Introduction
Cells form tissues in vivo through intimate interactions with extracellular matrices and surrounding cells [1-5]. Cancer tissues are char- acterized by the proliferation of fibroblasts, infiltration of immune cells, such as macro- phages, and induction of angiogenesis. These strongly depend on the microenvironment spe- cifically observed around cancer cells [6-10].
Cancer and other cells and intracellular compo- nents, including extracellular matrices, form a characteristic environment called cancer microenvironment. This microenvironment has recently been demonstrated to change dynami- cally and be strongly involved in cancer pro- gression. Specifically, various stimuli and nutri-
ents from the cancer microenvironment promote carcinogenesis. The cancer microenvironment is surrounded by various extracellular matrices which have bioactivity in vivo. Extracellular matrices include fibril-rich polymeric proteins (e.g., collagen and elastin), various glycopro- teins (e.g., fibronectin and laminin), and proteoglycans [11-16]. For example, interactions with cell surface receptors, such as integrins, occur in the extracellular matrices. The extracellular matrices influence cell motility and morphology, as well as cell proliferation and differentiation [17-20]. Therefore, the tumor cells are also likely to be influenced by the extracellular matrices at each stage from early carcinogenesis to proliferation, infiltration, and
Interleukin-6, Matrix Metallopeptidase-9 Expression in Three-dimensional Tumor Microenvironment using Oral Cancer Cells
Tamaki NAKANISHI
1and Koichi IMAI
21
Graduate School of Dentistry (Oral and Maxillofacial Surgery),
2Department of Biomaterials, Osaka Dental University, Osaka, Japan.
Synopsis
Cancer tissues strongly depend on the cancer microenvironment specifically observed around can- cer, where immune cell infiltration and angiogenesis, as well as fibroblast proliferation, are induced in comparison with normal tissues. Various three-dimensional culturing methods for solid cancer cells have already been reported as in vitro experimental methods to reflect the characteristics of cancers. The spheroid cultures, constructed with cancer cells, should more accurately reflect the biological characteristics of cancers than two-dimensional cultures. However, solid cancers appli- cable to spheroid culturing are still limited. Previously, we compared the results of inflammatory cytokine Interleukin-6 (IL-6) between two-dimensional culture and spheroid culture with HSC-4 cells derived from human oral squamous cell carcinoma collected from the tongue. In the present study, besides IL-6, the expression of Matrix Metalloproteinase-9 (MMP-9), which plays an important role in the infiltration and metastasis of cancer cells surrounded by ECM, was examined by real-time RT-PCR, and the sizes of spheroids were observed over time.
Key words: IL-6, MMP-9, ECM, RT-PCR, HSC-4 cells
ORIGINAL ARTICLE
metastasis. However, the ECM and intercellular interactions, required for the cancer microenvi- ronment, cannot be easily reproduced in vitro.
Specifically, the in vivo tumor cell environment cannot be easily reproduced using the conven- tional three-dimensional culture method [17-20].
Cancer cells constitute a three-dimensional structure in the microenvironment composed of interstitial tissues and normal cells in tumor tis- sues, and are proliferating under constant expo- sure to harsh conditions, such as low oxygen and limited nutrients.
Various spheroid culture methods have been developed to more accurately reflect the charac- teristics of cancer cells in vitro. Various swelling spheroid culture methods have been reported for ovarian, colon, and prostate cancers [24, 25].
However, not all solid cancers can be grown by spheroid culturing. Spheroid cultures more accurately reflect the biological characteristics of cancers than two-dimensional cultures. Cells exhibit various biological dynamics in three-dimensional spheroid cultures. Previously, we reproduced a three-dimensional tumor environment using the spheroids of HSC-4 cells collected from human squamous cell carcinoma to examine the expression of Interleukin-6 (IL-6) [26]. In the present study, besides IL-6,
the expression of Matrix Metalloproteinase-9 (MMP-9) , which plays an important role in the infiltration and metastasis of cancer cells, was investigated by real-time RT-PCR.
Materials & Methods 1. Cells and medium
Human lingual squamous cell carcinoma cell line HSC-4 (RIKEN RCB 1902, RIKEN Bio- Resource Reserch Center, Tshukuba, Japan), and human umbilical vein endothelial cell line HUVEC （ Human Umbilical Vein Endothelial Cells, LONZA, Basel, Switzerland ） were used.
HSC-4 cells were cultured in RPMI 1640 (Gibco, ThermoFisher Scientific Inc., MA, USA) supplemented with 10% FBS (Hyclone, GE Healthcare UK Ltd., England) and 1% penicil- lin/streptomycin (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan). HUVECs were cultured in EGM
TM-2 Endothelial Cell Growth Medium-2 BulletKit
TM(LONZA, Basel, Switzerland).
2. Vascular network formation
To promote cell adhesion and differentiation, HUVEC (Corning Inc., NY, USA) was cultured in a 96-well plate (Falcon 96 Well Cell Culture Plate, Corning Inc., NY, USA) coated with
Figure 1 Vascular network formation after immunostaining by ZOE fluorescence cell imager.
100μm
Matrigel
®(Corning Inc.) containing laminin, type IV collagen, and heparin sulfate proteogly- can. HUVECs were suspended in 100 μL of medium and seeded at 3.6 × 10
4cells/well. After static culturing at 37°C in a 5% CO
2incubator for 24 hours, the formation of a vascular network was confirmed with an inverted phase contrast microscope. The vascular network was
immunostained with a CD31 primary (Purified anti-human CD31 Antibody, BioLegend Inc., CA, USA) and secondary antibodies (Goat Anti-Mouse IgG, Dylight594, ThermoFisher Scientific Inc., MA, USA) to be observed with a ZOE fluorescence cell imager (Bio-Rad, CA, USA). The vascular network formation image is shown in Figure 1.
100μm Figure 2 ZOE fluorescence cell imager and inverted phase contrast microscope images due to three types of spheroid culture days.
Figure 3 An inverted phase contrast microscopic images of HSC-4 cells with a cell number of 1.0 × 103 seeded on the vascular network. (Left: single cells, Right: spheroids)
100μm
3. HSC-4 spheroid formation
HSC-4 cells were suspended in 150 μL of HUVEC culture medium and seeded in a 96-well spheroid microplate (96 Well Black With Clear Round Bottom Ultra Low Attachment Spheroid Microplate, Corning, NY,
USA) at 1.0 × 10
3cells/well. Three days later, the formation of HSC-4 spheroids was observed.
Live cells in the spheroids were stained with Calcein AM (ThermoFisher Scientific Inc., MA, USA) and observed at 4, 7, and 11 days using ZOE fluorescence cell imager (Figure 2). An- giogenesis was observed at 24 hours after the seeding of HUVECs. HSC-4 cells were prepared at 1.0 × 10
4cells/well, and divided into single- cell and spheroid groups (Figure 3). The single-cell group was two-dimensional cultures, while the spheroid group was 3-day cultures.
Each group was suspended and seeded in 50 μL of the HSC-4 culture medium. At 30 minutes after the seeding of the cells, type I collagen was added at 40 μL/well, followed by static culturing for 24 hours in a CO
2incubator to examine the expression levels of IL-6 and MMP-9 by real-time RT-PCR. In addition, the sizes of the spheroids were determined using a microscope over time.
Results
Figure 4 shows the expression levels of IL-6 and MMP-9 in the spheroids using real-time RT-PCR.
The expression levels of IL-6 and MMP-9 were
higher in the spheroid group than in the single- cell group, although there is no significant difference. The spheroids grew over time.
Discussion
The expression of MMP-9, besides IL-6, was enhanced in the spheroid cultures with HSC-4 cells collected from human squamous cell carcinoma, as reported previously. However, their expression levels were lower in the two-dimensional culture. Thus, the spheroid culture with HSC-4 cells could reproduce the microenvironment of cancer cells. IL-6, a repre- sentative inflammatory cytokine, is activated in various cancer cells and involved in cancer formation and metastasis. MMP-9 is involved in the destruction of the basement membrane made of type IV collagen, a vascular barrier relevant to cancer metastasis. Similar results were obtained with HSC-4 collected from human tongue squamous cell carcinoma.
Biological tissues exhibit a complicated in vivo environment, including intercellular inter- actions, cell-extracellular matrix interactions, nutrient transport, and cell migration. However, it’s been difficult to reproduce such an environ- ment in vitro by conventional two-dimensional culture. Cancer tissues have a complicated structure with various cells and ECM as back- grounds, besides normal tissues and cancer cells in the cancer tissues. Many studies have been conducted using an in vitro cancer microenvi-
Figure 4 Comparison of expression levels of IL-6 and MMP-9 in HSC-4 cells by real-time RT-PCR Both IL-6 and MMP-9 showed greater expression of spheroids than single cells
.
ronment [6-10, 26-30]. Three-dimensional cul- ture is widely employed with single scaffolds, such as collagen. However, as the extracellular matrices of living tissues have more complicated structures, they should be completely elucidated and multiple natural extracellular matrices should be applied, precluding the construction with artificial cell matrices. Therefore, important data on cancer tissues are likely to be obtained using spheroid cultures that construct three-dimensional tissues only with cells. Spe- cifically, the same extracellular environment as that in the body should be reproduced to analyze the functions of cancer cells. The results of the present study suggested the applicability of our method for reproducing biological environments in vitro.
Biological cancer microenvironments cannot easily be reproduced in conventional three-dimensional cultures using single collagen because of the lack of ECM and intercellular interactions. If cancer cells derived from the oral cavity can be cultured in a spheroid state, the effects of potential causative agents on the development of oral cancers may be elucidated.
Spheroid cultures generate microenvironments similar to biological tissues using intercellular interactions and natural extracellular matrices [31]. Spheroids serve as culture models most suitable for cell migration, differentiation, survival, and proliferation. In two-dimensional cultures, cells polarize only partially, while, in three-dimensional cultures, they polarize more similarly to in vivo. Thus, different gene expres- sion patterns are exhibited between two- and three-dimensionally proliferated cells. The above results can be applied to novel tests as alternatives to animal experiments, in order to simulate biological environments in vitro.
Acknowledgment
This study was carried out using the Institute of Dental Research, Osaka Dental University.
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(Received: November 12, 2018/
Accepted: December 23, 2018)
Corresponding author:
