Current standard-of-care systemic therapy options for locally advanced and metastatic bladder cancer (BC), which are predominantly based on cisplatin-gemcitabine combinations, are limited by significant treatment failure rates and frailty-based patient ineligibility. We previously addressed the urgent clinical need for better-tolerated BC therapeutic strategies using a drug screening approach, which identified outstanding antineoplastic activity of clofarabine in preclinical models of BC. To further assess clofarabine as a potential BC therapy component, we conducted head-to-head comparisons of responses to clofarabine versus gemcitabine in preclinical in vitro and in vivo models of BC, complemented by in silico analyses. In vitro data suggest a distinct correlation between the two antimetabolites, with higher cytotoxicity of gemcitabine, especially against several nonmalignant cell types, including keratinocytes and endothelial cells. Accordingly, tolerance of clofarabine (oral or intraperitoneal application) was distinctly better than for gemcitabine (intraperitoneal) in patient-derived xenograft models of BC. Clofarabine also exhibited distinctly superior anticancer efficacy, even at dosing regimens optimized for gemcitabine. Neither complete remission nor cure, both of which were observed with clofarabine, were achieved with any tolerable gemcitabine regimen. Taken together, our findings demonstrate that clofarabine has a better therapeutic window than gemcitabine, further emphasizing its potential as a candidate for drug repurposing in BC.Patient summaryWe compared the anticancer activity of clofarabine, a drug used for treatment of leukemia but not bladder cancer, and gemcitabine, a drug currently used for chemotherapy against bladder cancer. Using cell cultures and mouse models, we found that clofarabine was better tolerated and more efficacious than gemcitabine, and even cured implanted tumors in mouse models. Our results suggest that clofarabine, alone or in combination schemes, might be superior to gemcitabine for the treatment of bladder cancer.

Many xenobiotics are non-genotoxic carcinogens (NGC) in rodent liver. Their mode of action (MoA) and health risks for humans are unclear and no in-vitro tests are available to predict NGC. Human HepaRG™ cells in the differentiated (d-HepaRG) and non-differentiated state (nd-HepaRG) were studied as new approach methodology (NAM) for NGC. Cell-biological assays were performed with d-/nd-HepaRG and human hepatoma/hepatocarcinoma cell lines to characterize the benign/malignant phenotype. Reaction of d-/nd-HepaRG to several liver growth factors and NGC (phenobarbital, PB; cyproterone acetate, CPA; WY-14643) was compared to unaltered and premalignant rat hepatocytes in ex-vivo culture. Enzyme induction by NGC was checked by RT-qPCR/oligo-arrays. Growth, anchorage-independency, migration, clonogenicity, and in-vivo tumorigenicity of nd-HepaRG ranged between benign d-HepaRG and malignant hepatoma/hepatocarcinoma cells. All growth factors elevated DNA replication of d-/nd-HepaRG cells, similarly to unaltered/premalignant rat hepatocytes. NGC induced their prototypical enzymes in the rat and human cells, but elicited a growth response only in the unaltered/premalignant rat hepatocytes and not in human d-/nd-HepaRG cells. To conclude, a benign/premalignant phenotype of d-/nd-HepaRG cells and a reactivity towards several hepatic growth factors and NGC, as known from human hepatocytes, are essential components for an in-vitro model for early stage human hepatocarcinogenesis.The potential value as new approach methodology (NAM) for NGC is discussed.

Background: The heterogeneity of bladder cancers (BCs) is a major challenge for the development of novel therapies. However, given the high rates of recurrence and/or treatment failure, the identification of effective therapeutic strategies is an urgent clinical need. Objective: We aimed to establish a model system for drug identification/repurposing in order to identify novel therapies for the treatment of BC. Design, setting, and participants: A collection of commercially available BC cell lines (n = 32) was comprehensively characterized. A panel of 23 cell lines, representing a broad spectrum of BC, was selected to perform a high-throughput drug screen. Outcome measurements and statistical analysis: Positive hits were defined as compounds giving >50% inhibition in at least one BC cell line. Results and limitations: Amongst >1700 tested chemical compounds, a total of 471 substances exhibited antineoplastic effects. Clofarabine, an antimetabolite drug used as third-line treatment for childhood acute lymphoblastic leukaemia, was amongst the limited number of drugs with inhibitory effects on cell lines of all intrinsic subtypes. We, thus, reassessed the substance and confirmed its inhibitory effects on commercially available cell lines and patient-derived cell cultures representing various disease stages, intrinsic subtypes, and histologic variants. To verify these effects in vivo, a patientderived cell xenograft model for urothelial carcinoma (UC) was used. Well-tolerated doses of clofarabine induced complete remission in all treated animals (n = 12) suffering from both early- and late-stage disease. We further took advantage of another patientderived cell xenograft model originating from the rare disease entity sarcomatoid carcinoma (SaC). Similarly to UC xenograft mice, clofarabine induced subcomplete to complete tumour remissions in all treated animals (n = 8). Conclusions: The potent effects of clofarabine in vitro and in vivo suggest that our findings may be of high clinical relevance. Clinical trials are needed to assess the value of clofarabine in improving BC patient care. Patient summary: We used commercially available cell lines for the identification of novel drugs for the treatment of bladder cancer. We confirmed the effects of one of these drugs, clofarabine, in patient-derived cell lines and two different mouse models, thereby demonstrating a potential clinical relevance of this substance in bladder cancer treatment. (c) 2022 The Authors. Published by Elsevier B.V. on behalf of European Association of Urology. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/).

Clinical efficacy of oxaliplatin is frequently limited by severe adverse effects and therapy resistance. Acquired insensitivity to oxaliplatin is, at least in part, associated with elevated levels of glutathione (GSH). In this study we report on an oxaliplatin-based platinum(IV) prodrug, which releases L -buthionine- S , R -sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase, the rate-limiting enzyme in GSH biosynthesis. Two complexes bearing either acetate ( BSO-OxOAc ) or an albumin-binding maleimide ( BSO-OxMal ) as second axial ligand were synthesized and characterized. The in vitro anticancer activity of BSO-OxOAc was massively reduced in comparison to oxaliplatin, proving its prodrug nature. Nevertheless, the markedly lower intracellular oxaliplatin uptake in resistant HCT116/OxR cells was widely overcome by BSO-OxOAc resulting in distinctly reduced resistance levels. Platinum accumulation in organs of a colorectal cancer mouse model revealed higher tumor selectivity of BSO-OxMal as compared to oxaliplatin. This corresponded with increased antitumor activity, resulting in significantly enhanced overall survival. BSO-OxMal -treated tumors exhibited reduced GSH levels, proliferative activity and enhanced DNA damage (pH2AX) compared to oxaliplatin. Conversely, pH2AX staining especially in kidney cells was distinctly increased by oxaliplatin but not by BSO-OxMal . Taken together, our data provide compelling evidence for enhanced tumor specificity of the oxaliplatin(IV)/BSO prodrug.

More than a decade ago, studies on cellular cisplatin accumulation via active membrane transport established the role of the high affinity copper uptake protein 1 (CTR1) as a main uptake route besides passive diffusion. In this work, CTR1 expression, cisplatin accumulation and intracellular copper concentration was assessed for single cells revisiting the case of CTR1 in the context of acquired cisplatin resistance. The single-cell workflow designed for in vitro experiments enabled quantitative imaging at resolutions down to 1 µm by laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS). Cisplatin-sensitive ovarian carcinoma cells A2780 as compared to the cisplatin-resistant subline A2780cis were investigated. Intracellular cisplatin and copper levels were absolutely quantified for thousands of individual cells, while for CTR1, relative differences of total CTR1 versus plasma membrane-bound CTR1 were determined. A markedly decreased intracellular cisplatin concentration accompanied by reduced copper concentrations was observed for single A2780cis cells, along with a distinctly reduced (total) CTR1 level as compared to the parental cell model. Interestingly, a significantly different proportion of plasma membrane-bound versus total CTR1 in untreated A2780 as compared to A2780cis cells was observed. This proportion changed in both models upon cisplatin exposure. Statistical analysis revealed a significant correlation between total and plasma membrane-bound CTR1 expression and cisplatin accumulation at the single-cell level in both A2780 and A2780cis cells. Thus, our study recapitulates the crosstalk of copper homeostasis and cisplatin uptake, and also indicates a complex interplay between subcellular CTR1 localization and cellular cisplatin accumulation as a driver for acquired resistance development.

Receptor tyrosine kinase inhibitors have become a central part of modern targeted cancer therapy. However, their curative potential is distinctly limited by both rapid resistance development and severe adverse effects. Consequently, tumor-specific drug activation based on prodrug designs, exploiting tumor-specific properties such as hypoxic oxygen conditions, is a feasible strategy to widen the therapeutic window. After proof-of-principal molecular docking studies, we have synthesized two cobalt(iii) complexes using a derivative of the clinically approved Abelson (ABL) kinase and fibroblast growth factor receptor (FGFR) inhibitor ponatinib. Acetylacetone (acac) or methylacetylacetone (Meacac) have been used as ancillary ligands to modulate the reduction potential. The ponatinib derivative, characterized by an ethylenediamine moiety instead of the piperazine ring, exhibited comparable cell-free target kinase inhibition potency. Hypoxia-dependent release of the ligand from the cobalt(iii) complexes was proven by changed fluorescence properties, enhanced downstream signaling inhibition and increased in vitro anticancer activity in BCR-ABL- and FGFR-driven cancer models. Respective tumor-inhibiting in vivo effects in the BCR-ABL-driven K-562 leukemia model were restricted to the cobalt(iii) complex with the higher reduction potential and confirmed in a FGFR-driven urothelial carcinoma xenograft model. Summarizing, we here present for the first time hypoxia-activatable prodrugs of the clinically approved tyrosine kinase inhibitor ponatinib and a correlation of the in vivo activity with their reduction potential.

A high-throughput laser ablation–inductively coupled plasma–time-of-flight mass spectrometry (LA-ICP-TOFMS) workflow was implemented for quantitative single-cell analysis following cytospin preparation of cells. For the first time, in vitro studies on cisplatin exposure addressed human monocytes and monocyte-derived macrophages (undifferentiated THP-1 monocytic cells, differentiated M0 macrophages, as well as further polarized M1 and M2 phenotypes) at the single-cell level. The models are of particular interest as macrophages comprise the biggest part of immune cells present in the tumor microenvironment and play an important role in modulating tumor growth and progression. The introduced bioimaging workflow proved to be universally applicable to adherent and suspension cell cultures and fit-for-purpose for the quantitative analysis of several hundreds of cells within minutes. Both, cross-validation of the method with single-cell analysis in suspension for THP-1 cells and with LA-ICP-TOFMS analysis of adherent M0 cells grown on chambered glass coverslips, revealed agreeing platinum concentrations at the single-cell level. A high incorporation of cisplatin was observed in M2 macrophages compared to the M0 and M1 macrophage subtypes and the monocyte model, THP-1. The combination with bright-field images and monitoring of highly abundant endogenous elements such as phosphorus and sodium at a high spatial resolution allowed assessing cell size and important morphological cell parameters and thus straightforward control over several cell conditions. This way, apoptotic cells and cell debris as well as doublets or cell clusters could be easily excluded prior to data evaluation without additional staining.