Cancer Letters

Cancer Letters

Volume 367, Issue 1, 10 October 2015, Pages 34-42
Cancer Letters

Original Articles
Colorectal cancer-derived tumor spheroids retain the characteristics of original tumors

https://doi.org/10.1016/j.canlet.2015.06.024Get rights and content

Highlights

  • Establishment of three dimensional spheroids derived from colorectal cancer tissues.

  • The spheroids retain molecular and histological characteristics of patient's tumor.

  • The spheroids maintain stem cell composition for months.

  • Cetuximab efficacy test reflects the retaining human genetic mutation.

  • Providing new platform for the personalized anti-cancer therapeutics.

Abstract

Primary cultures of cancer cells are useful for developing personalized medicine. In this study, we characterized three lines of three-dimensional (3D) tumor spheroids established directly from tumor tissues of patients with colorectal cancers (CRCs). Each line mainly included EpCAM-positive cells and cells expressing putative cancer stem cell markers such as CD133, CD44, CD24, ALDH1, and LGR5. These characteristic stem cell markers remained identically for months in vitro. Short tandem repeat genotyping suggested that genetic fingerprints of these tumor spheroids were similar to those of the original tumor tissues from which they were derived. Mutational analysis showed that each line had the same mutation profile for APC, KRAS, MLH1, serine–threonine kinase 11, and TP53 as its parental tumor tissue. One line harboring an activating KRAS mutation was resistant to cetuximab while the remaining two lines harboring wild-type KRAS showed different responses to cetuximab. Immunohistochemical analysis showed that xenograft tumors derived from these lines retained the histopathological and mutational patterns of their parental tumors. Collectively, these results clearly showed that 3D tumor spheroids directly generated from tumor tissues of patients with CRCs preserved the characteristics of their parental tumor tissues and could be used for developing personalized medicines for CRCs.

Introduction

To date, development of novel anticancer drugs has largely depended on the use of two-dimensional (2D) monolayer cancer cell lines and 2D cell line-implanted xenograft models. These 2D cancer cell lines are easy to manipulate, thus facilitating high-throughput screening (HTS) [1]. However, these cell lines inadequately reflect the biological features of original tumor tissues, thus limiting their potential to predict in vivo cellular responses to potential anticancer drugs [2]. Therefore, alternative culture systems of cancer cells that realistically reflect the characteristics of parental tumors are needed for studying cancer biology and validating potential anticancer drugs.

Unlike 2D cancer cell lines, three-dimensional (3D) tumor spheroid culture systems preserve the characteristics of original tumors. These 3D tumor spheroids can be generated from both, established 2D cancer cell lines and fragments of tumor tissue. However, compared with 3D tumor spheroids derived from cancer cell lines, those derived from primary tumor tissues are better at mimicking the features of original tumors, such as tumor heterogeneity and complexity [3], [4], [5], [6], [7]. In addition, these primary tumor tissue-derived spheroids show some characteristics of cancer stem cells (CSCs) [1], [7].

Recent studies have shown that novel 3D tumor spheroids, termed cancer tissue-originated spheroids (CTOSs), can be directly generated from tumor fragments of various cancers, including colorectal cancers (CRCs), urothelial cancer, and lung cancer [8], [9], [10]. Importantly, CTOSs and CTOS-derived xenograft (CTOS-DX) tumors are histopathologically similar to their corresponding parental tumors [8], [9], [10]. In this study, we applied CTOS culturing method to report the characteristics of three CTOS lines generated from surgical tumor specimens of Korean patients with CRCs. DNA fingerprinting, gene mutation analysis, and histopathological analysis suggested that these CRC CTOS lines realistically reflected the characteristics of original tumors from which they were derived.

Section snippets

Patient tumor specimens and CTOS culture

Fresh surgical specimens from patients with CRCs were obtained from the Department of Surgery, Kyungpook National University Medical Center (KNUMC), Daegu, Korea, with the patients' informed consent. The study was approved by the Institutional Review Board (No. KNUMCBIO_12-1007) of the KNUMC. Histological assessment of the surgically resected tumor specimens was conducted by a pathologist. The specimens were then transferred to our laboratory in a test tube containing RPMI supplemented with

CRC CTOSs represent inter-patient heterogeneity

In this study, we successfully established three CTOS lines from freshly resected tumor tissues from three patients with CRCs (1 patient with colon cancer and two patients with rectal cancers; Table 1). Cultures of CTOSs grew as morphologically compact and round spheres or cell aggregates (Fig. 1, left). We observed that these cell aggregates underwent morphological changes to form highly compact and round spheres after culturing for 2 days. H & E staining suggested that these CTOSs were

Discussion

Before in vivo testing in animal models, potential anticancer drugs are routinely validated using conventional 2D monolayer cultures of various cancer cell lines, which is a simple and well-suited approach for HTS. However, this model poorly reflects the complex in vivo features of solid tumors, such as a hypoxic/necrotic center, stem cell properties, slow proliferation, and nutrient/drug gradient. In contrast, organotypic 3D tumor spheroid cultures derived directly from patients' tumor tissues

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant Number: HI11C1300). This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. NRF-2012R1A2A1A03670452). The biospecimens for this study were provided by National Biobank of Korea-Kyungpook National University Hospital

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