Sphere Culture of Murine Lung Cancer Cell Lines Are Enriched with Cancer Initiating Cells

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Date: Nov. 13, 2012
From: PLoS ONE(Vol. 7, Issue 11)
Publisher: Public Library of Science
Document Type: Article
Length: 6,836 words
Lexile Measure: 1490L

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Author(s): Brian J. Morrison, Jason C. Steel, John C. Morris *

Introduction

Lung cancer is the cause of almost 20% of all cancer deaths in the United States [1]. It accounts for more deaths than the next four most common cancers combined. Despite improvements in diagnosis and treatment, the overall 5-year survival for all patients with lung cancer is a dismal 15%, and this declines to less than 2% in patients with metastatic disease [2]. Hence, lung cancer is a major public health problem, and a better understanding of the disease biology and improvements in treatment are greatly needed.

Increasing evidence supports the concept of a specialized population of cells within tumors termed cancer initiating cells (CICs), alternatively "cancer stem cells" or tumor-initiating cells. These cells are thought to be responsible for the tumor's origin, maintenance, progression, and resistance to therapy. CICs display functional characteristics of stem cells such as the capacity for self-renewal and the ability to give rise to differentiated progeny. Putative CICs have been identified in myeloid leukemia [3], , and tumors of the brain [5], [6] and breast [7]. More recently, lung CICs have been isolated from human cell lines and patient samples [8]-[15]. Investigating lung CICs may increase understanding of the origin of lung cancer and may lead to novel therapeutic approaches targeting these cells.

CICs have been shown to form multicellular three-dimensional spheres in vitro when grown in non-adherent serum-free conditions [6], [16]. Under these conditions, most tumor cells undergo anoikis, a form of programmed cell death, whereas the rare CICs divide to generate tumor spheroids. The sphere assay and a recently proposed mathematical interpretation allow for the assessment of the symmetric division expansion rate of malignant stem cell-like cells, and the evaluation of the effects of treatment on the self-renewal and proliferative activity of these cells [17]. This assay is a powerful tool to assess the functional and phenotypic properties associated with CICs. In a study using patient lung cancer samples, sphere formation was found in seven of 19 tumors examined [10]. These cells were found to have in vitro and in vivo properties of CICs including self-renewal, proliferative and differentiation capacity, expression of CD133, enrichment for side population (SP) cells, expression of the embryonic "stemness" genes Oct-4 and NANOG, chemotherapy resistance, and tumorigenicity. Sphere culture has been used to enrich for lung cancer CIC populations [8], [9], [13], [14].

Figure 1. In vitro tumorsphere assays. Photomicrographs (10X) of passage 2 day 7 sphere cultures of (A ) TC-1 spheres, (B ) HM-LLC spheres and (C ) LM-LLC sphere media culture. Bar = 100 [micro]m. (D ) Total fold expansion and (E ) symmetric division frequency over eight passages for TC-1 spheres (red) and HM-LLC spheres (black), and two passages for LM-LLC sphere media culture (grey) shows significant differences; *P<0.001. (F-G ) Fold expansion over seven passages for TC-1 spheres (red) and HM-LLC spheres (black). (H ) Passage 2 spheres were cultured and counted for total number of cells on the days as...

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Gale Document Number: GALE|A477091362