Use of 3D Spheroid Models for the Assessment of RT Response in Head and Neck Cancer

Abstract

Radiotherapy (RT) is a key player in the treatment of head and neck cancer (HNC). The RT response, however, is variable and influenced by multiple tumoral and tumor microenvironmental factors, such as human papillomavirus (HPV) infections and hypoxia. To investigate the biological mechanisms behind these variable responses, preclinical models are crucial. Up till now, 2D clonogenic and in vivo assays have remained the gold standard, although the popularity of 3D models is rising. In this study, we investigate the use of 3D spheroid models as a preclinical tool for radiobiological research by comparing the RT response of two HPV-positive and two HPV-negative HNC spheroid models to the RT response of their corresponding 2D and in vivo models. We demonstrate that HPV-positive spheroids keep their higher intrinsic radiosensitivity when compared to HPV-negative spheroids. A good correlation is found in the RT response between HPV-positive SCC154 and HPV-negative CAL27 spheroids and their respective xenografts. In addition, 3D spheroids are able to capture the heterogeneity of RT responses within HPV-positive and HPV-negative models. Moreover, we demonstrate the potential use of 3D spheroids in the study of the mechanisms underlying these RT responses in a spatial manner by whole-mount Ki-67 and pimonidazole staining. Overall, our results show that 3D spheroids are a promising model to assess the RT response in HNC.

Keywords: 3D spheroid models; head and neck cancer; human papillomavirus; radiotherapy.

Figure 1

HPV-negative and HPV-positive HNC cells formed 3D spheroids. (A) Images of HPV-negative FADU and CAL27 spheroids and HPV-positive SCC154 and SCC47 spheroids taken on day 5. Scale bar, 100 μm. (B) Absolute tumor growth curves of HPV-negative FADU and CAL27 and HPV-positive SCC154 and SCC47 spheroids. Day 1 indicates the day of seeding. Data are presented as mean ± s.e.m., n = 3.

Figure 2

RT response in 3D spheroids of HPV-negative and HPV-positive cells assessed by tumor growth curves. (A) Relative tumor growth curves of HPV-negative FADU and CAL27 spheroids. The spheroids were treated with 4 Gy and 8 Gy at day 7 after seeding. (B) Relative tumor growth curves of HPV-positive SCC154 and SCC47 spheroids. The spheroids were treated with 4 Gy and 8 Gy at day 14 after seeding. (A,B) Relative curves were normalized to the spheroid area at the start of RT treatment. Day 1 indicates the start of treatment. Data are presented as mean ± s.e.m., n = 3. * p-values < 0.05 were determined by two-way ANOVA.

Figure 3

RT response of HPV-negative and HPV-positive cells assessed by clonogenic assays (A) and in vivo xenograft models (B). (A) HPV-negative (FADU and CAL27) and HPV-positive (SCC154 and SCC47) cells were irradiated with the indicated RT doses. Clonogenic cell survival is shown as the mean clonogenic survival fraction ± s.e.m., n = 3. (B) Relative tumor volume curves of HPV-negative CAL27 and HPV-positive SCC154 xenografts treated with vehicle with RT (5 × 2 Gy). Relative tumor growth curves were normalized to the tumor volume at the start of treatment day 1. Day 1 indicates the start of RT treatment. Data are presented as mean ± s.e.m., n = 3. (A,B) * p-values < 0.05 were determined by two-way ANOVA with Bonferroni correction for multiple testing.

Figure 4

Proliferative capacity (A) and hypoxia levels (B) of HPV-negative and HPV-positive spheroids. (A) Whole-mount staining of Ki-67-positive cells (left) and mean intensity (right) in HPV-negative (FADU and CAL27) and HPV-positive (SCC154 and SCC47) spheroids at the end of the experiment. (B) Whole-mount staining of pimonidazole-positive cells (left) and mean intensity (right) in HPV-negative (FADU and CAL27) and HPV-positive (SCC154 and SCC47) spheroids. (A,B) Data are presented as mean ± s.e.m., n = 2 at minimum. p-values < 0.05 were determined by two-tailed t-test. Scale bar, 100 μm.