Dataset Information

High-throughput screening in colorectal cancer tissue-originated spheroids.

ABSTRACT: Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In this system, we apply the CTOS method in an ex vivo platform from xenograft tumors, using machines to handle CTOS and reagents, and testing a CTOS reference panel of multiple CTOS lines for the hit drugs. CTOS passages in xenograft tumors resulted in minimal changes of morphological and genomic status, and xenograft tumor generation efficiently expanded the number of CTOS to evaluate multiple drugs. Our panel of colorectal cancer CTOS lines exhibited diverse sensitivities to the hit compounds, demonstrating the usefulness of this system for investigating highly heterogeneous disease.

SUBMITTER: Kondo J

PROVIDER: S-EPMC6317944 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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High-throughput screening in colorectal cancer tissue-originated spheroids.

Kondo Jumpei J Ekawa Tomoya T Endo Hiroko H Yamazaki Kanami K Tanaka Norio N Kukita Yoji Y Okuyama Hiroaki H Okami Jiro J Imamura Fumio F Ohue Masayuki M Kato Kikuya K Nomura Taisei T Kohara Arihiro A Mori Seiichi S Dan Shingo S Inoue Masahiro M

Cancer science 20181120 1

Patient-derived cancer organoid culture is an important live material that reflects clinical heterogeneity. However, the limited amount of organoids available for each case as well as the considerable amount of time and cost to expand in vitro makes it impractical to perform high-throughput drug screening using organoid cultures from multiple patients. Here, we report an advanced system for the high-throughput screening of 2427 drugs using the cancer tissue-originated spheroid (CTOS) method. In ...[more]

PMID: 30343529

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Project description:Identifying new substances that could potentially be used for tumor therapy and the precise analysis of their spectrum of action requires models that are as similar as possible to the tumor present in the patient. Traditionally, two-dimensional (2D) cell cultures are used. However, these only resemble solid tumors to a limited extent. More realistic in vivo models, such as tissue cultures, which are invaluable for the final analysis of the effect of new substances, are unsuitable for high-throughput screening (HTS), such as substance library screening. Therefore, we addressed which parameters need to be optimized to produce 3D cultures suitable for HTS using established tumor cell lines and ultra-low attachment plates, and we tested which experimental parameters need to be considered. In our studies, we have focused on cell lines from gliomas. Gliomas are incurable tumors of the central nervous system and are the subject of intensive research. Our studies used ten glioma cell lines from which we generated spheroids using ultra-low attachment plates. We then determined the spheroid size as a function of the initial cell number and the culture time. We analyzed cell viability using propidium iodide staining, evaluated the effects of temozolomide and radiation on spheroids, and compared the effect to that on 2D cultures. We found that spheroid size correlated linearly with the initial cell number. Fewer cells (250-500) generally resulted in better growth than a higher number. However, not all cell lines produced growing spheroids at all. The spheroids had an outer layer of living cells and an inner core of dead cells. The size of the inner core of dead cells was different in the various cell lines and developed differently during the incubation period. Radiation affected spheroids more than 2D cultures, especially at higher cell densities. Our results provide insight into using glioma cell lines to form spheroids as model systems. We have identified initial cell numbers as a critical parameter for their effective use in research, offering a hopeful outlook for tumor therapy research and drug development.

Project description:BackgroundOrganotypic tumor spheroids, a 3D in vitro model derived from patient tumor material, preserve tissue heterogeneity and retain structural tissue elements, thus replicating the in vivo tumor more closely than commonly used 2D and 3D cell line models. Such structures harbour tumorigenic cells, as revealed by xenograft implantation studies in animal models and maintain the genetic makeup of the original tumor material. The aim of our work was a morphological and proteomic characterization of organotypic spheroids derived from colorectal cancer tissue in order to get insight into their composition and associated biology.ResultsMorphological analysis showed that spheroids were of about 250 μm in size and varied in structure, while the spheroid cells differed in shape and size and were tightly packed together by desmosomes and tight junctions. Our proteomic data revealed significant alterations in protein expression in organotypic tumor spheroids cultured as primary explants compared to primary colorectal cancer tissue. Components underlying cellular and tissue architecture were changed; nuclear DNA/ chromatin maintenance systems were up-regulated, whereas various mitochondrial components were down-regulated in spheroids. Most interestingly, the mesenchymal cells appear to be substantial component in such cellular assemblies. Thus the observed changes may partly occur in this cellular compartment. Finally, in the proteomics analysis stem cell-like characteristics were observed within the spheroid cellular assembly, reflected by accumulation of Alcam, Ctnnb1, Aldh1, Gpx2, and CD166. These findings were underlined by IHC analysis of Ctnnb1, CD24 and CD44, therefore warranting closer investigation of the tumorigenic compartment in this 3D culture model for tumor tissue.ConclusionsOur analysis of organotypic CRC tumor spheroids has identified biological processes associated with a mixture of cell types and states, including protein markers for mesenchymal and stem-like cells. This 3D tumor model in which tumor heterogeneity is preserved may represent an advantageous model system to investigate novel therapeutic approaches.

Project description:Targeted intracellular delivery of biomolecules and therapeutic cargo enables the controlled manipulation of cellular processes. Laser-based optoporation has emerged as a versatile, non-invasive technique that employs light-based transient physical disruption of the cell membrane and achieves high transfection efficiency with low cell damage. Testing of the delivery efficiency of optoporation-based techniques has been conducted on single cells in monolayers, but its applicability in three-dimensional (3D) cell clusters/spheroids has not been explored. Cancer cells grown as 3D tumor spheroids are widely used in anti-cancer drug screening and can be potentially employed for testing delivery efficiency. Towards this goal, we demonstrated the optoporation-based high-throughput intracellular delivery of a model fluorescent cargo (propidium iodide, PI) within 3D SiHa human cervical cancer spheroids. To enable this technique, nano-spiked core-shell gold-coated polystyrene nanoparticles (ns-AuNPs) with a high surface-to-volume ratio were fabricated. ns-AuNPs exhibited high electric field enhancement and highly localized heating at an excitation wavelength of 680 nm. ns-AuNPs were co-incubated with cancer cells within hanging droplets to enable the rapid aggregation and assembly of spheroids. Nanosecond pulsed-laser excitation at the optimized values of laser fluence (45 mJ cm-2), pulse frequency (10 Hz), laser exposure time (30 s), and ns-AuNP concentration (5 × 1010 particles per ml) resulted in the successful delivery of PI dye into cancer cells. This technique ensured high delivery efficiency (89.6 ± 2.8%) while maintaining high cellular viability (97.4 ± 0.4%), thereby validating the applicability of this technique for intracellular delivery. The optoporation-based strategy can enable high-throughput single cell manipulation, is scalable towards larger 3D tissue constructs, and may provide translational benefits for the delivery of anti-cancer therapeutics to tumors.

Dataset Information

High-throughput screening in colorectal cancer tissue-originated spheroids.

Publications

High-throughput screening in colorectal cancer tissue-originated spheroids.

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