PEGylated liposome-encapsulated rhenium-188 radiopharmaceutical inhibits proliferation and epithelial-mesenchymal transition of human head and neck cancer cells in vivo with repeated therapy

Abstract

Human head and neck squamous cell carcinoma (HNSCC) is usually treated with chemoradiotherapy, but the therapeutic efficacy could be hampered by intrinsic radioresistance and early relapse. Repeated administrations of rhenium-188 (¹⁸⁸Re)-conjugated radiopharmaceutical has been reported to escalate the radiation doses for better control of advanced human cancers. Here we found that high dosage of ¹⁸⁸Re-liposome, the liposome-encapsulated ¹⁸⁸Re nanoparticles exhibited significant killing effects on HNSCC FaDu cells and SAS cells but not on OECM-1 cells. To investigate the biological and pharmaceutical responses of high ¹⁸⁸Re-liposomal dosage in vivo, repeated doses of ¹⁸⁸Re-liposome was injected into the orthotopic tumor model. FaDu cells harboring luciferase reporter genes were implanted in the buccal positions of nude mice followed by intravenous injection of ¹⁸⁸Re-liposome. The Cerenkov luminescence imaging (CLI) was performed to demonstrate an increased accumulation of ¹⁸⁸Re-liposome in the tumor lesion of nude mice with repeated doses compared to a single dose. Repeated doses also enhanced tumor growth delay and elongated the survival of tumor-bearing mice. These observations were associated with significant loss of Ki-67 proliferative marker and epithelial-mesenchymal transition (EMT) markers in excised tumor cells. The body weights of mice were not significantly changed using different doses of ¹⁸⁸Re-liposome, yet repeated doses led to lower blood counts than a single dose. Furthermore, the pharmacokinetic analysis showed that the internal circulation of repeated ¹⁸⁸Re-liposomal therapy was elongated. The biodistribution analysis also demonstrated that accumulations of ¹⁸⁸Re-liposome in tumor lesions and bone marrow were increased using repeated doses. The absorbed dose of repeated doses over a single dose was about twofold estimated for a 1 g tumor. Together, these data suggest that the radiopharmacotherapy of ¹⁸⁸Re-liposome can enhance tumor suppression, survival extension, and internal circulation without acute toxicity using repeated administrations.

Fig. 1. Effects of ¹⁸⁸Re-liposome on different HNSCC cell lines using low dose and high dose.

a Change of cell morphology and amount in FaDu cells, SAS cells, and OECM-1 cells treated with low dose (100 μCi) and high dose (300 μCi) of ¹⁸⁸Re-liposome. Scale bar: 100 μm. b Quantification of cell number in cells treated with low or high dose of ¹⁸⁸Re-liposome. Data were represented as means ± S.D. *p < 0.05 by a t-test

Fig. 2. Comparison of PEGylated ¹⁸⁸Re-liposomal accumulation in orthotopic HNSCC tumors after a single and repeated injections.

a The experimental scheme for ¹⁸⁸Re-liposome treatment. b CLI signals acquired by the IVIS system. c The ratios of photon flux were determined by normalizing the tumor (left side of mouth) to non-tumor (right side of mouth) regions. Red circles represented the region of interest (ROI) for tumor lesions (n = 6). Data were represented as means ± S.E.M. *p < 0.05 by a t-test

Fig. 3. Monitoring the therapeutic efficacy of ¹⁸⁸Re-liposome administrated in HNSCC tumor-bearing mice with different regimes.

a Reporter gene imaging of tumor growth responding to a single dose, repeated doses of ¹⁸⁸Re-liposome, and untreated control (n = 5). b Quantification of BLI signals. c Caliper measurement of tumor volumes (n = 5). Data were represented as means ± S.D. *p < 0.05 by an unpaired two-tailed t-test. d Representative photos of excised orthotropic tumors. e Analysis of animal survival using the Kaplan–Meier method with log-rank test (p < 0.001)

Fig. 4. Effects of ¹⁸⁸Re-liposome on the expression of molecular markers involved in cell proliferation and metastasis.

a Measurement of IHC stained Ki-67 expression in orthotopic tumors. Scale bar, 200 μm. b Quantification of Ki-67 positive cells according to the images of pseudo-colored analysis. Three fields were randomly selected for counting the Ki-67 expressing cells. Data were represented as means ± S.D. *p < 0.05 by a t-test. c The expression of EMT-related markers in orthotopic tumors with different treatments of ¹⁸⁸Re-liposome for 4 weeks. d Quantification of EMT-related markers detected in the blots using densitometry (n = 3). Data were represented as means ± S.D.

Fig. 5. Evaluation of toxicity caused by single dose and repeated doses of ¹⁸⁸Re-liposomal administrated in tumor-bearing mice.

a Measurement of body weights (n = 5). The data point of each curve represented the mean ± S.D. of body weights averaged from five mice. b Counting of RBC, PLT, and WBC. The blood was obtained after mice were treated with a single dose or repeated doses of ¹⁸⁸Re-liposome for 2 days and 6 days (n = 3). Data were represented as means ± S.D. *p < 0.05 by a t-test

Fig. 6. Biodistribution and pharmacokinetic analysis for comparing a single dose and repeated doses of ¹⁸⁸Re-liposomal injection.

a Biodistribution of single dose and repeated doses. (n = 5). S.I. small intestine, L.I. large intestine. b Comparison of tumor-to-muscle ratios between a single dose and repeated doses of ¹⁸⁸Re-liposomal injection at different time points. c Pharmacokinetic analysis. (n = 5). Data were represented as means ± S.D. *p < 0.05 by a t-test