Cell Line and Chemicals

Human hepatocarcinoma cells (HC-4), obtained from the Cell Resource Center for Biomedical Research, Cell Bank (Tohoku University, Japan) were used in this study. A mycoplasma contamination test was performed before transplantation cells into the animals, which was confirmed to be negative. Cells were maintained in Eagle Minimum Essential Medium (Eagle MEM; FUJIFILM Wako Pure Chemical Corporation, Japan) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA). 177Lu-chloride (177LuCl3; PDRadiopharma, Japan) and Lipiodol (Guerbet, France) are analytical grade and were used without purification.

In Vitro Cell Irradiation and Replication Assay

To confirm HCC radiosensitivity, we performed a gamma-irradiation replication assay, following our previously reported protocol [15]. The HC-4 cells were irradiated with gamma rays in a single-cell suspension using a gamma cell 40 Exactor (Nordion International; dose rate 0.60 Gy/min) (0, 3, 6, 9, or 12 Gy). Five groups categorized according to the radiation dose were prepared: 0 Gy (control group), 3 Gy (3 Gy group), 6 Gy (6 Gy group), 9 Gy (9 Gy group), and 12 Gy (12 Gy group). At days 3, 5, 7, and 14, cells were harvested, stained with trypan blue, and counted. The replication rate (RR) was calculated according to Eq. (1):

$$\text=\frac}_}}}_}}$$

(1)

Nirradiated​ is the number of viable cells in the irradiated group, and Ncontrol is that in the non-irradiated control group.

Synthesis of 177Lu-Oxine and Emulsification into Lipiodol

177Lu-oxine was prepared according to a published procedure [16]. Briefly, a measured activity (60 MBq, 2.49 µL) of 177LuCl3 was combined with 0.5 M oxine in ethanol and 20 mM ammonium acetate buffer (pH 6.5), heated at 50 °C for 30 min, then cooled and extracted with dichloromethane. After drying under nitrogen, the 177Lu-oxine residue was obtained. The radiochemical purity (≥ 99%) was confirmed by performing thin-layer chromatography (TEC-CONTROL Chromatography Strips (Black), Mirion Technologies (Capintec), Inc., Florham Park, New Jersey, USA) using methanol as the solvent. Dried 177Lu-oxine (37 MBq) was mixed with 0.6 mL lipiodol (50 °C) for 1 h in a 50 °C water bath to obtain 177Lu-lipiodol. Afterward, 0.6 mL saline was added, and the biphasic mixture was vigorously vortexed, then allowed to stand for 5 min. The lipiodol phase (containing 177Lu-oxine) was separated and measured with a curiemeter. To assess lipophilicity, a fixed amount of 177Lu-lipiodol was mixed with saline, centrifuged,

and the radioactivity in each phase was measured. The partition coefficient (log P) was calculated according to Eq. (2):

$$}_\left(\frac}}\right)$$

(2)

Tumor Embolization Study In Vivo

All animal experiments were approved by the Institutional Animal Care and Use Committee of Kansai Medical University (Approval Number: 24–057).

Tumor Embolization Study In Vivo

Five male F344 NJcl rnu/nu rats (6–8 weeks old) were subcutaneously inoculated with 5.0 × 106 HC-4 cells in each thigh. Tumors were allowed to grow for five weeks, reaching approximately 0.3 cm3 in volume. Three rats (RI-group) received 177Lu-lipiodol (2.8 MBq in 50 μL), and two rats (C-group) received non-labeled lipiodol (50 μL). Under isoflurane anesthesia, a small groin incision was made to expose the right femoral artery, which was directly punctured (29G needle), and the lipiodol emulsion was slowly injected under fluoroscopic guidance. The artery was ligated post-injection, and the incision was sutured.

SPECT/CT Imaging

All imaging was performed on an Inveon® multimodality system (Siemens Medical Solutions, USA). CT was acquired with a transaxial field of view (FOV) of 106.73 mm in a step-and-shoot mode, and SPECT was performed with a tungsten collimator (3-RWB-1.8). Reconstruction was performed using the MAP3D algorithm (PSF correction, 16 iterations, 6 subsets). Immediately after 177Lu-lipiodol administration and on days 3, 7, and 14, we evaluated the accumulation rate (AR) in the tumor and lungs relative to the whole-body ROI (ROIWB). Equations (3) and (4) define AR in the tumor and lungs, respectively:

$$}_}= \frac}_}}}_}}\times 100 \%$$

(3)

$$}_}= \frac}_}}}_}}\times 100 \%$$

(4)

ROItumor was defined as a 10 cm3 spheroid region centered on the tumor in the lower leg of the treatment side, ROIlung was defined as a 90 cm3 long rectangular region centered on the lung region, and ROIWB as an 1100 cm3 long rectangular region that contained the entire rat. The accumulation rate in the tumor and lung of the treatment group was evaluated over time.

Tumor volumes were measured on days 3, 7, 14, 21, and 28 via ultrasound. Rats were euthanized on day 28. Tumors and major organs (e.g., liver, spleen, lungs, kidneys, bone, blood) were harvested, weighed, and their radioactivity was measured using a gamma counter (WIZARDTM 3′ 1480, PerkinElmer Life Sciences) to yield the percentage of injected dose per gram of tissue (% ID/g). The ID/g was calculated using the dilution standard. Body weight was monitored as an indicator of systemic toxicity. Gross inspection of organs was performed to identify morphological abnormalities. Hematological parameters were not measured in this pilot study.

Evaluation of Antitumor Effect and Side Effects

To observe the treatment effect, the tumor volume of both thighs was measured by ultrasound on days 3, 7, 14, 21, and 28 after the treatment, and growth rate was obtained and growth rate ratio (T/U ratio) between the treated and untreated sides were calculated Eqs. (5) and (6). At the same time point, body weight was also measured, and the weight change rate was calculated according to Eq. (7) to assess transient toxicity.

$$\text= \frac}\,(\text0)}$$

(5)

$$\text/\text=\frac}}$$

(6)

$$\text=\frac}(\text0)}$$

(7)

On day 28, post-treatment dissection of all organs was visually inspected.

Statistical Analysis

Statistical analysis of the results was performed using repeated measures ANOVA, and Tukey’s post-hoc test was subsequently applied. Group differences between the RI-group and C-group at each time point were analyzed using independent sample t-tests, and the p-values were adjusted for multiple comparisons using Bonferroni correction. In vivo tumor growth rate was compared between the groups at each time point using the t-test and Bonferroni correction was applied to correct for the time effect. The statistical significance was set at p < 0.05.