Correlation of Somatostatin Receptor-2 Expression with Gallium-68-DOTA-TATE Uptake in Neuroblastoma Xenograft Models

Abstract

Peptide-receptor imaging and therapy with radiolabeled somatostatin analogs such as ⁶⁸Ga-DOTA-TATE and ¹⁷⁷Lu-DOTA-TATE have become an effective treatment option for SSTR-positive neuroendocrine tumors. The purpose of this study was to evaluate the correlation of somatostatin receptor-2 (SSTR2) expression with ⁶⁸Ga-DOTA-TATE uptake and ¹⁷⁷Lu-DOTA-TATE therapy in neuroblastoma (NB) xenograft models. We demonstrated variable SSTR2 expression profiles in eight NB cell lines. From micro-PET imaging and autoradiography, a higher uptake of ⁶⁸Ga-DOTA-TATE was observed in SSTR2 high-expressing NB xenografts (CHLA-15) compared to SSTR2 low-expressing NB xenografts (SK-N-BE(2)). Combined autoradiography-immunohistochemistry revealed histological colocalization of SSTR2 and ⁶⁸Ga-DOTA-TATE uptake in CHLA-15 tumors. With a low dose of ¹⁷⁷Lu-DOTA-TATE (20 MBq/animal), tumor growth inhibition was achieved in the CHLA-15 high SSTR2 expressing xenograft model. Although, in vitro, NB cells showed variable expression levels of norepinephrine transporter (NET), a molecular target for ¹³¹I-MIBG therapy, low ¹²³I-MIBG uptake was observed in all selected NB xenografts. In conclusion, SSTR2 expression levels are associated with ⁶⁸Ga-DOTA-TATE uptake and antitumor efficacy of ¹⁷⁷Lu-DOTA-TATE. ⁶⁸Ga-DOTA-TATE PET is superior to ¹²³I-MIBG SPECT imaging in detecting NB tumors in our model. Radiolabeled DOTA-TATE can be used as an agent for NB tumor imaging to potentially discriminate tumors eligible for ¹⁷⁷Lu-DOTA-TATE therapy.

Figure 1

mRNA expression level and Western blotting analyses of SSTR2 and NET. (a) RNA was isolated from different neuroblastoma cell lines, converted into cDNA, followed by RT-PCR with SSTR2 and NET specific primers. The GAPDH gene was used as a reference gene. (b) Protein lysates were prepared from different neuroblastoma cell lines. Protein samples were separated by polyacrylamide gel electrophoresis. Expression of SSTR2 and NET proteins was visualized using specific antibodies. β-Actin was used as internal loading control.

Figure 2

Representative micro-PET/CT images at 1 hour after injection of 10 MBq of  ⁶⁸Ga-DOTA-TATE in the CHLA-15 (a) and SK-N-BE(2) (b) tumor-bearing NOD/SCID mice. Images were presented in the axial (left) and coronal (right) orientations. The white arrows denote localized tumor on the shoulder. (c) Standardized Uptake Values (SUV) in CHLA-15 and SK-N-BE(2) xenografts were calculated using the formula: SUV = C_PET(T)/(Injected  dose/Bodyweight). The difference was significant between CHLA-5 and SK-N-BE(2) tumors (^∗∗P < 0.01). Two-tailed unpaired t-tests were performed to compare the SUV_mean values obtained.

Figure 3

Colocalization of SSTR2 and autoradiography on CHLA-15 xenografts. CHLA-15 and SK-N-BE(2) tumors were removed immediately after PET/CT imaging. The spatial distribution of the ⁶⁸Ga-DOTA-TATE uptake was visualized by autoradiography (white signal) in the serial sections of CHLA-15 (a) and SK-N-BE(2) (b) tumors. (c) Representative CHLA-15 tumor section was stained for SSTR2 (red fluorescence) and DAPI (blue fluorescence). (d) The merging image of SSTR2 immunostaining and autoradiograph of the same CHLA-15 tumor section. (e) A representative SK-N-BE(2) tumor section stained for SSTR2 (red fluorescence) and DAPI (blue fluorescence).

Figure 4

Antitumoral effects of ¹⁷⁷Lu-DOTA-TATE in the CHLA-15 neuroblastoma model. (a) Mice (n = 7) with subcutaneous CHLA-15 xenografts were treated with one dose of 20 MBq of ¹⁷⁷Lu-DOTA-TATE. Control mice (n = 7) received saline. Tumor volume was measured and plotted as shown. Values are stated as mean ± SE; ^∗P < 0.05. (b) Animal body weight was monitored in CHLA-15 tumor-bearing mice with/without ¹⁷⁷Lu-DOTA-TATE treatment.

Figure 5

(a) Example images of ¹²³I-MIBG SPECT/CT in the SK-N-BE(2), CHLA-15, and BE(2)C xenograft models. The white arrows denote localized tumor on the shoulder. (b) Scatter plots of the tumor-to-muscle (T/M) ratios of ¹²³I-MIBG uptake in the SK-N-BE(2), CHLA-15, and BE(2)C xenograft models derived from SPECT/CT imaging. One-way analysis of variance (ANOVA) with Tukey's test was used for statistical analysis. ^∗P<0.05; ^∗∗P < 0.01. (c) Scatter plots of the tumor-to-muscle (T/M) ratio of ¹²³I-MIBG autoradiography in the SK-N-BE(2), CHLA-15, and BE(2)C xenograft models. One-way ANOVA with Tukey's test was used for statistical analysis. ^∗P < 0.05; ^∗∗P<0.01. (d) Western Blot of NET with homogenized CHLA-15, SK-N-BE(2), and BE(2)C xenografts. Three tumors were randomly selected from three groups for Western blot analysis. (e) Quantitative analysis of NET protein expression in CHLA-15, SK-N-BE(2), and BE(2)C xenografts. NET protein expression in Western blot images was quantified densitometrically using ImageJ software (NIH, USA) and normalized with respect to the corresponding expression of β-actin. Comparison of NET expression between different groups was analyzed by the nonparametric Kruskal-Wallis analysis with Dunn's multiple comparison tests. No significant difference of NET expression was observed between CHLA-15, SK-N-BE(2), and BE(2)C xenografts.