Novel method of cell line establishment utilizing fluorescence-activated cell sorting resulting in 6 new head and neck squamous cell carcinoma lines

Abstract

Background: The purpose of this study was to present the establishment of new cell lines, which is important to cancer research.

Methods: Six new head and neck squamous cell carcinoma cell lines were established using a novel fluorescence-activated cell sorting (FACS) method in order to overcome the barrier of fibroblast overgrowth and the susceptibility of primary tumors to fail in vitro.

Results: Antibodies chosen for specific targeting of epithelial cells and fibroblasts successfully separated cells for line establishment in 6 of 12 attempts, providing an alternative method of establishing head and neck squamous cell carcinoma cell lines. Each attempt at cell line establishment resulted in an epithelial carcinoma population, which was genotyped and catalogued as a unique cell line, and a corresponding fibroblast population.

Conclusion: The selection of antibody markers could be optimized to aid in the establishment of any cancer cell line derived from any tumor tissue; this method is not limited to head and neck cancer. © 2015 Wiley Periodicals, Inc. Head Neck 38: E459-E467, 2016.

Keywords: cell line; fibroblasts; flow cytometry; squamous cell carcinoma.

Figure 1

An early attempt at cell line establishment from a primary tumor, using traditional partial-trypsinization methods. An island of epithelial cells is seen surrounded by fibroblast overgrowth.

Figure 2

FACS plots of each primary tumor that became a successful cancer cell line with the unstained plot on the left and the stained plot on the right. Cells expressing the EpCAM marker shift to the right along the X-axis, whereas cells expressing the FSP marker shift up along the Y-axis. Debris and other cell types do not stain for either antibody. Primary tumors shown are A) HN165; B) HN166; C) HN173; D) HN176 E) HN177; F) HN181.

Figure 3

Cells from the EpCAM+ / FSP− population were collected and grown in culture. These cells were established as new carcinoma lines A) UM-SCC-105; B) UM-SCC-106; C) UM-SCC-108; D) UM-SCC-109; E) UM-SCC-110; F) UM-SCC-111.

Figure 4

Cells from the EpCAM− / FSP+ population were collected and grown in culture. These cells were stored as tumor-associated fibroblasts (TAFs) in conjunction with the established cell lines A) UM-SCC-105; B) UM-SCC-106; C) UM-SCC-108; D) UM-SCC-109; E) UM-SCC-110; F) UM-SCC-111.

Figure 5

FACS plots of each established cell line when mixed with an equal number of TAFs and separated. The unstained plot is shown on the left and the stained plot is shown on the right. Cells expressing the EpCAM marker shift to the right along the X-axis, whereas cells expressing the FSP marker shift up along the Y-axis. Cell lines with their corresponding TAFs are A) UM-SCC-105; B) UM-SCC-106; C) UM-SCC-108; D) UM-SCC-109; E) UM-SCC-110; F) UM-SCC-111.

Figure 6

Flank xenografts of the newly established cell lines after transduction with luciferase. 100,000 transduced cells were injected into the left flank, while 100,000 transduced cells along with 100,000 non-transduced tumor-associated fibroblasts were injected into the right flank of NOD/SCID mice. Tumors initiated earlier when co-injected with fibroblasts and grew larger than tumors that did not have fibroblasts co-injected. Cell lines used are A) UM-SCC-105; B) UM-SCC-106; C) UM-SCC-110.