Tyrolean Cancer Research Institute

BackgroundUrothelial carcinoma (UC) is a biologically heterogeneous disease, and current molecular classifications have limited integration into clinical decision‐making. To further pursue precision oncology efforts in UC, we developed a molecular classification framework applicable to transcriptomic and proteomic data from non–muscle‐invasive bladder cancer (NMIBC), muscle‐invasive bladder cancer (MIBC) and urothelial cancer cell lines.MethodsUsing a whole‐transcriptome self‐organised map and regularised semi‐supervised clustering of 4439 bulk NMIBC and MIBC transcriptomes and proteomes, and 33 UC cell lines, we identified three molecular UC clusters. Making use of both in silico and in vitro approaches, we selected promising treatment approaches for each cluster.ResultsThe three developed clusters displayed distinct signatures of mRNA, proteins, biological processes, metabolism and essential driver genes. They also differed in prognosis and machine learning‐predicted treatment vulnerabilities and resistance. High‐risk, stroma‐rich Cluster #1 cancers were predicted to respond to selected cytotoxic drugs, ferroptosis inducers and PARP inhibitors. For the aggressive, fast‐proliferating, immune‐infiltrated Cluster #2 tumours with basal/squamous differentiation, cytotoxic agents and EGFR/ERBB‐ and MEK/ERK‐targeting therapies were proposed. Cluster #3 cancers of predominantly luminal papillary phenotype with scarce stroma and immune infiltration were enriched with NMIBC and low‐risk malignancies. For patients with Cluster #3 tumours, selected epigenetic drugs or EGFR/FGFR inhibitors may represent attractive treatment options.ConclusionsOur novel molecular taxonomy holds promise as a practical framework for patient risk stratification and clinical trials in UC. Our molecular classification scheme may facilitate personalised transcriptome‐ and proteome‐based risk assessment and clinical trial design for the development of various therapeutics.key pointsWe developed three UC clusters, applicable for MIBC and NMIBC, which were validated using transcriptomic- and proteomic datasets.Publically available UC cell lines were assigned to the clusters, to have in vitro models representing each cluster.The clusters differ in molecular and biological signatures, with distinct prognostic and therapeutic characteristics.

BackgroundDynamic remodelling of the tumour microenvironment (TME) plays a central role in prostate cancer (PCa) progression, immune evasion and therapy resistance. However, the co-existence of both tumour-promoting and tumour-restraining stromal elements necessitates extensive characterisation of the TME for effective targeting. Fibromuscular cell heterogeneity in PCa remains poorly characterised, in part due to challenges in isolating cells embedded within the desmoplastic stroma. This study therefore aimed to better characterise fibroblast and smooth muscle cell (SMC) populations as the major tissue-resident stromal cell subtypes within the PCa TME.MethodsA PCa single-cell RNA sequencing (scRNA-seq) dataset was re-analysed to define fibromuscular subtypes. Due to low fibroblast yields, an optimised tissue dissociation protocol was developed and benchmarked against two commercial kits via flow cytometry, immunostaining of clinical specimens and ex vivo culture. Dimensionality reduction and clustering were applied to the CD31− stromal fraction using a multiparameter surface marker panel. Annotation of the resulting clusters based on their surface marker profile was supported by integrating scRNA-seq and immuno-histological findings.ResultsThe optimised protocol yielded over twice the viable cells/mg tissue compared to two commercial kits, preserved surface marker integrity, enhanced successful cultivation of mesenchymal cells and recovered diverse stromal subpopulations from benign and malignant samples. Dimensionality reduction and clustering of flow cytometry counts identified 11 distinct CD31− stromal populations. Integration with transcriptomic data and immunofluorescence of clinical specimens identified spatially- and prognostically-distinct fibroblast subtypes, including inflammatory and myofibroblastic cancer-associated fibroblasts, pericytes linked to poor prognosis and a novel SMC subset associated with stromal activation.ConclusionThis study presents a robust workflow for improved isolation and characterisation of fibromuscular stromal cells in PCa. The multimodal approach enabled refined characterisation of phenotypically distinct and clinically-relevant stromal subpopulations within their spatial context providing a foundation for future TME-targeted therapies.

Colorectal cancer liver metastasis (CRLM) is associated with poor survival, primarily due to acquired therapy resistance. While novel therapies arise, translation is limited by the lack of tumor models accurately representing dynamic microenvironmental interplay. Here, we show that ex vivo normothermic machine perfusion (NMP) offers a novel preclinical framework to study the intratumoral dynamics of CRLM biology. Six resected metastatic human livers were preserved for two days and subjected to multi-omic profiling of serially sampled adjacent liver and metastatic tissue using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST). Tissue integrity was assessed and cross-validated by immunofluorescence (IF), high-resolution respirometry (HRR) and flow-cytometry. NMP was successfuly applied to metastatic livers with minimal surgical adaptations, preserving both intrinsic hepatic properties and tissue viability over an extended duration. Single-cell and spatial mapping confirmed preservation of CRLM phenotypic properties and demonstrated high clinical translatability by applicability of the intrinsic epithelial consensus molecular subtypes to metastasis. Spatially deconvoluted pathway activities reflected functional tissue-microenvironments. Transcriptomic profiles - including those of tumor-associated myeloid cells - were preserved during NMP. Finally, we demonstrate tumor-associated myeloid cell persistence as a driver of disease progression and poor survival in colorectal cancer. Our findings represent the basis for future innovative applications adopting NMP in the context of CRLM, providing a new preclinical tumor model avenue.

This research outlines the development of an innovative delivery system for administration of chlorido[N,N'-bis(salicylidene)-1,2-phenylenediamine]-iron(III) salophene - designated Iron(III) Salophene (Fe³⁺SP). The identity of Fe³⁺SP was confirmed using FTIR, and its lipophilicity (log P) was quantified by HPLC. Fe³⁺SP was then incorporated into lipid-based nanocarriers (LBNCs) with a lipid matrix of triglycerides of caprylic/capric acid (45.4 %; v/v), phosphatidylcholine (36.4 %; m/v) and benzyl alcohol (18.2 %; v/v) as solvent. Metabolic activity was investigated via the MTT assay on various cell lines, and cellular uptake was analyzed by confocal laser scanning microscopy using different dyes. The log P value of 1.8 for the Fe3+SP-complex confirmed sufficient lipophilicity for incorporation into lipid-based nanocarriers. Ultrasonic treatment significantly reduced particle size from 1000 nm to about 200 nm and improved particle uniformity from 1.0 to about 0.2 for blank and Fe³⁺SP LBNCs. Different stability studies showed consistent droplet sizes in various buffer systems (150-230 nm) over 14 days, and uniform particle distribution (∼0.2) confirmed stability in physiological media over 48 h. TEM analysis revealed that Fe³⁺SP maintains uniform morphology and enhances stability, leading to consistent particle size and shape, promoting cellular uptake. Compared to Fe³⁺SP in DMSO, the LBNC-loaded formulation exhibited a five-fold higher effect on cancer cell lines at a similar cellular uptake, indicating higher efficacy. These findings suggest that LBNCs offer a promising platform for the oral delivery of Fe³⁺SP, with significant advantages over conventional delivery systems.

ABSTRACTThis study aimed to improve the drug‐like properties of benzimidazole‐based Pt(II)‐N‐heterocyclic carbene (NHC) complexes, particularly by enhancing their water solubility and delivery to cancer cells. Accordingly, four new Pt(II) complexes of the benzimidazol‐2‐ylidene type, featuring monodentate carboxylato ligands, were prepared and their structures confirmed through a combination of spectroscopic and crystallographic techniques. Their stability in aqueous solution and cell culture medium was investigated by 1H NMR spectroscopy and HPLC‐MS analysis. Cytotoxicity was assessed using the MTT assay in ovarian cancer cell lines (A2780wt (cisplatin sensitive) and A2780cis (cisplatin resistant)) and a noncancerous bone marrow stromal cell line (HS‐5). Most complexes exhibited cytotoxicity comparable to or exceeding that of carboplatin, with preferential activity toward cancer cells. Loading of all four Pt(II) complexes into bacterial ghost cells (BGs) derived from two different nonpathogenic bacterial strains, Escherichia coli (E. coli) Nissle 1917 and E. coli NM522 notably enhanced the intracellular accumulation and cytotoxicity. Furthermore, mechanistic studies demonstrated that all tested compounds, regardless of formulation, induced apoptosis. Their potential to trigger immunogenic cell death was also evaluated, though only a modest effect was observed on selected hallmarks. Collectively, these findings highlight the potential of dicarboxylatoplatinum(II)‐NHC complexes, particularly loaded into BG‐based formulations, as promising anticancer drug candidates.

SS-, RR-, SR- and RR/SS-configured 1,3-diethyl-4,5-diphenyl-4,5-dihydro-1H-imidazol-2-ylidenes were introduced as new imidazoline-based N-heterocyclic carbene (NHC) ligands for the design of antitumor-active (NHC)gold(I) complexes (halido(NHC)gold(I) complexes: chlorido (5a-d), bromido (6a-d), iodido (7a-d); SS,SS-, RR,RR-, SR,SR-, and RR,SS-configured [(NHC)2Au(I)]+ complexes: 8a-d). X-ray structures of the SS-configured complexes 5a-7a showed bis-equatorially arranged phenyl rings and disturbed columnar structures with increased Au-Au distances (>5.6Å). The SR-configuration forced the phenyl ring in a synclinal position above the NHC plane allowing only the formation of separated dimers (5c-7c). In case of the [(NHC)2Au(I)]+ complex 8c, single molecules were observed in the crystals. The steric and dynamic conditions reduced ligand scrambling in solution and thus increased stability. The complexes showed higher growth inhibitory effects in ovarian (A2780wt (wild-type), A2780cis (Cisplatin-resistant)) than in breast cancer cells (MDA-MB-231, MCF-7) and circumvented the Cisplatin resistance in A2780cells (effects in A2780wt=A2780cis). Chlorido- and bromido(NHC)gold(I) complexes caused comparable effects, because of a fast Br/Cl exchange (6a-d → 5a-d). The iodido(NHC)gold(I) complexes 7a-d were more active, due to a partial degradation to 8a-d. The latter were the most cytotoxic compounds of this study. The configuration of the NHC ligand did not influence the cytotoxicity of the complexes. Enantiomers and diastereomers showed the same antimetabolic effects. On the examples of 5a-d and 8a-d, the cellular uptake was studied. The maximum gold levels in A2780wt and MDA-MB-231cells were achieved within 30min of incubation. At concentrations corresponding to the half maximal inhibitory concentration (IC50) values of the antiproliferative effect (5a-d: 20μM, 8a-d: 5μM), 5b, 5c, and 5d induced almost the same gold content in A2780wt cells, which was 30-50% lower than that of 5a. The trend of accumulation for [(NHC)2Au(I)]+ complexes was 8d<8a < 8b<8c. Furthermore, 5a-d inhibited the cyclooxygenase-1 (COX-1) and thioredoxin reductase (TrxR) and upregulated the Glutathione (GSH) level in A2780wt cells. Contrarily, 8a-d did not reduce COX-1 and TrxR activity, but led to moderate GSH down-regulation. The GSH level was not lowered in favour of Glutathione disulfide (GSSG), demonstrating that 8a-d influence the formation of GSH. © 2017 Elsevier Inc. All rights reserved.

The present structure-activity relationship study investigates the development of novel chemosensitizers targeting therapy-resistant cancer stem cells (CSCs). We used 4'-((2-propyl-1H-benzo[d]imidazole-1-yl)methyl)-[1,1'-biphenyl]-2-carboxylic acid, derived from the angiotensin II type 1 receptor blocker telmisartan, as a lead structure, demonstrating that the biphenyl moiety is essential for chemosensitizing activity. Introducing a methyl carboxylate or carboxamide instead of the COOH-group significantly enhanced this effect, leading to the development of highly potent compounds. These novel, noncytotoxic chemosensitizers effectively target CSCs and overcome drug resistance by interfering with CSC persistence mechanisms─hyperactivated STAT5 signaling and increased drug transporter activity─with demonstrated efficacy in leukemia, ovarian, and prostate cancers. The carboxamide of telmisartan (telmi-amide, 7c) significantly reduced tumor growth in an imatinib-resistant leukemia xenograft model, both as monotherapy and combined with imatinib, showing promising oral bioavailability and tolerability. In summary, telmisartan derivatives act as effective chemosensitizers and offer an innovative strategy for targeting CSCs in various malignant diseases.

Molecular similarities between embryonic and malignant cells can be exploited to target tumors through specific signatures absent in healthy adult tissues. One such embryonic signature tumors express is oncofetal chondroitin sulfate (ofCS), which supports disease progression and dissemination in cancer. Here, we report the identification and characterization of phage display-derived antibody fragments recognizing two distinct ofCS epitopes. These antibody fragments show binding affinity to ofCS in the low nanomolar range across a broad selection of solid tumor types in vitro and in vivo with minimal binding to normal, inflamed, or benign tumor tissues. Anti-ofCS antibody drug conjugates and bispecific immune cell engagers based on these targeting moieties disrupt tumor progression in animal models of human and murine cancers. Thus, anti-ofCS antibody fragments hold promise for the development of broadly effective therapeutic and diagnostic applications targeting human malignancies.