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. 2015 Jun 5;16(8):1231–1240. doi: 10.1080/15384047.2015.1056410

In vitro 3-dimensional tumor model for radiosensitivity of HPV positive OSCC cell lines

Mei Zhang 1,2, Barbara Rose 3,4, C Soon Lee 5,6,7, Angela M Hong 1,2,*
PMCID: PMC4623221  PMID: 26046692

Abstract

The incidence of oropharyngeal squamous cell carcinoma (OSCC) is increasing due to the rising prevalence of human papillomavirus (HPV) positive OSCC. HPV positive OSCC is associated with better outcomes than HPV negative OSCC. Our aim was to explore the possibility that this favorable prognosis is due to the enhanced radiosensitivity of HPV positive OSCC. HPV positive OSCC cell lines were generated from the primary OSCCs of 2 patients, and corresponding HPV positive cell lines generated from nodal metastases following xenografting in nude mice. Monolayer and 3 dimensional (3D) culture techniques were used to compare the radiosensitivity of HPV positive lines with that of 2 HPV negative OSCC lines. Clonogenic and protein assays were used to measure survival post radiation. Radiation induced cell cycle changes were studied using flow cytometry. In both monolayer and 3D culture, HPV positive cells exhibited a heterogeneous appearance whereas HPV negative cells tended to be homogeneous. After irradiation, HPV positive cells had a lower survival in clonogenic assays and lower total protein levels in 3D cultures than HPV negative cells. Irradiated HPV positive cells showed a high proportion of cells in G1/S phase, increased apoptosis, an increased proliferation rate, and an inability to form 3D tumor clumps. In conclusion, HPV positive OSCC cells are more radiosensitive than HPV negative OSCC cells in vitro, supporting a more radiosensitive nature of HPV positive OSCC.

Keywords: apoptosis, cell cycle, DNA damage, human papillomavirus, Oropharyngeal cancer, radiosensitivity, 3D scaffold

Abbreviations

3 dimensional

3D

5-bromo-2 485 0 -deoxy-uridine

BrdU

epithelial-mesenchymal transition

EMT

Gray

Gy

Human papillomavirus

HPV

Immunohistochemistry

IHC

In situ ybridization

ISH

Oropharyngeal squamous cell carcinoma

OSCC

P16INK4

p16

polymerase chain reaction

PCR

Propodium iodide

PI

Ribonuclease A

RNAse

Introduction

The incidence of oropharyngeal squamous cell carcinoma (OSCC) is increasing in Western countries due to the rising prevalence of human papillomavirus (HPV) positive OSCC.1,2 The biological characteristics of HPV positive OSCC are distinct from those of HPV negative OSCC with respect to expression of the p16INK4A (p16) and other gene profiles, including frequency of mutations in TP53.3,4 Epidemiologically, patients with HPV positive OSCC tend to be younger, are less likely to have a history of tobacco and alcohol use, and have a higher number of sexual partners, in particular oral sex partners, than patients with HPV negative OSCC.5 Furthermore, patients with HPV positive OSCC have a better prognosis, despite the fact that they often present with higher grade tumors and more advanced nodal disease.6 The reason for better outcomes seen in patients with HPV positive OSCC is not fully understood and could be related to intrinsic radiosensitivity of the HPV positive OSCC.7,8 Various OSCC cell lines have been established but only a few HPV positive OSCC cell lines are available for in vitro study.9-11

In this study, we established and characterized cell lines from the HPV positive OSCC of 2 patients. Corresponding HPV positive cell lines were subsequently generated from nodal metastases following xenografting of these cell lines into nude mice. The aims were to compare the intrinsic radiosensitivity of these HPV positive cell lines and HPV negative OSCC cell lines using monolayer culture and an in vitro 3 dimensional (3D) tumor model and to characterize their cell cycle response after irradiation.

Results

Validation of HPV status

All cell lines used in this study were first checked for HPV status by HPV E6-based multiplex real-time PCR assay and p16 immunohistochemistry (IHC). The RT-PCR results indicated that RPAT1 and RPAT2 were HPV type 16 positive and UM-SCC4 and WSU-HN6 were HPV negative. The results were further confirmed by P16 IHC. For the HPV positive cell lines, we performed additional studies using in situ hybridization (ISH) and IHC of HPV16/18 E6 and HPV16 E7 proteins. As shown in Fig. S1, we detected HPV DNA signals on RPAT1 and RPAT2 cells by ISH. The expression level of HPV E6 protein was high, while HPV E7 was produced in 30% of RPAT2 cells and less than 10% of RPAT1 cells. We used p16 as a marker to monitor the HPV status from early to late passages of cell culture. The level of p16 expression was stable with passaging.

Figure 1:

Figure 1:

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(A–D) Representative microscopic images of OSCC cells in monolayer culture. A. UM-SCC4 and WSU-HN6. B. Morphological features of HPV positive RPAT1 and RPAT1L monolayer grown in 10% FBS/RPMI. C. Morphological features of HPV positive RPAT2 and RPAT2L monolayer grown in 2% and 10% FBS/RPMI; the reseeding of spheroid surrounded by cells with mesenchymal phenotype. Scale bar as indicate. Arrows indicated the cells with mesenchymal phenotype. (D) RPAT2L in monolayer and the formation of multilayered clump. (E) Representative images of IHC staining of Vimentin and CD44 on HPV positive OSCC. Magnification for A-D 4 x, scale bar = 200 µm; Magnification for (E) is 4 x, scale bar = 20 µm.

Distinct morphology and growth pattern of HPV positive OSCC cells in monolayer culture

Morphologically, both RPAT1 and RPAT2 exhibited a heterogeneous appearance in terms of size and shape; in contrast, the 2 HPV negative cell lines (UM-SCC4 and WSU-HN6) were more homogeneous (Fig. 1A). The appearance of RPAT1 and RPAT2 was dependent on cell density and the percentage of FBS in the medium. Clustered cobblestone-shaped cells became spindle-shaped on switching from 2% FBS medium to 10% and when reaching confluence (Figs. 1B–D). As indicated by the arrows, they were capable of forming spheroids in low FBS medium and the reseeded colonies grew in a spindle shape. There was no contact inhibition for HPV positive OSCC cells and the cells formed dome-shaped clusters. RPAT1L maintained the morphological and growth patterns of the primary cell line RPAT1. The cells were highly mobile, therefore losing colony forming ability. Interestingly, RPAT2L cells were more uniform in shape and size than RPAT2 but still maintained the capability of growth without contact inhibition, the ability to form spontaneous spheroids and a distinct mesenchymal like out-growth from the spheroid (Fig. 1C). Vimentin and CD44 IHC were positive on RPAT1 and RPAT2 and confirmed this observation of mesenchymal-like out-growth (Fig. 1E). Interestingly the HPV positive metastatic cells lost these stem cell functions.

RPAT1, RPAT2 and RPAT1L grew at a moderately slow rate with cell doubling time of 2–3 days as represented by the growth curve in Fig. 2. RPAT2L showed a similar growth rate to HPV negative cells with a doubling time of 24 hours. However, the HPV negative cells reached a plateau around day 6 after confluence, while HPV positive RPAT2L had sustained growing capacity without any contact inhibition caused by confluence (Fig. 2).

Figure 2:

Figure 2:

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Growth curves comparing HPV positive cells and HPV negative cells. The cell numbers were counted every day for 8 days after seeding. Values represent the means ± SD of 3 independent experiments.

3D culture model

The 3D cultures were observed regularly by light and fluorescent microscopy and representative images of cells grown on scaffolds are shown in Figure 3A and B. Using H&E staining of paraffin sections of the cultures, we found that RPAT2L demonstrated formation of multiple layers of tumor clusters in 2 weeks. Serial sections at different levels revealed 3D tumor clusters in the middle of the scaffold with dimensions of at least 3 × 3 mm, and the tumor sections were well defined and continuous (Figs. 3C–F). The thickest section reached 500 µm (Fig. 3D). Cells were ovoid to spindle-shaped with indistinct borders and had a high nuclear to cytoplasm ratio similar to that of the primary OSCC in the patient (Fig. 3G). RPAT2L formed tumor masses with proliferating cells in the periphery, resting cells in the middle layer and necrotic cells in the center (areas shown by arrows). In conjunction with the resting cells, stromal-like structures formed in the core area surrounding the scaffold, represented in H&E sections as pale pink staining (Fig. 3C and 3E). Similar to the slower growth rate seen in monolayer cultures, RPAT2 and RPAT1 only formed very small 3D tumor clumps after 2 weeks incubation (Fig. 3H), whereas the highly mobile RPAT1L traveled along the surface to form thin layer of cells covering a large area of scaffold. In comparison, UM-SCC4 cells were much smaller and formed thinner clusters tightly around the scaffold; the clusters were disconnected and randomly distributed (Figs. 3I–J).

Figure 3:

Figure 3:

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A-B Representative images of RPAT2L cells grow on scaffold with Light microscopic (A) and fluorescent microscopic images of Hoechst 33258 stained cells (B). Scale bar = 200 µm. C-F and I-J are Microscopic images of H&E staining on paraffin sections of 3D culture after 2 week in culture. RPAT2L 3D culture (C–F) was compared to its corresponding patient's H&E (G) and the 3D culture of UM-SCC4 (I, J). C, E and I displays the cross sections of the 3D culture at 4x (scale bar = 1mm) and D, F and J showing the area of interest at 40x (scale bar = 100 µm). Arrows indicate the non-viable cell central region. H is an example of the small cell cluster formed with slow growing RPAT2 cells (scale bar = 100 µm). (K) Cell proliferation on scaffold was measured from day 1 to day 10 by Alamar Blue assay. The intensity of reduced Alamar Blue was shown as fold changes. The data was generated on 3 individual experiments with triplicate scaffold, normalized with standard curve.

Due to the sensitivity of the Alamar Blue assay, there was no significant change for slow growing cells. Further quantitative studies using the Alamar Blue assay were carried out on RPAT2L and UM-SCC4. RPAT2L grew faster than UM-SCC4 under 3D conditions. The intensity of the reduced Alamar Blue produced by RPAT2L increased rapidly from day 1 to day 3. On the other hand, the intensity of reduced Alamar Blue by UM-SCC4 changed slowly, and intensity detected on day 10 was similar to the that of the day 3 culture of RPAT2L (Fig. 3K).

The HPV positive cell lines were more sensitive to radiation than the negative cell lines

All cell lines except RPAT1L were suitable for clonogenic assay. The clonogenic assays revealed that survival after 2 Gy (SF2) of RPAT1 and RPAT2 was 0.367 and 0.372 respectively, whereas the SF2 for UM-SCC4 and WSU-HN6 cells was 0.59 and 0.64 respectively (p = 0.007, Fig. 4A). The SF2 for RPAT2L was 0.6. The survival after 10 Gy was 0.0007, 0.0006 and 0.0019 for RPAT1, RPAT2 and RPAT2L respectively; whereas the survival fraction was 0.034 for UM-SCC4 and 0.019 for WSU-HN6 (p = 0.019).

Figure 4:

Figure 4:

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Survival fraction of monolayer clonogenic assay and 3D culture protein assay. Values represent the means ± SD of 3 independent experiments.

Fig. 4B shows the radiosensitivity of RPAT2L and UM-SCC4 in 3D culture. Using total protein measurement of the irradiated cells in 3D culture, the survival after 2 Gy was 0.74 for RPAT2L and 0.92 for UM-SCC4 (p = 0.023) and 0.13 to 0.48 respectively after 10 Gy (p = 0.045). These results show the same trend as radiation survival in monolayer culture.

Radiation-induced distinctive cell cycle changes between HPV negative and HPV positive cells

To determine the potential mechanisms of HPV-related radiation sensitivity, cell cycle progression post radiation was evaluated by flow cytometry. Without radiation treatment, all HPV positive cell lines maintained a high proportion of cells in G1 phase (>80%, compared to less than 50% for HPV negative cells p ≤ 0.01). In HPV positive cell cultures, there were no significant cell cycle changes at 24 hours and 48 hours post radiation (Fig. 5). However, an increase in apoptotic death (sub-G1) was detected at 48 hours post radiation for RPAT2L reaching 44% after 10 Gy. In contrast, HPV negative cells showed a marked accumulation of the G2 population 24 hours post radiation (p < 0.01). This G2 cell cycle blockage was dose-dependent and reduced after 24 hours, resuming a normal level 48 hours post radiation. Associated with these cell cycle changes was a non significant level of dose-dependent increase in the sub-G1 population. However, the radiation-induced sub-G1 change was significantly different between RPAT2L (44%) and WSU-HN6 (18%, p < 0.01).

Figure 5:

Figure 5:

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Flow cycle distribution present in G0 (sub-G1), G1, S and G2/M at 24 h and 48 h after radiation. Each point represents the mean of at least 2 separate experiments.

The effect of radiation on established 3D scaffold culture was influenced by the dynamic cell proliferation status

The effect of radiation on cell proliferation and DNA synthesis in 3D culture was studied by BrdU labeling (Fig. 6). When labeled with BrdU, both HPV positive and HPV negative cells showed a distribution of BrdU-incorporated S phase cells at the periphery. In the RPAT2L 3D culture, BrdU positive cells increased markedly by spreading deeper into the basal layer 4 hours post 10 Gy radiation; the proportion of BrdU positive cells returned to normal by day 2 associated with a reduction of the thickness of the tumor clumps. In contrast, most BrdU-labeled cells were restricted to the superficial layer post radiation for HPV negative cell line UM-SCC4. Fig. 7B demonstrates the trend of the BrdU positive index at different times post radiation. Before radiation, the BrdU index was 40.5% and 24.3% for RPAT2L and UM-SCC4 respectively. RPAT2L showed a rapid reaction to 10 Gy of radiation, and the BrdU Index had more than doubled 4 hours post radiation (Fig. 6B light blue bars). In contrast, BrdU incorporation of UM-SCC4 cells did not show rapid change at 4 hours, and was moderately decreased at 24 hours (Fig. 6B dark blue bars). The BrdU index of both cell lines subsequently returned to pre-radiation levels.

Figure 6:

Figure 6:

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Radiation response of 10 Gy single dose on 3D culture by BrdU labeling. (A) Representative images of IHC staining of BrdU at 4 h, 24 h and 48 h after radiation, by comparing UM-SCC4 and RPAT2L. Scale bar = 100 µm. (B) BrdU incorporation as % of control after radiation. Values are means of ± SD of at least 3 separate experiments.

Radiation can inhibit the initiation of 3D growth

Radiation exposure affected initiation of 3D tumor growth. When seeded after a single dose of 10 Gy, most HPV positive RPAT2L cells did not recover from radiation damage. The irradiated cells lost the ability to form clusters; on day 3 dead cells started to detach from the scaffold; by day 7 only 1–2 layers of cells remained on the scaffold, most of which showed signs of stress such as condensed nuclei or an enlarged cytoplasm (Fig. S2). The effect of radiation on 3D tumor growth was not remarkable for the UM-SCC4 HPV negative cells, which still showed small tumor clumps at day 7.

Discussion

Understanding the tumor microenvironment is essential in the study of radiobiology,12-14 therefore 3D culture may be a better in vitro model than monolayer culture to study radiosensitivity. In this study, we successfully established cell lines from the HPV positive OSCC from 2 patients. Subsequently, corresponding cell lines were generated from nodal metastases following xenografting of these cell lines into nude mice. We examined the HPV status by PCR, ISH and IHC of HPV E6, E7 and p16, then compared the morphological characteristics and radiosensitivity of HPV positive cell lines with those of 2 established HPV negative cell lines using monolayer and 3D culture with a synthetic scaffold. This is the first time we have presented the study results from these interesting HPV positive cell lines.

The appearance of the established HPV positive cell lines was heterogeneous, changing from cobblestone to spindle shape in response to a change in the culture medium from 2% to 10% PBS in both monolayer culture and 3D culture. This change in morphology is perhaps related to the epithelial-mesenchymal transition (EMT) phenomenon. Cancer stem cell studies have revealed that the tumor metastasis process is linked to EMT. In this process, cancer cells transform their morphological phenotype prior to invasion into stroma.15-17 Our IHC results confirmed the morphological EMT with strong expression of Vimentin and CD44 on RPAT1 and RPAT2. These mesenchymal proteins were not expressed after the cells migrated to lymph nodes on RPAT1L and RPAT2L. On the other hand, the HPV negative OSCC lines used in this study did not demonstrate any variation in size and shape. This may explain the association with higher nodal stage at diagnosis in patients with HPV positive OSCC. Our findings are consistent with previous reports that histologically, HPV positive and HPV negative OSCC have distinct appearances, with HPV positive OSCCs likely to have a basaloid appearance, and the HPV negative OSCCs being more epitheloid.18-20

Various 3D culture methods have been used for HPV studies. Traditionally, raft culture has been used for study of HPV infection in keratinocytes as it represents the differentiation potential of the epithelial cells.21-23 As early as the 1990s, spheroid culture was explored for cell morphology. Knuechel et al studied differentiation patterns comparing 2D and 3D- culture systems.24 In our 3D culture system, a porous scaffold was used to provide a more realistic and dynamic environment for cells. Our results showed that the 3D growth had the advantage of allowing the RPAT2L to form tumor masses with the different layers displaying the differentiation potential of the cells from the stem cell-like basal layer. The interconnected surface of the scaffold also provided an environment suitable for cell mobility which may relate to their tendency for lymph node metastasis.18,19,25

We have previously shown that HPV status predicts outcome in OSCC treated with surgery plus adjuvant radiotherapy, as well as with definitive radiation therapy ± chemotherapy after adjusting for other known prognostic factors.26 One potential explanation for the better prognosis seen in HPV positive OSCC is the better response to radiation therapy. Using both the clonogenic assay and total protein measurement, we showed that the HPV positive cell lines were more sensitive to radiation than the HPV negative cell lines. The results are consistent with the increased radiation sensitivity reported by other groups,9,11 provide in vitro data to support the enhanced radiosensitivity of HPV positive OSCC.

HPV positive and negative cells have distinctive cell cycle regulation which may explain their difference in radiosensitivity. Flow cytometry indicated that HPV positive cells had a constant high percentage of G1/S population, whereas the HPV negative cells showed time and dose dependent accumulation of G2/M, reaching peak level at 24 hours post radiation. Accurate distinction of cells in late G1 from S phase could be difficult with flow cytometry analysis.27 The high percentage of G1 phase in HPV positive cells detected by flow cytometry could be the combination of G1 and in transition to S phase. After irradiation, p53 is activated and acts in conjunction with specific cycle-dependent kinases to cause cell cycle arrest in G1 phase to allow for DNA repair, or apoptosis if the DNA damage is irreparable and to check for DNA integrity before initiating cell division.28 Therefore the constant high proportion of HPV positive cells in the G1/S phase of the cell cycle could contribute to the enhanced radiosensitivity by apoptotic death. Another form of cell death after irradiation is mitotic death which occurs when cells with unrepaired DNA damage enter the M phase of the cell cycle. G2 delay is a feature of cellular response to radiation to allow for repair of radiation injury before entering mitosis.29,30 The lack of G2/M block in HPV positive cells after irradiation would make these cells more likely to undergo mitotic death after irradiation. However, the cell cycle changes observed in our study are inconsistent with previous studies by Rieckman and Kimple.8,9 Interestingly, in those 2 studies the proportion of G2 population varied greatly between the cell lines tested. Furthermore it is suggested by Kimple's group that significant G2 arrest was caused by expression of both E6 and E7; E6 expression alone being unable to induce G2 arrest.8 Both of our cell lines RPAT1 and RPAT2 showed intense E6 protein and less E7 expression, indicating multiple cell cycle control checkpoints were disrupted by HPV E6 and E7, thus allowing the cells to cycle into S phase with DNA damage.31-33

Both the HPV positive cell lines were from 2 patients with smoking history. Our previous study showed that only one third of patients with HPV positive OSCC had a smoking history compared with almost all patients with HPV negative OSCC.34 In that study, we did not identify any interaction between HPV status and smoking and smoking was a significant predictor of overall survival but not locoregional recurrence or disease specific survival. Further research is needed to establish HPV positive cell lines from never smoker to understand the in vitro radiosensitivity.

In conclusion, HPV positive OSCC cells are more radiosensitive than HPV negative OSCC cells. In HPV positive cells, there was a loss of G1/S cell cycle control and apoptosis induction after irradiation; in contrast there was G2 delay in HPV negative cells. The enhanced radiosensitivity, cell cycle changes and the high potential to migrate were related to HPV E6 and E7 status. By using the 3D culture technique, we were able to explore the radiation response in a dynamic and structured approach and discovered that the HPV positive cancer cells were fast growing and highly mobile. The two established HPV positive cell lines and the 3D model described in this study might provide a valuable research platform in the study of OSCC.

Materials and Methods

Establishing HPV positive OSCC cell lines

Cell lines (RPAT1, RPAT2) were established from HPV positive OSCCs of 2 patients. This study was approved by the institution ethics committee and informed consent was obtained from the patients. RPAT1 was from a 48-year old male ex-smoker with T3N2M0 OSCC and RPAT2 was from a 63-year old male smoker with T3N2M0 OSCC. The cell lines were generated using standard procedures. Briefly, a 0.5 × 0.5cm2 fresh biopsy specimen of the primary site was transferred from the operation theater to the laboratory in sterile phosphate buffered saline (PBS). The tissues were mechanically dissociated, seeded in 252 cm flasks (T25 Corning) and incubated in RPMI 1640 medium (GIBCO, Australia) supplemented with 10% fetal bovine serum (FBS; Interpath, Australia), 2mM glutamine and penicillin-streptomycin (100 unit/ml-100 µg/ml). In monolayer culture, RPAT1 and RPAT2 retained persistent proliferative capacity for more than 50 passages. The two cell lines were examined by the study pathologist to confirm the presence of malignant cells.

Two more cell lines, RPAT1L and RPAT2L were subsequently established from RPAT1 and RPAT2 by xenografting into nude mice. Briefly, the RPAT1 and RPAT2 cells were inoculated subcutaneously into the dorsal side of Balb/c nude mice with matrigel. Five mice were used for each cell line. The mice were housed under specific pathogen-free conditions at the Center of Animal Study at University of NSW (Sydney, Australia). The mice were weighed and measured with a calliper weekly for 6 weeks. The animals were then sacrificed and the injection site, lymph node, liver, spleen, kidney, heart and lung were examined. Tumor masses from metastatic axillary lymph nodes were collected to establish the RPAT1L and RPAT2L cell lines using the method described above. In addition, 2 established HPV negative OSCC cell lines (UM-SCC4 and WSU-HN6) were used.35,36 All cell lines tested HPV positive by E6-based multiplex real-time PCR assay and p16 IHC as described previously.26

Cell cultures in monolayer

All cultures were routinely maintained in exponential growth as monolayers in 5% or 10% FBS/Advanced RPMI 1640, incubated at 37°C in a humidified atmosphere containing 5% CO2/95% air and passaged twice a week by trypsinization. HPV positive cell lines were maintained in our lab for more than 10 years. Monolayer cell doubling time was determined by cell counting at regular intervals. The cells were plated in 6-well plates and incubated in media supplemented with 5% or 10% PBS, trypsinized daily and trypan blue-excluding cells were counted in a hemocytometer. The doubling time was calculated from the regression equation of the curve. All experiments were performed between passages 10 and 40.

Three dimensional cell culture

RPAT1, RPAT2, RPAT1L, RPAT2L and UM-SCC4 were used to establish 3D cultures using Variotis™ Tissue Scaffolds (Biometic, Sydney, Australia). These are synthetic scaffolds comprised of highly interconnected and porous polyester-based material, with a pore size in excess of 100 µm. The 3D cultures were initiated from 5 × 105 cells per scaffold (0.5 × 0.5 × 0.7cm3) with a random seeding technique as described previously.37 The scaffold cultures were incubated in 6-well plates in 5% to 10% FBS in Advanced RPMI 1640 medium which was changed every 1 to 2 days and maintained for different time periods to form 3D structures. The 3D culture growth rate was quantitatively measured by an Alamar Blue assay. After seeding and incubation for 1 to 10 days, the 3D culture was incubated with Alamar Blue reagent for 4 hours. 100 µl of supernatant was aliquoted into a 96-well plate and read on a microplate reader at 540 and 620 nm.

Monolayers: Irradiation protocol and radiation cell survival analysis

Prior to irradiation, cells in the monolayer culture were plated in T25 flasks and incubated at 37°C overnight. The density of the cells (500–5000 cells per flask) was adjusted to allow for plating efficiency and clonogenic survival after irradiation. Cells were irradiated with single doses from 0 to 10 Gy at a dose rate of 6 Gy/min, using a 6 MV photon linear accelerator (Varian Clinac 21Ex). Radiation dose homogeneity was estimated at ±5% using existing clinical depth-dose data for the linear accelerator. Following irradiation, the medium was replaced and the cells were incubated for 10–14 days to allow for colony formation. The colonies were then fixed with ethanol and stained methylene blue solution. Colonies containing more than 50 cells were counted. The survival fractions were used to generate radiation survival curves.

Monolayer cultures with or without irradiation were observed using phase-contrast microscopy and fluorescent microscopy to detect radiation induced apoptosis characterized by nuclear staining with Hoechst 33258. Hematoxylin and eosin (H&E) staining was also performed on paraffin sections of cell pellets and radiation induced apoptosis identified by characteristic nuclear condensation.

Cell cycle distribution and apoptosis analysis

The cell cycle distribution of monolayer cultures was assessed by flow cytometry using Propidium iodide (PI) staining after radiation by flow cytometry. Asynchronous cells were irradiated as above; all cells were harvested at 24, 48 and 72 hours post-radiation, washed with PBS after trypsinization, fixed with ice-cold 70% ethanol, and stored at 4°C until performing the cell cycle analysis. Fixed cells were centrifuged and resuspended in 0.5 ml PBS containing 50 µg/ml (PI), and 80 µg/ml ribonuclease A (RNAse). After incubation for 30 minutes at 37°C, 10,000 events were analyzed for distribution of G1, S and G2 phases using a FACSCalibur (Becton Dickinson, USA). Sub-G1 population was considered as apoptotic, with DNA content lower than G1.

3D cultures: Irradiation protocol and radiation cell survival analysis

RPAT2L and UM-SCC4 were selected for 3D radiation study. For 3D cultures a single dose of radiation was given as above. At various time points post irradiation, the irradiated 3D scaffolds and controls were washed with PBS and fixed in formalin acetic acid (FAA) then embedded in paraffin. Serial sections were cut to examine the formation of 3D cultures at different levels and H&E staining was performed. The radiation effect on 3D culture was defined by several methods. Firstly, phase-contrast microscopy and fluorescent microscopy were used to compare morphologic changes such as nuclear condensation with Hoechst staining in HPV positive and negative cultures. Secondly the effect of irradiation on total protein level was measured. The cells were irradiated with single doses of 0, 2, 5 and 10 Gy; they were then seeded onto the scaffold for 1, 3 and 7 days. The cultures were then lysed with buffer, and total protein was measured with a Bio-Rad protein assay kit (Life Science Research, Australia). Thirdly, we tested the effect of radiation on established 3D cultures by 5-bromo-2′-deoxy-uridine (BrdU) incorporation to visualize DNA synthesis on individual cells. Briefly, the 3D cultures were incubated for 2 weeks before irradiation with single dose of 10 Gy. Following irradiation, the medium was replaced and the 3D cultures were incubated for 4 hours, 24 hours and 48 hours. 20 mM BrdU solution was added 4 hours before the cultures were harvested by washing with PBS and fixed in FAA. The BrdU-labeled cells in a section were counted after staining with anti-BrdU antibody (#5292, Cell Signaling) at 1:4000 dilution and the BrdU index was calculated with BrdU positive cells among total cells counted.

Immunohistochemistry and In situ hybridization

Five micron sections of monolayer culture cell pellets or 3D scaffold culture were deparaffinized and stained with H&E for histology. The primary antibodies used for IHC were purified mouse anti-Human P16 (Thermo Scientific), mouse monoclonal HPV16/18 E6 (ab70, Abcam), HPV16 E7 (sc-51951, SantaCruz), CD44 (#3570S, Cell Signaling) and Vimentin (#M7020, Dako). IHC was performed using an autostainer (Dako, Melbourne, Australia) per the manufacturer's procedures. Following treatment with antigen retrieval buffer pH6 (Dako), sections were incubated for 60 minutes with primary antibody: p16 (1:200), HPV16/18 E6 (1:250), HPV16 E7 (1:20), CD44 (1:500), EpCAM (1:250) and Vimentin (1:400). Envision Dual Link System Peroxidase (Dako) was used as secondary antibody and sections were counter-stained with hematoxylin. For negative controls the primary antibodies were replaced with buffer.

In situ hybridization (ISH) was performed using GenPoint Tyramide Signal Amplification System with HPV 16/18 Biotinylated DNA Probe (Dako Y1412) according to the manufacturer's instructions. Briefly, after treatment with antigen retrieval buffer pH6 (Dako) for 40 minutes, sections were digested with 0.01% Pepsin (Dako 3002) for 5 minutes. This was followed by hybridization and stringent washing in a hybridizer at 90°C for 5 minutes to denature then hybridization at 37°C with the probe overnight. Sections were then washed in stringent buffer at 58°C for 30 minutes then incubated with Biotin Tyramide and secondary streptavidin-HRP for 15 minutes. The sections were subsequently counter-stained with hematoxylin. Negative control plasmid DNA (Dako X1415) was used for negative controls. Staining was visualized with an OLYMPUS BX53 Microscope and DP73 Camera and scored by the study pathologist (CSL).

Statistical analysis

Each experiment was carried out in triplicate and repeated 2 to 4 times independently. All data are expressed as mean ± SD. Student's t test was used for the significance of differences in mean value; P value ≤ 0.05 was considered as significant.

Acknowledgments

The authors are grateful for the assistance from the following scientists: Dr. Phillip Boughton (University of Sydney, Australia) and Ms. Christine Poon for providing the Variotis™ Tissue Scaffolds; A/Prof. J Guy Lyons (University of Sydney, Australia) for providing the UM-SCC4 and WSU-HN6 cell lines; Ms. Trina Lum (Department of Pathology, Royal Prince Alfred Hospital, Sydney, Australia) for helping with the Vimentin immunostaining; Dr. Michele Madigan (University of Sydney, Australia) for imaging and Ms. Deanna Jones for reading the manuscript.

Supplemental Material

Supplemental data for this article can be accessed on the publisher's website.

Figures S1 and S2.zip

Funding

This work was funded by Royal Australian and New Zealand College of Radiologists and Department of Radiation Oncology, Chris O'Brien Lifehouse at RPA.

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