Homogeneous pancreatic cancer spheroids mimic growth pattern of circulating tumor cell clusters and macrometastases: displaying heterogeneity and crater-like structure on inner layer

Feng, Hao; Ou, Baochi; Zhao, Jingkun; Yin, Shuai; Liu, Aiguo; Oechsle, Eva; Thasler, Wolfgang E.

doi:10.1007/s00432-017-2434-2

Cited by 16 publications

(12 citation statements)

References 45 publications

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“…Based on these limitations, we considered the forced floating method using ULA plates as the most suitable and straightforward method to generate RT4 spheroids for cytotoxicity assays. Our findings corroborate with the literature reporting promising results using ULA method with different types of tumor cells (Vinci et al, 2012 ; Howes et al, 2014 ; Feng et al, 2017 ; Selby et al, 2017 ).…”

Section: Resultssupporting

confidence: 93%

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

et al. 2017

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Introduction: Cell-based assays using three-dimensional (3D) cell cultures may reflect the antitumor activity of compounds more accurately, since these models reproduce the tumor microenvironment better.Methods: Here, we report a comparative analysis of cell behavior in the two most widely employed methods for 3D spheroid culture, forced floating (Ultra-low Attachment, ULA, plates), and hanging drop (HD) methods, using the RT4 human bladder cancer cell line as a model. The morphology parameters and growth/metabolism of the spheroids generated were first characterized, using four different cell-seeding concentrations (0.5, 1.25, 2.5, and 3.75 × 104 cells/mL), and then, subjected to drug resistance evaluation.Results: Both methods generated spheroids with a smooth surface and round shape in a spheroidization time of about 48 h, regardless of the cell-seeding concentration used. Reduced cell growth and metabolism was observed in 3D cultures compared to two-dimensional (2D) cultures. The optimal range of spheroid diameter (300–500 μm) was obtained using cultures initiated with 0.5 and 1.25 × 104 cells/mL for the ULA method and 2.5 and 3.75 × 104 cells/mL for the HD method. RT4 cells cultured under 3D conditions also exhibited a higher resistance to doxorubicin (IC50 of 1.00 and 0.83 μg/mL for the ULA and HD methods, respectively) compared to 2D cultures (IC50 ranging from 0.39 to 0.43).Conclusions: Comparing the results, we concluded that the forced floating method using ULA plates was considered more suitable and straightforward to generate RT4 spheroids for drug screening/cytotoxicity assays. The results presented here also contribute to the improvement in the standardization of the 3D cultures required for widespread application.

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Section: Resultssupporting

confidence: 93%

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

et al. 2017

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“…Such results are consistent with the observation in Ishikawa endometrial cells that a unicellular hollow spheroid is the starting point for differentiation of a multicellular hollow spheroid (Fleming, 2019). Multicellular hollow spheroids may well proliferate inward to form solid spheroids, a process observed for pancreatic spheroids (Feng et al, 2017) but not yet observed in Ishikawa spheroids. What has been observed is that hollow spheroids can proliferate outward by forming additional attached hollow spheroids (Fleming, 2018a).…”

Section: Mitonucleons Are Also Observed In Ishikawa Epithelial Tubulessupporting

confidence: 90%

“…As the gas vacuole within the mitonucleon in a single monolayer cell expands, it pressures polyploid nuclei against the cytoplasmic membrane and the developing spheroid detaches from the monolayer, having been converted from an anchored into a mobile "cell". Researchers studying spheroid formation in pancreatic cancer cell lines (Feng et al 2017), elicited structures capable of growth as various kinds of spheroids by an alternative method called high-throughput single-cell derived sphere formation. The method is like cloning but uses a sophisticated device to guarantee single cell distribution in conditions favoring non-attachment (Chen et al, 2015).…”

Section: Mitonucleons Are Also Observed In Ishikawa Epithelial Tubulesmentioning

confidence: 99%

Tubular membranes extended between monolayer cells, from solid spheroids, and from clustered hollow spheroids in Ishikawa endometrial cell cultures can carry chromatin granules and mitonucleons

Fleming

2019

Preprint

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Membrane tubule extensions, recently recognized an important communication element in mammalian cells are demonstrated to form in Ishikawa endometrial epithelial cells growing in monolayers, and to extend from solid spheroids and from clustered hollow spheroids. Two kinds of chromatin cargoes travel through these tubules. Chromatin granules can pass through an endometrial tubule bridge extending from one monolayer fragment to another. The passage of granules over time from one of the fragments appears to support the self-assembly of nuclei in the other colony fragment. Similarly, in a process detected by observing an open-ended membrane tubule extending from a solid cell spheroid a nucleus formed over a period of 3 hours.Indications are that chromatin granules such as those observed in the amitotic processes of epithelial dome cell formation and of hollow spheroid cell formation are contributing to the build up of nuclei. Mitonucleons, a transient subcellular organelle consisting of fused mitochondria intimately associated with aggregated chromatin are also observed to pass through tubular membrane extensions. Multiple membrane extensions can be shown to to extend from clusters of unicellular polyploid hollow spheroids whose formation is described for the first time in this paper.

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“…Nevertheless, the similarities between these clumps and CTC clusters (with respect to cell–cell contacts and expression of CTC-specific markers) have not been fully addressed. Likewise, complex methods for the development of multicellular and single cell-derived tumor spheroids from established pancreatic cell lines (using hanging drop and ultra-low attachment plates) have been recently reported as means to mimic the growth pattern of CTC clusters ( 28 ), but the size and morphology of these spheroids (large compact or hollow spheres) have little similarities to isolated CTC clusters.…”

Section: The Problemmentioning

confidence: 99%

In Vitro Model-Systems to Understand the Biology and Clinical Significance of Circulating Tumor Cell Clusters

2018

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The isolation of clusters of circulating tumor cells (CTCs) from cancer patients has recently challenged the accepted view that the initiation of secondary tumors during metastasis involves the dissemination of individual cancer cells. As such clusters appear to be more aggressive than single tumor cells, CTC clusters are now considered a main player in the metastatic process, and many studies are exploring their diagnostic, prognostic, and clinical significance. However, several technical challenges limit advances in this area. Here, we suggest the use of established cancer cell lines that grow as cell clusters in suspension as a complementary approach that can help in understanding the biology of CTC clusters and their clinical significance. We argue that the many similarities between these “surrogate” clusters and the CTC clusters isolated from patients (e.g., in terms of size, morphology, heterogeneous expression of epithelial and mesenchymal markers, and type of cell–cell junctions) make these cell lines ideal systems for the development of strategies aimed at preventing or slowing down the metastatic process by targeting CTC clusters.

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Homogeneous pancreatic cancer spheroids mimic growth pattern of circulating tumor cell clusters and macrometastases: displaying heterogeneity and crater-like structure on inner layer

Cited by 16 publications

References 45 publications

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

Comparative Analysis of 3D Bladder Tumor Spheroids Obtained by Forced Floating and Hanging Drop Methods for Drug Screening

Tubular membranes extended between monolayer cells, from solid spheroids, and from clustered hollow spheroids in Ishikawa endometrial cell cultures can carry chromatin granules and mitonucleons

In Vitro Model-Systems to Understand the Biology and Clinical Significance of Circulating Tumor Cell Clusters

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