Strain-Release-Driven Modular Synthesis of Oxetane-Based Amide Bioisosteres: Concise, Robust and Scalable Approach.

Amide bioisoterism is a widely used strategy in drug development to fine-tune physicochemical, pharmacokinetic, and metabolic properties, eliminate toxicity and gain intellectual property rights in uncharted chemical space. Of these, oxetane-amines offer particularly exciting possibilities as bioisosteres, although they are less frequently investigated than warranted due to the lack of simple and widely applicable synthetic methods. Herein, we report a two-step, practical, modular, robust, and scalable method for the construction of oxetane-containing amide bioisosteres that relies on the readily available oxetan-3-one. This operationally simple procedure exploits the enhanced reactivity of the keto group of the commercially available oxetan-3-one to form amine-benzotriazole intermediates, which springloaded adducts are then reacted with various aliphatic and aromatic organometallic reagents under mild conditions to afford various amino-oxetanes in good to high yields. The simplicity and broad applicability of the method greatly facilitates the synthesis of derivatives that were previously difficult or impossible to produce. The usefulness of this method in the field medicinal chemistry was also demonstrated by eliminating the well-known metabolic problem of ketoconazole.

Synthesis and Systematic Investigation of Lepidiline A and Its Gold(I), Silver(I), and Copper(I) Complexes Using In Vitro Cancer Models and Multipotent Stem Cells.

The imidazole alkaloid lepidiline A from the root of Lepidium meyenii has a moderate to low in vitro anticancer effect. Our aim was to extend cytotoxicity investigations against a panel of cancer cells, including multidrug-resistant cancer cells, and multipotent stem cells. Lepidiline A is a N-heterocyclic carbene precursor, therefore a suitable ligand source for metal complexes. Thus, we synthesized lepidiline A and its copper(I), gold(I), and silver(I) complexes and tested them against ovarian, gastrointestinal, breast, and uterine cancer cells and bone marrow-derived and adipose-derived mesenchymal stem cells. Lepidiline A and its copper complex demonstrated moderate cytotoxicity, while silver and gold complexes exhibited significantly enhanced and consistent cytotoxicity against both cancer and stem cell lines. ABCB1 in the multidrug-resistant uterine sarcoma line conferred significant resistance against lepidiline A and the copper-lepidiline A complex, but not against the silver and gold complexes. Our results indicate that only the copper complex induced a significant and universal increase in the production of reactive oxygen species within cells. In summary, binding of metal ions to lepidiline A results in enhanced cytotoxicity with the nature of the metal ion playing a critical role in determining its properties.

Characterisation of Canine and Feline Breast Tumours, Their Metastases, and Corresponding Primary Cell Lines Using LA-REIMS and DESI-MS Imaging.

Breast cancer, a complex disease with a significant prevalence to form metastases, necessitates novel therapeutic strategies to improve treatment outcomes. Here, we present the results of a comparative molecular study of primary breast tumours, their metastases, and the corresponding primary cell lines using Desorption Electrospray Ionisation (DESI) and Laser-Assisted Rapid Evaporative Ionisation Mass Spectrometry (LA-REIMS) imaging. Our results show that ambient ionisation mass spectrometry technology is suitable for rapid characterisation of samples, providing a lipid- and metabolite-rich spectrum within seconds. Our study demonstrates that the lipidomic fingerprint of the primary tumour is not significantly distinguishable from that of its metastasis, in parallel with the similarity observed between their respective primary cell lines. While significant differences were observed between tumours and the corresponding cell lines, distinct lipidomic signatures and several phospholipids such as PA(36:2), PE(36:1), and PE(P-38:4)/PE(O-38:5) for LA-REIMS imaging and PE(P-38:4)/PE(O-38:5), PS(36:1), and PI(38:4) for DESI-MSI were identified in both tumours and cells. We show that the tumours' characteristics can be found in the corresponding primary cell lines, offering a promising avenue for assessing tumour responsiveness to therapeutic interventions. A comparative analysis by DESI-MSI and LA-REIMS imaging revealed complementary information, demonstrating the utility of LA-REIMS in the molecular imaging of cancer.

Segmentation and Multi-Timepoint Tracking of 3D Cancer Organoids from Optical Coherence Tomography Images Using Deep Neural Networks.

Recent years have ushered in a transformative era in in vitro modeling with the advent of organoids, three-dimensional structures derived from stem cells or patient tumor cells. Still, fully harnessing the potential of organoids requires advanced imaging technologies and analytical tools to quantitatively monitor organoid growth. Optical coherence tomography (OCT) is a promising imaging modality for organoid analysis due to its high-resolution, label-free, non-destructive, and real-time 3D imaging capabilities, but accurately identifying and quantifying organoids in OCT images remain challenging due to various factors. Here, we propose an automatic deep learning-based pipeline with convolutional neural networks that synergistically includes optimized preprocessing steps, the implementation of a state-of-the-art deep learning model, and ad-hoc postprocessing methods, showcasing good generalizability and tracking capabilities over an extended period of 13 days. The proposed tracking algorithm thoroughly documents organoid evolution, utilizing reference volumes, a dual branch analysis, key attribute evaluation, and probability scoring for match identification. The proposed comprehensive approach enables the accurate tracking of organoid growth and morphological changes over time, advancing organoid analysis and serving as a solid foundation for future studies for drug screening and tumor drug sensitivity detection based on organoids.

Copper(II) Complexes with Isomeric Morpholine-Substituted 2-Formylpyridine Thiosemicarbazone Hybrids as Potential Anticancer Drugs Inhibiting Both Ribonucleotide Reductase and Tubulin Polymerization: The Morpholine Position Matters.

The development of copper(II) thiosemicarbazone complexes as potential anticancer agents, possessing dual functionality as inhibitors of R2 ribonucleotide reductase (RNR) and tubulin polymerization by binding at the colchicine site, presents a promising avenue for enhancing therapeutic effectiveness. Herein, we describe the syntheses and physicochemical characterization of four isomeric proligands H2L3-H2L6, with the methylmorpholine substituent at pertinent positions of the pyridine ring, along with their corresponding Cu(II) complexes 3-6. Evidently, the position of the morpholine moiety and the copper(II) complex formation have marked effects on the in vitro antiproliferative activity in human uterine sarcoma MES-SA cells and the multidrug-resistant derivative MES-SA/Dx5 cells. Activity correlated strongly with quenching of the tyrosyl radical (Y•) of mouse R2 RNR protein, inhibition of RNR activity in the cancer cells, and inhibition of tubulin polymerization. Insights into the mechanism of antiproliferative activity, supported by experimental results and molecular modeling calculations, are presented.

Mesenchymal Stem Cells Increase Drug Tolerance of A431 Cells Only in 3D Spheroids, Not in 2D Co-Cultures.

Mesenchymal stem cells (MSCs) are an integral part of the tumor microenvironment (TME); however, their role is somewhat controversial: conflicting reports suggest that, depending on the stage of tumor development, MSCs can either support or suppress tumor growth and spread. Additionally, the influence of MSCs on drug resistance is also ambiguous. Previously, we showed that, despite MSCs proliferating significantly more slowly than cancer cells, there are chemotherapeutic drugs which proved to be similarly toxic to both cell types. Here we established 2D co-cultures and 3D co-culture spheroids from different ratios of GFP-expressing, adipose tissue-derived MSCs and A431 epidermoid carcinoma cells tagged with mCherry to investigate the effect of MSCs on cancer cell growth, survival, and drug sensitivity. We examined the cytokine secretion profile of mono- and co-cultures, explored the inner structure of the spheroids, applied MSC-(nutlin-3) and cancer cell-targeting (cisplatin) treatments separately, monitored the response with live-cell imaging and identified a new, double-fluorescent cell type emerging from these cultures. In 2D co-cultures, no effect on proliferation or drug sensitivity was observed, regardless of the changes in cytokine secretion induced by the co-culture. Conversely, 3D spheroids developed a unique internal structure consisting of MSCs, which significantly improved cancer cell survival and resilience to treatment, suggesting that physical proximity and cell-cell connections are required for MSCs to considerably affect nearby cancer cells. Our results shed light on MSC-cancer cell interactions and could help design new, better treatment options for tumors.

DNA-dependent phase separation by human SSB2 (NABP1/OBFC2A) protein points to adaptations to eukaryotic genome repair processes.

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all domains of life and play essential roles via stabilizing and protecting single-stranded (ss) DNA as well as organizing multiprotein complexes during DNA replication, recombination, and repair. Two mammalian SSB paralogs (hSSB1 and hSSB2 in humans) were recently identified and shown to be involved in various genome maintenance processes. Following our recent discovery of the liquid-liquid phase separation (LLPS) propensity of Escherichia coli (Ec) SSB, here we show that hSSB2 also forms LLPS condensates under physiologically relevant ionic conditions. Similar to that seen for EcSSB, we demonstrate the essential contribution of hSSB2's C-terminal intrinsically disordered region (IDR) to condensate formation, and the selective enrichment of various genome metabolic proteins in hSSB2 condensates. However, in contrast to EcSSB-driven LLPS that is inhibited by ssDNA binding, hSSB2 phase separation requires single-stranded nucleic acid binding, and is especially facilitated by ssDNA. Our results reveal an evolutionarily conserved role for SSB-mediated LLPS in the spatiotemporal organization of genome maintenance complexes. At the same time, differential LLPS features of EcSSB and hSSB2 point to functional adaptations to prokaryotic versus eukaryotic genome metabolic contexts.

Complex formation of ML324, the histone demethylase inhibitor, with essential metal ions: Relationship between solution chemistry and anticancer activity.

N-(3-(dimethylamino)propyl-4-(8-hydroxyquinolin-6-yl)benzamide (ML324, HL) is a potent inhibitor of the iron-containing histone demethylase KDM4, a recognized potential target of cancer therapeutics. Herein, we report the proton dissociation and complex formation processes of ML324 with essential metal ions such as Fe(II), Fe(III), Cu(II) and Zn(II) using UV-visible, fluorescence, electron paramagnetic resonance and 1H NMR spectroscopic methods. The electrochemical behaviour of the copper and iron complexes was characterized by cyclic voltammetry and spectroelectrochemistry. The solid phase structure of ML324 analysed by X-ray crystallography is also provided. Based on the solution equilibrium data, ML324 is present in solution in H2L+ form with a protonated dimethylammonium moiety at pH 7.4, and this (N,O) donor bearing ligand forms mono and bis complexes with all the studied metal ions and the tris-ligand species is also observed with Fe(III). At pH 7.4 the metal binding ability of ML324 follows the order: Fe(II) < Zn(II) < Cu(II) < Fe(III). Complexation with iron resulted in a negative redox potential (E'1/2 = -145 mV vs. NHE), further suggesting that the ligand has a preference for Fe(III) over Fe(II). ML324 was tested for its anticancer activity in chemosensitive and resistant human cancer cells overexpressing the efflux pump P-glycoprotein. ML324 exerted similar activity in all tested cells (IC50 = 1.9-3.6 µM). Co-incubation and complexation of the compound with Cu(II) and Zn(II) had no impact on the cytotoxicity of ML324, whereas Fe(III) decreased the toxicity in a concentration-dependent manner, and this effect was more pronounced in the multidrug resistant cells.

ABCC1 and glutathione metabolism limit the efficacy of BCL-2 inhibitors in acute myeloid leukemia.

The BCL-2 inhibitor Venetoclax is a promising agent for the treatment of acute myeloid leukemia (AML). However, many patients are refractory to Venetoclax, and resistance develops quickly. ATP-binding cassette (ABC) transporters mediate chemotherapy resistance but their role in modulating the activity of targeted small-molecule inhibitors is unclear. Using CRISPR/Cas9 screening, we find that loss of ABCC1 strongly increases the sensitivity of AML cells to Venetoclax. Genetic and pharmacologic ABCC1 inactivation potentiates the anti-leukemic effects of BCL-2 inhibitors and efficiently re-sensitizes Venetoclax-resistant leukemia cells. Conversely, ABCC1 overexpression induces resistance to BCL-2 inhibitors by reducing intracellular drug levels, and high ABCC1 levels predicts poor response to Venetoclax therapy in patients. Consistent with ABCC1-specific export of glutathionylated substrates, inhibition of glutathione metabolism increases the potency of BCL-2 inhibitors. These results identify ABCC1 and glutathione metabolism as mechanisms limiting efficacy of BCL-2 inhibitors, which may pave the way to development of more effective therapies.

Effective targeting of breast cancer by the inhibition of P-glycoprotein mediated removal of toxic lipid peroxidation byproducts from drug tolerant persister cells.

Therapy resistance has long been considered to occur through the selection of pre-existing clones equipped to survive and quickly regrow, or through the acquisition of mutations during chemotherapy. Here we show that following in vitro treatment by chemotherapy, epithelial breast cancer cells adopt a transient drug tolerant phenotype characterized by cell cycle arrest, epithelial-to-mesenchymal transition (EMT) and the reversible upregulation of the multidrug resistance (MDR) efflux transporter P-glycoprotein (P-gp). The drug tolerant persister (DTP) state is reversible, as cells eventually resume proliferation, giving rise to a cell population resembling the initial, drug-naïve cell lines. However, recovery after doxorubicin treatment is almost completely eliminated when DTP cells are cultured in the presence of the P-gp inhibitor Tariquidar. Mechanistically, P-gp contributes to the survival of DTP cells by removing reactive oxygen species-induced lipid peroxidation products resulting from doxorubicin exposure. In vivo, prolonged administration of Tariquidar during doxorubicin treatment holidays resulted in a significant increase of the overall survival of Brca1-/-;p53-/- mammary tumor bearing mice. These results indicate that prolonged administration of a P-gp inhibitor during drug holidays would likely benefit patients without the risk of aggravated side effects related to the concomitantly administered toxic chemotherapy. Effective targeting of DTPs through the inhibition of P-glycoprotein may result in a paradigm shift, changing the focus from countering drug resistance mechanisms to preventing or delaying therapy resistance.

Nucleotide binding is the critical regulator of ABCG2 conformational transitions.

ABCG2 is an exporter-type ABC protein that can expel numerous chemically unrelated xeno- and endobiotics from cells. When expressed in tumor cells or tumor stem cells, ABCG2 confers multidrug resistance, contributing to the failure of chemotherapy. Molecular details orchestrating substrate translocation and ATP hydrolysis remain elusive. Here, we present methods to concomitantly investigate substrate and nucleotide binding by ABCG2 in cells. Using the conformation-sensitive antibody 5D3, we show that the switch from the inward-facing (IF) to the outward-facing (OF) conformation of ABCG2 is induced by nucleotide binding. IF-OF transition is facilitated by substrates, and hindered by the inhibitor Ko143. Direct measurements of 5D3 and substrate binding to ABCG2 indicate that the high-to-low affinity switch of the drug binding site coincides with the transition from the IF to the OF conformation. Low substrate binding persists in the post-hydrolysis state, supporting that dissociation of the ATP hydrolysis products is required to reset the high substrate affinity IF conformation of ABCG2.

Covalent fragment mapping of KRasG12C revealed novel chemotypes with in vivo potency.

G12C mutant KRas is considered druggable by allele-specific covalent inhibitors due to the nucleophilic character of the oncogenic mutant cysteine at position 12. Discovery of these inhibitors requires the optimization of both covalent and noncovalent interactions. Here, we report covalent fragment screening of our electrophilic fragment library of diverse non-covalent scaffolds equipped with 40 different electrophilic functionalities to identify fragments as suitable starting points targeting Cys12. Screening the library against KRasG12C using Ellman's free thiol assay, followed by protein NMR and cell viability assays, resulted in two potential inhibitor chemotypes. Characterization of these scaffolds in in vitro cellular- and in vivo xenograft models revealed them as promising starting points for covalent drug discovery programs.

Comparison of Different Clinical Chemotherapeutical Agents' Toxicity and Cell Response on Mesenchymal Stem Cells and Cancer Cells.

Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME.

P-Glycoprotein Activity at Diagnosis Does Not Predict Therapy Outcome and Survival in Canine B-Cell Lymphoma.

Various mechanisms are known to be involved in the development of multidrug resistance during cancer treatment. P-glycoprotein (P-gp) decreases the intracellular concentrations of cytotoxic drugs by an energy-dependent efflux mechanism. The aim of this study was to investigate the predictive value of P-gp function based on the evaluation of P-gp activity in tumor cells obtained from canine B-cell lymphoma patients at diagnosis. P-gp function of 79 immunophenotyped canine lymphoma samples was determined by flow cytometry using the Calcein assay. Dogs were treated with either the CHOP or the L-CHOP protocol, a subset of relapsed patients received L-asparaginase and lomustine rescue treatments. Among the 79 dogs, the median overall survival time was 417 days, and the median relapse-free period was 301 days. 47 percent of the samples showed high P-gp activity, which was significantly higher in Stage IV cancer patients compared to Stage II + III and V. Whereas staging was associated with major differences in survival times, we found that the intrinsic P-gp activity of tumor cells measured at diagnosis is not predictive for therapy outcome. Further studies are needed to identify the intrinsic and acquired resistant mechanisms that shape therapy response and survival in B-cell canine lymphoma patients.

Cytotoxicity of cinchona alkaloid organocatalysts against MES-SA and MES-SA/Dx5 multidrug-resistant uterine sarcoma cell lines.

Since the first application of natural quinine as an anti-malarial drug, cinchona alkaloids and their derivatives have been exhaustively studied for their biological activity. In our work, we tested 13 cinchona alkaloid organocatalysts, synthesised from quinine. These derivatives were screened against MES-SA and Dx5 uterine sarcoma cell lines for in vitro anticancer activity and to investigate their potential to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR). Decorating quinine with hydrogen-bond donor units, such as thiourea and (thio)squaramide, resulted in decreased half-maximal growth inhibition values on both cell lines (1.3-21 µM) compared to quinine and other cinchona alcohols (47-111 µM). Further cytotoxicity studies conducted in the presence of the P-gp inhibitor tariquidar indicated that several analogues, especially cinchona amines and squaramides, but not thiosquaramide, were expelled from MDR cells by P-gp. Similarly to the established P-gp inhibitor quinine, 6 cinchona analogues were shown to inhibit calcein-AM efflux. Interestingly, quinine and didehydroquinine exhibited a marginally increased toxicity against the multidrug resistant Dx5 cells. Collateral sensitivity of the MDR cell line was more pronounced when the cinchona thiosquaramide was complexed with Cu(II) acetate. Based on the results, cinchona derivatives are good anticancer candidates for further drug development.

Structure-Activity Relationships of 8-Hydroxyquinoline-Derived Mannich Bases with Tertiary Amines Targeting Multidrug-Resistant Cancer.

A recently proposed strategy to overcome multidrug resistance (MDR) in cancer is to target the collateral sensitivity of otherwise resistant cells. We designed a library of 120 compounds to explore the chemical space around previously identified 8-hydroxyquinoline-derived Mannich bases with robust MDR-selective toxicity. We included compounds to study the effect of halogen and alkoxymethyl substitutions in R5 in combination with different Mannich bases in R7, a shift of the Mannich base from R7 to R5, as well as the introduction of an aromatic moiety. Cytotoxicity tests performed on a panel of parental and MDR cells highlight a strong influence of experimentally determined pKa values of the donor atom moieties, indicating that protonation and metal chelation are important factors modulating the MDR-selective anticancer activity of the studied compounds. Our results identify structural requirements increasing MDR-selective anticancer activity, providing guidelines for the development of more effective anticancer chelators targeting MDR cancer.

Efficient Synthesis of Acylated, Dialkyl α-Hydroxy-Benzylphosphonates and Their Anticancer Activity.

An efficient method applying acyl chlorides as reagents was developed for the acylation of the hindered hydroxy group of dialkyl α-hydroxy-benzylphosphonates. The procedure did not require any catalyst. A few acylations were also performed with the SC-enantiomer of dimethyl α-hydroxy-benzylphosphonate, and the optical purity was retained. A part of the acyloxyphosphonates was tested against eight tumor cell lines of different tissue origin at c = 50 µM concentration. The compounds elicited moderate cytostatic effect against breast, skin, prostate, colon, and lung carcinomas; a melanoma cell line; and against Kaposi's sarcoma cell lines. Then, dose-dependent cytotoxicity was assayed, and benzoylation of the α-hydroxy group was identified as a moiety that increases anticancer cytotoxicity across all cell lines. Surprisingly, a few analogues were more toxic to multidrug resistant cancer cell lines, thus evading P-glycoprotein mediated drug extrusion.

Identifying Novel Inhibitors for Hepatic Organic Anion Transporting Polypeptides by Machine Learning-Based Virtual Screening.

Integration of statistical learning methods with structure-based modeling approaches is a contemporary strategy to identify novel lead compounds in drug discovery. Hepatic organic anion transporting polypeptides (OATP1B1, OATP1B3, and OATP2B1) are classical off-targets, and it is well recognized that their ability to interfere with a wide range of chemically unrelated drugs, environmental chemicals, or food additives can lead to unwanted adverse effects like liver toxicity and drug-drug or drug-food interactions. Therefore, the identification of novel (tool) compounds for hepatic OATPs by virtual screening approaches and subsequent experimental validation is a major asset for elucidating structure-function relationships of (related) transporters: they enhance our understanding about molecular determinants and structural aspects of hepatic OATPs driving ligand binding and selectivity. In the present study, we performed a consensus virtual screening approach by using different types of machine learning models (proteochemometric models, conformal prediction models, and XGBoost models for hepatic OATPs), followed by molecular docking of preselected hits using previously established structural models for hepatic OATPs. Screening the diverse REAL drug-like set (Enamine) shows a comparable hit rate for OATP1B1 (36% actives) and OATP1B3 (32% actives), while the hit rate for OATP2B1 was even higher (66% actives). Percentage inhibition values for 44 selected compounds were determined using dedicated in vitro assays and guided the prioritization of several highly potent novel hepatic OATP inhibitors: six (strong) OATP2B1 inhibitors (IC50 values ranging from 0.04 to 6 µM), three OATP1B1 inhibitors (2.69 to 10 µM), and five OATP1B3 inhibitors (1.53 to 10 µM) were identified. Strikingly, two novel OATP2B1 inhibitors were uncovered (C7 and H5) which show high affinity (IC50 values: 40 nM and 390 nM) comparable to the recently described estrone-based inhibitor (IC50 = 41 nM). A molecularly detailed explanation for the observed differences in ligand binding to the three transporters is given by means of structural comparison of the detected binding sites and docking poses.

Metal Complexes of a 5-Nitro-8-Hydroxyquinoline-Proline Hybrid with Enhanced Water Solubility Targeting Multidrug Resistant Cancer Cells.

Multidrug resistance (MDR) in cancer is one of the major obstacles of chemotherapy. We have recently identified a series of 8-hydroxyquinoline Mannich base derivatives with MDR-selective toxicity, however with limited solubility. In this work, a novel 5-nitro-8-hydroxyquinoline-proline hybrid and its Rh(η5-C5Me5) and Ru(η6-p-cymene) complexes with excellent aqueous solubility were developed, characterized, and tested against sensitive and MDR cells. Complex formation of the ligand with essential metal ions was also investigated using UV-visible, circular dichroism, 1H NMR (Zn(II)), and electron paramagnetic resonance (Cu(II)) spectroscopic methods. Formation of mono and bis complexes was found in all cases with versatile coordination modes, while tris complexes were also formed with Fe(II) and Fe(III) ions, revealing the metal binding affinity of the ligand at pH 7.4: Cu(II) > Zn(II) > Fe(II) > Fe(III). The ligand and its Rh(III) complex displayed enhanced cytotoxicity against the resistant MES-SA/Dx5 and Colo320 human cancer cell lines compared to their chemosensitive counterparts. Both organometallic complexes possess high stability in solution, however the Ru(II) complex has lower chloride ion affinity and slower ligand exchange processes, along with the readiness to lose the arene ring that is likely connected to its inactivity.

Mesenchymal-Stromal Cell-like Melanoma-Associated Fibroblasts Increase IL-10 Production by Macrophages in a Cyclooxygenase/Indoleamine 2,3-Dioxygenase-Dependent Manner.

Melanoma-associated fibroblasts (MAFs) are integral parts of melanoma, providing a protective network for melanoma cells. The phenotypical and functional similarities between MAFs and mesenchymal stromal cells (MSCs) prompted us to investigate if, similarly to MSCs, MAFs are capable of modulating macrophage functions. Using immunohistochemistry, we showed that MAFs and macrophages are in intimate contact within the tumor stroma. We then demonstrated that MAFs indeed are potent inducers of IL-10 production in various macrophage types in vitro, and this process is greatly augmented by the presence of treatment-naïve and chemotherapy-treated melanoma cells. MAFs derived from thick melanomas appear to be more immunosuppressive than those cultured from thin melanomas. The IL-10 increasing effect is mediated, at least in part, by cyclooxygenase and indoleamine 2,3-dioxygenase. Our data indicate that MAF-induced IL-10 production in macrophages may contribute to melanoma aggressiveness, and targeting the cyclooxygenase and indoleamine 2,3-dioxygenase pathways may abolish MAF-macrophage interactions.