 secretion of MΦ exposed to CM of NCI-H295R or JIL-2266 cells. N=4 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eF\u003c/strong\u003e) \u003cem\u003eCD163\u003c/em\u003e RNA and (\u003cstrong\u003eG\u003c/strong\u003e) CD163 protein expression in MΦ exposed to CM of NCI-H295R cells cultured in the presence or absence of the steroidogenesis inhibitor metyrapone (10 µM). N=6 biological replicates. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eH\u003c/strong\u003e) SgRNA knockdown of the glucocorticoid receptor (GR; gene: \u003cem\u003eNR3C1\u003c/em\u003e) in THP-1 MΦ. (\u003cstrong\u003eI\u003c/strong\u003e) CD163 protein expression in THP-1 \u003cem\u003eNR3C1\u003c/em\u003e KO and control cells in response to dexamethasone or NCI-H295R CM exposure. PBMC=peripheral blood mononuclear cell; CM=conditioned media; GC: glucocorticoid; NCI-R=NCI-H295R; mety=metyrapone\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/012b0348ab6927983f6486aa.png"},{"id":79575552,"identity":"bc7ca40b-6ed2-4d97-babe-60a2b97b7b38","added_by":"auto","created_at":"2025-03-31 11:15:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":262339,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlucocorticoids induce MΦ polarization towards a C1Q+ CD163+ phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Volcano plot showing differentially expressed genes (DEGs) in MΦ exposed to GM-CSF+IFNγ vs GM-CSF+IFNγ+dexamethasone (100 nM). Analysis of RNA data was performed with Nanostring nCounter. FDR adjusted p-values were calculated using the Benjamin\u0026amp;Yekutieli correction. N=3 biological replicates. (\u003cstrong\u003eB-E\u003c/strong\u003e) Expression of (B) \u003cem\u003eCD163\u003c/em\u003e, (C) \u003cem\u003eMARCO\u003c/em\u003e, (D) \u003cem\u003eC1QA\u003c/em\u003e and (E) \u003cem\u003eC1QB\u003c/em\u003e in MΦ exposed to GM-CSF+IFNγ vs GM-CSF+IFNγ+dexamethasone. N=3 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eF-H\u003c/strong\u003e) \u003cem\u003eC1QA\u003c/em\u003eRNA, (G) \u003cem\u003eC1QB\u003c/em\u003e RNA and (H) C1QA protein expression in MΦ exposed to GM-CSF+IFNγ and CM of NCI-H295R cells cultured in the presence or absence of the steroidogenesis inhibitor metyrapone (10 µM) vs controls. N=6 biological replicates. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eI\u003c/strong\u003e) Co-Immunofluorescence of C1QA and CD163 was performed in adrenocortical tissue. One representative image is shown. \u003cstrong\u003e(J\u003c/strong\u003e) Correlation of \u003cem\u003eC1QA\u003c/em\u003e and \u003cem\u003eCD68\u003c/em\u003e gene expression across different cancer entities. Data was obtained from TCGA. NCI-R=NCI-H295R; mety=metyrapone; CM=conditioned media\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/6567bdd5855a5c08a2f27718.png"},{"id":79573604,"identity":"164f5131-1cea-47a5-9e17-3fc10b5e7df2","added_by":"auto","created_at":"2025-03-31 11:07:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":186423,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGC-induced MΦ are highly efficient in C1q mediated phagocytosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Gene set enrichment analysis (GSEA) of DEGs in MΦ exposed to CM of steroidogenic NCI-H295R cells in comparison to controls. (\u003cstrong\u003eB\u003c/strong\u003e) Visualization of the enrichment results on phagocytosis (p\u0026lt;2.2e-16; FDR: 0.011718). (\u003cstrong\u003eC\u003c/strong\u003e) Representative flow cytometry plots and (\u003cstrong\u003eD\u003c/strong\u003e) quantification of phagocytic indices of MΦ exposed to CM of steroidogenic NCI-H295R cells and controls. CD64+ CSFE+ double positive cells were considered phagocytic macrophages. Phagocytotic indices were calculated as followed: % [CSFE+ CD64+ cells] * MFI [CSFE+ CD64+ cells). N=3 biological replicates are shown in Mean +/- SD. (\u003cstrong\u003eE\u003c/strong\u003e) Phagocytic indices of MΦ exposed to dexamethasone and controls. N=3 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eF\u003c/strong\u003e) Phagocytosis of “M1-like” MΦ after exposure to 10 µg/ml or 20 µg/ml purified human C1q. N=3 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eG\u003c/strong\u003e) MerTK and CD163 protein expression in MΦ polarized with GM-CSF/M-CSF and polarizing factors as indicated. (\u003cstrong\u003eH\u003c/strong\u003e) Phagocytosis of GC-polarized MΦ after exposure to the MerTK inhibitor UNC1062 (100 nM), anti-Gas6 antibody (20 µg/ml) or goat control IgG (20 µg/ml). N=3 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using One-way ANOVA. NCI-R=NCI-H295R. mety=metyrapone\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/9e7b293eef07f06dd0b86147.png"},{"id":79575555,"identity":"1a9eee54-122f-46bb-9ecd-83f72c83ad60","added_by":"auto","created_at":"2025-03-31 11:15:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":208323,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProstaglandin E\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e (PGE\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e) is an alternative inducer of CD163+ MΦ polarization derived from non-cortisol-secreting ACC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Heatmap of eicosanoid species in the CM of steroidogenic NCI-H295R and non-steroidogenic JIL-2266 cells detected by LC-MS/MS. (\u003cstrong\u003eB\u003c/strong\u003e) Total amount of PGE\u003csub\u003e2\u003c/sub\u003e in the supernatants of ACC cell lines after 48\u0026nbsp;h of culture quantified by ELISA. N=3 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using Welch´s t-test. (\u003cstrong\u003eC\u003c/strong\u003e) Immunoblot of CD163 protein expression in MΦ exposed to PGE\u003csub\u003e2\u003c/sub\u003e (varying concentrations as indicated). (\u003cstrong\u003eD\u003c/strong\u003e) \u003cem\u003eCD163\u003c/em\u003e RNA and (\u003cstrong\u003eE\u003c/strong\u003e) CD163 protein expression in MΦ exposed to CM of JIL-2266 cultured in the presence or absence of the COX-2 inhibitor celecoxib (200 nM). N=3 biological replicates. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eF\u003c/strong\u003e) IL-10 secretion of MΦ exposed to CM of JIL-2266 cells cultured in the presence or absence of the COX-2 inhibitor celecoxib. N=4 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using Welch´s t-test. (\u003cstrong\u003eG\u003c/strong\u003e) \u003cem\u003ePTGS2\u003c/em\u003e mRNA (N=3), (\u003cstrong\u003eH\u003c/strong\u003e) COX-2 and GR protein expression and (\u003cstrong\u003eI\u003c/strong\u003e) PGE\u003csub\u003e2\u003c/sub\u003e release (N=5) in JIL-2266 cells treated with dexamethasone (varying concentrations as indicated). Replicates are shown in Mean +/- SD. Statistical analysis was performed using One-way-ANOVA. (\u003cstrong\u003eJ\u003c/strong\u003e) \u003cem\u003ePTGS2\u003c/em\u003e mRNA (N=3), (\u003cstrong\u003eK\u003c/strong\u003e) COX-2 and GR protein expression and (\u003cstrong\u003eL\u003c/strong\u003e) PGE\u003csub\u003e2\u003c/sub\u003e release (N=5) in JIL-2266 cells treated with relacorilant (varying concentrations as indicated). Replicates are shown in Mean +/- SD. Statistical analysis was performed using One-way-ANOVA. (\u003cstrong\u003eM\u003c/strong\u003e) Immunoblot of CD163 protein expression in MΦ exposed to CM of steroidogenic NCI-H295R cells treated with metyrapone, celecoxib or a combination of both. GR=glucocorticoid receptor; dexa=dexamethasone; rela=relacorilant; cele=celecoxib; mety=metyrapone; NCI-R=NCI-H295R.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/f7c91cc998a31e8dc491983e.png"},{"id":79573616,"identity":"7555d00f-bc50-4d28-b72d-b80965fd2708","added_by":"auto","created_at":"2025-03-31 11:07:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2541684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIFNγ treatment of C1Q+ MΦ strongly induces CXCL9 expression which positively correlates with T cell infiltration and better survival in ACC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003eCXCL9\u003c/em\u003e RNA expression in MΦ exposed to GM-CSF+IFNγ vs GM-CSF+IFNγ+dexamethasone (100 nM). N=3 biological replicates are shown in Mean +/- SD. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eB-C\u003c/strong\u003e) CXCL9 secreted from MΦ exposed to GM-CSF+IFNγ vs GM-CSF+IFNγ and dexamethasone (100 nM; N=3) or (C) hydrocortisone (500 ng/ml; N=4). Biological replicates are shown in Mean +/- SD. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eD-E\u003c/strong\u003e) Correlation of \u003cem\u003eC1QA\u003c/em\u003e and \u003cem\u003eCD4\u003c/em\u003eexpression in ACC tumors within the (D) TCGA dataset (N=76) or (E) NCT MASTER dataset (N=88). (\u003cstrong\u003eF\u003c/strong\u003e) Pan-cancer correlation of \u003cem\u003eC1QA\u003c/em\u003e and \u003cem\u003eCD4\u003c/em\u003eexpression data obtained from TCGA. (\u003cstrong\u003eG-H\u003c/strong\u003e) Kaplan-Meier plots of PFS according to tumoral (G) \u003cem\u003eCD68\u003c/em\u003e or (H) \u003cem\u003eC1QA\u003c/em\u003e expression. Higher \u003cem\u003eCD68\u003c/em\u003eor \u003cem\u003eC1QA\u003c/em\u003e gene expression was significantly associated with favorable PFS. RNA expression and survival data was extracted from the TCGA dataset (N=76). (\u003cstrong\u003eI\u003c/strong\u003e) CXCL9 concentrations in plasma samples of ACC patients before and during immune-checkpoint-inhibition (ICI) therapy with indication of TAM numbers determined by IHC analysis of respective tumor tissue samples. N=7. Statistical analysis was performed using paired t-test. (\u003cstrong\u003eJ\u003c/strong\u003e) Percentage of CD163+ cells quantified from IHC stainings of respective tissue samples of ACC patients who responded to ICI therapy compared to those who did not respond. Statistical analysis was performed using Mann-Whitney t-test. GC=glucocorticoid; PFS=progression free survival; dexa=dexamethasone; HC=hydrocortisone\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/0a3dd4bb892e493842173e09.png"},{"id":83059993,"identity":"cc0adf68-9a7f-4c49-a1ec-cdd37809e8a3","added_by":"auto","created_at":"2025-05-19 14:33:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4966615,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/44a0ed62-8649-4043-b3dd-06488374b4e6.pdf"},{"id":79573605,"identity":"d06c29eb-a037-45d9-b34c-f92de6f0cf82","added_by":"auto","created_at":"2025-03-31 11:07:23","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10938,"visible":true,"origin":"","legend":"Table S1","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/70d0ebaed12df66e8467c3a0.xlsx"},{"id":79573608,"identity":"2eb8a3f0-8cf0-4ada-afad-c3e85d75a852","added_by":"auto","created_at":"2025-03-31 11:07:23","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1465428,"visible":true,"origin":"","legend":"Graphical abstract","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/0474d32575be7dbc3223ec6e.tif"},{"id":79573609,"identity":"79635f20-ce9c-41b2-880e-f9f2e374a250","added_by":"auto","created_at":"2025-03-31 11:07:24","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":2730614,"visible":true,"origin":"","legend":"Supplementary material","description":"","filename":"Supplementarymaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/981cb2cc592c5ae58235fa1f.pdf"},{"id":79573614,"identity":"89b4ca0f-737f-4d26-92b9-fd7686865fb9","added_by":"auto","created_at":"2025-03-31 11:07:24","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1463942,"visible":true,"origin":"","legend":"western blots_uncropped_merged","description":"","filename":"Westernblotsuncropped.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6213228/v1/504720af9ecc6d70a526736c.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"Tumoral glucocorticoids induce a phagocytic CD68+/CD163+/C1Q+ macrophage phenotype primed for IFNγ-driven CXCL9 secretion","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlucocorticoids (GCs) are a class of steroid hormones predominantly synthesized and secreted by the \u003cem\u003ezona fasciculata\u003c/em\u003e of the adrenal cortex in response to hypothalamic-pituitary-adrenal (HPA) axis activation. Within cancer tissues, GCs have emerged as an important component of the tumor microenvironment (TME), originating from three main sources: endogenous production \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, recycling of inactive metabolites \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e or as a consequence of therapeutic administration. Notably, GCs in the context of cancer are believed to promote tumor cell proliferation, survival and migration while concurrently suppressing antitumor immunity through functional modulation of immune cells \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdrenocortical carcinoma (ACC) is a rare, yet highly aggressive tumor of the adrenal cortex, with an estimated annual incidence ranging from approximately 0.5 to 2.0 cases per million individuals \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. While non-functional ACCs are commonly diagnosed incidentally or due to mass effects, the majority of ACCs is hormonally functional leading to distinct clinical presentations caused by hormonal hypersecretion. These clinical manifestations include Cushing\u0026rsquo;s syndrome in GC-producing tumors or virilization in androgen-secreting ACCs. Immune checkpoint inhibitors (ICI) that have profoundly changed the management of many malignancies have demonstrated limited efficacy in ACC. So far, a combined analysis of twenty studies with a total of 250 patients reported an objective response in only 14% of patients with an overall survival of 13.9 months \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, a clinically meaningful overall response rate of 52% and an overall survival (OS) of almost 21 months in one recent study suggest that a subset of patients may benefit from ICI therapy \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTumor associated macrophages (TAMs) exhibit a remarkable plasticity and can assume a dual role in the progression of cancer. Pro-inflammatory 'M1-like' (classically activated) macrophages (MΦ) are known for their antitumoral functions including cytotoxicity and immune stimulation, e.g. by the release of pro-inflammatory cytokines. Meanwhile, anti-inflammatory 'M2-like' (alternatively activated) MΦ often promote tumor growth fostering a profoundly immune-suppressed TME that negatively impacts on cancer outcome \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e or the responsiveness to ICI therapy \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Even though this simplified classification proposed by Mills \u003cem\u003eet al.\u003c/em\u003e broadly describes the inflammatory capacity of MΦ, this terminology is considered outdated as the current understanding suggests a spectrum of diverse MΦ phenotypes, each with a unique contribution to inflammation \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTherapeutic modulation of MΦ differentiation such as inhibition of colony stimulating factor 1 (CSF-1) or its receptor (CSF1R), has emerged as a promising approach to increase the utility of ICI in unresponsive or refractory tumors. However, clinical trials investigating CSF1R blockade, either as monotherapy or in combination with ICIs, have largely failed to produce significant clinical responses \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. This highlights the need for a more sophisticated approach that goes beyond broad depletion of TAMs. Specifically in ACC, therapeutic approaches targeting MΦ remain completely unexplored, primarily due to the limited understanding of MΦ infiltration and function in this rare endocrine malignancy. Current \u003cem\u003ein vitro\u003c/em\u003e studies propose the activation of the glucocorticoid receptor (GR) or reprogramming of the mitochondrial metabolism by GCs to profoundly suppress pro-inflammatory (M1) MΦ polarization and promote anti-inflammatory (M2) polarization \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, in the context of cancer, the impact of tumor-derived GCs on the function of TAMs remains largely unknown.\u003c/p\u003e \u003cp\u003eThe utilization of ACC as a model to study the role of GC activity in cancer may shed light on the relevance of endogenously produced or administered GCs across several malignancies. We here set out to identify the contribution of tumoral GCs on MΦ polarization in cancer and describe how therapeutic targeting of the underlying mechanisms may be relevant for clinical treatment concepts in regard to ACC and other solid tumors.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTissue samples\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTissue samples from patients with ACC were collected as part of the European Network for the Study of Adrenal Tumors (ENSAT) registry study or the MASTER (Molecularly Aided Stratification for Tumor Eradication Research) program. The ENSAT study has been approved by the ethics committees of the Ludwig-Maximilians-University; Munich; Germany (RRID:SCR_011358) (approval number: 379/10) and the Julius-Maximilians-University W\u0026uuml;rzburg (approval number: 88/11). Written informed consent was received from each patient before surgery and the study was conducted in accordance with the ethical principles of the declaration of Helsinki. The MASTER program (ClinicalTrials.gov: NCT05852522), a prospective observational study conducted by the German Cancer Research Center (DKFZ), the National Center for Tumor Diseases (NCT), and the German Cancer Consortium (DKTK), leverages whole-genome/exome sequencing (WGS/WES), RNA sequencing (RNA-seq), DNA methylation profiling, proteomics, and phosphoproteomics to guide treatment in young adults with advanced malignancies and patients with incurable rare cancers \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Anonymized non-small cell lung cancer (NSCLC) and colorectal cancer samples (CRC) were obtained from the Human Tissue and Cell Research (HTCR) foundation.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eImmunohistochemistry (IHC) was conducted as described \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In short, formalin-fixed, paraffin-embedded (FFPE) tumor sections were deparaffinized in xylene and dehydrated with increasing dilutions of ethanol. Antigen retrieval was performed in 10 mM citric acid monohydrate buffer at pH 6.5. Endogenous peroxidase activity was blocked with methanol containing 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 10 min at RT. Non-specific binding was blocked using 20% human AB serum for 1 h at RT. Primary antibodies (\u003cb\u003esuppl. table 1\u003c/b\u003e) were applied overnight at 4\u0026deg;C. Signals were developed using the HiDef Detection Polymer System Detection Amplifier (Medac, Germany) and HiDef Detection Polymer System HRP Polymer Detector (Medac, Germany). DAB (DAB Liquid Kit; Dako) was applied and the sections were counterstained with hematoxylin. Stained slides were dehydrated in 100% EtOH and dried at 60\u0026deg;C for 1 h. Slides were scanned using the Microscopes International uScopeMXII-20 Digital Microscope (RRID:SCR_026399) with 20x magnification and positive cell detection was performed using the software QuPath (RRID:SCR_018257).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eCell culture and drug treatment\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eNCI-H295R cells (RRID:CVCL 0458) were obtained from Cytion and cultured in DMEM/F12 supplemented with 1x Insulin-Transferrin-Selenium (Gibco) and 2.5% Nu-Serum (Corning). JIL-2266 cells were established and characterized in our laboratory and cultured as previously described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. All cell lines were cultured at 37\u0026deg;C and 5% CO2. Cells were checked regularly for mycoplasma contamination using the Venor\u0026reg; GeM Classic Mycoplasma Detection Kit (Minerva Biolabs). Ferroptosis was induced using RSL3 (Selleckchem). Inhibitors (liproxstatin-1, metyrapone or celecoxib (all Sigma)) were added 1 h prior cell death initiation or the start point of cell culture supernatant collection. Dexamethasone (Jenapharm\u0026reg;) and hydrocortisone (Pfizer) were used for exogenous glucocorticoid treatment. The glucocorticoid receptor antagonist relacorilant was obtained from Corcept Therapeutics (Menlo Park, CA, USA). PGE\u003csub\u003e2\u003c/sub\u003e was obtained from Selleckchem. Cas9 expressing THP-1 (RRID:CVCL_0006) monocytes were obtained from Prof. J\u0026ouml;rg Wischhusen, University Hospital W\u0026uuml;rzburg and cultured in RPMI 10% FBS, 1% P/S and 50 \u0026micro;M \u0026szlig;-mercaptoethanol. For differentiation, THP-1 cells were plated into 12-well plates and exposed to 10 ng/ml PMA for 48 h before further polarizing factors were added for a duration of 48 h. For NR3C1 KO cells, THP-1 cells were first differentiated for 2 days using 10 ng/ml PMA, followed by detachment of cells using accutase. Cells were counted and 2.5*10^6 cells were transfected with 1.6 \u0026micro;g sgRNA (IDT) using the LONZA\u0026trade; Nucleofector 2b Device (RRID:SCR_022262) (program V-001). After 5 h medium was changed to THP-1 medium containing 2.5 ng/ml PMA and 50 \u0026micro;M β-mercaptoethanol. 48 h after transfection, cytokines or CM was added for 48 h.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eMacrophage isolation and polarization\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eHuman PBMCs from healthy blood donors were isolated by density gradient using Lymphoprep\u0026trade; (StemCell) and transferred to 12-well plates at a density of 5*10^6 cells/ml in macrophage medium (RPMI-1640 (Gibco) supplemented with 10% FBS (Sigma), 1% penicillin-streptomycin (Sigma) and 1% L-glutamine (Gibco)). Monocytes attached to the culture vessel after 24 h were incubated in macrophage medium supplemented with either GM-CSF (Gibco; 100 ng/ml) or M-CSF (Gibco; 40 ng/ml) for 'M1-like' or 'M2-like' MΦ differentiation, respectively. On day 7, further polarizing factors IFNγ (Gibco; 10 ng/ml), IL-4 (Gibco; 20 ng/ml), LPS (Invitrogen; 100 ng/ml) or IL-10 (Gibco; 150 ng/ml) were added for a total of 6 days with medium change and PBS washing every other day. A schematic representation of the macrophage polarization protocol and their phenotype characteristics is depicted in the \u003cb\u003esupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. For the generation of conditioned media (CM), ACC cell lines were cultured for 48 h at 3.5*10^5 cells/well of a 6 well plate. Supernatant was collected, sterile-filtered and mixed with macrophage medium (30% v/v). This medium was added to the monocytes from day 1 onwards. Medium was changed on day 6 and then every other day.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eImmuno-Cytochemistry\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePBMCs were isolated and macrophages were differentiated and polarized according to the protocol stated above. For immunostaining, cells were fixed with 4% PFA for 10 min at 4\u0026deg;C. Subsequently, cells were blocked with PBS/4% BSA for 30 min and incubated with the primary antibodies (\u003cb\u003esuppl. table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) overnight at 4\u0026deg;C. Secondary antibodies (\u003cb\u003esuppl. table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) were added for 1 h RT in the dark. Pictures were taken using the EVOS M7000 Imaging System (RRID:SCR_025070).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFFPE tumor sections were deparaffinized in xylene and dehydrated with increasing dilutions of ethanol. Antigen retrieval was performed in 10 mM citric acid monohydrate buffer at pH 6.5. Non- specific binding was blocked using 20% human AB serum for 30 min at RT. Primary antibodies (\u003cb\u003esuppl. table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) were applied overnight at 4\u0026deg;C. Secondary antibodies (\u003cb\u003esuppl. table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e) were incubated for 1 h at RT followed by Hoechst staining (1:2000 in PBS) for 8 min. Slides were mounted with Prolong Gold Antifade reagent (ThermoFisher).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhagocytosis assay\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eACC cells were stained with CSFE (BioTracker 488 Green CSFE Cell Proliferation Kit, Sigma) and seeded in 12 well plates at a density of 3,5*10^5 cells/well. After 24 h incubation at 37\u0026deg;C, cells were treated with RSL3 for 2 h. Medium was discarded and the wells were washed with PBS twice. MФ were added to the cultured early ferroptotic ACC cells at a ratio of 10:1 tumor cell:MФ overnight. Flow cytometry was used to quantify percentages of CSFE\u0026thinsp;+\u0026thinsp;CD64\u0026thinsp;+\u0026thinsp;macrophages. For inhibition studies, MФ were pre-incubated with the selective MerTK inhibitor UNC1062 (100 nM; MCE: HY-117548), anti-Gas6 antibody (20 \u0026micro;g/ml; R and D Systems Cat# AB885, RRID:AB_354376), a goat isotype IgG control (20 \u0026micro;g/ml; Thermo Fisher Scientific Cat# 02-6202, RRID:AB_2532946) or purified C1q (Merck; C1740) for 30 minutes before they were added to ferroptotic tumor cells.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eFlow Cytometry\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFerroptotic cell death was monitored with Annexin V/propidium iodide (PI) staining (BioLegend). For phagocytosis assays, the co-cultured ferroptotic NCI-H295R cells and macrophages were trypsinized and washed with PBS. Anti-CD64 antibody was added at a dilution of 1:100 in PBS and cells were incubated for 30 min at RT. Cells were washed with PBS and incubated with secondary antibody for 30 min at RT protected from light. Cells were washed and resuspended in 200 \u0026micro;l FACS buffer. The percentage of CSFE\u0026thinsp;+\u0026thinsp;CD64\u0026thinsp;+\u0026thinsp;cells was determined using FACS-Fortessa at the Ludwig Maximilian University Hospital Munich Flow Cytometry Core Facility (RRID:SCR_026395). CSFE\u0026thinsp;+\u0026thinsp;cells were detected in the FITC channel, CD64\u0026thinsp;+\u0026thinsp;cells in the APC channel and cell populations quantified with FlowJo (RRID:SCR_008520).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCells were lysed in RIPA buffer (Sigma) supplemented with 1% Protease Inhibitor (Sigma), 1% Phosphatase B Inhibitor and 2% Phosphatase C Inhibitor (Santa Cruz). Protein concentrations were quantified using the Pierce\u0026trade; BCA Protein Assay Kit. Absorbance values were measured at the BMG Labtech FLUOstar Omega (RRID:SCR_025024) microplate reader at 562 nm. Protein was loaded onto a 4\u0026ndash;20% Mini-PROTEAN\u0026reg; TGX\u0026trade; Precast Protein Gel (BioRad) and separated by SDS-PAGE. Proteins were transferred by semi-dry blot onto a nitrocellulose membrane (Cytiva) that was subsequently blocked in 5% skimmed milk in TBS-Tween at RT for 1 h. Primary antibodies (suppl. table 1) were diluted in 5% skimmed milk in TBS-Tween and incubated over night at 4\u0026deg;C. Membranes were washed and incubated at RT for 1 h with horseradish-peroxidase (HRP)-labeled secondary antibodies. Immunoblots were developed and visualized using Clarity\u0026trade; Western ECL Substrate at the Bio-Rad Chemidoc XRS Gel Imaging System (RRID:SCR_019690) with Image Lab Software (RRID:SCR_014210). GAPDH expression was used as loading control.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eqPCR\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal RNA was extracted from cells using Maxwell\u0026reg; RSC simplyRNA tissue Kit (Promega) and reversetranscribed into cDNA using GoScript\u0026trade; Reverse Transcription Mix (Promega). qPCR was performed using TaqMan Real-Time-PCR Assays (PTGS2: Hs00153133m1; ACTB: Hs99999903_m1) and TaqMan Gene Expression Master Mix (both ThermoFisher) in a final volume of 25 \u0026micro;l. qPCR was carried out at the Agilent Stratagene Mx3000P qPCR cycler (RRID:SCR_026398). qPCR conditions consisted of an initial denaturation step of 3 min at 95˚C, followed by 39 cycles of 30 sec denaturation at 95˚C, 30 sec annealing at 60˚C and 30 sec elongation at 72\u0026deg;C. mRNA was quantified using the 2ΔΔCq method and βactin was used as internal control.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eNanostring nCounter gene expression analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGene expression analysis was performed on RNA isolated either from in vitro differentiated macrophages or from FFPE ACC tissue sections. RNA was extracted using the AllPrep DNA/RNA FFPE Kit (Qiagen). RNA quantity and quality were assessed with a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific) and samples meeting predefined quality criteria were further analyzed for gene expression quantification with the NanoString nCounter Analysis System (RRID:SCR_021712). A panel (NanoString Technologies (RRID:SCR_023912)) consisting of 473 immune related genes plus eleven housekeeping genes was used according to the manufacturer\u0026acute;s instructions. Analysis was performed by nSolver Analysis Software (RRID:SCR_003420). Samples passing imaging quality control thresholds (fields of view read\u0026thinsp;\u0026gt;\u0026thinsp;75%, binding density of 0.05\u0026ndash;2.25, positive control linearity\u0026thinsp;\u0026gt;\u0026thinsp;0.95, and positive control detection limit\u0026thinsp;\u0026gt;\u0026thinsp;2) were normalized using technical controls and housekeeping genes, based on positive control normalization with the geometric mean as the normalization factor. For comparisons, a one-tailed Student\u0026rsquo;s t-test p-value and a false discovery rate (FDR) was calculated by the Benjamini\u0026ndash;Yekutieli method. Genes were ranked by fold change (FC) and FDR, with thresholds set at FC\u0026thinsp;\u0026ge;\u0026thinsp;2 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.1.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eELISA\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eProstaglandin E\u003csub\u003e2\u003c/sub\u003e (PGE\u003csub\u003e2\u003c/sub\u003e), IL-10 and CXCL9 concentrations in cell supernatants were measured by ELISA (PGE\u003csub\u003e2\u003c/sub\u003e: Cayman Chemical; 514010; Gas6; Invitrogen; BMS2291; IL-10: R\u0026amp;D Systems; D1000B; CXCL9/MIG: Invitrogen, Item No 900-K87K). If not stated otherwise, concentrations were normalized to total protein levels isolated as stated above.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eSteroid profiles of ACC tissues were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS) as previously described \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Oxylipin measurements in supernatants of ACC cells and ACC tissues were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e and outlined in the Supplementary Methods.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSurvival Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo assess the prognostic significance of gene expression levels, we performed survival analysis using clinical and RNA seq data from The Cancer Genome Atlas (TCGA) project and a local ACC biobank (NCT MASTER). Patients were stratified into high- and low-expression groups based on the median expression level of each gene of interest. Survival curves were generated using Kaplan-Meier estimates, exported from the either the cBioPortal platform (TCGA) or generated using R Project for Statistical Computing (RRID:SCR_001905) version R/4.0.0 (NCT MASTER). Differences in survival between groups were assessed using the log-rank test. For the use of data from a public database, an approval exemption has been obtained from the LMUs\u0026acute; ethics committee.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGene Set Enrichment Analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor pathway and functional enrichment analysis, we employed Gene Set Enrichment Analysis (GSEA) using the WebGestalt: WEB-based GEne SeT AnaLysis Toolkit (RRID:SCR_006786). Differentially expressed genes identified from our RNA dataset were inputted into WebGestalt to assess their association with known biological pathways, gene ontology (GO) categories, and functional gene sets.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIf not stated otherwise, three independent replicates were performed per experiment. For experiments using human MΦ each biological replicate equals a different biological blood donor. Statistical analyses were performed using GraphPad Prism (RRID:SCR_002798) version 9.0. If not stated otherwise, biological replicates are shown in Mean +/- SD and the statistical analyses were performed using a two-tailed Student\u0026rsquo;s t test for paired comparisons or one way analysis of variance (ANOVA) for multiple comparisons. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, ns\u0026thinsp;=\u0026thinsp;not significant.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMacrophages in ACC exhibit a CD68\u0026thinsp;+\u0026thinsp;CD163\u0026thinsp;+\u0026thinsp;phenotype irrespective of tumoral glucocorticoid excess\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUsing chromogenic immunohistochemistry (IHC), we investigated the proportion of TAMs in ACC tissue samples from a total of 25 patients. We observed a heterogenous but overall abundant presence of CD68+ (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and CD163\u0026thinsp;+\u0026thinsp;cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Intra-tumoral TAM counts varied from 3.1\u0026ndash;40.1% (median\u0026thinsp;=\u0026thinsp;15.0%) for CD68\u0026thinsp;+\u0026thinsp;cells and 3.0\u0026ndash;37.6% (median\u0026thinsp;=\u0026thinsp;16.0%) for CD163\u0026thinsp;+\u0026thinsp;cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, immunofluorescence microscopy revealed the near-total co-localization of CD68 and CD163 in ACC tissue samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Tissue cortisol levels measured using LC-MS/MS did not correlate with the total TAM population assessed by IHC staining (Spearman: p\u0026thinsp;=\u0026thinsp;0.95; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Likewise, we did not observe a significant correlation between intra-tumoral cortisol concentrations and the total tumoral expression of the 'M2-like' macrophage marker CD163 and CD206, as determined by immunoblotting of respective tumor lysates (Spearman: p\u0026thinsp;=\u0026thinsp;0.58; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, G). In a separate cohort of 56 ACC patients stratified by clinical and biochemical hormone status, tumoral RNA expression of \u003cem\u003eCD68\u003c/em\u003e, \u003cem\u003eCD163\u003c/em\u003e, and \u003cem\u003eCD206\u003c/em\u003e showed no significant difference between patients with or without clinical cortisol excess (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH-J). Moreover, when tumors were categorized as inactive, androgen-overproducing, GC-overproducing, or combined androgen and GC excess, no differential expression of these markers was observed across the four groups (\u003cb\u003eFig. S2A-C\u003c/b\u003e). Altogether these findings suggest that MΦ are a prevalent immune cell population in ACC heavily skewed towards a CD68\u0026thinsp;+\u0026thinsp;CD163\u0026thinsp;+\u0026thinsp;phenotype independently of tumoral GC overproduction.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e, \u003cb\u003eboth cortisol-secreting and non-cortisol-secreting ACC cells induce a distinct CD163\u0026thinsp;+\u0026thinsp;macrophage phenotype\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo directly study the impact of ACC tumor cells on MΦ polarization \u003cem\u003ein vitro\u003c/em\u003e, we next isolated monocytes from healthy donor blood samples and exposed them to conditioned medium (CM; 33% v/v) of two different ACC cell lines as outlined in the experimental setup shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. CM from the cortisol-secreting ACC cell line NCI-H295R and the non-cortisol-secreting ACC cell line JIL-2266 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) was collected after 48 h of culture and added to human monocytes from day one onwards. Given that the MΦ differentiation factors GM-CSF and M-CSF are known early drivers of M1 and M2 MΦ differentiation, respectively \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, exposure of MΦ to CM was investigated in the absence of exogenous growth factors. Additionally, CM of ACC cells was added to MΦ exposed to GM-CSF\u0026thinsp;+\u0026thinsp;IFNγ ('M1-like' MΦ polarization; mimicking T cell activation, e.g. induced by immune checkpoint inhibition) to examine potential inhibitory effects in the context of ICI therapy. Irrespective of whether it was derived from cortisol-secreting or non-cortisol-secreting ACC cells, CM treatment induced an upregulation of CD163 expression in MΦ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D; \u003cb\u003eFig. S3A\u003c/b\u003e). Remarkably, IL-10 secretion was pronouncedly higher in MΦ polarized with non-secreting JIL-2266 CM (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), suggesting a difference in the MΦ phenotype induced by the two cell lines. Even though \u003cem\u003eCSF-1\u003c/em\u003e mRNA (encoding M-CSF) levels and M-CSF secretion were higher in JIL-2266 cells (\u003cb\u003eFig. S3B, C\u003c/b\u003e), they were negligibly low. To test the hypothesis that MΦ polarization driven by steroidogenic NCI-H295R cells was predominantly caused by tumor cell secreted cortisol, we next cultured this ACC cell line in the presence of the steroidogenesis inhibitor metyrapone (cortisol concentrations in supernatants shown in \u003cb\u003eFig. S3D\u003c/b\u003e), before CM was collected and applied to isolated monocytes. Accordingly, blockade of cortisol synthesis in tumor cells decreased the 'M2-like' polarization of MΦ, as shown by morphological changes (\u003cb\u003eFig. S3E\u003c/b\u003e) and CD163 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, G). In THP-1 MΦ, knockout of the glucocorticoid receptor (GR; gene: \u003cem\u003eNR3C1\u003c/em\u003e) efficiently attenuated CD163 expression upon treatment with dexamethasone or NCI-H295R CM, further supporting a GC-GR mediated mechanism (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH, I). Taken together, these experiments show that GC secretion by ACC tumor cells induces a CD163\u0026thinsp;+\u0026thinsp;MΦ phenotype that is functionally distinct from CD163\u0026thinsp;+\u0026thinsp;MΦ polarized by non-cortisol-secreting ACC cell lines.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGlucocorticoids induce MΦ polarization towards a C1Q\u0026thinsp;+\u0026thinsp;CD163\u0026thinsp;+\u0026thinsp;phenotype\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo more precisely define the phenotype of MΦ induced by GCs in the context of ICI therapy, we next analyzed the RNA profile of \u003cem\u003ein vitro\u003c/em\u003e differentiated and polarized MΦ via a predesigned Nanostring nCounter panel (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). MΦ were treated with a combination of GM-CSF (day 1), IFNγ (day 7) and the synthetic glucocorticoid dexamethasone (day 1). Co-exposure to dexamethasone and GM-CSF\u0026thinsp;+\u0026thinsp;IFNγ resulted in a strong upregulation of the 'M2-like' marker \u003cem\u003eCD163\u003c/em\u003e and \u003cem\u003eMARCO\u003c/em\u003e compared to GM-CSF\u0026thinsp;+\u0026thinsp;IFNγ monotreatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Moreover, genes encoding subunits of the complement component C1Q, in particular \u003cem\u003eC1QA\u003c/em\u003e and \u003cem\u003eC1QB\u003c/em\u003e were among the most discriminatively upregulated transcripts in comparison to 'M1-like' MΦ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, D, E). Remarkably, we found significantly elevated expression of \u003cem\u003eC1QA\u003c/em\u003e and \u003cem\u003eC1QB\u003c/em\u003e also in MΦ cultured in the presence of steroidogenic NCI-H295R CM, but not JIL-2266 CM \u003cb\u003e(Fig. S4A, B)\u003c/b\u003e. Additionally, upregulation of \u003cem\u003eC1QA\u003c/em\u003e and \u003cem\u003eC1QB\u003c/em\u003e in MΦ exposed to CM of NCI-H295R was abrogated when ACC cells were cultured in the presence of the steroidogenesis inhibitor metyrapone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-H, \u003cb\u003eFig. S4C\u003c/b\u003e). Co-immunofluorescence analysis revealed a positive cytosolic staining of C1QA in cells that were also positive for CD163, supporting the presence of a C1Q\u0026thinsp;+\u0026thinsp;CD163\u0026thinsp;+\u0026thinsp;MΦ phenotype in adrenocortical tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). In ACC patients, we found \u003cem\u003eC1QA\u003c/em\u003e gene expression most upregulated in tumors with high CD163 expression levels (\u003cb\u003eFig. S4D\u003c/b\u003e). A pan-cancer analysis of TCGA data demonstrated that the \u003cem\u003eC1QA/CD68\u003c/em\u003e gene expression ratio was notably elevated in ACC samples compared to other tumor types, further supporting the hypothesis of a C1Q-dominated MΦ phenotype in GC-producing tumors, such as ACC (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). In summary, our findings indicate that local GC synthesis induces a CD163\u0026thinsp;+\u0026thinsp;MΦ type characterized by high expression of the complement component C1Q.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eGC-induced MΦ are highly efficient in C1q mediated phagocytosis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTo evaluate the functional relevance of the GC-induced C1Q\u0026thinsp;+\u0026thinsp;MΦ phenotype, we next conducted a gene set enrichment analysis (GSEA) of RNA data obtained from the Nanostring nCounter analysis. Interestingly, GSEA showed a statistically significant enrichment (p\u0026thinsp;\u0026lt;\u0026thinsp;2.2e-16; FDR: 0.01) of gene sets associated with phagocytosis in MΦ polarized with CM of the steroidogenic ACC cell line NCI-H295R (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Following this finding, we next co-cultured GC-induced MΦ with dying ACC cells and assessed their phagocytic capacity (experimental setup shown in \u003cb\u003eFig. S5A\u003c/b\u003e). As we have previously shown that ferroptosis is a prominent mode of cell death in adrenocortical cells \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, tumor cell death was initiated by treatment with the ferroptosis inducer RSL3, which led to a strong increase in phosphatidylserine (PS) exposure, as detected by Annexin V positivity (\u003cb\u003eFig. S5B, C).\u003c/b\u003e Consistent with the GSEA results, MΦ treated with NCI-H295R CM exhibited an enhanced phagocytic index compared to control treatment when co-cultured with ferroptotic ACC cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D). Similarly, MΦ polarized with the synthetic GC dexamethasone demonstrated a significantly increased phagocytic index relative to 'M1-like' MΦ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Furthermore, these MΦ displayed a notable, albeit non-significant, upregulation of genes encoding MHC class II molecules, pointing towards an improved capacity for antigen presentation. (\u003cb\u003eFig. S5D, E)\u003c/b\u003e. Given that secreted C1q has been shown to target dying cells for phagocytosis by recognizing phosphatidylserine (PS) on their surface \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e we next investigated whether adding serum-derived C1q to 'M1-like' MΦ enhances their phagocytic capacity. We observed that exposure of the ACC cell/MΦ co-culture to C1q increased the percentage of CSFE\u0026thinsp;+\u0026thinsp;MΦ in a dose-dependent manner, supporting the hypothesis of C1q-mediated phagocytosis by GC-polarized MΦ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF; \u003cb\u003eFig. S5F\u003c/b\u003e). We also observed that GCs induced the expression of MerTK on MΦ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG), an alternative mediator of phagocytosis via the MerTK/Gas6/PS axis. Intriguingly, phagocytosis by C1Q\u0026thinsp;+\u0026thinsp;MΦ was neither impaired by treatment with the selective MerTK antagonist UNC1062 nor by administration of a neutralizing antibody against its ligand Gas6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). In conclusion, these experiments demonstrate that GCs induce a highly phagocytic MΦ phenotype, primarily mediated by increased expression and secretion of the complement component C1q, rather than through the MerTK pathway.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eProstaglandin E\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003e(PGE\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003cb\u003e) is an alternative inducer of CD163\u0026thinsp;+\u0026thinsp;MΦ polarization derived from non-cortisol-secreting ACC cells\u003c/b\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTo decipher the component in the CM of the non-cs ACC cell line (JIL-2266) that was likewise capable of inducing CD163\u0026thinsp;+\u0026thinsp;MΦ polarization, we next performed a targeted LC/MS-based analysis of oxylipids of both CM. In comparison to the supernatant of steroidogenic ACC cells, we detected exceptionally elevated levels of the eicosanoid prostaglandin E\u003csub\u003e2\u003c/sub\u003e (PGE\u003csub\u003e2\u003c/sub\u003e) in the CM of the non-cortisol-secreting JIL-2266 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This observation was confirmed by PGE\u003csub\u003e2\u003c/sub\u003e ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Treatment of MΦ with PGE\u003csub\u003e2\u003c/sub\u003e in the absence of exogenous growth factors led to a concentration-independent upregulation of the MΦ marker CD163 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Accordingly, exposure to PGE\u003csub\u003e2\u003c/sub\u003e inhibited IFNγ-induced 'M1-like' polarization, while addition of the COX-2 inhibitor celecoxib prior CM collection blocked PGE\u003csub\u003e2\u003c/sub\u003e production (\u003cb\u003eFig. S6A\u003c/b\u003e) and restored 'M1-like' polarization of macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-F; \u003cb\u003eFig. S6B\u003c/b\u003e). In contrast to dexamethasone, however, PGE\u003csub\u003e2\u003c/sub\u003e or JIL-2266 CM treated MΦ showed lesser expression of \u003cem\u003eCD163\u003c/em\u003e and C1Q complement components, while the expression of \u003cem\u003eCXCL1,2\u003c/em\u003e and \u003cem\u003e8\u003c/em\u003e was most prominently increased in comparison to 'M1-like' MΦ, albeit without reaching statistical significance (\u003cb\u003eFig. S6C, D\u003c/b\u003e). Previous reports suggest GC secretion and conversion of arachidonic acid into PGE\u003csub\u003e2\u003c/sub\u003e via COX-2 (gene: \u003cem\u003ePTGS2\u003c/em\u003e) to be interconnected \u003csup\u003e\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In line, we found high expression of COX-2 in the \u003cem\u003ezona glomerulosa\u003c/em\u003e and \u003cem\u003ezona reticularis\u003c/em\u003e of the normal adrenal gland but not in the cortisol producing \u003cem\u003ezona fasciculata\u003c/em\u003e (\u003cb\u003eFig. S6E\u003c/b\u003e). \u003cem\u003eIn vitro\u003c/em\u003e, exposure of non-steroidogenic ACC cells to increasing concentrations of dexamethasone decreased \u003cem\u003ePTGS2\u003c/em\u003e mRNA and COX-2 protein expression and led to a substantial reduction in PGE\u003csub\u003e2\u003c/sub\u003e secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-I). Conversely, treatment of ACC cells with the highly specific GR antagonist relacorilant induced an increase in intracellular \u003cem\u003ePTGS2\u003c/em\u003e and COX-2 levels and a consequent increase in secreted PGE\u003csub\u003e2\u003c/sub\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ-L). These results indicate that MΦ polarization by PGE\u003csub\u003e2\u003c/sub\u003e may be predominantly associated to hormonally inactive tumors but could gain significance upon therapeutic inhibition of steroidogenesis. Supporting this idea, simultaneous treatment of steroidogenic ACC cells with metyrapone and celecoxib most effectively reduced 'M2-like' polarization following steroidogenic CM exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). Taken together, these results demonstrate that in the absence of GCs or upon GR blockade, PGE\u003csub\u003e2\u003c/sub\u003e can induce the polarization of CD163\u0026thinsp;+\u0026thinsp;MΦ. Blockade of both, tumor cell steroidogenesis and PGE\u003csub\u003e2\u003c/sub\u003e synthesis, might most efficiently reduce 'M2-like' polarization of tumoral macrophages.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eIFNγ treatment of C1Q\u0026thinsp;+\u0026thinsp;MΦ strongly induces CXCL9 expression which positively correlates with T cell infiltration and better survival in ACC\u003c/b\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUpon IFNγ stimulation, we found the T cell chemoattractants \u003cem\u003eCXCL9\u003c/em\u003e and CXCl\u003cem\u003e10\u003c/em\u003e among the most up-regulated genes in MΦ pre-exposed to dexamethasone in comparison to 'M1-like' MΦ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This finding was confirmed by CXCL9 ELISA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Likewise, exposure to hydrocortisone (HC; 500 ng/ml) strongly increased CXCL9 secretion of MΦ upon treatment with IFNγ (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Yet, full establishment of the GC-induced C1Q\u0026thinsp;+\u0026thinsp;MΦ phenotype appeared to be critical in this context, as application of dexamethasone at a later timepoint reduced the expression of C1QA and the increase in CXCL9 production (\u003cb\u003eFig. S7A, B\u003c/b\u003e). Accordingly, MΦ that were exposed to IFNγ and dexamethasone at the same timepoint did not produce higher amounts of CXCL9 in comparison to 'M1-like' MΦ (\u003cb\u003eFig. S7C\u003c/b\u003e). As CXCL9 and CXCL10 are highly potent T cell attractants by binding to its receptor CXCR3, mainly expressed on T cells \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e, we next analyzed the connection between this MΦ phenotype and T cell infiltration in ACC. In two independent datasets, the complement component \u003cem\u003eC1QA\u003c/em\u003e strongly correlated with \u003cem\u003eCD3D\u003c/em\u003e, \u003cem\u003eCD8A\u003c/em\u003e and most significantly with \u003cem\u003eCD4\u003c/em\u003e gene expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E; \u003cb\u003eFig. S7D-G\u003c/b\u003e). A pan-cancer analysis of TCGA data revealed this correlation to be true across several cancer entities (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Consistently, \u003cem\u003eCD68\u003c/em\u003e expression was positively correlated with better OS within both datasets (\u003cb\u003eFig. S7H, I\u003c/b\u003e) and better PFS within the TCGA dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). \u003cem\u003eC1QA\u003c/em\u003e gene expression was correlated with better OS and PFS in the TCGA dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, \u003cb\u003eFig. S7J\u003c/b\u003e), however this correlation was not observed in the MASTER dataset (\u003cb\u003eFig. S7K\u003c/b\u003e). Finally, in ACC patients undergoing immune checkpoint inhibition therapy (pembrolizumab or nivolumab), plasma CXCL9 concentrations were significantly elevated after the initiation of immunotherapy compared to baseline, with a more pronounced increase in CXCL9 levels observed in plasma of ACC patients with high intra-tumoral macrophage numbers (\u0026gt;\u0026thinsp;10%) than in those with low TAM counts (\u0026lt;\u0026thinsp;10%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI). Moreover, in this small cohort, a higher percentage of CD163\u0026thinsp;+\u0026thinsp;cells within respective tumor tissue samples was associated with response to ICI therapy, yet this trend was non-significant. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Together, these data suggest that the presence of C1Q+/CD163\u0026thinsp;+\u0026thinsp;macrophages in GC-producing tissues, such as ACC, may increase immunotherapy outcomes through enhancing CXCL9-dependent T cell chemoattraction in response to immunotherapy-induced IFNγ release.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWith the success of immunotherapies in many cancer entities, mechanisms that confer resistance and prevent treatment success have come into focus in molecular oncology. The potency of GCs to suppress T cell activity has been proposed to profoundly impact immunotherapy outcomes but the actual consequences of GC action in the tumoral immune microenvironment are poorly understood \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. To better characterize these local effects of glucocorticoids we here exploited the cell-autonomous secretion of GCs present in the majority of ACC, an immunologically cold tumor responding poorly to immunotherapy \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe show that tumor associated macrophages dominate the tumor immune microenvironment in ACC irrespective of the amount of tumoral steroid hormone secretion. We find that GCs lead to a specific 'M2-like' MΦ phenotype characterized by expression of the complement component C1Q and a profoundly increased phagocytic capacity. In the absence of GCs or upon GR blockade, the eicosanoid PGE\u003csub\u003e2\u003c/sub\u003e might promote polarization of 'M2-like' MΦ that however lack C1Q expression. Lastly, C1Q\u0026thinsp;+\u0026thinsp;MΦ efficiently produce the chemokine CXCL9 upon stimulation with IFNγ and are strongly associated with T cell infiltration in ACC and \u0026ndash; more importantly - across cancer entities.\u003c/p\u003e \u003cp\u003eTumoral T cell infiltration is generally considered the most crucial factor determining prognosis in cancer patients and the efficacy of immunotherapy \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. In ACC however, T cell infiltration has been shown to be scarce with only a median of 7.7 cells/HPF for CD3\u0026thinsp;+\u0026thinsp;T cells \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Intriguingly, in a recent publication addressing the efficacy of the PD-1 inhibitor camrelizumab in combination with the tyrosine kinase inhibitor apatinib, tumoral infiltration with CD8 T cells prior therapy initiation was not correlated with the hormonal status of ACC patients \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTAMs have been shown to play a critical role in determining the tumoral immune control \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. In most cancers, MΦ infiltration is linked to worse clinical outcome, supporting the notion of a tumor-promoting role of TAMs \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Surprisingly, in ACC we observed a higher abundance of MΦ, as specified by high expression of \u003cem\u003eCD68\u003c/em\u003e, to significantly correlate with improved OS and PFS. As TAMs predominantly originate from circulating monocytes that undergo differentiation into MΦ within the TME, they dynamically respond to cues from their microenvironment \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. As a consequence, the particular polarization of TAMs ultimately determines tumoral T cell infiltration \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. By utilizing both, clinical data and tissue mass spectrometry of fresh frozen ACC tissue specimens, we did not find any significant correlation between tumoral cortisol levels and MΦ numbers. Furthermore, TAMs exhibited a CD68+/CD163+ 'M2-like' phenotype in both, GC secreting and hormonally inactive tumors. In contrast to our results, a deconvolution analysis of TCGA data by Baechle \u003cem\u003eet al.\u003c/em\u003e observed a small, yet significantly increased presence of M1 MΦ and a non-significant trend towards more M2 MΦ in non-cortisol-secreting ACCs \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This small discrepancy with our study may be due to the fact that the clinical annotation of GC secretion in TCGA was used for classification by Baechle \u003cem\u003eet al.\u003c/em\u003e while we also had the opportunity to measure intra-tumoral GCs and to quantify MΦ in corresponding tissues directly. Furthermore, the lack of suitable 'M1-like' MΦ markers for IHC analysis (e.g. CD80 or CD86 which are also strongly expressed on 'M2-like' MΦ and other immune cells) did not allow us to directly address 'M1-like' MΦ in our ACC tumor cohorts.\u003c/p\u003e \u003cp\u003eDetailed RNA expression analysis from our \u003cem\u003ein vitro\u003c/em\u003e polarized MΦ revealed a significant upregulation of genes encoding the complement component C1Q in MΦ treated with dexamethasone or CM of steroidogenic ACC cells. Fluorescence microscopy confirmed the co-expression of C1QA and CD163 in adrenal macrophages, suggesting that C1Q\u0026thinsp;+\u0026thinsp;MΦ are induced by active steroidogenesis of adrenocortical cells, rather than by other tumor cell-derived factors. However, we did not observe higher \u003cem\u003eC1QA\u003c/em\u003e or \u003cem\u003eC1QB\u003c/em\u003e RNA expression in tumors with clinical and biochemical GC excess, indicating that MΦ may adopt this phenotype independently of the amount of tumoral GC production. Notably, C1Q\u0026thinsp;+\u0026thinsp;MΦ have previously been identified in other solid cancers, including melanoma, breast cancer and hepatocellular carcinoma \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Interestingly, a recent study also detected GC production in several non-adrenal tumor types. In these entities, GCs were found to be produced by recycling of inactive metabolites via 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by HSD11B1) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. By utilizing TCGA data, we found a significantly increased \u003cem\u003eC1QA/CD68\u003c/em\u003e gene expression ratio in tumor samples with high \u003cem\u003eHSD11B1\u003c/em\u003e expression across these cancer types (\u003cb\u003eFig. S8A\u003c/b\u003e), further supporting the link between local GC production and C1Q\u0026thinsp;+\u0026thinsp;macrophages in diverse tumor microenvironments.\u003c/p\u003e \u003cp\u003eCurrent knowledge on the impact of C1Q\u0026thinsp;+\u0026thinsp;MΦ on T cell function and patient prognosis is ambiguous, highlighting their complex and context-specific roles. A recent multi-omics study identified a PD-L1+/MHCII+/C1Q\u0026thinsp;+\u0026thinsp;macrophage phenotype as immunostimulatory to T cells and associated with favorable clinical outcomes in breast cancer patients \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Additionally, a FOLR2\u0026thinsp;+\u0026thinsp;TAM population (identified by high levels of \u003cem\u003eAPOE\u003c/em\u003e, \u003cem\u003eAPOC1\u003c/em\u003e, \u003cem\u003eC1QA\u003c/em\u003e and \u003cem\u003eC1QC\u003c/em\u003e) was recently identified to be associated with CD8\u0026thinsp;+\u0026thinsp;T cell infiltration in human breast cancer \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Conversely, the presence of tumoral (secreted) C1q has also been linked to the promotion of tumor growth in two distinct mouse models \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. In contrast, we did not observe C1q treatment of ACC cells to affect their proliferation over 48 hours (\u003cb\u003eFig. S8DB\u003c/b\u003e). Moreover, the proliferation of CD8\u0026thinsp;+\u0026thinsp;T cell in the presence or absence of CD3/CD28 activation remained unaltered when exposed to either C1Q\u0026thinsp;+\u0026thinsp;MΦ or 'M1-like' MΦ supernatant (\u003cb\u003eFig. S8C\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, we demonstrate that C1Q\u0026thinsp;+\u0026thinsp;macrophages are capable of producing high amounts of the T cell chemoattractant CXCL9 in response to IFNγ, a hallmark of ICI therapy \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. As GC excess in ACC patients was not associated with altered PFS or OS following ICI therapy \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, the immunosuppressive effects of GCs on T cells may not be the primary obstacle for ICI efficacy in ACC, pointing to a more complex interplay of factors influencing treatment outcomes. In addition, a recently published study on the efficacy of the PD-1 inhibitor camrelizumab in combination with apatinib in pre-treated ACC patients revealed a strong connection between the response to ICI therapy and the presence of baseline CXCR3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, underlining the relevance of this macrophage-produced cytokine on the CXCL9/CXCR3-dependent T cell chemoattraction in ACC.\u003c/p\u003e \u003cp\u003eOur data suggest that C1q-mediated phagocytosis plays a central role in the tumoricidal activity of MΦ in ACC. In line, Wilmouth \u003cem\u003eet al.\u003c/em\u003e recently demonstrated a highly phagocytic MΦ phenotype to be present in a spontaneous ACC model in male mice \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. In this study, the androgen dependent polarization of tumoricidal phagocytic MΦ completely reversed the development of adrenal tumors. Bulk RNA sequencing data from their mouse tumors identified a strong up-regulation of \u003cem\u003eC1QA/B/C\u003c/em\u003e in ACC bearing mice, which is in strong agreement with the results observed in our study. However, in the context of cancer, phagocytosis of tumor cells may be a double-edged sword. On the one hand, the clearance of tumor cells and presentation of tumoral antigens can increase the adaptive antitumoral immune response. On the other hand, clearance of cancer cells may prevent the release of damage-associated molecular patterns, thus contributing to immune evasion and tumor progression. Undoubtedly, unraveling the consequences of tumor cell clearance by MΦ in this context requires further investigation.\u003c/p\u003e \u003cp\u003eFinally, we here identified PGE\u003csub\u003e2\u003c/sub\u003e as a possible mediator of MΦ polarization in ACC. We hypothesize that the release of PGE\u003csub\u003e2\u003c/sub\u003e is confined to hormonally inactive ACCs while in GC-secreting tumors PGE\u003csub\u003e2\u003c/sub\u003e synthesis is transcriptionally suppressed in a GR-dependent manner. Our findings add to recent reports that describe PGE\u003csub\u003e2\u003c/sub\u003e as a negative regulator of immune responses which contributes to tumoral immune evasion in several tumor models \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. However, even though \u003cem\u003ein vitro\u003c/em\u003e PGE\u003csub\u003e2\u003c/sub\u003e appeared as a promising alternative mechanism of 'M2-like' polarization induced by non-cortisol-secreting ACC cells, in ACC tumors we found PGE\u003csub\u003e2\u003c/sub\u003e levels to be generally low compared to other solid tumor entities (\u003cb\u003eFig. S8D\u003c/b\u003e), underscoring that even marginal concentrations of GCs may be sufficient to suppress COX-2 expression and subsequent PGE\u003csub\u003e2\u003c/sub\u003e production, but induce the polarization of a C1Q\u0026thinsp;+\u0026thinsp;CD163\u0026thinsp;+\u0026thinsp;macrophage phenotype.\u003c/p\u003e \u003cp\u003eUndeniably, our study has certain limitations. One of the main restrictions is the small number of clinically annotated patient samples. This is primarily due to the rarity of this endocrine malignancy. Additionally, the well-established negative impact of GC excess on patient outcomes \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e may seem contradictory to the favorable impact of GC-induced C1Q\u0026thinsp;+\u0026thinsp;MΦ observed in this study. However, our data also underlines that C1Q macrophage polarization occurs under physiological cortisol concentrations within the adrenal gland, suggesting that the detrimental impact of GC excess on the immune cell compartment in ACC patients may outweigh the beneficial impact of C1Q macrophages on tumor cell clearance. Furthermore, under conditions of ICI therapy, there is currently no association between response to therapy and tumoral GC overproduction. Besides, in the NCT MASTER dataset, \u003cem\u003eC1QA\u003c/em\u003e expression was not correlated with improved OS, likely reflecting the dual effects of GCs: enhanced \u003cem\u003eC1QA\u003c/em\u003e expression alongside their adverse systemic impact. Finally, the primary limitation of this work is the lack of \u003cem\u003ein vivo\u003c/em\u003e studies. Those would be critical for confirming our results within a physiological context and assessing their potential translational impact. Yet, immunocompetent animal models of both cortisol producing and hormonally inactive ACC are scarce.\u003c/p\u003e \u003cp\u003eDespite these open questions, the results obtained in this study contribute to a better understanding and characterization of the GC-induced MΦ phenotype in ACC and other cancers. Our results highlight two important points. First, therapies that inhibit MΦ (such as CSF1R taregting therapies) have to be used very cautiously in the future especially when combined with T cell-based treatments (e.g. CAR-T or ICI). Depletion of MΦ may eradicate key mediators of tumor cell clearance e.g. by efficient phagocytosis and crucial facilitators of T cell infiltration, a major determinant of immunotherapy efficacy. Second, the presence of MΦ within ACC tissues combined with a high percentage of CXCR3\u0026thinsp;+\u0026thinsp;T cells in the blood, may serve as a potential indicator for predicting the response to ICI therapy.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eACC: adrenocortical carcinoma\u003c/p\u003e\n\u003cp\u003eCHX: cycloheximide\u003c/p\u003e\n\u003cp\u003eCOX-2: cyclooxygenase 2\u003c/p\u003e\n\u003cp\u003eCXCL9: CXC motif chemokine ligand 9\u003c/p\u003e\n\u003cp\u003eFDR: false discovery rate\u003c/p\u003e\n\u003cp\u003eGC(s): glucocorticoid(s)\u003c/p\u003e\n\u003cp\u003eGM-CSF: Granulocyte macrophage-colony stimulating factor\u003c/p\u003e\n\u003cp\u003eGR: glucocorticoid receptor\u003c/p\u003e\n\u003cp\u003eHC: hydrocortisone\u003c/p\u003e\n\u003cp\u003eICI: immune checkpoint inhibition\u003c/p\u003e\n\u003cp\u003eIF: immunofluorescence\u003c/p\u003e\n\u003cp\u003eIHC: immunohistochemistry\u003c/p\u003e\n\u003cp\u003eLip-1: Liproxstatin 1\u003c/p\u003e\n\u003cp\u003eM\u0026Phi;: macrophage\u003c/p\u003e\n\u003cp\u003eM-CSF: macrophage-colony stimulating factor\u003c/p\u003e\n\u003cp\u003eOS: overall survival\u003c/p\u003e\n\u003cp\u003ePBMC: peripheral blood mononuclear cell\u003c/p\u003e\n\u003cp\u003ePBS: phosphate buffered saline\u003c/p\u003e\n\u003cp\u003ePFA: paraformaldehyde\u003c/p\u003e\n\u003cp\u003ePFS: progression-free survival\u003c/p\u003e\n\u003cp\u003ePGE\u003csub\u003e2\u003c/sub\u003e: prostaglandin E\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003ePMA: phorbol-12-myristate-13-acetate\u003c/p\u003e\n\u003cp\u003ePS: phosphatidylserine\u003c/p\u003e\n\u003cp\u003eRSL3: (1S, 3R) Ras-selective lethal small molecule 3\u003c/p\u003e\n\u003cp\u003eTAM: tumor associated macrophage\u003c/p\u003e\n\u003cp\u003eTIME: tumor immune microenvironment\u003c/p\u003e\n\u003cp\u003eTME: tumor microenvironment\u003c/p\u003e\n\u003cp\u003eWB: western blot\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank\u0026nbsp;Prof. J\u0026ouml;rg Wischhusen, University Hospital W\u0026uuml;rzburg, for generating and sharing the Cas9 expressing THP-1 cells.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eWork in the author\u0026rsquo;s laboratories was supported by the German Research Organization (Deutsche Forschungsgemeinschaft, DFG) within the collaborative research centers (SFB Transregio) no. TRR205 (project number 314061271) to LSL, NB, MP, MR, JPFA, MF, IW and MKr.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003eConceptualization: AT, IW, MKr\u003c/p\u003e\n\u003cp\u003eMethodology: AT, TM, AG, FR, MH, MKi, NB, MP, NP\u003c/p\u003e\n\u003cp\u003eInvestigation: AT, TM, FR, MKi, NB, MP, NP, SF\u003c/p\u003e\n\u003cp\u003eVisualization: AT, TM, MKi, NP, MH\u003c/p\u003e\n\u003cp\u003eSupervision: IW, MKr,\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;original draft: AT, MKr\u003c/p\u003e\n\u003cp\u003eWriting\u0026mdash;review \u0026amp; editing: AT, TM, PS, SA, MKi, AG, FR, LSL, NB, MP, NP, MH, ST, SF, HG, DH, MF, MR, JPFA, IW, MKr\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final version of the manuscript\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eData and materials availability\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The complete RNA datasets generated during the current study can be provided on reasonable request. Requests should be submitted to the corresponding author. The use of the THP-1 monocytic line expressing Cas9 is covered by a material transfer agreement (MTA) between Prof. Dr. J\u0026ouml;rg Wischhusen and Dr. Isabel Weigand.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAcharya N, Madi A, Zhang H, Klapholz M, Escobar G, Dulberg S \u003cem\u003eet al.\u003c/em\u003e Endogenous Glucocorticoid Signaling Regulates CD8\u0026thinsp;+\u0026thinsp;T Cell Differentiation and Development of Dysfunction in the Tumor Microenvironment. Immunity 2020; 53: 658\u0026ndash;671.e6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSidler D, Renzulli P, Schnoz C, Berger B, Schneider-Jakob S, Fl\u0026uuml;ck C \u003cem\u003eet al.\u003c/em\u003e Colon cancer cells produce immunoregulatory glucocorticoids. Oncogene 2011; 30: 2411\u0026ndash;2419.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaves MD, Otsuka S, Taylor MA, Donahue KM, Meyer TJ, Cam MC \u003cem\u003eet al.\u003c/em\u003e Tumors produce glucocorticoids by metabolite recycling, not synthesis, and activate Tregs to promote growth. J Clin Invest 2023; 133: e164599.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCohen N, Mouly E, Hamdi H, Maillot M-C, Pallardy M, Godot V \u003cem\u003eet al.\u003c/em\u003e GILZ expression in human dendritic cells redirects their maturation and prevents antigen-specific T lymphocyte response. Blood 2006; 107: 2037\u0026ndash;2044.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamdi H, Godot V, Maillot M-C, Prejean MV, Cohen N, Krzysiek R \u003cem\u003eet al.\u003c/em\u003e Induction of antigen-specific regulatory T lymphocytes by human dendritic cells expressing the glucocorticoid-induced leucine zipper. Blood 2007; 110: 211\u0026ndash;219.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKerkhofs TMA, Verhoeven RHA, Van der Zwan JM, Dieleman J, Kerstens MN, Links TP \u003cem\u003eet al.\u003c/em\u003e Adrenocortical carcinoma: A population-based study on incidence and survival in the Netherlands since 1993. Eur J Cancer 2013; 49: 2579\u0026ndash;2586.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLib\u0026eacute; R. Adrenocortical carcinoma (ACC): diagnosis, prognosis, and treatment. Front Cell Dev Biol 2015; 3: 45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbabneh O, Ghazou A, Alawajneh M, Alhaj Mohammad S, Bani-Hani A, Alrabadi N \u003cem\u003eet al.\u003c/em\u003e The Efficacy and Safety of Immune Checkpoint Inhibitors in Adrenocortical Carcinoma: A Systematic Review and Meta-Analysis. Cancers 2024; 16: 900.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu Y, Cai L, Peng X. Camrelizumab plus apatinib for previously treated advanced adrenocortical carcinoma: A single-arm, open-label, phase 2 trial. J Clin Oncol 2024. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1200/JCO.2024.42.16_suppl.4511\u003c/span\u003e\u003cspan address=\"10.1200/JCO.2024.42.16_suppl.4511\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMills CD, Kincaid K, Alt JM, Heilman MJ, Hill AM. M-1/M-2 macrophages and the Th1/Th2 paradigm. J Immunol Baltim Md 1950 2000; 164: 6166\u0026ndash;6173.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Geng X, Hou J, Wu G. New insights into M1/M2 macrophages: key modulators in cancer progression. Cancer Cell Int 2021; 21: 389.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Elsas MJ, Middelburg J, Labrie C, Roelands J, Schaap G, Sluijter M \u003cem\u003eet al.\u003c/em\u003e Immunotherapy-activated T cells recruit and skew late-stage activated M1-like macrophages that are critical for therapeutic efficacy. Cancer Cell 2024; 42: 1032\u0026ndash;1050.e10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSreejit G, Fleetwood AJ, Murphy AJ, Nagareddy PR. Origins and diversity of macrophages in health and disease. Clin Transl Immunol 2020; 9: e1222.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnders CB, Lawton TMW, Smith HL, Garret J, Doucette MM, Ammons MCB. Use of integrated metabolomics, transcriptomics, and signal protein profile to characterize the effector function and associated metabotype of polarized macrophage phenotypes. J Leukoc Biol 2022; 111: 667\u0026ndash;693.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMantovani A, Allavena P, Marchesi F, Garlanda C. Macrophages as tools and targets in cancer therapy. Nat Rev Drug Discov 2022; 21: 799\u0026ndash;820.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZizzo G, Hilliard BA, Monestier M, Cohen PL. Efficient clearance of early apoptotic cells by human macrophages requires \u0026ldquo;M2c\u0026rdquo; polarization and MerTK induction. J Immunol Baltim Md 1950 2012; 189: 3508\u0026ndash;3520.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAuger J-P, Zimmermann M, Faas M, Stifel U, Chambers D, Krishnacoumar B \u003cem\u003eet al.\u003c/em\u003e Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids. Nature 2024; 629: 184\u0026ndash;192.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorak P, Heining C, Kreutzfeldt S, Hutter B, Mock A, H\u0026uuml;llein J \u003cem\u003eet al.\u003c/em\u003e Comprehensive Genomic and Transcriptomic Analysis for Guiding Therapeutic Decisions in Patients with Rare Cancers. Cancer Discov 2021; 11: 2780\u0026ndash;2795.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeigand I, Altieri B, Lacombe AMF, Basile V, Kircher S, Landwehr L-S \u003cem\u003eet al.\u003c/em\u003e Expression of SOAT1 in Adrenocortical Carcinoma and Response to Mitotane Monotherapy: An ENSAT Multicenter Study. J Clin Endocrinol Metab 2020; 105: 2642\u0026ndash;2653.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLandwehr L-S, Schreiner J, Appenzeller S, Kircher S, Herterich S, Sbiera S \u003cem\u003eet al.\u003c/em\u003e A novel patient-derived cell line of adrenocortical carcinoma shows a pathogenic role of germline MUTYH mutation and high tumour mutational burden. Eur J Endocrinol 2021; 184: 823\u0026ndash;835.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBechmann N, Watts D, Steenblock C, Wallace PW, Sch\u0026uuml;rmann A, Bornstein SR \u003cem\u003eet al.\u003c/em\u003e Adrenal Hormone Interactions and Metabolism: A Single Sample Multi-Omics Approach. Horm Metab Res Horm Stoffwechselforschung Horm Metab 2021; 53: 326\u0026ndash;334.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng Y, Bilyal A, Chen L, Schnitzler MM y, Kocabiyik J, Gudermann T \u003cem\u003eet al.\u003c/em\u003e Endothelial epoxyeicosatrienoic acid release is intact in aldosterone excess. \u003cem\u003eAtherosclerosis\u003c/em\u003e 2024; 398. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.atherosclerosis.2024.118591\u003c/span\u003e\u003cspan address=\"10.1016/j.atherosclerosis.2024.118591\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeigand I, Schreiner J, R\u0026ouml;hrig F, Sun N, Landwehr L-S, Urlaub H \u003cem\u003eet al.\u003c/em\u003e Active steroid hormone synthesis renders adrenocortical cells highly susceptible to type II ferroptosis induction. Cell Death Dis 2020; 11: 1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePa\u0026iuml;dassi H, Tacnet-Delorme P, Garlatti V, Darnault C, Ghebrehiwet B, Gaboriaud C \u003cem\u003eet al.\u003c/em\u003e C1q Binds Phosphatidylserine and Likely Acts as a Multiligand-Bridging Molecule in Apoptotic Cell Recognition. J Immunol Baltim Md 1950 2008; 180: 2329.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang YY, Arnold T, Michlmayr A, Rainprecht D, Perticevic B, Spittler A \u003cem\u003eet al.\u003c/em\u003e Serum-dependent processing of late apoptotic cells for enhanced efferocytosis. Cell Death Dis 2014; 5: e1264\u0026ndash;e1264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark A, Lee Y, Kim MS, Kang YJ, Park Y-J, Jung H \u003cem\u003eet al.\u003c/em\u003e Prostaglandin E2 Secreted by Thyroid Cancer Cells Contributes to Immune Escape Through the Suppression of Natural Killer (NK) Cell Cytotoxicity and NK Cell Differentiation. Front Immunol 2018; 9: 1859.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim W, Park C, Shim MK, Lee YH, Lee YM, Lee Y. Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoidinduced leucine zipper. Br J Pharmacol 2014; 171: 735\u0026ndash;745.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarolina E, Kato T, Khanh VC, Moriguchi K, Yamashita T, Takeuchi K \u003cem\u003eet al.\u003c/em\u003e Glucocorticoid Impaired the Wound Healing Ability of Endothelial Progenitor Cells by Reducing the Expression of CXCR4 in the PGE2 Pathway. Front Med 2018; 5. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fmed.2018.00276\u003c/span\u003e\u003cspan address=\"10.3389/fmed.2018.00276\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTokunaga R, Zhang W, Naseem M, Puccini A, Berger MD, Soni S \u003cem\u003eet al.\u003c/em\u003e CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation - A target for novel cancer therapy. Cancer Treat Rev 2018; 63: 40\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhadka S, Druffner SR, Duncan BC, Busada JT. Glucocorticoid regulation of cancer development and progression. Front Endocrinol 2023; 14: 1161768.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGalon J, Costes A, Sanchez-Cabo F, Kirilovsky A, Mlecnik B, Lagorce-Pag\u0026egrave;s C \u003cem\u003eet al.\u003c/em\u003e Type, density, and location of immune cells within human colorectal tumors predict clinical outcome. Science 2006; 313: 1960\u0026ndash;1964.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFridman WH, Pag\u0026egrave;s F, Saut\u0026egrave;s-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 2012; 12: 298\u0026ndash;306.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, Massobrio M, Regnani G \u003cem\u003eet al.\u003c/em\u003e Intratumoral T cells, recurrence, and survival in epithelial ovarian cancer. N Engl J Med 2003; 348: 203\u0026ndash;213.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLandwehr L-S, Altieri B, Schreiner J, Sbiera I, Weigand I, Kroiss M \u003cem\u003eet al.\u003c/em\u003e Interplay between glucocorticoids and tumor-infiltrating lymphocytes on the prognosis of adrenocortical carcinoma. J Immunother Cancer 2020; 8: e000469.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu K, Lin K, Li X, Yuan X, Xu P, Ni P \u003cem\u003eet al.\u003c/em\u003e Redefining Tumor-Associated Macrophage Subpopulations and Functions in the Tumor Microenvironment. Front Immunol 2020; 11. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2020.01731\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2020.01731\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMedrek C, Pont\u0026eacute;n F, Jirstr\u0026ouml;m K, Leandersson K. The presence of tumor associated macrophages in tumor stroma as a prognostic marker for breast cancer patients. BMC Cancer 2012; 12: 306.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHouse IG, Savas P, Lai J, Chen AXY, Oliver AJ, Teo ZL \u003cem\u003eet al.\u003c/em\u003e Macrophage-Derived CXCL9 and CXCL10 Are Required for Antitumor Immune Responses Following Immune Checkpoint Blockade. Clin Cancer Res Off J Am Assoc Cancer Res 2020; 26: 487\u0026ndash;504.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVermare A, Gu\u0026eacute;rin MV, Peranzoni E, Bercovici N. Dynamic CD8\u0026thinsp;+\u0026thinsp;T Cell Cooperation with Macrophages and Monocytes for Successful Cancer Immunotherapy. Cancers 2022; 14: 3546.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBaechle JJ, Hanna DN, Sekhar KR, Rathmell JC, Rathmell WK, Baregamian N. Integrative computational immunogenomic profiling of cortisol-secreting adrenocortical carcinoma. J Cell Mol Med 2021; 25: 10061\u0026ndash;10072.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Sun L, Chen Z, Liu W, Xu Q, Liu F \u003cem\u003eet al.\u003c/em\u003e The immune-metabolic crosstalk between CD3\u0026thinsp;+\u0026thinsp;C1q\u0026thinsp;+\u0026thinsp;TAM and CD8\u0026thinsp;+\u0026thinsp;T cells associated with relapse-free survival in HCC. Front Immunol 2023; 14: 1033497.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBulla R, Tripodo C, Rami D, Ling GS, Agostinis C, Guarnotta C \u003cem\u003eet al.\u003c/em\u003e C1q acts in the tumour microenvironment as a cancer-promoting factor independently of complement activation. Nat Commun 2016; 7: 10346.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRevel M, Saut\u0026egrave;s-Fridman C, Fridman W-H, Roumenina LT. C1q\u0026thinsp;+\u0026thinsp;macrophages: passengers or drivers of cancer progression. Trends Cancer 2022; 8: 517\u0026ndash;526.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Guo W, Guo Z, Yu J, Tan J, Simons DL \u003cem\u003eet al.\u003c/em\u003e PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer. Cell Rep Med 2024; 5. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.xcrm.2024.101420\u003c/span\u003e\u003cspan address=\"10.1016/j.xcrm.2024.101420\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNalio Ramos R, Missolo-Koussou Y, Gerber-Ferder Y, Bromley CP, Bugatti M, N\u0026uacute;\u0026ntilde;ez NG \u003cem\u003eet al.\u003c/em\u003e Tissue-resident FOLR2\u0026thinsp;+\u0026thinsp;macrophages associate with CD8\u0026thinsp;+\u0026thinsp;T cell infiltration in human breast cancer. Cell 2022; 185: 1189\u0026ndash;1207.e25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoumenina LT, Daugan MV, No\u0026eacute; R, Petitprez F, Vano YA, Sanchez-Salas R \u003cem\u003eet al.\u003c/em\u003e Tumor Cells Hijack Macrophage-Produced Complement C1q to Promote Tumor Growth. Cancer Immunol Res 2019; 7: 1091\u0026ndash;1105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWong CW, Huang YY, Hurlstone A. The role of IFN-γ-signalling in response to immune checkpoint blockade therapy. Essays Biochem 2023; 67: 991\u0026ndash;1002.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRemde H, Schmidt-Pennington L, Reuter M, Landwehr L-S, Jensen M, Lahner H \u003cem\u003eet al.\u003c/em\u003e Outcome of immunotherapy in adrenocortical carcinoma: a retrospective cohort study. Eur J Endocrinol 2023; 188: 485\u0026ndash;493.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilmouth JJ, Olabe J, Garcia-Garcia D, Lucas C, Guiton R, Roucher-Boulez F \u003cem\u003eet al.\u003c/em\u003e Sexually dimorphic activation of innate antitumor immunity prevents adrenocortical carcinoma development. Sci Adv 2022; 8: eadd0422.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMao Y, Sarhan D, Steven A, Seliger B, Kiessling R, Lundqvist A. Inhibition of tumor-derived prostaglandin-e2 blocks the induction of myeloid-derived suppressor cells and recovers natural killer cell activity. Clin Cancer Res Off J Am Assoc Cancer Res 2014; 20: 4096\u0026ndash;4106.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThumkeo D, Punyawatthananukool S, Prasongtanakij S, Matsuura R, Arima K, Nie H \u003cem\u003eet al.\u003c/em\u003e PGE2-EP2/EP4 signaling elicits immunosuppression by driving the mregDC-Treg axis in inflammatory tumor microenvironment. Cell Rep 2022; 39: 110914.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNastos C, Papaconstantinou D, Paspala A, Pararas N, Vryonidou A, Pikouli A \u003cem\u003eet al.\u003c/em\u003e The impact of adrenocortical carcinoma hormone secreting status as a predictor of poor survival: a systematic review and meta-analysis. Langenbecks Arch Surg 2024; 409: 316.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6213228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6213228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGlucocorticoids (GCs) play a multifaceted role in modulating immune responses in cancer. Adrenocortical carcinoma (ACC) is a rare endocrine malignancy that produces excessive glucocorticoids in ~\u0026thinsp;60%, providing a unique model to study intra-tumoral GC activity. Here, we report that ACC tumors are strongly infiltrated by CD68+/CD163+ 'M2-like' macrophages, independent of cortisol overproduction. \u003cem\u003eIn vitro\u003c/em\u003e, GC exposure drives the polarization of macrophages towards a C1Q\u0026thinsp;+\u0026thinsp;subtype with enhanced phagocytic activity, mediated by upregulated expression and secretion of the complement component C1q. IFNγ stimulation of C1Q\u0026thinsp;+\u0026thinsp;macrophages significantly enhanced the production and secretion of the T cell chemoattractant CXCL9, surpassing the concentrations produced by classical pro-inflammatory 'M1-like' macrophages. Notably, the presence of intra-tumoral macrophages correlated with increased T cell infiltration, improved patient survival and response to ICI therapy in ACC. Collectively, this study identifies a GC-driven CD68+/CD163+/C1Q\u0026thinsp;+\u0026thinsp;macrophage phenotype with high phagocytic capacity and IFNγ-induced cytokine secretion, suggesting a potential role in T cell recruitment and the enhancement of immunotherapy efficacy in ACC and other solid tumors.\u003c/p\u003e","manuscriptTitle":"Tumoral glucocorticoids induce a phagocytic CD68+/CD163+/C1Q+ macrophage phenotype primed for IFNγ-driven CXCL9 secretion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 11:07:18","doi":"10.21203/rs.3.rs-6213228/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8583dc99-42e9-46d7-aeef-fd99a1108859","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45917614,"name":"Biological sciences/Immunology/Tumour immunology/Immunosurveillance/Immunoediting"},{"id":45917615,"name":"Biological sciences/Cancer/Cancer microenvironment"},{"id":45917616,"name":"Health sciences/Oncology/Cancer/Endocrine cancer/Adrenal tumours"},{"id":45917617,"name":"Health sciences/Biomarkers/Prognostic markers"},{"id":45917618,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages/Phagocytes"}],"tags":[],"updatedAt":"2025-05-19T14:25:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-31 11:07:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6213228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6213228","identity":"rs-6213228","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}