Elsevier

Oral Oncology

Volume 108, September 2020, 104807
Oral Oncology

A rapid quantification of invasive phenotype in head and neck squamous cell carcinoma: A novel 3D pillar array system

https://doi.org/10.1016/j.oraloncology.2020.104807Get rights and content

Highlights

  • Detecting and evaluating invasive phenotype of head and neck squamous cell carcinoma is challenging.

  • 3D pillar array system allows rapid and easy detection of invasive cancer cell by measuring cell attachment on the pillar.

  • 3D pillar array system also allows testing of the impact of drug treatments on the changes in invasive phenotype.

Abstract

Background

The widely used in vitro invasion assays for head and neck squamous cell carcinoma (HNSCC) are wound healing, transwell, and organotypic assays. However, these are still lab-intensive and time-consuming tasks. For the rapid detection and high throughput screening of invasiveness in 3D condition, we propose a novel spheroid invasion assay using commercially available pillar platform system.

Materials and methods

Using the pillar-based spheroid invasion assay, migration and invasion was evaluated in three patient-derived cells (PDCs) of HNSCC. Immunofluorescence of live cells was used for the quantitative measurement of migratory and invaded cells attached to the pillar. Expression of epithelial-mesenchymal transition (EMT)-related gene (snai1/2) was measured by qRT-PCR. We also tested the impact of drug treatments (cisplatin, docetaxel) on the changes in the invasive phenotype.

Results

All PDCs successfully formed spheroid at 4 days and can be measured invasiveness within 7 days. Intriguingly, one PDC (#1) obtained from the advanced stage showed robust migration, invasion and higher transcription of snai1/2, compared with the other two PDCs. Furthermore, the invasion ratio of the control spheroids was about 70% while the invasion ratios of drug-treated spheroids were lower than 50%, and the difference showed statistical significance (p < 0.01).

Conclusion

The presented spheroid invasion assay using pillar array could be useful for the evaluation of cancer cell behavior and physiology in response to diverse therapeutic drugs.

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)

  • A. Jemal et al.

    Global cancer statistics

    CA Cancer J Clin

    (2011)
  • A.S. Crystal et al.

    Patient-derived models of acquired resistance can identify effective drug combinations for cancer

    Science

    (2014)
  • C.L. Chaffer et al.

    A perspective on cancer cell metastasis

    Science

    (2011)
  • M.K. Chung et al.

    CD271 confers an invasive and metastatic phenotype of head and neck squamous cell carcinoma through the upregulation of slug

    Clin Cancer Res

    (2018)
  • S. Saito et al.

    Stromal fibroblasts are predictors of disease-related mortality in esophageal squamous cell carcinoma

    Oncol Rep

    (2014)
  • S. Raulic et al.

    Stanniocalcin 2 expression is regulated by hormone signalling and negatively affects breast cancer cell viability in vitro

    J Endocrinol

    (2008)
  • T. Liu et al.

    Carcinoma-associated fibroblasts promoted tumor spheroid invasion on a microfluidic 3D co-culture device

    Lab Chip

    (2010)
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