Introduction

The incidence of head and neck squamous cell carcinoma (HNSCC) ranks the sixth most common human cancer globally, and over 600,000 cases are newly diagnosed annually1. HNSCC has high mortal rate (~350,000 death each year) because of sound recurrent and metastatic rates2,3. Additionally, surgical treatment or histological diagnosis of local invasion of human HNSCC usually leads to severe side effects including anatomic destruction, dysphasia, aphonia, and aphasia, which are caused by tumorous distribution around important physiological structures such as the spinal cord and carotid artery4,5. The intrinsic radioresistance is also related to the recurrence of HNSCC after chemoradiotherapy6,7. As adjuvant radiotherapy and chemotherapy remain a primary option for the treatment of HNSCC, development of optimal approaches for improvement of the therapeutic efficacy of HNSCC and maintenance of life quality is critical.

Rhenium-188 (188Re) is a high-energy β-particle radionuclide (2.12 MeV) with 15% γ-rays (155 keV) obtained from an alumina-based 188W/188Re generator8. The short average penetration distance of β-particles (around 3.8 mm) in soft tissues endows 188Re as an ideal radionuclide for tumor ablation, including the palliative therapy of bone metastasis with minimal harmful effects to surrounding normal tissues9,10. Polyethylene glycol (PEG)-decorated 188Re-liposome is a nano-sized biocompatible radiopharmaceutical that has been used for evaluating the theranostic efficacy in different human cancers, including colorectal cancer, glioblastomas, lung cancer, ovarian cancer, and esophageal cancer preclinically11,12,13,14,15. We have shown that 188Re-liposome could be accumulated in orthotopic HNSCC tumor lesions, but the therapeutic efficacy was moderate16. Radioresistance is a feature of HNSCC and is related to the tumor relapses after chemoradiotherapy6. Modification of treatment regime may be important to improve the therapeutic efficacy of 188Re-liposome.

Dose escalation of 188Re-conjugated radiopharmaceutical has been used for treatment of different human cancers. Palmedo et al.17 have found that an escalated dose of 188Re-HEDP (over 2.6GBq) offers 60–75% pain palliation in prostate cancer patients with osseous metastases with the occurrence of thrombocytopenia and leukopenia up to 8 weeks. Additionally, a perspective phase II clinical trial using 64 hormone-refractory prostate cancer patients concluded that enhanced pain palliation, reduced prostatic specific antigen (PSA), increased progression-free, and overall survival when patients received double injections rather than a single injection of 188Re-HEDP18. Repeated intratumoral injection of 188Re microspheres into the hepatoma animal model also achieves better therapeutic efficacy19. Whether repeated doses of 188Re-liposome can also enhance the therapeutic efficacy in HNSCC is of interest to study.

Epithelial–mesenchymal transition (EMT) is an important process of tumor metastasis. During EMT, epithelial cells can transit to mesenchymal phenotypes accompanied by high motility, which is caused by a loss of junction, cytoskeletal reorganization, and morphological change20. Such a transition is associated with vigorous reprogramming of gene expression, including E-cadherin, vimentin, zinc-finger E-box-binding 1 (ZEB-1), basic Helix-Loop-Helix Transcription Factor 1 (TWIST1), and Zinc-finger protein SNAI1 (SNAIL) transcription factors21. Intraperitoneal injection of 188Re-liposome has recently been reported to block EMT and reactivate p53 function in ovarian tumors22. A recent report showed that 188Re-liposome could induce the expression of let-7 microRNA in HNSCC orthotopic tumors16. Let-7 is known to inhibit EMT by suppressing the high-mobility group AT-hook 2 (HMGA2) gene that activates the expression of SNAIL and TWIST to inhibit tumor growth and metastasis23,24,25. Whether 188Re-liposome also influences the expression of EMT-related markers in HNSCC is of interest to investigate.

In this study, we showed that high doses of 188Re-liposome exhibited different killing efficacies on cultured HNSCC cells. To compare the low dose and high dose of 188Re-liposome on the therapeutic efficacy of HNSCC in vivo, we used a single therapy and repeated therapy to assess the biological and pharmaceutical responses in an orthotopic tumor model. Additionally, the systemic toxicity and markers of tumor proliferation and EMT, as well as dosimetry were examined and compared in tumor lesions treated with different dosages of 188Re-liposome. The significance of 188Re-liposome-based radiopharmacotherapy was discussed.

Results

Killing effects of 188Re-liposome on different HNSCC cell lines

HNSCC includes malignancies initiating from different locations of the oral cavity. Here we administrated 188Re-liposome on three human HNSCC cell lines, including FaDu cells, SAS cells, and OECM-1 cells to examine the killing effects using different doses. It was found that cell killings of FaDu cells and SAS cells were more significant than that of OECM-1 cells using high dose (300 μCi) of 188Re-liposome (Fig. 1a, b). This is an important implication for clinical application of 188Re-liposome on different types of human HNSCC.

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

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

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Effects of single dose and repeated doses of 188Re-liposome on tumor targeting

To investigate the response of HNSCC to 188Re-liposome in vivo, we established an orthotopic tumor model in immune-deficient nude mice using FaDu cells. The experimental regimes of a single i.v. injection or repeated i.v. injections of 188Re-liposome into tumor-bearing mice were schemed (Fig. 2a). The time interval for repeated injections of 188Re-liposome was 6 days as considered for the half-life of 188Re and clinical feasibility. Cerenkov luminescent imaging (CLI), an optical signal raised by charged particles using medical isotopes26, was firstly used to compare the ratios of 188Re-liposome accumulation in tumor-bearing mice between a single dose and repeated doses. Implantation of orthotopic tumor into the buccal position of each mouse exhibited a time-dependent increase of CLI signals after injection of 188Re-liposome (Fig. 2b). The signal intensity of each tumor lesion was normalized to a non-tumor region of the same tumor-bearing mouse. The results showed that repeated doses of 188Re-liposome tended to exhibit higher tumor-to-non-tumor ratio than a single dose of 188Re-liposome up to 48 h (Fig. 2c). The luminescent signals substantially disappeared right before the second injection (6 days after the first injection) of 188Re-liposome (Supplementary Data 1).

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

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

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Comparison of therapeutic efficacy between single injection and repeated treatment of 188Re-liposome on HNSCC animal model

The therapeutic efficacy of 188Re-liposome was subsequently investigated using the bioluminescent imaging in FaDu-3R tumors that expressed luciferase activity. Repeated injections of 188Re-liposome exhibited better tumor suppressive effects than a single injection of 188Re-liposome in tumor-bearing mice (Fig. 3a). The results were also quantified by measuring the photon fluxes in untreated controls, a single dose, and repeated doses of 188Re-liposome (Fig. 3b). Additionally, repeated doses of 188Re-liposome exhibited slower tumor growth rates than a single dose and untreated control using caliper measurement of tumor volumes (Fig. 3c). The orthotopic tumors were also excised from tumor-bearing mice after 4 weeks of tumor growth to demonstrate the enhanced tumor suppressive effects by repeated doses of 188Re-liposome (Fig. 3d). Furthermore, the animal survival of repeated 188Re-liposome treatment was greater than that of a single 188Re-liposome treatment and untreated controls (Fig. 3e). The median survival times of tumor-bearing mice with repeated injections, a single injection, and untreated control were 64, 45, and 32 days, respectively.

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

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)

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Effects of 188Re-liposome on expression of markers for proliferation and EMT using a single dose or repeated doses

Tumors were also resected for IHC staining of Ki-67 proliferative marker. The level of Ki-67 was significantly suppressed by repeated doses of 188Re-liposome compared to a single dose and untreated controls demonstrated by the pseudo-colored image method (Fig. 4a, b). We also examined whether the expressions of EMT-related markers in tumors were affected by injection of 188Re-liposome. Compared to a single dose of 188Re-liposome, repeated dose apparently induced E-cadherin levels, inhibited N-cadherin and Twist1/2 levels (Fig. 4c). 188Re-liposome could equally suppress the levels of ZEB-1, vimentin, and Slug markers using a single dose or repeated dose (Fig. 4c). Interestingly, we also found that the level of γ-H2AX, a DNA damage marker was increased by repeated doses of 188Re-liposome compared to a single dose (Fig. 4c). These blots were also quantified using dosimetry (Fig. 4d).

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

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.

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Comparison of toxic effects in tumor-bearing mice treated with a single dose and repeated doses of 188Re-liposome

We also compared the potent adverse effects in tumor-bearing mice treated with a single dose or repeated doses of 188Re-liposome. The changes of body weights were not significantly different by comparing the untreated controls and 188Re-liposome-injected groups (Fig. 5a). Moreover, compared to a single dose of 188Re-liposome, the counts of WBC, RBC, and platelets were significantly suppressed by repeated doses at different time points (Fig. 5b). A single injection of 188Re-liposome could also suppress the counts of WBC but not RBC and platelets.

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

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

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Comparison of biodistribution and pharmacokinetics in tumor-bearing mice treated with a single dose and repeated doses of 188Re-liposome

Biodistribution analysis was performed in two groups of mice injected with a single dose or repeated doses of 188Re-liposome. Compared to other organs, repeated doses exhibited higher accumulation rates of 188Re-liposome in bone marrows and tumors than a single dose (Fig. 6a and Supplementary Data 2). Moreover, the tumor-to-muscle ratio (T/M) of repeated doses to a single dose of 188Re-liposome was about twofold calculated from the biodistribution data up to 48 h (Fig. 6b). In regards of pharmacokinetics, liposome-free 188Re-BMEDA (640 μCi/150 μL) was used as a control to compare the circulation of 188Re-liposome using a single injection or repeated injections into mice. The results showed that the injection of 188Re-liposome exhibited slower clearance and longer retention than 188Re-BMEDA, and these effects were even greater in repeated doses than in a single dose of 188Re-liposome (Fig. 6c). The pharmacokinetic-related parameters were compared between intravenous injections of 188Re-BMEDA and 188Re-liposome (Table 1). Notably, the AUC of repeated doses was 1.65-fold to that of a single dose.

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

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

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Table 1 Pharmacokinetic analysis for single dose and repeated dose of 188Re-liposome treatment on HNSCC tumor-bearing mice
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Dosimetric analysis for a single dose and repeated doses of 188Re-liposome administrating in the HNSCC tumor model

The estimated internal radiation doses were compared between a single dose and repeated doses of 188Re-liposome (Supplementary Data 3). Compared to a single dose of 188Re-liposome, repeated doses caused over twofold absorbed dose in the bladder wall (0.497 verses 0.0891mGy/MBq), red marrow (0.395 verses 0.145 mGy/MBq), and small intestine (0.377 verses 0.0899mGy/MBq). The effective doses were 0.177 and 0.245 mSv/MBq for a single dose and repeated doses of 188Re-liposome, respectively. The estimated tumor absorbed doses of a single dose and repeated doses were 0.136 and 0.264 mGy/MBq using a 1 g spheroid model, respectively. The absorbed doses for different sizes of spheroid tumor mass treated with 188Re-liposome with a single dose or repeated doses were also performed (Supplementary Data 4).

Discussion

HNSCC includes different cell types in head and neck regions that exhibit heterogeneous responses to radiation therapy. Here we used high dose of 188Re-liposome to treat three different HNSCC cell lines, and demonstrated that significant cell killing was only found in FaDu cells and SAS cells but not in OECM-1 cells. Despite FaDu cells were further used for the establishment of orthotopic tumor model, OECM-1 cells were also attempted to be implanted into nude mice. However, this cell type failed to form tumors (data not shown). This is consistent with a previous report that OECM-1 tumor is barely formed27. Little is known about the mechanisms of 188Re-liposome mediated cell death. It has been reported that mutation of p53, retinoblastoma (pRb), NOTCH, phosphoinositol-3 kinase (PI3K), phosphatase and tensin homolog (PTEN), AKT kinase, and epithelial growth factor receptor (EGFR) pathways are commonly found in the occurrence of HNSCC28. Interestingly, targeting survivin also induce both apoptotic and autophagic cell death in HNSCC29. Whether different cell killing efficacies mediated by 188Re-liposome is associated with these pathways will be important to further investigate.

188Re is a cost-effective isotope with theranostic potent30. Due to its affordable price, a dose-escalation study using 188Re-HEDP has been reported to evaluate its effects on pain palliation of prostate cancer patients with osseous metastases17. However, a phase I dose-escalation trial has been used to determine the dose-limiting toxicity (DLT), and the suppression of red marrow was the only DLT to be observed31. Administration of relatively high doses of 188Re may be a potent candidate for radioimmunotherapy, although increased renal and liver uptakes were also detected31. Additionally, the short physical half-life of 188Re (T1/2 = 16.9 h) is an important property for repeated treatment32. 188Re-HEDP has been applied for pain relief therapy of bone metastases secondary to breast cancer and prostate cancer33. The results of clinical trials suggest that the use of 188Re-conjugated radiopharmaceuticals with repeated doses for cancer treatment should be feasible. Using animal models, it is possible to compare the effects of a single dose and repeated doses of 188Re-conjugated radiopharmaceutical on various human cancers for clinical consideration. Here we used the radioresistant FaDu cancer cells34 to demonstrate that repeated therapy of 188Re-liposome was more effective than a single therapy in the suppression of tumors formed by these cells. Repeated treatments with original doses rather than an escalated single dose were adopted to avoid exceeding 80% MTD. These preclinical results are partially consistent with previous reports that repeated treatments of 188Re-conjugated radiopharmaceutical will provide better tumor control in clinical trials18,35.

The improved therapeutic effects of repeated doses of 188Re-liposome can be elucidated by the tumor accumulation of this radiopharmaceutical and the molecular responses of the orthotopic tumor. The hypopharyngeal cancer FaDu cells can induce angiogenesis in nude mice36. The enhanced permeability and retention (EPR) effect should not be impaired by the first injection of repeated 188Re-liposome doses as both single injection and repeated injections of 188Re-liposome could accumulate in tumor lesions. Interestingly, repeated doses of 188Re-liposome could change the expression of several EMT-related molecules. A recent report showed that 188Re-liposome could induce E-cadherin and suppress vimentin in human ovarian cancer cells22. In this study, this effect was further demonstrated in an HNSCC tumor model by examining additional EMT-related markers, which were also enhanced by repeated doses of 188Re-liposome. Notably, we found that the level of γ-H2AX, a DNA double-strand breaks marker was also higher in tumors treated with repeated doses of 188Re-liposome. It has been reported that ZEB-1 could promote DNA repair and lead to radioresistance in cancer cells37. Hence, suppression of ZEB-1 by repeated doses of 188Re-liposome may suppress the DNA repair effects and increase the radiosensitivity of orthotopic tumors. As EMT may have a pivotal role in the recurrence of HNSCC38, inhibition of EMT by repeated therapy of 188Re-liposome may also reduce the probability of recurrence.

The side effects were the primary concerns when the repeated therapy of 188Re-liposome was adopted. It is assumed that optimal time interval between the first dose and second dose may reduce potent toxicity without loss of therapeutic efficacy. In this study, the time interval of repeated 188Re-liposomal injections was 6 days (approximately over 9 half-lives of decay). No significant reduction of body weight was detected after repeated therapy; therefore, this treatment should not cause acute toxicity. On the other hand, the counts of RBC, WBC, and PLT were suppressed by 188Re-liposome; and repeated doses exhibited stronger effects than a single dose. Hence, repeated doses of 188Re-liposome may increase the possibility of hematologic impairment. These results were partially consistent with 188Re-HEDP that showed clinically unimportant decreases in WBC and platelet using repeated doses of 186Re-HEDP with a time interval at 8–12 weeks39.

The circulation period of repeated 188Re-liposomal doses was longer than that of a single dose as shown by pharmacokinetic analysis, suggesting that the enhanced therapeutic efficacy is associated with longer retention of 188Re-liposome after repeated injections. On the other hand, it may imply that elongated circulation of 188Re-liposome increases bone marrow dose and reduces blood counts. It is consistent with a previous report that bone marrow toxicity was a main limiting factor of 186Re-HEDP40, at least in part.

The OLINDA/EXA code based dosimetric calculation revealed that the bladder wall, small intestine, and red marrow received over twofold of absorbed dose after repeated therapy of 188Re-liposome. The ratios of enhancement were 5.58, 4.19, and 2.72 for the bladder wall, small intestine, and red marrow, respectively. As the tolerance doses of the bladder wall (50–70 Gy) and small intestine (20–45 Gy) are higher than for red marrow (2–10 Gy) in radiotherapy41,42, using repeated doses of 188Re-liposome may be acceptable for clinical purposes. The effective dose of both single dose and repeated doses calculated for a male adult model remains far lower than a single diagnostic procedure using radionuclide (1–10 mSv) or background radiation amount (about 3 mSv/year)43.

In summary, current data indicate that in cultured cells, high dose of 188Re-liposome would perform various killing effects on different origins of HNSCC cells. For in vivo study, repeated doses of 188Re-liposome exhibited greater tumor ablation and survival than a single dose administrated in the HNSCC tumor model. Extended circulation time of 188Re-liposome might contribute to increased accumulation of this radiopharmaceutical in tumor lesions after repeated administration to enhance the tumor suppression. Although acute toxicity was not detected, a significant decrease of blood cells could be a limiting factor when using repeated therapy of 188Re-liposome for HNSCC treatment. Good hospitalization or prevention of immunological/hematological impairment should be considered for repeated therapy of 188Re-liposome in clinical application.

Materials and methods

Cell lines, plasmid, and cell counts

Human FaDu hypopharyngeal carcinoma cells (American Type Culture Collection, Manassas, VA, USA) and FaDu-3R cells harboring a pLT-3R construct with multiple reporter genes were maintained as a previous report16. Cells were maintained in RPMI-1640 (Life Technologies Inc., Carlsbad, CA, USA) medium. Human tongue carcinoma SAS cell line was a kind gift obtained from Prof. Muh-Hua Yang (National Yang-Ming University, Taipei, Taiwan) and was cultured in Dulbecco’s modified Eagle’s medium (DMEM). Oral squamous cell carcinoma OECM-1 was a kind gift from Dr. Yu-Jen Chen (Department of Radiation Oncology, MacKay Memorial Hospital, Taipei, Taiwan) and was cultured in RPMI-1640. All cell lines were supplemented with 10% fetal bovine serum (FBS), 2 mM l-glutamine, 50 U/mL of penicillin and 50 μg/mL of streptomycin (Invitrogen Inc., Carlsbad, CA), and were incubated at 37 °C in a humidified incubator with 5% CO2 and passaged every two days. Cell counts were also used for evaluation of cell viability before and after drug treatment. Briefly, cells (5 × 105) were seeded in 10-cm dishes and incubated overnight. The medium was then replaced by fresh medium containing different concentrations of 188Re-liposome. Cell images were acquired after cells were exposed to 188Re-liposome for 72 h, and cell numbers were counted using hemocytometry.

Preparation of 188Re-liposome for intravenous injection

The procedures of 188Re-liposome preparation of validation have been described before11. The mean loading efficiency of 188Re-liposome was ~70–80% determined by (Total radioactivity eluted)/(Remnant radioactivity in chromatographic column). Each injection used 23.68MBq (640 μCi) corresponding to 80% maximum tolerated dose (MTD) as described previously14.

Establishment of HNSCC orthotopic tumor model

The orthotopic implantation of FaDu-3R cells in BALB/c nude mice has been described previously16. Animal experiments had been approved by the Institutional Animal Care and Utilization Committee (IACUC) of National Yang-Ming University (No. 1041106).

Evaluation of tumor uptake and therapeutic efficacy of 188Re-liposome in tumor-bearing mice

After administration of 188Re-liposome, CLI was performed to acquire signals using the In Vivo Imaging System (IVIS 50, Perkin Elmer Inc., Waltham, MA, USA). For evaluation of therapeutic efficacy, the tumor viability and growth rate were assessed using the luciferase based reporter gene imaging and caliper measurement, respectively. The tumor volume was determined by the formula: (width2 × length)/2 after caliper measurement. For survival analysis, the end point of each datum was established when tumor volume reached 1000 mm3 by caliper measurement, or when the body weight reduced over 25% from the first day of treatment.

Immunohistochemical (IHC) staining

The paraffin embedded tissue sections were prepared and incubated with anti-Ki-67 antibody (MAB4190, EMD Millipore, Billerica, MA, USA) at 4 °C overnight followed by horseradish peroxidase (HRP)-conjugated secondary antibodies. All sections were scanned by the Aperio digital Pathology Slide Scanner (Leica Biosystems, Buffalo Grove, IL, USA). The images were subjected to the ImmunoRatio automated counting tool to estimate the Ki-67 positivity index of the nuclei44.

Western blot analysis and antibodies

Tumors were harvested from the tumor-bearing mice after 4 weeks of treatment and then lysed in T-PER™ Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA) containing 1% protease inhibitor cocktail (Sigma-Aldrich Co., St. Louis, MO, USA). The procedures of Western blot analysis have been reported previously45. The primary antibodies used in this study included anti-N-cadherin (GTX100443), anti-E-cadherin (GTX100443), anti-Twist1/2 (GTX127310), anti-ZEB-1 (GTX105278), anti-vimentin (GTX100619), anti-Slug (GTX128796), anti−γ-H2AX (GTX628789; GeneTex, Inc., Irvine, CA, USA), and anti-GAPDH (MA5-15738; Thermo Fisher Scientific, Waltham, MA, USA).

Measurement of blood cell counts

Blood samples were acquired from mice at different time points after the treatment of 188Re-liposome by orbital sinus sampling. Red blood cells (RBC), white blood cells (WBC), and platelets were recorded by XT-1800i, an automated hematology analyzer (Sysmex Co., Chuo-ku, Kobe, Hyogo, Japan).

Analysis of biodistribution and pharmacokinetic

The tumor-bearing mice were randomly assigned to three groups for injection of 188Re-liposome or 188Re-BMEDA followed by biodistribution and pharmacokinetic analysis as reported previously with slightly modification16. For biodistribution, mice were killed by CO2 asphyxiation after intravenous injection of 188Re-liposome followed by harvesting of different organs. Samples were weighted and counted by a γ-scintillation counter (1470 WIZARD Gamma Counter, Wallac, Finland). The results were represented as the percentage injected dose per gram tissue (% ID/g). For pharmacokinetic analysis, the blood samples were collected from mice using the tail vein puncture with microliter capillary tubes at 0.083, 025, 0.5, 1, 2, 4, 8, 12, 16, 20, 24, 28, 36, 44, and 52 h. Samples were then counted by a γ-scintillation counter and calculated by the WinNonLin software (v6.6, Pharsgiht Corp., Mountain View, California, USA) using a non-compartment model.

Dosimetric evaluation of 188Re-liposomal absorbed dose in vivo

The dosimetry of percentage injected dose activity per weight tissue (%ID/g) in human was extrapolated from the biodistribution data of mice using the guideline of Medical Internal Radiation Dosimetry (MIRD) pamphlets implanted in the OLINDA/EXM software46,47. The number of disintegration of tumor was used to calculate the absorbed dose in tumor (1 g) using the sphere model.

Statistical analysis

The statistical differences were analyzed by a two-tailed t-test (GraphPad Prism 6.0; GraphPad Software, San Diego, CA, USA). All data were represented as mean ± S.D. or mean ± S.E.M. Use of statistic methods and sample numbers were also described in each figure legend. The Kaplan–Meier method with the log-rank test was used to compare survival rates among different treatments. The level of statistical significance was set to p < 0.05 for all tests.