Elsevier

Experimental Cell Research

Volume 319, Issue 14, 15 August 2013, Pages 2216-2229
Experimental Cell Research

Research Article
Colon cancer mesenchymal stem cells modulate the tumorigenicity of colon cancer through interleukin 6

https://doi.org/10.1016/j.yexcr.2013.06.003Get rights and content

Highlights

  • We isolated mesenchymal stem cell (MSC)-like cells from colon cancer tissues.

  • We found that these isolated MSC-like cells are nontumorigenic.

  • We found that these isolated MSC-like cells could promote tumor growth.

  • We found that IL-6 from MSC-like cells is important for tumor cell growth.

  • We found that IL-6/Notch-1/CD44 signaling axis occurs in colon tumor cells.

Abstract

Multipotent mesenchymal stem cells (MSCs) have been isolated from several tumors and are implicated to play critical roles to increase malignant cell growth, invasion and metastasis. Here, we show that the MSC-like cells were isolated from human colon cancer tissues. These isolated hCC-MSCs (human colon cancer-derived mesenchymal stem cells) shared similar characteristic features with bone marrow-derived MSCs, which include cell morphology, surface antigens and specific gene expression. Additionally, the hCC-MSCs could differentiate into osteocytes or adipocytes under appropriate culture conditions. The conditioned medium collected from the cultured hCC-MSCs was shown to enhance the migration and invasive activity of HCT-116 colon cancer cells in vitro. Besides, transplantation of HCT-116 cells along with hCC-MSCs in nude mice increased the tumor growth and metastasis. Further study revealed that IL-6 present in the hCC-MSC-conditioned medium sufficiently induced the levels of Notch-1 and CD44 in HCT-116 and HT-29 cells, which contribute to enhance tumorigenic activity of HCT-116 and HT-29 cells. By using immunohistochemical staining, the intense co-expression of IL-6, Notch-1 and CD44 was predominantly detected in human colon cancer tissues. Taken together, our findings suggest the importance of the IL-6/Notch-1/CD44 signaling axis in the interaction between hCC-MSCs and colon cancer cells.

Introduction

Mesenchymal stem cells (MSCs) are multipotent progenitor cells that are present in numerous normal tissues, including adipose tissue, bone marrow and liver. MSCs could differentiate into bone and adipose cells under specific culture conditions [1], [2], [3], [4]. In addition to normal tissues, MSC-like cells have recently been isolated from tumor tissues, where they may participate in the stroma formation within the tumor. For instance, multipotent MSCs were identified in lipomas, bone sarcomas and gastric tumor tissues and in the tumor stroma of a prostate cancer xenograft in a mouse model [5], [6], [7], [8]. All these MSCs isolated from these tumor tissues have similar phenotypes, such as the spindle-shaped morphology, unique surface markers, stem cell-associated gene expression, and the potency to differentiate into adipose and bone cell lineages. Although the detailed molecular markers for MSCs are still not completely defined, several stem cell transcription factors (Oct-4 and Nanog), self-renewal regulatory factors (such as Bmi1), surface antigens (CD44, CD90 and CD73) and abundant VEGF could be commonly detected in MSCs [9], [10], [11]. Thus, besides cancer stem cells that initiate and maintain tumor formation, the MSCs in tumor tissues may also act as important contributors in tumor promotion or progression.

In fact, many studies have emphasized the association between MSCs and tumor formation or progression [12]. It was reported that tumor-stromal microenvironment may recruit or mobilize adult stem cells into tumor tissues in certain tumors [13]. Using a noninvasive micro-PET imaging and in situ analysis in a mouse model clearly revealed that the implanted MSCs could migrate into tumor tissues during tumor formation, and subsequently grew into the tumor stroma [14]. MSCs within tumor lesions might have an impact on mutagenesis of tumor cells [15], [16]. In addition, MSCs within the tumor stroma could also secrete cytokines and chemokines that mediate tumor cells growth and metastasis [17]. Karnoub et al. [18] demonstrated that the chemokine CCL5 secreted from human bone marrow-derived MSCs could enhance breast cancer metastasis. Similarly, Prantl et al. [19] found that prostate tumor cells mixed with adipose tissue-derived MSCs led to greatly enhance tumor progression. In contrast, MSCs might have different roles in tumor progression. Khakoo et al. [20] showed that MSCs possessed potent antitumorigenic effects on Kaposi's sarcoma in an animal model.

IL-6 is an inflammatory cytokine, which has been implicated in the progression of a variety of malignancies, including colon cancer. Patients with colon cancer have higher levels of IL-6 in serum than the healthy control group [21]. In addition, IL-6 could stimulate the growth of colorectal carcinoma cells through a paracrine mechanism [22]. Despite the close correlation of IL-6 levels with the progression of colon cancer, the molecular events linking IL-6 to colon cancer are not fully understood. In general, IL-6 may initiate multiple signaling cascades, including STAT, PI3K/AKT and ERK/MAPK pathways. Recently, it was found that IL-6 could induce clonogenic growth and invasiveness of SW48 colon cancer cells via the signals of the Ras/MAPK and PI3K/AKT, and the enhanced malignant phenotypes might be related to the up-regulation of specific matrix metalloproteinases (MMPs) induced by IL6 [23].

Notch is a family of transmembrane receptor proteins, which consists of four members (Notch-1, Notch-2, Notch-3 and Notch-4). Activation of the Notch-mediated signaling requires ligand binding, including Jagged-1, Jagged-2, Delta-like-1, Delta-like-3 and Delta-like-4 [24]. After ligand binding, Notch proteins undergo sequentially proteolytic cleavage, liberate the intracellular domain, and then translocate to the nucleus, which in turn activate the downstream target genes by associating with a cellular DNA-binding protein CBF-1. The signaling via Notch plays important roles in cell survival and differentiation and has been involved in pathogenesis of several tumors. Aberrant Notch signaling has been frequently detected in human malignancies. Although a wealth of data suggests a critical role for Notch in the tumor initiation and progression, the function of Notch as an oncogene or a tumor suppressor gene seems to depend on the spatial or temporal phenotype in given tumor types [25]. In human colon cancer, growing evidence has suggested that active Notch signaling enhances the growth, survival and colony formation of tumor cells in vitro, as well as induces tumor progression and metastasis in vivo [26].

CD44 is a cell-surface glycoprotein with various isoforms, which is expressed in many cells, including hematopoietic, endothelial and epithelial cells [27]. The production of different CD44 isoforms is mainly due to alternative splicing of the encoding transcript and post-translational modifications of the gene products. CD44 is a major receptor for hyaluronan (HA) and can also interact with other extracellular ligands, such as collagen, laminin, fibronetin, and MMPs [28]. A large body of evidence has suggested that CD44 plays important roles in cell adhesion, cell migration and signaling transduction during embryonic development, hematopoiesis, immune response, and in several types of carcinoma [29], [30]. Recently, CD44 has been implicated as a cancer stem cell marker in colon, breast, gastric, and head and neck carcinomas [29], [30], [31], [32]. As for colon cancer, the isolated CD133+/CD44+ subpopulation cells from HCT-116 colon cancer cells displayed self-renewal and differentiation capacity in vitro, and initiated the formation of xerograft tumors more efficiently than the non-CD133+/CD44+ cell population [31], [32].

Herein, we isolated and characterized MSC-like cells, hCC-MSCs, derived from primary human colon cancer tissues. Besides the effect of hCC-MSCs on the progression of colon cancer, the potential molecular events between hCC-MSCs and colon cancer were determined in the study.

Section snippets

Isolation and primary culture of MSCs from colon cancer tissues

Fresh colon cancer tissues were obtained during surgery. This study was approved by the institutional review board (IRB) of Chang-Gung Memorial Hospital. Written informed consent was received from all participants. A total of twenty-three primary colon cancer tissue specimens from patients with cancer stage IIA to IVA were collected. The specimens were first immersed in 95% ethanol to avoid contamination, and then washed with phosphate-buffered saline (PBS) and antibiotics. The surface of the

Isolation and characterization of MSC-like cells from human colon cancers

Culture of cells from fresh human colon cancer tissues under standard MSC nutritional conditions resulted in isolation of MSC-like cells (hCC-MSCs) with typical MSC morphology (Fig. 1A). In the study, hCC-MSCs were isolated from 8 out of 23 fresh human colon cancer samples. Like hBM-MSCs, the isolated hCC-MSCs initially displayed long, dispersed, spindle-shaped and fibroblast-like morphology, and began to form cell clusters after culture for about two weeks. Immunophenotyping showed that the

Discussion

The MSCs are known for their ability of self-renewal and differentiation into a variety of cell lineages [35], [36]. Interestingly, several studies have shown the importance of MSCs in tumor tissues. Bone marrow-derived MSCs were recently found to participate in the tumor microenvironment that mediates tumor growth and progression [12]. However, whether MSC-like cells exist in colon cancer lesions or the potential roles of these MSC-like cells in colon cancer progression remain to be addressed.

Conclusions

We have isolated MSC-like cells from primary human colon cancer tissues. Our study strengthens the concept of the association between MSC-like cells and tumor cells in colon carcinogenesis and demonstrates the importance of IL-6/Notch-1/CD44 regulatory signaling in colon cancer progression. Blockade of the IL-6/Notch-1/CD44 signaling axis might provide a solution to disrupt the stemness of colon cancer cells and prevent tumor progression.

Conflict of interest

None for all authors.

Acknowledgments

This work was supported by the grants from Chang Gung Memorial Hospital, Taiwan (CMRPG660191) and ChangHua Christian Hospital (100- CCH-IRP-19).

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