Laser ablation inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS) enables the quantitative imaging of metal-based therapeutics with cellular resolution. Despite its analytical power, systematic screening of metal-containing anticancer agents remains limited by the substantial time, cost, and labor required for cell handling, staining, and analysis. Here, we present a barcoding strategy that allows us to pool multiple experiments together and analyze them simultaneously. Our labeling approach combines wheat germ agglutinin (WGA) with lanthanide-labeled anti-WGA barcodes, enabling robust discrimination of experimental conditions. Following LA-ICP-TOFMS measurement, pooled data sets can be processed using MeXpose, a data processing pipeline for single cells, to accurately assign each cell to its original barcode, i.e., experiment. In this study, we applied this novel strategy to investigate the uptake of BOLD-100 and oxaliplatin in HCT116 wild-type (WT) and oxaliplatin-resistant (OxR) colorectal cancer cells using different drug concentrations. Pooling 10 experiments into a single analytical run allowed us to reduce consumable use (mainly argon for the ICP and antibody usage if stained), measurement time, and downstream processing while increasing data consistency. Barcoding-enabled single-cell analysis confirmed a substantial reduction in oxaliplatin uptake in oxaliplatin-resistant HCT116 cells compared with the WT cell line, whereas BOLD-100 uptake was affected to a much lesser extent. These results demonstrate the utility of this strategy for the efficient and scalable assessment of metal-based therapeutics.

Systemic application of Toll-like receptor 7/8 (TLR7/8) agonists against cancer is severely limited due to uncontrolled immune activation. In this study, a platinum(IV)-based prodrug strategy is developed for the systemic administration of a TLR7/8 agonist, selectively activated in the malignant tissue simultaneously with the immunogenic cell death inducer oxaliplatin. Two oxaliplatin(IV)-based complexes are synthesized comprising the TLR7/8 agonist gardiquimod: Ox-Gardi-PEG, containing polyethylene glycol as the second axial ligand, and Ox-Gardi-Mal, a maleimide-bearing derivative to exploit the tumor-targeting effects of serum albumin. In vitro, cytotoxicity and immune pathway-inducing potency of Ox-Gardi-PEG and Ox-Gardi-Mal are diminished under standard cell culture conditions compared to free oxaliplatin and gardiquimod, respectively, and markedly enhanced under reducing conditions, underscoring the activation-by-reduction concept. In vivo, Ox-Gardi-Mal shows superior and TLR7/8 signaling-dependent anticancer efficacy and prolongs overall survival of cancer-bearing mice, while mitigating hematotoxic effects associated with oxaliplatin. Therapy significantly elevates expression of MHC-I on antigen-presenting immune cell subsets, increases the frequency of activated plasmacytoid dendritic cells and tumor-infiltrating CD8+ T cells, as well as depleted primarily immunosuppressive M2 macrophages. These results demonstrate that tumor-targeted oxaliplatin(IV)-based prodrugs carrying TLR7/8 agonists offer a potent dual-release strategy for improved immunochemotherapy, while minimizing excessive immune responses associated with systemic TLR7/8 activation.

We report on the synthesis and characterization of four novel Ru(II) polypyridyl complexes incorporating bipyridine or phenanthroline ancillary ligands. Substituted 1,2,4-oxadiazoles are introduced here for the first time as chelating ligands for Ru(II), and the structure of a representative complex unequivocally established by single-crystal X-ray diffraction. The antiproliferative activity of the metal complexes in human colorectal (HCT116 and oxaliplatin- and BOLD-100-resistant derivatives) and ovarian (A2780 and A2780cis) cancer cell lines was evaluated. The presence of phenanthroline ancillary ligands induced the highest cytotoxicity, with platinum-resistant cells displaying enhanced sensitivity compared to parental lines. Cellular uptake of the metal complexes was quantified by ICP-MS. Finally, in vivo tests were conducted in BALB/c mice using the most promising compound, to evaluate its toxicity profile and its ability to inhibit CT-26 tumor growth. Interestingly, both duplex and G-quadruplex DNA can be excluded as potential molecular targets, as assessed by FRET-melting assays, UV-Vis spectroscopy, and circular dichroism.

BACKGROUND:Despite the widespread clinical use of prostate-specific membrane antigen (PSMA)-targeted imaging and therapy in prostate cancer, the biological functions underlying PSMA-associated tumour aggressiveness remain incompletely understood. METHODS:PSMA-PET-guided sampling of paired PSMA-high and PSMA-low tumour regions was used for integrated proteomic and epigenomic analyses. PSMA-ATGL correlation was validated by immunohistochemistry in 90 treatment-naïve patients. Metabolic profiling was performed using Seahorse Mito Fuel Flex Test assays under unperturbed conditions and pharmacological or genetic perturbation of ATGL and PSMA. Therapeutic vulnerability was assessed using NG-497 and siRNA-mediated knockdown. RESULTS:PSMA-high tumours exhibited lipolytic reprogramming characterised by ATGL upregulation, confirmed by a robust PSMA-ATGL correlation in the validation cohort (P < 0.0001). Despite shared fatty acid dependency, LNCaP cells exhibited intrinsic metabolic inflexibility while 22RV1 cells displayed high adaptability. Both pharmacological and genetic ATGL inhibition most potently impaired LNCaP proliferation. Enzalutamide upregulated ATGL in both cell lines, revealing reciprocal regulation between AR signaling and the PSMA/ATGL axis. CONCLUSIONS:ATGL-mediated lipolysis represents a promising therapeutic target in prostate cancer, with heightened sensitivity in tumours lacking metabolic flexibility to utilize alternative pathways.

High-grade gliomas (HGGs) are the most aggressive adult brain tumors, with a dismal median survival of approximately 15 months, highlighting the need for novel therapeutic strategies. In a prior immunotherapy trial using dendritic cells against glioblastoma, miR-216b emerged as a potential predictive biomarker. Thus, we hypothesize that miR-216b impacts glioma aggressiveness and thereby therapeutic success. Here, we demonstrate that miR-216b is significantly downregulated in the majority of Isocitrate dehydrogenase 1/2 (IDH) wild-type HGG tissue samples (n = 42) and cell models (n = 18). Functional assays revealed that miR-216b overexpression impairs glioma cell proliferation, migration, and stemness characteristics. Transcriptomic and target prediction analyses identified CDK4, a key cell cycle regulator, as a direct target of miR-216b, confirmed via luciferase reporter assays. Correspondingly, upregulating miR-216b (mimic) via transfection decreased CDK4 mRNA and protein levels accompanied by a p21-dependent increase of cells in G0/G1 phase. In addition, miR-216b expression correlated with increased sensitivity to the CDK4/6 inhibitor Abemaciclib. Notably, miR-216b levels were significantly higher in less aggressive IDH-mutant gliomas (n = 21), linking its downregulation to malignancy grade. Collectively, our findings discovered miR-216b as a tumor suppressor in HGGs, modulating CDK4 expression and affecting the responsiveness to CDK4/6 inhibitors. The observed results support the potential of miR-216b as both a prognostic and predictive indicator in HGGs.

Reactive oxygen species (ROS)-induced aberrant oncogenic signalling has been proposed to mediate the progression and development of pleural mesothelioma (PM). In this study, we demonstrate how ROS promote oncogenic signalling, especially in the context of cell migration and immune evasion via YB-1 phosphorylation in mesothelial and PM cell models. Xanthine (X)- and xanthine oxidase (XO)-generated ROS exposure led to increased migration and a more elongated cell shape in mesothelial and PM cells in live-cell videomicroscopy analyses. These effects were associated with the enhanced phosphorylation of ERK, AKT, and YB-1 and the elevated gene expression of PD-L1 and PD-L2, which were analysed with immunoblotting and quantitative real-time RT-PCR, respectively. The pharmacological inhibition of AKT (ipatasertib), MEK (trametinib), and RSK (BI-D1870) resulted in the reversal of ROS-induced effects, with the strongest effects observed upon the inhibition of YB-1 phosphorylation by BI-D1870. The results suggest that ROS exposure has a strong impact on cell migration and immune evasion not only in PM cells but also in mesothelial cells, from which PM arises. Interfering with ROS-responsive kinase pathways, particularly YB-1 phosphorylation, could counteract pro-migratory and immune-evasive effects in PM.

Understanding the localized fate of metal-based therapeutics is critical for optimizing anticancer efficacy and mitigating systemic toxicities, including nephrotoxicity, neurotoxicity, and ototoxicity. While platinum-based drugs are known to interact with both healthy and tumor cells as well as the extracellular matrix (ECM), conventional methods often lack the spatial resolution required to distinguish between these compartments. Here, we present an analytical framework integrating immunohistochemistry (IHC) with high-resolution laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). While preserving spatial architecture, this approach enabled the partitioning of intracellular and extracellular platinum fractions in a three-dimensional multicellular tumor spheroid model (MDA-MB-468), revealing that 23% of the total platinum mass remained associated with the added collagen matrix. The robustness of this framework was further validated in murine spleen and kidney tissues. By applying area-based normalization to account for varying cell densities, we successfully differentiated distribution trends within complex regions, such as the splenic red and white pulp. This strategy provides a quantitative tool for assessing drug penetration and compartmentalized biodistribution in complex biological matrices.

Many cancer-associated deaths result from metastases rather than primary tumors. Growing evidence suggests that DNA methylation alterations are crucial for inducing a plastic phenotype that allows cancer cells to adapt to the metastatic microenvironment. Brain metastases of melanoma (MBM) and glioblastoma (GB) share a neuroectodermal origin and the brain as tissue of residence, but their epigenetic regulation is poorly understood. Aiming at elucidating shared and tumor-distinct features, we analyzed the methylation of MGMT regulatory elements. We focused on MGMT because MGMT promoter methylation is used as a predictive marker for temozolomide response in GB, but its role in MBM has been discussed controversially. By targeting 12 CpG dinucleotides (CpGs) in the promoter, 68 CpGs in intergenic enhancers, and 31 CpGs in intragenic enhancers, we identified shared features, including an L-shaped relationship between promoter methylation and MGMT protein expression and an inverse L-shaped relationship between intragenic enhancer methylation and MGMT protein expression. GB exhibited higher methylation, particularly in promoter and intergenic enhancers, and stronger associations between methylation and overall survival than MBM. These results highlight both conserved and tumor-specific MGMT regulation, reflecting the complexity of epigenetic control in brain malignancies and emphasizing divergent evolution between MBM and GB.

T-cell leukemias and lymphomas (TCL) form a heterogeneous group of rare and often aggressive malignancies. Because of the rarity and heterogeneity of TCL subtypes, clinical trials are challenging to conduct, making pharmacogenomic studies in cell line panels critical for the discovery of targeted therapeutics. The scarcity of data repositories with integrated multiomics and drug screening data hinders the preclinical evaluation of drug vulnerabilities and the identification of molecular markers predictive of responses to monotherapies and combinations. To address this gap, we conducted comprehensive pharmacogenomic profiling on a panel of 38 TCL cell lines, representing major clinical TCL subtypes to capture the molecular and phenotypic diversity. The TCL-38 multiomics data resource includes harmonized genetic, molecular, and epigenetic profiles, with comprehensive annotations and standardized drug response assessment of each cell line. This resource, together with machine learning predictions, was leveraged to identify TCL subtype-specific therapeutic vulnerabilities, including single-agent sensitivities and synergistic drug combinations, which were linked to genetic or epigenetic features as potential predictive biomarkers. This integrated and openly available resource (https://aittokallio.group/tcl38) could help advance the currently limited treatment options for patients with TCL.Significance: Integrated and harmonized multiomics analyses and drug screening across a heterogeneous panel of T-cell leukemias and lymphomas provide a resource to uncover drug targets and predictive biomarkers to improve patient outcomes.

BRAF-altered pediatric high-grade gliomas (pHGG) harbor a dismal prognosis. Although targeted therapy with BRAF and MEK inhibitors provides initial benefit, resistance emerges rapidly in clinical practice indicating an urgent need for improved therapeutic strategies. As BRAF mutations frequently co-occur with homozygous CDKN2A/B loss, we investigated CDK4/6 inhibition as a rational therapeutic strategy. Using BRAF-mutant cell and human-to-organoid transplant (HOT) models, we assessed the impact of CDK4/6 and MEK inhibitors as mono- or combination therapies on viability, apoptosis, senescence, and molecular signaling. Activation of signaling pathways in primary and recurrent matched samples pre- and post-combined MEK and BRAF treatment was examined by RNA sequencing. In vivo efficacy was evaluated in orthotopic and subcutaneous patient-derived xenograft (PDX) models, as well as in one clinical case. BRAF-mutant, CDKN2A/B-deficient pHGGs displayed strong sensitivity to the CDK4/6 inhibitor abemaciclib in addition to the MEK inhibitor trametinib. These tumors were particularly vulnerable to combined abemaciclib and trametinib treatment, which induced senescence and apoptosis, and uniquely suppressed mTOR activity. Both HOT and PDX models exhibited tumor regression, prolonged survival and sustained response even after therapy discontinuation. These effects were accompanied by a decreased mesenchymal-like cellular phenotype, as indicated by lower CD44 expression and a shift toward a more rounded cell morphology. Interestingly, trametinib- and dabrafenib post-treatment samples exhibited further increase in CD44 levels, along with upregulation of PI3K and hypoxia signaling, indicating therapy-associated reinforcement of mesenchymal transition. The combination of trametinib and ribociclib was translated into clinical application, by showing good response of a patient with BRAF-altered and therapy-refractory pHGG. Our study demonstrated enhanced and prolonged effects of combined CDK4/6 and MEK inhibition in BRAF/CDKN2A-co-altered recurrent pHGG. We further provide evidence that the aggressive mesenchymal cell compartment is particularly targeted by this treatment combination, warranting further preclinical and clinical investigation.

Antibody-drug conjugates (ADCs), particularly enfortumab vedotin (EV) in combination with pembrolizumab, have emerged as transformative treatments for advanced bladder cancer (BC). However, platinum-based chemotherapy, especially gemcitabine-cisplatin (GC), remains a widely used, first-line standard globally. This study investigated the impact of GC-therapy on subsequent ADC responsiveness. Using novel, patient-derived BC models, in vitro selection for acquired GC-resistance consistently resulted in downregulation of the EV target, Nectin-4, and corresponding EV cross-resistance in GC-resistant (GC/R) cells. This decrease in membranous Nectin-4 expression was validated in two independent human BC cohorts: (1) matched transurethral resections and radical cystectomy/lymph node specimens during neoadjuvant therapy and (2) primary tumors and distant metastases during adjuvant GC chemotherapy. Transcriptomic profiling indicated that GC-resistance and Nectin-4 downregulation were linked to a transcriptional shift toward an epithelial-to-mesenchymal transition (EMT)-like phenotype. Large-scale drug screening and transcriptomic data highlighted potential vulnerabilities in GC/R cells, notably associated with the transforming growth factor beta (TGF-beta) signaling pathway. These findings suggest that platinum-based chemotherapy may promote EMT-like transcriptional reprogramming in BC, associated with Nectin-4 loss and EV resistance. As such, assessing membranous Nectin-4 expression prior to EV therapy might be particularly relevant in postplatinum settings. Additionally, identifying alternative, chemotherapy-induced druggable vulnerabilities can inform more effective treatment strategies.

Cisplatin, carboplatin, and oxaliplatin (OXA) are widely used platinum-based anticancer drugs, but their clinical application is limited by severe adverse effects, including nephrotoxicity, ototoxicity, and neurotoxicity, as well as drug resistance. Oxaliplatin (Eloxatin) is the only platinum compound showing superior clinical efficacy in the treatment of colorectal cancer, yet its use is restricted by dose-dependent toxicity. To address these limitations, OXA and four derivatives were loaded into bacterial ghosts (BGs) as novel drug carriers. BGs derived from the nonpathogenic strains Escherichia coli Nissle 1917 (EcN) and Escherichia coli NM522 (Ec NM522) were loaded by simple resuspension and incubation. Platinum content was quantified by inductively coupled plasma mass spectrometry, while the physicochemical properties of the formulations were characterized by fast protein liquid chromatography, transmission and scanning electron microscopy, electron energy-loss spectroscopy, and flow cytometry. The stability of OXA-loaded BG formulations was evaluated in different media and under various storage conditions for up to 120 days. The formulations demonstrated high batch-to-batch reproducibility, and bacterial encapsulation preserved the in vitro anticancer activity of the platinum compounds.

Abstract Background Tumor-associated macrophages (TAMs) are key drivers of the immunosuppressive tumor microenvironment (TME), supporting tumor progression through diverse functions. However, mechanistic studies of TAM polarization remain limited by the lack of physiologically relevant human model systems that capture stromal-immune interactions and macrophage heterogeneity. Methods We established advanced human co-culture systems that integrate healthy donor-derived macrophages with patient-derived organoids and tumoroids (PDOs and PDTs), as well as matched normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). These multicellular models enabled the investigation of interactions among stromal, epithelial, and immune cells within tumor and adjacent normal tissue environments. Results The co-culture systems recapitulated distinct macrophage states associated with tumor and adjacent normal environments and identified fibroblasts as major regulators of macrophage phenotypes. CAFs promoted macrophage metabolic remodeling characterized by altered lipid handling and enrichment of TAM-like signatures. Mechanistically, we identified thrombospondin 1 (TSP1) as a CAF-secreted factor linked to metabolic priming. Recombinant TSP1 induced transient lipid accumulation followed by mitochondrial remodeling. In tumor co-culture conditions, CD36 inhibition reduced lipid accumulation in macrophages, supporting a role for TSP1-linked lipid crosstalk in stromal-immune interactions. Conclusion Our study establishes advanced patient-derived co-culture models as a platform to investigate human TAM biology and stromal-immune interactions in CRC. Using these systems, we identify a fibroblast-associated TSP1-lipid axis linked to macrophage metabolic remodeling and TAM-like polarization, highlighting stromal metabolic communication as a potential targetable feature of the CRC microenvironment. Graphical abstract

Understanding the localized fate of metal-based therapeutics is critical for optimizing anticancer efficacy and mitigating systemic toxicities, including nephrotoxicity, neurotoxicity, and ototoxicity. While platinum-based drugs are known to interact with both healthy and tumor cells as well as the extracellular matrix (ECM), conventional methods often lack the spatial resolution required to distinguish between these compartments. Here, we present an analytical framework integrating immunohistochemistry (IHC) with high-resolution laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). While preserving spatial architecture, this approach enabled the partitioning of intracellular and extracellular platinum fractions in a three-dimensional multicellular tumor spheroid model (MDA-MB-468), revealing that 23% of the total platinum mass remained associated with the added collagen matrix. The robustness of this framework was further validated in murine spleen and kidney tissues. By applying area-based normalization to account for varying cell densities, we successfully differentiated distribution trends within complex regions, such as the splenic red and white pulp. This strategy provides a quantitative tool for assessing drug penetration and compartmentalized biodistribution in complex biological matrices.

Cancer is the leading cause of disease-related deaths among children in high-income countries. Tumor heterogeneity and lack of mechanism-of-action-based therapeutic options are key challenges to overcome in order to improve pediatric cancer patients survival. Here, we report the EU-IMI-2 funded public-private partnership - ITCC-Pediatric Preclinical Proof-of-Concept Platform (ITCC-P4)-, which has built a large repertoire of patient-derived xenograft (PDX) models, representing all major solid pediatric cancer types, for in vivo drug testing. Three-hundred-fifty-three PDX models from diagnostic and relapsed pediatric cancers have been established and molecularly characterized, together with matched germline/tumor samples. As proof-of-concept, we present in vivo drug screening data in neuroblastoma and rhabdomyosarcoma models. PDX data, accessible at http://r2platform.com/itcc-p4, allow the selection of models based on oncogenic drivers and/or potential biomarkers for preclinical testing. Operated by a non-profit entity (www.itccp4.com), this sustainable platform aids academic and industrial researchers in developing and prioritizing innovative therapies for pediatric cancer. ### Competing Interest Statement Stefan Pfister, Co-founder and shareholder Heidelberg Epignostix GmbH Natalie Jaeger is a full-time employee of Heidelberg Epignostix GmbH Martin Sill, Co-founder and shareholder Heidelberg Epignostix GmbH Jens Hoffmann: Shareholder EPO Experimental Pharmacology & Oncology Berlin-Buch GmbH Justyna Wierzbinska and Andreas Schlicker are employees of Bayer AG. Andreas Schlicker is a shareholder of Bayer AG. Petra Hamerlik provides consultancy for LindonLight Collective and Rakobina Therapeutics. Stefano Cairo is now a full-time employee of Champions Oncology, Rockville, Maryland, USA David Shields is an employee of Pfizer Inc and holds shares in the company. Maureen M. Hattersley is an employee of AstraZeneca and holds shares in the company. Employees from the following pharmaceutical companies also contributed as co-authors to the ITCC-P4 consortium project, as stated in their affiliations: LILLY, ROCHE, PFIZER, BAYER ,PHARMA MAR, CHARLES RIVER, JANSSEN, AZ, AMGEN, SERVIER, SANOFI.

In this work, a panel of twelve ruthenium(II) and osmium(II) derived N,O,O-tridentate complexes (1a-2f) with a variation of longer, branched and unbranched alkyl substituents was synthesized and characterized via NMR, HRMS, elemental analysis and X-ray diffraction analysis. Resilience to dissociation in biologically relevant solution was determined over 72 hours, revealing most stable complexes to derive from naphthoquinones bearing tert-butyl- and neopentyl-substituents. Osmium derived complexes were found to be generally more inert than their ruthenium counterparts. Cytotoxicity was examined, revealing IC50 values in the nanomolar to lower micromolar range for derivatives 1a-2f in three human cancer lines and a typical pattern of selectivity for SW480 cells. Cellular accumulation correlated with in vitro cytotoxicity; however, longer and branched substituents did not improve the cellular accumulation. Cell cycle experiments showed consistent cell cycle inhibition in both SW480 and CH1/PA-1 cells for ruthenium-based compounds only. Indolamin-2,3-dioxygenase 1 (IDO1) inhibition assays in SKOV3 cells revealed significant inhibitory potential of Ru-Ethyl, in clear distinction to other ruthenium and osmium complexes.

Cancer is the leading cause of disease-related deaths among children in high-income countries. Tumor heterogeneity and lack of mechanism-of-action-based therapeutic options are key challenges to overcome to improve pediatric cancer patient survival. To address these challenges, we formed the EU-IMI-2 funded public-private partnership "ITCC-Pediatric Preclinical Proof-of-Concept Platform" (ITCC-P4), which built a large repertoire of patient-derived xenograft (PDX) models representing all major high-risk solid pediatric cancer types for in vivo drug testing. A total of 353 PDX models were established from diagnostic and relapsed pediatric cancers and molecularly characterized, together with matched germline/tumor samples. Serial PDX models were also established, spanning diagnostic/posttreatment, primary/relapse, and metastasis-derived pairs. Proof-of-concept in vivo drug screening data in neuroblastoma and rhabdomyosarcoma models identified potential predictive biomarkers for targeted therapy. Molecular data from the PDX models, accessible at https://r2platform.com/itcc-p4, allowed the selection of models for preclinical testing based on oncogenic drivers and/or potential biomarkers. Operated by a non-profit entity, this sustainable platform aids academic and industrial researchers in developing and prioritizing innovative therapies for pediatric cancer.

Ovarian cancer (OC) is the most lethal gynecological malignancy, with platinum resistance posing a major therapeutic challenge. To explore alternatives, we synthesized silver- and gold-based N-heterocyclic carbene (NHC) complexes differing only in their central metal ion and evaluated their activity in platinum-resistant OC. Structure-activity relationships revealed distinct metal-dependent behaviors. Silver complexes showed little variation with ligand modifications, whereas gold complexes displayed pronounced differences. Two bis-NHC-gold compounds were of particular interest: In an isogenic OC resistance model (A2780 and A2780/cis), [(NHC2)2Au]Br showed cross-resistance, while [(NHC1)2Au]Br induced collateral sensitivity. These effects were independent of intracellular accumulation, apoptosis induction, or TrxR inhibition. Instead, proteomic and metabolic analyses demonstrated that [(NHC1)2Au]Br inhibited oxidative phosphorylation, forcing a metabolic shift to aerobic glycolysis. As A2780/cis cells already rely on maximal glycolysis, [(NHC1)2Au]Br caused an energy collapse. These findings highlight a metabolic vulnerability in cisplatin-resistant OC that may be exploited for the development of novel therapeutic candidates.