Evaluation of [18F]F-DPA as a target for TSPO in head and neck cancer under normal conditions and after radiotherapy

Abstract

Background: Many malignant tumours have increased TSPO expression, which has been related to a poor prognosis. TSPO-PET tracers have not comprehensively been evaluated in peripherally located tumours. This study aimed to evaluate whether N,N-diethyl-2-(2-(4-([¹⁸F]fluoro)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide ([¹⁸F]F-DPA) can reflect radiotherapy (RT)-induced changes in TSPO activity in head and neck squamous cell carcinoma (HNSCC).

Methods: RT was used to induce inflammatory responses in HNSCC xenografts and cells. [¹⁸F]F-DPA uptake was measured in vivo in non-irradiated and irradiated tumours, followed by ex vivo biodistribution, autoradiography, and radiometabolite analysis. In vitro studies were performed in parental and TSPO-silenced (TSPO siRNA) cells. TSPO protein and mRNA expression, as well as tumour-associated macrophages (TAMs), were also assessed.

Results: In vivo imaging and ex vivo measurement revealed significantly higher [¹⁸F]F-DPA uptake in irradiated, compared to non-irradiated tumours. In vitro labelling studies with cells confirmed this finding, whereas no effect of RT on [¹⁸F]F-DPA uptake was detected in TSPO siRNA cells. Radiometabolite analysis showed that the amount of unchanged [¹⁸F]F-DPA in tumours was 95%, also after irradiation. PK11195 pre-treatment reduced the tumour-to-blood ratio of [¹⁸F]F-DPA by 73% in xenografts and by 88% in cells. TSPO protein and mRNA levels increased after RT, but were highly variable. The proportion of M1/M2 TAMs decreased after RT, whereas the proportion of monocytes and migratory monocytes/macrophages increased.

Conclusions: [¹⁸F]F-DPA can detect changes in TSPO expression levels after RT in HNSCC, which does not seem to reflect inflammation. Further studies are however needed to clarify the physiological mechanisms regulated by TSPO after RT.

Keywords: Head and neck cancer; PET; Radiotherapy; TSPO; [18F]F-DPA.

Fig. 1

Time schedule and set-up of the three in vivo experiments performed in this study. [¹⁸F]FDG and [¹⁸F]F-DPA imaging of FaDu tumour bearing mice was performed on consecutive days a 1 or b 2 weeks (wk) after radiotherapy (RT). c [¹⁸F]F-DPA imaging of Cal33 tumour bearing mice was performed 1 week after RT. TAM tumour-associated macrophages, IHC immunohistochemistry, WB Western blot, ARG autoradiography

Fig. 2

a In vivo uptake of [¹⁸F]F-DPA (summed 20–40 min post injection) in non-irradiated (Ctrl) and irradiated (RT, 2 × 5 Gy) FaDu tumours 1 and 2 weeks (wk) after RT. b The [¹⁸F]FDG uptake (summed 60–80 min post injection) was determined in same tumours on the previous day. c PET/CT images of [¹⁸F]F-DPA and [¹⁸F]FDG uptake in non-irradiated and irradiated FaDu tumours 1 week after treatment. d The uptake of [¹⁸F]F-DPA in Cal33 tumours 1 week after RT. Data is expressed as percentage of injected dose per millilitre tissue (% ID/mL) in whole tumours, mean ± SD, n = 3–6/group. Each data point represents one tumour. *p < 0.05, **p < 0.01 are considered to be statistically significant compared to controls, by two-tailed Student’s t test

Fig. 3

a Time-activity curves (TACs) derived from volumes of interests drawn over whole tumours after injection of [¹⁸F]FDG and [¹⁸F]F-DPA. The [¹⁸F]FDG TAC is shown from a non-irradiated (Ctrl) tumour, whereas the [¹⁸F]F-DPA TACs are shown for non-irradiated, irradiated (RT, 2 × 5 Gy), and pre-treated (Pretrt., 1 mg PK11195) tumours. b Expression of TSPO protein in non-irradiated and irradiated FaDu tumours 1 and 2 weeks (wk) after treatment. Data is expressed as relative protein expression normalized to housekeeping gene GAPDH, mean ± SD, n = 4–5/group. Each data point represents one tumour. TSPO Translocator protein, GAPDH Glyceraldehyde 3-phosphate dehydrogenase

Fig. 4

Ex vivo biodistribution of ¹⁸F-radioactivity in FaDu and Cal33 tumour bearing mice 40 min after [¹⁸F]F-DPA injection. a Whole body biodistribution in non-irradiated (Ctrl) and FaDu tumour bearing mice irradiated locally to tumour (RT, 2 × 5 Gy). Data is expressed as percentage of injected dose per gram tissue (% ID/g), mean ± SD, n = 3/group. *p < 0.05 is considered to be statistically significant compared to controls, by two-tailed Student’s t test. SI small intestine, LI large intestine, Subcut. subcutaneous, BAT brown adipose tissue. b, c Uptake of [¹⁸F]F-DPA in blood components, muscle and tumour from non-irradiated, irradiated, and FaDu tumour bearing mice pre-treated with 1 mg of PK11195 30 min prior tracer injection. Data is expressed as percentage of injected dose per gram tissue (% ID/g), or as muscle-to-blood (M/B) and tumour-to-blood (T/B) ratios, mean ± SD, n = 5–10/group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 is considered to be statistically significant compared between each group, by one-way ANOVA. d, e Uptake of [¹⁸F]F-DPA in blood components, muscle and tumour from non-irradiated and irradiated Cal33 tumour bearing mice. Data is expressed as % ID/g, or as T/B ratios, mean ± SD, n = 4/group. **p < 0.01 is considered to be statistically significant compared to controls, by two-tailed Student’s t test

Fig. 5

a RadioTLC chromatograms of plasma and tumour homogenates from non-irradiated (Ctrl), irradiated (RT, 2 × 5 Gy), and FaDu tumour bearing mice pre-treated (Pretrt.) with 1 mg PK11195 (i.p. injection) 30 min prior to tracer injection. Analyses were carried out 40 min after [¹⁸F]F-DPA administration 1 week after RT. Chromatogram of parental [¹⁸F]F-DPA included for reference. Values are average percentages of unchanged tracer from two independent experiments. R_f Retention value. b Ex vivo tumour autoradiography from non-irradiated and irradiated mice. Autoradiography was performed directly after in vivo imaging 1 week after RT (upper row). Sections incubated in vitro with [¹⁸F]F-DPA (2 nmol/L) for 1 h and pre-treated with PK11195 (1000 nmol/L) in vitro are shown in the middle and lower row, respectively. Brain sections from a 17-month-old APP/PS1-21 transgenic mouse (model of Alzheimer’s disease) were used as positive controls under same conditions

Fig. 6

a Proportion of monocytes, immature/migratory macrophages and macrophages in stage M1 and M2 in non-irradiated and irradiated FaDu tumours. Data is expressed as percentage of total monocyte/macrophage population, mean ± SD, n = 4. *p < 0.05 is considered to be statistically significant compared to controls, by two-tailed Student’s t test. Flow cytometry charts depicting the population-proportions from b non-irradiated and c irradiated FaDu tumours. Q1 monocytes, Q2 immature/migratory macrophages, Q3 M1 stage macrophages, Q4 M2 stage macrophages. The gating strategy for selecting the subpopulations is shown in Supplementary Fig. 4

Fig. 7

a In vitro uptake of [¹⁸F]F-DPA in non-irradiated (Ctrl), irradiated (RT, 5 × 2 Gy) and PK11195 pre-treated FaDu cells (Pretrt., 10 μM, 30 min prior and during the tracer incubation). Data is expressed as ¹⁸F-radioactivity in relation to viable cells, mean ± SD, n = 14 (Ctrl and RT), n = 4 (pre-treated). b Data shown in a divided into [¹⁸F]F-DPA uptake with higher (358 GBq/μmol) and lower (10 GBq/μmol) molar activity in Ctrl and RT FaDu cells. c Expression of TSPO and γH2Ax (DNA damage marker) proteins in cells shown in a. Data is expressed as relative protein expression normalized to housekeeping gene GAPDH, mean ± SD, n = 14. d TSPO mRNA expression levels in cells shown in a. The bars represent TSPO mRNA relative to the average level of housekeeping genes TBP and RPLP0, data is expressed as mean ± SD, n = 14. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 are considered to be statistically significant compared to controls, by two-tailed Student’s t test. TSPO Translocator protein, TBP TATA-binding protein, RPLP0 60S acidic ribosomal protein P0

Fig. 8

a Uptake of [¹⁸F]F-DPA in non-irradiated (Ctrl) and irradiated (RT, 5 × 2 Gy), non-targeting (NT siRNA) and TSPO silenced (TSPO siRNA), FaDu cells. Data is expressed as ¹⁸F-radioactivity uptake in relation to viable cells, mean ± SD, n = 7 b Expression of TSPO and γH2Ax (DNA damage marker) proteins. Data is expressed as relative protein expression normalized to NT siRNA control (GAPDH used as housekeeping gene), mean ± SD, n = 7. c TSPO mRNA expression was analysed by RT-qPCR. The bars represent TSPO mRNA relative to the average of housekeeping genes TBP and RPLP0, data is expressed as mean ± SD, n = 7. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ¹⁸F-radioactivity in non-irradiated NT siRNA or TSPO siRNA cells vs irradiated cells, respectively, by two-way ANOVA. TSPO Translocator protein, TBP TATA-binding protein, RPLP0 60S acidic ribosomal protein P0