Abstract
Three-dimensional (3D) cell culture systems have been used to obtain multicellular spheroidal cell aggregates, or spheroids, from cancer cells. However, it is difficult to efficiently prepare large tumor-derived spheroids from cancer cells. To circumvent this problem, we here used a tool equipped with removal membrane, called Spheroid Catch, for the selection and enrichment of large-sized and/or size-matched spheroids from human squamous cell carcinoma (SAS cells) without loss of recovery. After a five-round process of selection and enrichment, we successfully isolated a subpopulation of SAS cells with augmented spheroid-forming capability, named eSAS: the efficiency of spheroid formation is 28.5% (eSAS) vs 16.8% (parental SAS). Notably, we found that some of eSAS cells survived after exposure of high doses of cisplatin in 3D culture. Moreover, orthotopic implantation by injecting eSAS cells into the tongues of nude mice showed reduced survival rate and increased tumor growth compared with those of nude mice injected with SAS cells. These results suggest that spheroids exhibiting properties of higher spheroid forming capacity can be efficiently collected by using Spheroid Catch. Indeed, genome-wide cDNA microarray and western blot analyses demonstrated higher mRNA and protein levels of hedgehog acyltransferase (HHAT), which is associated with stem maintenance in cell carcinoma by catalysing the N-palmitoylation of Hedgehog proteins, in eSAS cells than in SAS cells. We propose that Spheroid Catch could be useful for the study of spheroids, and potentially organoids, in the basic and clinical sciences, as an alternative method to other type of cell strainers.
Highlights
The formation of multicellular spheroidal cell aggregates, or spheroids, is a conspicuous characteristic of cancer stem cells (CSCs) and tumor-initiating cells (TICs) that possess the ability for self-renewal, proliferation and generation of downstream progenitor cells to promote tumor growth [1]
Recent advances in three-dimensional (3D) cell culture systems that mimic in vivo physiology allow observation of spheroid formation by a variety of cancer cell lines [4,5,6,7]. 3D culture is used for efficient antitumor drug screening to exclude false-positive compounds from entry into clinical trials [8]
SAS cells cultured in spheroid-forming medium (SFM) on a spheroid-forming plate (SFP) were collected and transferred to Spheroid Catch inserted into a collection tube (Figure 1A-i, -ii and -iii)
Summary
The formation of multicellular spheroidal cell aggregates, or spheroids, is a conspicuous characteristic of cancer stem cells (CSCs) and tumor-initiating cells (TICs) that possess the ability for self-renewal, proliferation and generation of downstream progenitor cells to promote tumor growth [1]. Since CSCs and TICs within the tumor mass have been proposed to mediate chemoresistance and cancer recurrence [2, 3], the development of an efficient experimental system to study the molecular mechanisms of tumor-derived spheroids in cultured cancer cells would facilitate considerably the study of the mechanisms involved in chemoresistance and cancer recurrence. For many cancer cell lines, the efficiency of spheroid formation is low and/or the size of the spheroids is small, which hampers detailed investigation of the molecular mechanisms of spheroids in vivo [1]. The development of a convenient and simple technique that allows selection of large-sized and/or size-matched spheroids in targeted cancer cell lines is under active investigation
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