Human papillomavirus-positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) has increased in incidence and has a much better prognosis than HPV-negative (HPV−) OPSCC with radiotherapy alone, but exactly why is unknown. The present study therefore aimed to further examine the sensitivity and possible changes in gene expression of several HPV+ and HPV− OPSCC, including various novel cell lines, upon ionizing irradiation (IR). Previously established HPV+ UM-SCC-47, UPCI-SCC-90, CU-OP-2, CU-OP-3 and HPV− UM-SCC-4, UM-SCC-6, UM-SCC-74a, UM-SCC-19 and newly established CU-OP-17 and CU-OP-20, characterised here, were subjected to 0–6 Gy. Surviving fractions of each cell line were tested by clonogenic assays, and irregularities in cell cycle responses were examined by flow cytometry, while changes in gene expression were followed by mRNA sequencing. HPV+ OPSCC cell lines showed greater variation in sensitivity to ionizing irradiation (IR) and tended to be more sensitive than HPV− OPSCC cell lines. However, their IR sensitivity was not correlated to the proportion of cells in G2 arrest, and HPV− cell lines generally showed lower increases in G2 after IR. Upon IR with 2 Gy, mRNA sequencing revealed an increase in minor HPV integration sites in HPV+ cell lines, and some changes in gene expression in OPSCC cell lines, but not primarily those associated with DNA repair. To conclude, HPV+ OPSCC cell lines showed greater variation in their sensitivity to IR, with some that were radioresistant, but overall the HPV+ OPSCC group still tended to be more sensitive to IR than the HPV− OPSCC group. In addition, HPV+ OPSCC lines were more frequently in G2 as compared to HPV− cell lines, but the increase in G2 arrest upon IR in HPV+ OPSCC was not correlated to sensitivity to IR. Increases in minor HPV integration sites and changes in gene expression were also demonstrated after irradiation with 2 Gy.
The incidence of human papillomavirus-positive (HPV+) tonsillar and base of tongue squamous cell carcinomas (TSCC/BOTSCC), the major subsites of oropharyngeal squamous cell cancer (OPSCC), (but not other OPSCC subsites) is still increasing in most western countries (
Patients with HPV+ TSCC/BOTSCC usually respond well to treatment and have better long-term survival compared to HPV− TSCC/BOTSCC and HNSCC in general, irrespective if they receive radiotherapy alone, or radio-chemotherapy (i.e. 80 vs. 40–50% 5-year overall survival) (
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In 2014, to our knowledge, there are eight published HPV+ HNSCC cell lines: UPCI-SCC-90, UPCI-SCC-154, UPCI-SCC-152, UM-SCC-104, UM-SCC-47, 93-VU-147T, UD-SCC-2 and UT-SCC-45 (
Five HPV+ and five HPV− OPSCC cell lines were included in this study.
To derive novel lines, TSCC biopsies were obtained, during the diagnostic procedure (where ultimately diagnosis is finalised by multidisciplinary meetings) from patients, prior to treatment at Cardiff and Vale University Health Board, in concordance with Ethical and NHS R&D approval (reference number 13/WA/0002), by written consent. The derivation of HPV+ CU-OP-2 and CU-OP-3 has been described previously (
HPV+ UM-SCC-47 and HPV− UM-SCC-6, UM-SCC-19, UM-SCC-74a and UM-SCC-4 were obtained from Professor Thomas Carey at the University of Michigan USA. HPV+ UPCI-SCC-90 was purchased from Deutsche Sammlung von Mikoorganismen und Zellkulturen (DSMZ), Leibniz, Germany. All these cell lines are described in the data base
Human epithelial keratinocytes (HEKn) were purchased from Thermo Fisher Scientific, Inc. and grown as described previously in EpiLife media (
All cell lines were tested for absence of mycoplasma, by standardised PCR, using the Venor®GeM detection kit (Minerva Biolabs), which is specific to the highly conserved 16s rRNA coding region, thereby detecting a wide range of mycoplasma species.
The presence of HPV in CU-OP-17 and CU-OP-20 was confirmed by a bead-based multiplex assay for 27 HPV types as described in detail previously (
Cells were cultured at low cell densities, more specifically in ranges of 2,500-12,500 cells per plate on 6-cm culture dishes (VWR) and incubated at 37°C with 5% CO2 for 24 h. Cell density per cell line, was defined before the initiation of clonogenic assays. Cell density, was defined by individual cell line density tests (using ranges of cells between 1,000-20,000 cells/well lasting for ~10-15 days depending on the cell line. After 24 h the cells were irradiated with 0–6 Gy (Gammacell-1000 MDS Nordion; a caesium-137 source) and the media were changed after 7 days. The assays were stopped 10–15 days later (depending on the growth of the cell lines) and the cells stained with crystal violet, and the colonies counted using a Colony counter (ColonyDoc-It Imaging Station, UVP).
All experiments were performed in triplicates. The plating efficiency (PE) and survival fraction (SF) of each cell line per IR dose was calculated. Cells were classified as radiosensitive SF <0.40 or radio-resistant SF >0.40 as described before (
Cells were cultured without feeder cells in 6-cm culture dishes (VWR) and treated with 0–6 Gy 24 h after seeding. Untreated cells were collected 24 h after seeding, while irradiated cells were collected 8, 24 and 48 h after treatment. Approximately 500,000 cells/cell line were fixed in 1 ml of 70% ethanol in fluorescence-activated cell sorting (FACS) tubes and stored at −20°C for at least 1 h. Cells were then washed with PBS, incubated with 100 µl of RNase A (10 µg/ml) (Sigma-Aldrich; Merck KGaA) for 45 min at 37°C, centrifuged at 270 × g, and resuspended in 200 µl propidium iodide (PI) solution (50 µg/ml) (Sigma-Aldrich; Merck KGaA) and incubated for 15 min at 37°C.
The PI-stained cells were analysed using a BD Accuri C6 (BD Biosciences) low-pressure flow cytometer (absorbance 488 nm). Data were extracted as FCS files and the cell cycle distribution was analysed with FlowJo analysis software (version 10; FlowJo LLC), using the cell cycle tool, based on the Watson pragmatic algorithm (
Cells (untreated and 2 Gy) were collected 24 h after treatment and RNA was extracted with QiaAMP Mini kit (Qiagen) according to the manufacturer's instructions.
Library preparation and validation for mRNA sequencing was performed through a commercial service/collaboration with Wales Gene Park (Cardiff University, UK). Library preparation, including depletion of ribosomal RNA was carried out using the Illumina® TruSeq® Stranded Total RNA with Ribo-Zero Gold™ kit (Illumina Inc.) according to the instructions of the manufacturer.
A 75-base paired-end dual index read format was used on the Illumina® HiSeq2500 in high-output mode by the Wales Gene Park (Cardiff University, UK). Sequencing data were analysed by the bioinformatics service by Dr Peter Giles at the Wales Gene Park (Cardiff University, UK). Trimmed reads were mapped against a combined human sequence genome hg19 and an HPV16 genome reference sequence NC_001526 using STAR (Alex Dobin, Git Hub) to generate circos plots. For both exons and transcripts, gene expression counts were calculated, using Subread feature Counts Version 1.5.1 (
The DEseq2 analysis tool was used to identify differentially expressed genes (statistical analysis of count matrices for systematic changes between conditions) (
Circos plots were used to visualise and identify human (hg19) and viral (HPV-16) mRNA fusion transcripts (
Explant cultures using fresh primary biopsies from two patients with TSCC were attempted as described previously (
The sensitivity of the panel of 10 OPSCC cell lines to IR was assessed using clonogenic survival assays. The SFs of all OPSCC cell lines to 0.5–6 Gy respectively are shown in
Special focus was put on an indicated clinically relevant dose of 2 Gy (
There were significant differences in SF within the HPV+ and HPV− groups after IR with 2 Gy. HPV+ UM-SCC-47 and CU-OP-20 were more radiosensitive to 2 Gy compared to CU-OP-2 and CU-OP-3, and UPCI-SSC-90 was more radiosensitive than CU-OP-3 (for all at least P<0.05) (
To gain insight into the mechanisms underlying the differences in sensitivity to IR, the cell lines were treated with IR and the effects on cell cycle distribution were assessed by flow cytometry. IR induced cell cycle effects primarily on HPV+ OPSCC as compared to HPV− OPSCC and mainly in the proportion of cells in G2, and the data are presented in detail below.
No significant changes were observed in cell cycle distribution 8 h after 2–6 Gy for any cell line. However, after 24 h (2 Gy), a significant increase in cells in G2 phase was observed for two HPV+ OPSCC cell lines: UM-SCC-47 (the most radiosensitive line, P=0.0135) and CU-OP-3 (the most radioresistant line, P=0.0075) (
Compared to untreated controls, at 2 Gy after 24 h, a significant increase in cells in G2 was observed in one HPV− OPSCC cell line, UM-SCC-4, P=0.0222 (
Roughly, 50–60% of HPV+ and HPV− OPSCC cells were initially in G1, and few changes occurred 8–48 h after IR (
Compared to untreated controls some changes in the proportion of cells in S phase occurred early (8 h), but not later (24–48 h) after IR in both HPV+ and HPV− OPSCC cell lines (
No significant changes were observed in the cell cycle of HEKn between the non-treated samples and the 2 Gy and 6 Gy samples (8, 24 and 48 h) (
RNA sequencing was used to identify the presence on human: Viral fusion transcripts before and after treatment with 2 Gy IR. This data was visualised using circos plots, which demonstrated fusion transcripts as described previously for UM-SCC-47, UPCI-SCC-90, CU-OP-2 and CU-OP-3 (
The RNA-sequencing data was also examined to determine whether there were consistent differences in response to IR between the HPV+ and HPV− cell lines. An unsupervised analysis comparing treated and untreated cells was initially performed. After correction for multiple testing, 519 transcripts or genes were indicated as significant (data not shown). This indicated 2 Gy had a significant effect on transcription of multiple genes. The top 19 genes with significant differences after treatment of the OPSCC lines with 2 Gy are presented in
The present study investigated responses to ionising irradiation (IR) in five human papillomavirus-positive (HPV+) and five HPV− oropharyngeal squamous cell carcinoma (OPSCC) cell lines. Clonogenic survival assays, flow cytometry and RNA sequencing were used to assess survival, cell cycle distribution and HPV integration. We also report derivation and characterisation of two novel OPSCC cell lines. HPV+ OPSCC cell lines showed greater variation in radiosensitivity than was apparent among the HPV− cell lines, and we observed a tendency for greater sensitivity to IR in HPV+ lines, although the correlation between radiosensitivity and HPV status was not perfect. HPV+ cells more frequently demonstrated an increase in G2 arrest following IR as compared to the HPV− OPSCC cell lines. It was interesting and potentially clinically relevant to note that radiation treatment resulted in a marked increase in novel HPV: Human fusion transcripts, which may suggest that radiation could facilitate integration of HPV DNA into novel genomic sites. However, due to the limited number of available cell lines and without further studies, a definite conclusion cannot be made after the current study. RNA sequencing did not indicate major changes in transcription of genes associated with DNA repair, DNA damage or stress mechanisms.
Following irradiation of HPV+ cell lines (UM-SCC-47, UPCI-SCC-90, CU-OP-2, CU-OP-3, and CU-OP-20) and the HPV− cell lines (UM-SCC-6, UM-SCC-4, UM-SCC-19, UM-SCC-74a and CU-OP-17), our observations show some important differences from earlier reports (
The wider variability in sensitivity to IR in the HPV+ group is in line with one previous report, where the authors also therefore suggest caution in de-intensification of therapy via dose reduction (
Of note, the HPV+ CU-OP-20, which according to our circos plots lacked major integrated HPV sites, was among the most radiosensitive HPV+ OPSCC cell line. The data are of course very limited, but would be consistent with suggestions that HPV+ OPSCC with episomal HPV DNA may have an even better prognosis (
Similar to other publications, we found that upon IR the proportion of HPV+ cell lines often exhibited an increase in G2 arrest as compared to HPV− OPSCC lines, while changes in the proportion of cells in G1 and S-phase were not as apparent for any of the OPSCC cell lines (
In this study, we also demonstrated an increase in HPV minor integration sites upon IR. We suggest that this could be due to an increase in DNA lesions following IR, and that this promotes integration of HPV DNA (possibly previously episomal) into new genomic locations.
mRNA sequencing IR treated and untreated cells also allowed investigation of changes in transcription of genes correlated to DNA repair, cell cycle arrest and apoptosis. Our data did not indicate an increase in transcription of specific DNA repair genes after IR, which could be interpreted as consistent with the suggestion that the IR sensitivity of HPV+ OPSCC cell lines could be due to impaired capability to repair double-stranded DNA breaks (
There are limitations inherent in our study; only 10 OPSCC cell lines were assessed. Nonetheless this is a larger sample than investigated in several previous studies. By including five OPSCC cell lines previously not tested with IR, we could show a tendency, but not a significantly increased sensitivity to IR in HPV+ as compared to HPV− OPSCC cell lines, and this may challenge a previous dogma (
Furthermore as already mentioned, another limitation was the special focus on one irradiation dose 2 Gy. However, we could confirm that upon IR, the proportion of cells in G2 increased much more in HPV+ OPSCC cell lines as compared to HPV− OPSCC, but in this study the increase in G2 was not correlated to radiosensitivity as reported previously (
To summarise, in this report five HPV+ and five HPV− OPSCCs cell lines, including five lines not previously assessed, were examined for radiosensitivity. HPV+ OPSCC lines demonstrated a wide range of sensitivity to IR, and importantly not all cell lines were radiosensitive, although they still tended to be more sensitive to irradiation than HPV− OPSCC lines. Furthermore, upon IR, HPV+ OPSCCs more often increased the proportion of cells in G2 arrest as compared to HPV− OPSCC cell lines, but the increases in G2 arrest were not correlated to radiosensitivity. Lastly, upon treatment with 2 Gy some increases in minor HPV integration sites were noted and changes in gene expression were demonstrated, but not in genes primarily associated with DNA repair.
To conclude, our data suggest that HPV+ OPSCC cell lines may possibly vary more in radiosensitivity than previously anticipated, and despite they are generally more sensitive than HPV− OPSCC cell lines, individual variations may exist within both the HPV+ and the HPV− OPSCC groups. Furthermore, in spite of the fact that 10 cell lines were tested, the data are limited and additional studies are warranted.
We are very thankful for the support provided by Cancer Research Wales as well as the patients who took part in this study. Tina Dalianis and Nicole Wild are acknowledged for carefully reading the manuscript and providing valuable suggestions, and Ramona Ursu for technical help with the bead based multiplex assay.
Grant funding from Cancer Research Wales (grant no. CRW14-506688) supported this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
RNA-seq data discussed in this publication is available in the NCBIs Gene Expression Omnibus (GEO) and is accessible through GEO series accession number GSE153966 (
SH performed the majority of the experiments, interpreted the data, calculated the statistics and contributed to the writing of the manuscript. EP and JJ collaborated with SH and performed some experiments. PG contributed by analyzing and providing the raw data. DO, AAH and ME contributed by identifying and consenting patients and by providing the biopsy samples for the establishment of the cell lines. NP and SM assisted all the authors, with the planning of some experiments, performed combinational analyses, and assisted in final interpretation and presentation of the data. NP and SH were involved in the work from its initiation and bringing into completion and were involved in writing and finalizing the work and the manuscript. All authors critically read and approved the manuscript.
The study with regard to patient material was performed according to permission from the Cardiff and Vale University Health board, in concordance with Ethical and NHS R&D approval (reference no. 13/WA/0002).
Not applicable.
Authors report no competing interests to disclose.
Surviving fraction (SF) of HPV+ and HPV− cell lines following ionizing irradiation. (A) SF of HPV+ cell lines. At 2 Gy the difference in survival between the most radiosensitive (UM-SCC-47) and the most radioresistant (CU-OP-3) cell lines was significant (****P<0.0001). Additional significant differences are noted only in the text of the results section. ns, not significant. Error bars indicate standard deviations. (B) SF of HPV− cell lines to IR. At 2 Gy the difference in survival between the most radiosensitive (UM-SCC-19) and the most radioresistant (CU-OP-17) cell lines was significant (****P<0.0001). Additional significant differences are noted only in the text of the results section. ns, not significant. Error bars indicate standard deviations. (C) SF after treatment with 2 Gy for all HPV+ and HPV− SCC cell lines and the human epithelial keratinocyte cell line HEKn. As example of statistical significance, the comparison of UM-SCC-47 to CU-OP-17 is presented (****P<0.0001). For this specific comparison a one-way ANOVA with a Sidak post-test was used. HPV, human papillomavirus.
Distribution of cells in G1, S-phase and G2 at different time points after ionizing irradiation (2 and 6 Gy) for HPV+ cell lines: (A) UM-SCC-47, (B) UPCI-SCC-90, (C) CU-OP-2, (D) CU-OP-20 and (E) CU-OP-3. All indicated statistical significance values in the figure apply only to cells in G2 phase. The statistics present visually the significance between the untreated G2 phase and the treated G2 phases at the presented time points (*P<0.05, **P<0.01, ***P<0.01 and ****P<0.001). Untreated samples presented represent time zero. N/A, not available; ns, not signifcant. All other statistical differences for G1 and S are presented in the text of the results section. HPV, human papillomavirus.
Distribution of cells in the G1, S-phase and G2 at different time points after ionizing irradiation of HPV− cell lines with 2 and 6 Gy: (A) UM-SCC-19, (B) UM-SCC-6, (C) UM-SCC-4, (D) UM-SSC-74a and (E) CU-OP-17. All indicated statistical significance values in the figure apply only to cells in G2 phase. The statistics present visually the significance between the untreated G2 phase and the treated G2 phases at the presented time points (*P<0.05 and **P<0.01). Untreated samples presented represent time zero. ns, not signifcant. All other statistical differences for G1 and S are presented in the text of the results section. HPV, human papillomavirus.
CU-OP-17 and CU-OP-20 cell lines and the corresponding patient characteristics.
Study number | Sex (M/F) | Age (years) | Smoking status |
Site | TNM stage |
P16 IHC | P53 status |
HPV type | Treatment |
Recurrence | Cell line population doublings |
---|---|---|---|---|---|---|---|---|---|---|---|
CU-OP-17 | M | 68 | Y (100 pack/yr) | Tonsil | T4aN1M0 | Y | WT | Negative | Induction chemo, then CRT, then neck dissection | None | 125 |
CU-OP-20 | M | 55 | Never | Tonsil | T2N2aM0 | Y | WT | 16 | Neck dissection followed by CRT to primary | None | 120 |
Pack-year (pack/year) refers to the number of cigarette packs smoked per day multiplied by the number of years smoked.
(T, size and extent of tumour; N, number of nearby lymph nodes that have cancer; M, cancer metastasized).
WT, wild-type, according to mRNA sequencing.
Chemo-radiotherapy (CRT).
Approximate population doublings (PD) of the established cell lines. IHC, immunohistochemistry; HPV, human papillomavirus.
Surviving fraction (SF) of HPV+ cell lines after treatment with 0.5–6 Gy.
Surviving fraction (%) | |||||
---|---|---|---|---|---|
HPV+ cell lines sensitive to IR | HPV+ cell lines resistant to IR | ||||
Treatment dose (Gy) | UMSCC-47 | UPCI-SCC-90 | CU-OP-20 | CU-OP-2 | CU-OP-3 |
0.5 | 78.4 | 72.5 | 66.8 | 93.6 | 90.5 |
1 | 44.4 | 48.5 | 46.9 | 64.1 | 83.3 |
2 |
14.1 | 28.3 | 20.4 | 44.7 | 60.4 |
4 | 1.3 | 7.5 | 3.8 | 12.2 | 19.1 |
6 | 0.3 | 1.7 | 0.8 | 7.2 | 12.1 |
SF <40% defined as sensitive to IR according to (
Surviving fraction (SF) of HPV− OPSCC cell lines and HEKn after treatment with 0.5–6 Gy.
Surviving fraction (%) | ||||||
---|---|---|---|---|---|---|
HPV− cell line sensitive to IR | HPV− cell lines resistant to IR | Non-cancer cell line response to IR | ||||
Treatment dose (Gy) | UMSCC-19 | UMSCC-6 | UMSCC-74a | UMSCC-4 | CU-OP-17 | HEKn |
0.5 | 81.7 | 87.4 | 85.3 | 92.3 | 94.3 | 79.8 |
1 | 69.1 | 62.9 | 70.1 | 85.1 | 84.2 | 70.6 |
2 |
32.4 | 44.9 | 47.7 | 60.1 | 71.8 | 32.7 |
4 | 15.5 | 11.3 | 8.9 | 15.6 | 28.8 | 17.6 |
6 | 3.8 | 2.4 | 0.9 | 2.3 | 10.3 | 1.6 |
SF, <40% defined as sensitive to IR according to (