Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air–liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.
Niphathesine C and related pyridine alkaloids are well known natural products with interesting antimicrobial activities, characterized by a pyridine ring and a lipophilic side chain with a terminal nitrogen-containing functional group. This paper describes the synthesis of analogues of these alkylpyridine alkaloids with variation of the heterocyclic ring and the terminal functional group. Key steps of the syntheses are a Sonogashira reaction of appropriate aryl iodides with undec-10-ynol or undec-10-ynoic acid derivatives. The resulting compounds were tested in an agar diffusion assay against several bacteria and fungi.
The complex and dynamic microenvironment of tumors influences their development, progression, and response to therapy. In addition to cancer cells, solid tumors consist of a tumor microenvironment (TME) containing fibroblasts, immune cells, blood and lymphatic vessels, and the extracellular matrix. A wide variety of secreted proteins maintain the heterotypic interactions between the cell types in the TME. However, it is not well understood how the TME responds to treatment at the proteome level. Here, we developed a unique nascent proteomic approach for precision-cut tumor slices to address this issue. Precision-cut tumor slices (PCTS) are a technology in which tumor tissues are cut to a defined thickness of 150-300 µm and cultured ex vivo for a certain time. PCTS maintain both the three-dimensional architecture and tumor heterogeneity and preserve their TME with respect to different cell types and the extracellular matrix. Our approach for PCTS nascent proteome analysis combines pulsed-SILAC (stable isotope labeling with amino acids in cell culture) with click chemistry to selectively isolate and quantify newly synthesized proteins in the TME upon drug treatment. PCTS were generated from patient-derived xenografts and primary human ovarian tumors. After a depletion step, the PCTS were cultured in AHA-SILAC medium and treated with cisplatin. PCTS and culture media containing secreted proteins were harvested separately. Newly synthesized proteins were enriched via click chemistry and analyzed using mass spectrometry. A maximum labelling efficiency of >60% was achieved. Human PCTS showed a higher labelling efficiency than mouse xenografts. The PCTS of different tumors showed varying labeling efficiencies, indicating patient heterogeneity. Nascent proteome analysis enables the investigation of drug resistance and response in different patients. Cisplatin treatment resulted in the downregulation of >200 proteins in responsive tumors. A PCTS resistant to cisplatin did not show this response. Moreover, the corresponding patient had a worse clinical outcome than patients whose tumors were sensitive ex vivo. GSEA revealed the involvement of components such as cadherin binding and DNA translation. Protein-protein interactions can be predicted via STRING analysis. Tumor response or resistance was validated using viability assays and immunohistochemical staining for biomarkers of DNA damage and cell death. In conclusion, we established an ex vivo nascent proteome analysis method to study drug response within the complex TME, which can predict the tumor in vivo drug response. By combining the PCTS culture system with pulsed SILAC-AHA treatment, this approach allows the tracking of compositional and dynamic changes within the proteome and monitoring of the direct proteome response on a rapid timescale. It can be used to study cellular communication, predict therapeutic outcomes, and identify new therapeutic targets. Citation Format: Julia Thiel, Lina-Marie Wagner, Karim Aljakouch, Julia Schüler, Bernd Winkler, Kathrin Böpple, Thomas E. Mürdter, Georg Sauer, German Ott, Walter E. Aulitzky, Matthias Schwab, Jeroen Krijgsveld, Meng Dong. Nascent proteome analysis of drug response in precision-cut tumor slices [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2020.
1. The utility of 1-aminobenzotriazole (ABT), incorporated in food, has been investigated as an approach for longer term inhibition of cytochrome P450 (P450) enzymes in mice. 2. In rats, ABT inhibits gastric emptying, to investigate this potential limitation in mice we examined the effect of ABT administration on the oral absorption of NVS-CRF38. Two hour prior oral treatment with 100 mg/kg ABT inhibited the oral absorption of NVS-CRF38, T was 4 hours for ABT-treated mice compared to 0.5 hours in the control group. 3. A marked inhibition of hepatic P450 activity was observed in mice fed with ABT containing food pellets for 1 month. P450 activity, as measured by the oral clearance of antipyrine, was inhibited on day 3 (88% of control), week 2 (83% of control) and week 4 (80% of control). 4. T values for antipyrine were comparable between ABT-treated mice and the control group, alleviating concerns about impaired gastric function. 5. Inclusion of ABT in food provides a minimally invasive and convenient approach to achieve longer term inhibition of P450 activity in mice. This model has the potential to enable pharmacological proof-of-concept studies for research compounds which are extensively metabolised by P450 enzymes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.