Involvement of let-7 microRNA for the therapeutic effects of Rhenium-188-embedded liposomal nanoparticles on orthotopic human head and neck cancer model

Abstract

Human head and neck squamous cell carcinoma (HNSCC) is usually treated by surgical resection with adjuvant radio-chemotherapy. In this study, we examined whether the radiopharmaceutical 188Re-liposome could suppress the growth of HNSCC followed by an investigation of molecular mechanisms. The orthotopic HNSCC tumor model was established by human hypopharyngeal FaDu carcinoma cells harboring multiple reporter genes. The drug targeting and therapeutic efficacy of 188Re-liposome were examined using in vivo imaging, bio-distribution, pharmacokinetics, and dosimetry. The results showed that 188Re-liposome significantly accumulated in the tumor lesion compared to free 188Re. The circulation time and tumor targeting of 188Re-liposome were also longer than that of free 188Re in tumor-bearing mice. The tumor growth was suppressed by 188Re-liposome up to three weeks using a single dose treatment. Subsequently, microarray analysis followed by Ingenuity Pathway Analysis (IPA) showed that tumor suppressor let-7 microRNA could be an upstream regulator induced by 188Re-liposome to regulate downstream genes. Additionally, inhibition of let-7i could reduce the effects of 188Re-liposome on suppression of tumor growth, suggesting that let-7 family was involved in 188Re-liposome mediated suppression of tumor growth in vivo. Our data suggest that 188Re-liposome could be a novel strategy for targeting HNSCC partially via induction of let-7 microRNA.

Keywords: 188Re-liposome; HNSCC; let-7 microRNA; microarray analysis; orthotopic tumor model.

Figure 1. Reporter gene imaging for tracking the growth of human HNSCC in xenograft tumor model

A. Orthotopic implantation of FaDu-3R cells at buccal cavity and detection of formed tumor using the IVIS optical imaging system; B. in vivo detection of orthotopic HNSCC xenograft tumor with HSV1-tk activity using nanoSPECT/CT and 3D reconstruction of the images. The ¹²³I-FIAU was used as the radiotracer; C. use of IVIS optical imaging system to monitor the tumor growth weekly. Wk₀ indicated the initial imaging acquired immediately after tumor implantation; D. quantification of the photon flux to plot the tumor growth curve in vivo.

Figure 2. Accumulation of PEGylated ¹⁸⁸Re-liposome in orthotopic HNSCC tumor lesion

A. γ-rays emission of ¹⁸⁸Re-liposome was detected in the HNSCC tumor lesion in vivo at different time points using nanoSPECT/CT imaging, Circles represented the region of interest (ROI); B. β particles emission caused Cerenokov bioluminescent imaging by ¹⁸⁸Re-liposome was detected using the IVIS optical imaging system. The arrows indicated positions of implanted tumors.

Figure 3. Determination of the biodistribution and pharmacokinetics of PEGylated ¹⁸⁸Re-liposome used in the orthotopic HNSCC tumor model

A. Illustration of timeline for execution of biodistribution and pharmacokinetics analysis after administration of ¹⁸⁸Re-liposome or ¹⁸⁸Re-BMEDA into the tumor-bearing mice; B. biodistribution of ¹⁸⁸Re-BMEDA; C. biodistribution of ¹⁸⁸Re-liposome. The arrow indicated the accumulation of ¹⁸⁸Re-liposome in tumors. N=5 for each time point; D. comparison of pharmacokinetics of ¹⁸⁸Re-BMEDA and ¹⁸⁸Re-liposome by detecting the radioactivity in blood. N=5 for each group.

Figure 4. Evaluation of therapeutic efficacy of PEGylated ¹⁸⁸Re-liposome on HNSCC xenograft tumor model

A. Illustration of timeline for monitoring the therapeutic efficacy of radiopharmaceuticals on FaDu-3R cells formed tumors using the bioluminescent imaging (BLI); B. the ¹⁸⁸Re-BMEDA (upper panel) and the ¹⁸⁸Re-liposome (lower panel) were injected with the 80% maximal tolerated dose (MTD, 640 μCi), and the tumor growth was tracked by BLI. (N=9 for each group); C. quantification of the tumor bearing mice treated with either ¹⁸⁸Re-BMEDA or ¹⁸⁸Re-liposome. The data was normalized to the first BLI acquisition and performed as the fold change. Data are presented as mean ± SD. (*: p<0.05, **p<0.01); D. comparison of body weight changes between tumor bearing mice treated with ¹⁸⁸Re-BMEDA and ¹⁸⁸Re-liposome; E. The survival curves of ¹⁸⁸Re-BMEDA and ¹⁸⁸Re-liposome treated mice. The endpoint was set up as the 20% weight loss or death. The results were analyzed by the log-rank test (p < 0.01).

Figure 5. MicroRNA microarray analysis after the HNSCC tumor bearing mouse was treated with PEGylated ¹⁸⁸Re-liposome

A. The microarray data were analyzed by IPA, and sorted by the activation z-score >3; B. Analysis of microRNA microarray results by GSEA. The let-7 microRNA family was mostly enriched. (Permutation:1000, sorted by p-value, calculated from the log 2 ratio); C. demonstration of let-7 family members (let-7b, let-7e and let-7i) induced by ¹⁸⁸Re-liposome but not liposome empty vector using qPCR. Each datum was the mean of four repeats. *: p < 0.05.

Figure 6. Knockdown of let-7i by LNA™ compromises the effects of ¹⁸⁸Re-liposome on suppressing the growth of human HNSCC tumors in vivo

A. Quantitative PCR analysis for measuring the level of let-7i before and after LNA™ transfection; B. knockdown of let-7i (Let-7i LNA+R) and over-expression of let-7i (Let7i OV+R) increased and decreased the luciferase reporter gene activity, respectively. *: p< 0.05; C. comparison of cell growth rates with or without transfection of LNA™; D. comparison of cell viability in FaDu-3R cells using the MTT assay after transfection of let-7i LNA for 48 hours or left untransfected; E. comparison of plating efficiency between let-7i LNA transfected cells and untransfected cells using the colony formation assay. The colonies were formed and counted after 14 days of incubation (*: p<0.05, **: p<0.01); F. BLI imaging for tracking the FaDu-3R tumor growth among control, ¹⁸⁸Re-liposome treatment, LNA™ treatment and combination of both. Tumors were formed by normal or LNA™ transfected FaDu-3R cells; G. visualization of tumor size change under different treatments for 19 days; H. tumor growth curves determined by caliperly measuring tumor volume at each time point, and then normalized to the tumor size at day 0. (N=6); *: p < 0.05.