2026 年 49 巻 2 号 p. 310-315
Triple-negative breast cancer (TNBC) is characterized by the absence of hormone receptors and human epidermal growth factor receptor 2 (HER2) expression, rendering hormone therapy and HER2-targeted treatments ineffective. Consequently, conventional chemotherapy remains the primary therapeutic option, despite its severe side effects and poor prognosis. Hybrid liposomes (HL) composed of phospholipids and polyethylene glycol (PEG)-based surfactants have been reported to have therapeutic effects on various cancers without containing anticancer drugs. In this study, we investigated whether photodynamic therapy (PDT) with HL containing indocyanine green (HL/ICG) enhanced the therapeutic effect of HL alone in a mouse model of subcutaneous implantation of triple-negative breast cancer (TNBC) cells in vivo. HL/ICG selectively accumulated in tumors in mice implanted with 4T1-Luc cells, a TNBC cell line. Histological analysis of resected tumor tissues following HL/ICG-mediated PDT revealed a significant increase in cells positive for oxidative stress markers, indicating elevated intracellular oxidative damage. Additionally, a marked presence of apoptotic cells was observed, suggesting that PDT effectively induced programmed cell death in tumor tissues. These results indicate that PDT with HL/ICG induces oxidative stress-mediated apoptosis in tumors derived from 4T1-Luc cells and significantly enhances the therapeutic efficacy of HL alone in vivo, highlighting its potential as a promising strategy for the treatment of TNBC.
Breast cancer is the most common malignancy among Japanese women, with over 90000 new cases annually. Treatment strategies depend on tumor invasion and molecular subtype, but prognosis varies due to heterogeneity.1,2) Triple-negative breast cancer (TNBC), accounting for approx. 15% of cases3) lacks estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2), rendering hormone and targeted therapies ineffective and leading to poor outcomes.
Photodynamic therapy (PDT) generates reactive oxygen species (ROS) via light activation of photosensitizers.4) Although talaporfin sodium is clinically used, its non-specific accumulation causes photosensitivity, highlighting the need for tumor-selective agents. There is active development of new PDT agents with high tumor affinity, as well as ongoing research on nanoparticle-based drug delivery systems.5,6) Hybrid liposomes (HL)7) exhibit antitumor activity without conventional drugs by inducing cancer cell fusion, caspase activation, and apoptosis,8–24) with favorable safety and clinical benefits reported.24)
Recent in vitro studies demonstrated that PDT using HL/ICG, which incorporates indocyanine green (ICG), achieved greater inhibition of TNBC cells compared with HL alone.25) Indocyanine green (ICG) was chosen for this study because it is Food and Drug Administration (FDA)-approved, highly biocompatible, and exhibits strong absorption in the near-infrared (NIR) region. This spectral property lies within the tissue optical window and permits deeper penetration of light into biological tissues. Furthermore, ICG is suitable for photodynamic therapy (PDT) because irradiation induces the formation of reactive oxygen species (ROS), particularly singlet oxygen. However, its in vivo efficacy remains unclear.
The objective of this study was to confirm that HL/ICG-based PDT enhances therapeutic efficacy beyond that achieved by HL alone in a TNBC mouse model.
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was purchased from NOF Corporation (Tokyo, Japan), Polyoxyethylene (25) dodecyl ether (C12(EO)25) from Nikko Chemicals Co., Ltd. (Tokyo, Japan), and ICG from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other reagents were of reagent grade.
Preparation of HL and HL/ICGHL were prepared by mixing DMPC and C12(EO)25 (90 : 10 mol%) in 5% glucose solution, followed by ultrasonic irradiation at 45°C for 1 min/mL. The dispersion was sterilized by filtration (0.20 μm) and stored at 25°C. HL/ICG were prepared similarly with 1 mol% ICG and stored at 4°C.
Cell CultureMouse TNBC 4T1-Luc cells (RIKEN Cell Bank, Japan) were cultured in RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum at 37°C in 5% CO2.
Observation of HL/ICG Accumulation in Tumors Using the TNBC Mouse ModelFemale BALB/c-R/J mice26) (8–12 weeks old) with severe immunodeficiency were used. Mice were bred in-house and provided by Professor Seiji Okada (Kumamoto University). All procedures involving animals were conducted in accordance with the Guidelines for Animal Experimentation under Japanese law and were approved by the Animal Experimentation Committee of Sojo University. 4T1-Luc cells (1 × 106) were injected subcutaneously, and tumor volume was calculated as major axis × (minor axis)2 × 0.5. HL/ICG (DMPC 203 mg/kg) was administered via tail vein when tumors exceeded 700 mm3. Fluorescence imaging was performed using an in vivo imaging system (AEQUORIA; excitation 775/50 nm, emission 845/55 nm, Hamamatsu Photonics K.K., Shizuoka, Japan). Tumors were harvested 24 h post-injection for observation.
PDT Using HL/ICG in a TNBC Mouse ModelOn day 11 post-injection, mice were stratified and randomly assigned to experimental groups (n = 8) based on tumor volume. HL/ICG (DMPC dose: 203 mg/kg) was administered via tail vein injection. After 24 h, tumors were irradiated with an 808 nm laser light source (100 mW/cm2) for 1 min to activate ICG. This administration and irradiation protocol was repeated for 2 weeks. Therapeutic efficacy was evaluated based on tumor volume and tumor weight.
Oxidative Stress in Subcutaneous Tumors after HL/ICG-PDTAfter treatment, subcutaneous tumors were excised and fixed in Bouin’s solution. Oxidative stress was evaluated by immunohistochemical detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG). During PDT with ICG, singlet oxygen (1O2), ROS, is generated. Singlet oxygen preferentially oxidizes guanine (G) among the nucleobases, forming 8-oxo-7,8-dihydroguanine (8-oxoG) and, in DNA, its lesion marker 8-OHdG.27) Tumor sections were deparaffinized, treated with 1% hydrogen peroxide in methanol to block endogenous peroxidase, and blocked with skim milk. Sections were incubated with a monoclonal anti-8-OHdG antibody (Ab), followed by a biotin-labeled secondary Ab. Staining was performed using 3,3′-diaminobenzidine and counterstained with hematoxylin. Stained sections were examined under an optical microscope (SMZ745T, Nikon Corporation, Tokyo, Japan).
Apoptosis in Subcutaneous Tumors after HL/ICG-PDTAfter completion of the treatment experiment, subcutaneous tumors were excised and fixed in 10% neutral buffered formalin. Tumor tissues were paraffin-embedded and sectioned for histological analysis. Apoptotic cells were detected using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (S7100, Merck Millipore, Darmstadt, Germany), following the manufacturer’s protocol for the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Stained sections were observed using an optical microscope.
Statistical AnalysisAll data are presented as mean ± standard deviation (S.D.). Statistical significance was determined using Student’s t-test, with p < 0.05 considered statistically significant.
HL/ICG was intravenously administered to mice bearing subcutaneous 4T1-Luc tumors, and its tumor accumulation was assessed using in vivo fluorescence imaging. As shown in Fig. 1, 24 h after administration, ICG-derived fluorescence had nearly disappeared in nanoparticles composed of DMPC/ICG and C12(EO)25/ICG, as well as in free ICG. By contrast, strong fluorescence signals persisted at the tumor site in mice treated with HL/ICG. Excised tumor tissues from the HL/ICG group also exhibited significantly stronger ICG fluorescence compared with other treatment groups. Previous studies reported that HL/ICG maintains a stable membrane structure for over 1 month and has a membrane size (<100 nm)25) that enables evasion of clearance by the reticuloendothelial system (RES).28) Centrifugal ultrafiltration of HL/ICG using a 50 kDa membrane produced no visible ICG coloration in the filtrate, and fluorescence attributable to free ICG was not detected when the filtrate was analyzed with a fluorescence spectrophotometer (unpublished data). These findings suggest that most of the ICG is encapsulated within the nanoparticles. HL selectively fuses with cancer cell membranes,16) which exhibit higher fluidity than normal cells, and accumulates in tumor tissues via the enhanced permeability and retention (EPR) effect.29) These synergistic properties likely contributed to the pronounced accumulation of HL/ICG at tumor sites.
n = 2 animals per group. Scale bar = 1 cm. DMPC dose: 203.4 mg/kg; C12(EO)25 dose: 43.3 mg/kg; ICG dose: 2.6 mg/kg.
To assess the therapeutic efficacy of HL/ICG-mediated PDT, treatment experiments were performed in mice bearing subcutaneous 4T1-Luc tumors. Control groups included HL alone, HL-mediated PDT (HL-PDT), and HL/ICG without PDT. Tumor volume progression during the treatment period is shown in Fig. 2. Compared with the control group, all treatment groups exhibited suppressed tumor growth, with HL/ICG-PDT producing the most significant inhibition of tumor volume increase.
Data are presented as mean ± S.D. n = 8 animals per group. Scale bar = 1 cm. DMPC dose: 203.4 mg/kg; C12(EO)25 dose: 43.3 mg/kg; ICG dose: 2.6 mg/kg. Two-tailed Student’s t-tests were performed to compare the indicated groups. The resulting p-values were as follows: (a) Control vs. HL, p = 1.38 × 10−5; (b) Control vs. HL-PDT, p = 1.50 × 10−5; (c) Control vs. HL/ICG, p = 8.19 × 10−6; (d) Control vs. HL/ICG-PDT, p = 4.04 × 10−7; (e) HL vs. HL/ICG-PDT, p = 2.72 × 10−4; (f) HL-PDT vs. HL/ICG-PDT, p = 3.66 × 10−4; (g) HL/ICG vs. HL/ICG-PDT, p = 7.22 × 10−4.
Tumor weights and representative images of resected tumors are shown in Fig. 3. As illustrated in Fig. 3A, tumor weights decreased in all treatment groups compared with the controls, with HL/ICG-PDT achieving the greatest reduction. The photographs in Fig. 3B further demonstrate visibly smaller tumors in the HL/ICG-PDT group. These findings indicate that HL/ICG efficiently accumulates in tumor tissue and exerts a potent antitumor effect when activated by PDT.
n = 8 animals per group. (A) Tumor weights in 4T1-Luc subcutaneous mouse model treated with HL/ICG-PDT. Data are presented as mean ± S.D. Two-tailed Student’s t-tests were performed to compare the indicated groups. The resulting p-values were as follows: (a) Control vs. HL, p = 9.31 × 10−5; (b) Control vs. HL-PDT, p = 8.97 × 10−6; (c) Control vs. HL/ICG, p = 9.37 × 10−11; (d) Control vs. HL/ICG-PDT, p = 7.67 × 10−12; (e) HL vs. HL/ICG-PDT, p = 6.19 × 10−3; (f) HL-PDT vs. HL/ICG-PDT, p = 2.69 × 10−5; (g) HL/ICG vs. HL/ICG-PDT, p = 5.03 × 10−6. (B) Representative photographs of tumors from the 4T1-Luc subcutaneous mouse model treated with HL/ICG-PDT. Scale bar = 1 cm.
Taken together, these results suggest that HL combined with ICG for PDT represents a promising therapeutic strategy, offering synergistic antitumor effects alongside tumor-selective delivery and the inherent therapeutic potential of HL.
HL/ICG-PDT Induces Robust Tumor-Selective Oxidative StressImmunohistochemical staining for 8-OHdG in subcutaneous tumors after HL/ICG-PDT is shown in Fig. 4. No oxidative stress-positive cells were detected in the Control, HL, HL-PDT, or HL/ICG groups. By contrast, numerous 8-OHdG-positive cells were observed in the HL/ICG-PDT group, indicating effective induction of oxidative stress via ROS generation in 4T1-Luc cells. The absence of 8-OHdG-positive cells in other groups confirms that both ICG-loaded HL nanoparticles and light irradiation are essential for efficient ROS production.
n = 8 animals per group. Scale bar = 200 μm. Brown indicates 8-OHdG-positive cells.
These findings suggest that the tumor-suppressive effect of HL/ICG-PDT is not attributable to light exposure or drug administration alone, but rather to photodynamically induced oxidative stress causing cellular damage. Since 8-OHdG is a well-established marker of DNA oxidative damage, HL/ICG-PDT likely induces nucleic acid-level injury in tumor cells.
Overall, these results demonstrate that HL/ICG-PDT is a promising therapeutic strategy combining tumor-selective HL accumulation with light-triggered oxidative stress.
Robust Apoptotic Response in Tumors Following HL/ICG-PDTApoptosis induction in subcutaneous tumors by HL/ICG-PDT was assessed using TUNEL staining (Fig. 5). Numerous TUNEL-positive cells were observed in the HL, HL-PDT, HL/ICG, and HL/ICG-PDT groups, indicating apoptosis under these treatment conditions. Notably, the HL/ICG-PDT group showed clear apoptotic expression alongside oxidative stress, suggesting apoptosis as a key mechanism of tumor cell death induced by PDT. By contrast, no TUNEL-positive cells appeared in the control group. The presence of apoptotic cells in HL monotherapy, HL-PDT, and HL/ICG groups implies that HL accumulation and ICG photosensitivity contribute to apoptosis induction. The possibility of non-apoptotic cell death in HL/ICG PDT remains to be elucidated. We also evaluated ferroptosis as a potential non-apoptotic mode of cell death. However, in an in vitro lipid peroxidation assay, HL/ICG-PDT did not enhance lipid peroxidation (unpublished data), suggesting that ferroptosis is unlikely. These data support apoptosis as a principal mechanism for HL/ICG-PDT, and further mechanistic studies are warranted to assess non-apoptotic pathways.
n = 8 animals per group. Scale bar = 200 μm. Arrows indicate apoptotic cells.
This study is the first to show that HL/ICG-PDT exerts a significant therapeutic effect in a TNBC mouse model. HL/ICG-PDT combines light-induced cytotoxicity with tumor-selective drug delivery through HL’s selective fusion with cancer cell membranes and the enhanced EPR effect in the tumor microenvironment. This dual-targeting mechanism differentiates HL/ICG from conventional nanocarriers, representing a new paradigm in cancer therapy. These findings highlight HL/ICG-PDT as a promising strategy, warranting further optimization and mechanistic studies for clinical translation.
This research was supported in part by a Grant-in-Aid for Scientific Research (No. 22K20516) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors declare no conflict of interest.
