Epithelial ovarian cancer (OvCa) metastasizes early within the abdominal cavity, and rarely spreads hematogenously. The peritoneum and the omentum, which is a large pad of fat tissue covering the bowel, are the most common sites of metastasis. Currently, there are no treatments specific to metastatic OvCa, which affects 80% of all patients, and new drug discovery for this indication has been very slow. Unfortunately, no drug has been approved for the treatment of OvCa since 1999. Current high-throughput drug screening strategies rely on cell lines cultured on plastic, which, since they are separated from the tumor microenvironment, are poorly representative of OvCa pathology. Recently, we developed an organotypic 3D culture assembled from primary human omental fibroblasts, mesothelial cells, and extracellular matrix, which is representative of the tumor microenvironment observed in patients with OvCa. Co-culture of labeled primary human OvCa cells with the 3D culture mimics the early steps of metastasis, adhesion and invasion. We propose to adapt the 3D culture to a robust and reproducible assay in a 384-well format feasible for high-throughput screening (HTS). Our preliminary experiments have shown that using a 3D culture with a 96-well format for HTS is feasible and yields reproducible results.
Our first aim focuses on assay optimization, and will be performed using the 450 compound NIH clinical collection in the University of Chicago (U of C) HTS core facility. We will optimize controls, signal-to-background ratio, reproducibility, and work on acquiring a detailed understanding of control parameters (e.g. primary cell purity, incubation times, day-to-day variations) for the adaptation to HTS. Positive hits will be defined as having equal or greater efficiency than carboplatin, currently the most efficient OvCa drug. Plans for the second aim include optimization of the 3D culture for the secondary screening using a ten-point dose response curve. Working with the U of C HTS facility, we will further develop three assays measuring (i) OvCa apoptosis, (ii) proliferation, and (iii) adhesion/invasion through matrigel in order to confirm the efficacy of the compounds identified. Our goal is to establish a robust and reproducible assay that is amenable to automated analysis of the 300,000 compounds available through the MLPCN centers. Compounds which prevail through the primary and secondary screenings and 2 of the 3 confirmatory assays will ultimately be developed further. We believe that HTS using primary cancer cells growing in an organ-specific microenvironment has the potential to identify lead compounds and improve our knowledge of OvCa biology. Furthermore, the successful adaption of a 3D culture assay for HTS will serve as a proof- of-concept for other drug screens using organotypic cultures.
""""""""Adaptation of an Organotypic 3 Dimensional Culture for High-Throughput Screening"""""""" There are few drugs that are effective against ovarian cancer, and their action is generally temporary. One reason that progress finding new drugs has been slow is because pharmaceutical programs use cancer cell lines cultured on plastic, often decades old, for screening. We propose to adapt an organotypic 3D culture of the human omentum, with primary ovarian cancer cell lines for screening compounds through the NIH Molecular Libraries Roadmap Initiative in collaboration with the Molecular Libraries Production Centers Network (MLPCN). Once efficacy of a compound is confirmed in vitro and in vivo a lead compound will be further developed in close collaboration between chemists, biologists and physicians.
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| Kenny, Hilary A; Lal-Nag, Madhu; White, Erin A et al. (2015) Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy. Nat Commun 6:6220 |
| Mitra, A K; Chiang, C Y; Tiwari, P et al. (2015) Microenvironment-induced downregulation of miR-193b drives ovarian cancer metastasis. Oncogene 34:5923-32 |
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| Lengyel, Ernst (2010) Ovarian cancer development and metastasis. Am J Pathol 177:1053-64 |
