Influence of the human papillomavirus on the radio-responsiveness of cancer stem cells in head and neck cancers

Abstract

A growing proportion of head and neck cancers (HNC) result from HPV infection. Between HNC aetiological groups (HPV positive and HPV negative) clinical evidence demonstrates significantly better treatment response among HPV positive cancers. Cancer stem cells (CSCs) are identified in HNC tumour populations as agents of treatment resistance and a target for tumour control. This study examines dynamic responses in populations of a CSC phenotype in HNC cell lines following X-irradiation at therapeutic levels, and comparing between HPV statuses. Variations in CSC density between HPV groups showed no correlation with better clinical outcomes seen in the HPV positive status. CSC populations in HPV positive cell lines ranged from 1.9 to 4.8%, and 2.6 to 9.9% for HPV negative. Following 4 Gy X- irradiation however, HPV negative cell lines demonstrated more frequent and significantly greater escalation in CSC proportions, being 3-fold that of the HPV positive group at 72 hours post irradiation. CSC proportions of tumour populations are not fixed but subject to change in response to radiation at therapeutic dose levels. These findings imply a potential effect of aetiology on radio-responsiveness in CSCs, illustrating that clonogen treatment response may be more informative of therapy outcomes than inherent population density alone.

Figure 1

Gating strategy for cell counting by flow cytometry. (A) Initial selection of viable cells by forward vs side scatter. (B) Selection of single cells from gated area in A, by forward scatter-area vs height. (C) Gating by negative controls for ALDH and CD44. (D) CD44+/ALDH + cells in upper right quadrant. FloJo Software v10.0.7 Ashland, www.FlowJo.com.

Figure 2

Baseline proportions of CSC population in HNSCC cell lines by HPV status. Analysis repeats n = 7. Error bars by standard error of mean. GraphPad Prism v8.2.1 www.graphpad.com.

Figure 3

CSC proportions in treated cells and controls across 3 days following 4 Gy X-ray dose. Analysis repeats n = 7. Error bars by standard error of mean. GraphPad Prism v8.2.1 www.graphpad.com.

Figure 4

Ratio of response in CSC population following 4 Gy by HPV status. Analysis repeats n = 7. Error bars by standard error of mean. GraphPad Prism v8.2.1 www.graphpad.com.

Figure 5

Comparison of CD44+/ALDH + measurements for UM-SCC-47 by T25 and 6 well plate protocols. Analysis repeats n = 7. Error bars by standard error of mean. GraphPad Prism v8.2.1 www.graphpad.com.