A rapid quantification of invasive phenotype in head and neck squamous cell carcinoma: A novel 3D pillar array system
Introduction
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and the 5-year survival of a patient with the advanced stage is approximately 50% [1]. Metastasis is a leading cause of the poor prognosis of HNSCC patients, and its underlying pathogenesis and mechanism are still the focus of intense investigation [2], [3]. It is well recognized that the metastatic process has multiple steps: loss of cell-to-cell adhesion with increased cell motility (cell migration), invasion across the extracellular matrix (ECM), and the basement membrane of the blood vessels (intravasation), and extravasation into metastatic foci [4]. To develop or validate the diagnostic and therapeutic strategies (e.g., anti-cancer drug screening), several in vitro laboratory or in vivo animal models have been used to recapitulate multi-factorial interactions between tumor and surrounding tumor microenvironment (TME). Conventional in vitro experimental tools, such as scratch assay or modified Boyden chamber assay, are easier to perform with a simple design, which allows a more controlled environment for repetitive and reproducible measurement of cancer cell migration and invasion, when compared with in vivo animal model [5], [6], [7], [8]. However, the direct correlation of outcomes using in vitro assays with the biology of human cancer is disputed, mostly because of the limitations of a two-dimensional (2D) cell culture system, which lacks spatial and temporal components of a three-dimensional (3D) solid tumor. Considering that cancer cells grow and metastasize in the body with a 3D organization interacting with neighboring cancer cells as well as TME, the invasion assay should reflect the 3D condition to precisely evaluate the invasive phenotype of cancer [9], [10].
In the current study, we describe a novel, 3D pillar array system, in which Matrigel™ surrounded cancer spheroid on the tip of each pillar. For rapid quantification of the changes of invasive phenotype of cancer cells from the spheroids, simple staining and automated optical analysis is performed. To evaluate the clinical efficacy of this system, patient-derived cells (PDCs) of HNSCC patients were used after the validation experiments with immortalized cancer cell lines.
Section snippets
Patient-derived cell culture
Acquisition of PDC samples and the relevant experimental protocol was approved by the Institutional Review Board of Samsung Medical Center (SMC IRB file number 2015-06-132-008), and informed consent was obtained from patients. Also, this work was performed in compliance with all relevant ethical regulations and guidelines for research using human specimens. To introduce a spheroid into pillar/well chip platform, PDCs were acquired from HNSCC patients following informed consent. Detailed
Invasion assay of A549 cell line in the pillar array
As illustrated in Fig. 2, A549 cell line, which was usually used for 2D invasion assay, migrated from spheroid and attached on the surface of the pillar. To verify invasion assay in the pillar array, A549 having high mobility for invasion assay [15], [16] was used in the first experiment. In no drug treatment, cells surrounding spheroids attached to the pillar. Thus, those cells surrounding spheroid were faintly stained (Fig. 2A). Based on the fluorescence intensity, spheroid was identified,
Discussion
To date, various techniques for evaluating cancer cell invasiveness have been addressed and validated for the progression and prognosis of HNSCC. Notably, recent advances in bioengineering technology enable diverse platforms, mimicking the interaction between tumor and TME [25], [26] For example, cancer organoids or spheroids are formed via a hanging-drop method or within a U-shaped well plate. Then, the spheroids are imbedded into ECM, such as collagen or Matrigel™, to study changes in
Author contribution statement
Bokhyun Song, Soo Yoon Kim, Hye Jin Kim, and Da-Yong Shin performed the all experiments including culturing patient derived cells and prepared the figures 3–6.
Bosung Ku designed pillar array and supported experiments using pillar array.
Dong Woo Lee, Sung Yong Choi, and Man Ki Chung designed the study, interpreted the experimental data, prepared the all figures and wrote the main manuscript text.
All authors reviewed and confirmed the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported in part by a grant (NRF-2016R1D1A1B03931296) awarded by the Ministry of Education, and a grant (No. 2018R1C1B5045068) from the National Research Foundation of Korea (NRF) under the Korea government (MSIT).
References (34)
- et al.
Cancer metastasis: building a framework
Cell
(2006) - et al.
Breast cancer cell colonization of the human bone marrow adipose tissue niche
Neoplasia
(2015) - et al.
ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells
Am J Pathol
(2012) - et al.
Automatic 3D cell analysis in high-throughput microarray using micropillar and microwell chips
J Biomol Screen
(2015) - et al.
Epithelial-to-mesenchymal transition and cancer stem(-like) cells in head and neck squamous cell carcinoma
Cancer Lett
(2013) - et al.
Molecular requirements for epithelial-mesenchymal transition during tumor progression
Curr Opin Cell Biol
(2005) - et al.
Drug efficacy comparison of 3D forming and preforming sphere models with a micropillar and microwell chip platform
SLAS Discov
(2019) - et al.
Single cell-based automated quantification of therapy responses of invasive cancer spheroids in organotypic 3D culture
Methods
(2017) - et al.
Inhibition of MMP-2-mediated cellular invasion by NF-κB inhibitor DHMEQ in 3D culture of breast carcinoma MDA-MB-231 cells: A model for early phase of metastasis
Biochem Biophys Res Commun
(2017) - et al.
Targeted next-generation sequencing of locally advanced squamous cell carcinomas of the head and neck reveals druggable targets for improving adjuvant chemoradiation
Eur J Cancer
(2016)
Global cancer statistics
CA Cancer J Clin
Patient-derived models of acquired resistance can identify effective drug combinations for cancer
Science
A perspective on cancer cell metastasis
Science
CD271 confers an invasive and metastatic phenotype of head and neck squamous cell carcinoma through the upregulation of slug
Clin Cancer Res
Stromal fibroblasts are predictors of disease-related mortality in esophageal squamous cell carcinoma
Oncol Rep
Stanniocalcin 2 expression is regulated by hormone signalling and negatively affects breast cancer cell viability in vitro
J Endocrinol
Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device
Lab Chip
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SY Choi and DW Lee contributed to the present manuscript as co-1st authors.