Lung cancer is the leading cause of cancer-related mortality worldwide, with over 1.3 million deaths per year.1 Results of CT screening studies for lung cancer indicate that we will see increasing numbers of patients who will be diagnosed with early peripheral lung cancers.2 Surgical resection by lobectomy with systematic hilar and mediastinal lymph node dissection offers the best opportunity for cure and is the gold standard of treatment for early-stage non–small cell lung cancer (NSCLC). Unfortunately, compromised cardiopulmonary function or medical comorbidities may make patients unsuitable candidates for pulmonary resection.3 This necessitates the development of alternative treatments. Percutaneous radiofrequency ablation (RFA) therapy has been applied to the tumors in various locations, including the liver,4,5 kidney,6 bone,7 and lung.8 However, percutaneous RFA therapy for lung cancer has a risk of pneumothorax, hemothorax, and pleural effusion.9,10 Although cryoablation causes cellular damage through a complex combination of cellular events during tissue freezing and thawing, percutaneous cryoablation also has the same problems as RFA by a percutaneous approach.11 Bronchoscopic photodynamic therapy (PDT) is being utilized to treat NSCLC. When photosensitizers are exposed to light of a specific wavelength, they produce singlet oxygen instead of heat-mediated cellular cytotoxicity. PDT has clinical indications in selected patients with early-stage central endobronchial tumors for radical cure and endobronchial luminal obstruction to improve respiratory function.12,13 Transbronchial laser ablation with a high output power has also been applied for the treatment of endobronchial tumors14,15; however, peripheral lung tumors cannot be treated by the existing lasers because of the diameter of the laser fiber. To access the peripheral lung, thinner laser fibers are required. For example, if we were to use an ultrathin bronchoscope (BF-XP 160F, outer diameter, 2.8 mm, forceps channel, 1.2 mm; Olympus, Tokyo, Japan) with a 21 G needle catheter (inner diameter, 514 μm), the diameter of the laser fiber would need to be <500 μm. Thin laser fibers are not suitable for high-powered ablation because of the limitation of the output power. To develop a flexible ultrathin bronchoscopy-guided laser ablation treatment using the combination of a low-powered laser and a topical injection of photosensitizer to enhance the laser effect, we focused on a near-infrared (NIR) laser and indocyanine green (ICG). NIR laser has high tissue penetration because the spectral range is referred to as the “optical window” of tissues owing to the lack of efficient absorbers in this spectral range.16 ICG is known to be a Food and Drug Administration (FDA)-approved drug and can absorb NIR laser and convert the absorbed light energy into heat inside the ICG molecule.17 The aim of the present study was to evaluate the efficacy and the feasibility of the photothermal therapy by the combination of a low-power NIR laser and a topical injection of ICG.
MATERIALS AND METHODS
Photothermal Enhancement of ICG
An 808 nm laser with 250 mW output was irradiated using different dilutions of ICG (Diagnogreen; Daiichi Sankyo, Tokyo, Japan) and the temporal thermal effect was monitored. Different dilute concentrations of ICG, 0.5 and 0.02 g/L, and saline were prepared. About 1 mL of these solutions in a conical tube were irradiated with a laser from a distance of 1 cm for 10 minutes. The distance between the tip of the laser fiber and the conical tube was adjusted to the spot size, which can be smaller than the diameter of the conical tube. A NIR laser diode with a wavelength of 808 nm (LRD-0808-PFR-02000-01; Laserglow Technologies, Toronto, ON, Canada) was used in this study. A laser power meter console (PM100D; Thorlabs, Newton, NJ) was used in this study. During the irradiation, the surface temperatures of the solutions were monitored using a thermal camera (Thermo Shot F30S; Nippon Avionics Co., Tokyo, Japan).
In Vitro Study
Cultured Lung Cancer Cell Preparation
The human NSCLC cell lines, including A549 (adenocarcinoma) and NCI-H460 (large cell carcinoma), were provided by Dr Brian Wilson, Department of Biophysics, Faculty of Medicine, University of Toronto. MGH-7 (squamous cell carcinoma) was provided by Dr Ming Tsao of the Department of Pathology, Princess Margaret Hospital. Tumor cells were cultured with cell culture medium in humidified incubators at 37°C with 5% CO2. Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies Inc., Burlington, ON, Canada) was used for A549 and Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies Inc.) was used for H460 and MGH-7.
The Hyperthermic Protocol Quantified by the MTS Assay
A 100 µL of medium containing 3.5×104 of human lung cancer cells per tube was placed in conical tubes. A constant-temperature shaking water bath (SWB25; Thermo Fisher Scientific Inc., Waltham, MA) was used as the heat source. The conical tubes were submerged in the water bath under hyperthermic conditions with temperatures and time durations in the range of 49 to 61°C for 5 to 120 minutes. A conical tube heated to >80°C was used as a positive control and a conical tube maintained in a 37°C incubator was used as a negative control. After the treatment, we immediately added 20 µL per well of MTS/PMS solution (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI) and returned the tubes to the 37°C incubator. One hour later, the cell suspension was taken and placed in 96-well plates (Sarstedt Inc., Newton, NC). Live cells were measured using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay according to the manufacturer’s instructions. The absorbance at 490 nm was recorded using an ELISA plate reader (µQuant; BioTek Instruments Inc., Winooski, VT). The absorbance at 630 nm was used as a reference wavelength to eliminate background contributed by cell debris, fingerprints, and other nonspecific absorbance. The mean absorbance from wells containing cell-free medium was used as the baseline and was deducted from the absorbance of other cell-containing wells.18 All samples were assayed in quadruplicate.
The Hyperthermic Treatment and Reculturing
Cultures were maintained in a 5% CO2 incubator in 25 cm2 culture flasks (BD Falcon, Franklin Lakes, NJ) to prevent contamination from leakage during the heating process. The microscopic images of cultured cells were digitally recorded before treatment. A constant-temperature shaking water bath (SWB25; Thermo Fisher Scientific Inc.) was used as the heat source. Flasks were submerged in the water bath under the hyperthermic conditions with temperatures and time durations in the range of 51 to 57°C for 5 to 20 minutes. After the treatment, the flasks were immediately removed and the microscopic images were recorded; then, the flasks were returned to the 37°C incubator. Observations were made at 3 different sites within each flask every day for 7 days after the thermal treatment by taking digital microscopic images of the cultured cells.
In Vivo Study
ICG Laser Hyperthermia Using a Nude Mouse Subcutaneous Human Lung Cancer Xenograft Model
Experimental animals: human lung cancer cells (H460 cancer cell line) were grown in male nude mice by injecting 100 µL of tumor suspension containing 1×106 cells with growth factor–reduced Matrigel (BD Biosciences, Mississauga, ON, Canada) into the right flank under general anesthesia with 2% (vol/vol) isoflurane inhalation. After the tumor reached 5 mm in diameter, the experiment was conducted.
Photothermal therapy: 6 male nude mice with human lung cancer using a xenograft model were used for the study. Three mice were treated by laser therapy with a topical injection of 50 µL of ICG at a concentration of 0.5 g/L before irradiation. Another 3 mice were treated by laser therapy with a topical injection of 50 µL of saline before irradiation. Tumors were irradiated with a laser with 500 mW output at 808 nm with a 10 mm spot size for 10 minutes. Surface temperatures of the tumors were monitored by a thermal camera. After the treatment, the images of the tumors were digitally recorded every day until euthanization and the tumor areas were calculated using Image J software (version 1.46r). When the tumor diameter reached 1 cm, the mice were killed and the tumor was harvested. If the tumor diameter did not reach 1 cm, the mouse was killed 60 days after treatment. The care and handling of the animals were carried out in accordance with a protocol approved by the University Health Network’s Animal Resource Centre.
Statistical analyses were carried out using GraphPad Prism version 6.0 (GraphPad Software Inc., San Diego, CA). Survivals were estimated by the Kaplan-Meier analysis. The Log-rank test was used to identify differences between the ICG/laser and the saline/laser groups. A P-value <0.05 was considered to indicate statistical significance.
Photothermal Enhancement of ICG
ICG (1 mL of 0.5 g/L) was heated 20°C for 1 minute and finally heated to a temperature of >30°C from the base temperature by irradiation of the 808 nm laser with 250 mW output (Fig. 1). Considering the body temperature, it is equivalent to 67°C in vivo. The terminal temperature increase of 0.02 g/L ICG was 15°C. During the irradiation, the surface temperature of saline was almost unchanged.
In Vitro Study
The temperature-dependent and time-dependent cytotoxic effects of thermal therapy were clarified. The results of this study are shown in Figure 2. Error bars indicate results over a range of 4 samples for each data point. The viability of the H460 human lung cancer cells was lost at 57°C after 20 minutes of heating, and at 59°C and 61°C after 5 minutes of heating; at this point, the graph reached a plateau. The responses to the hyperthermic treatment of 3 cancer cell lines are shown in Figure 3. The cancer cell viability was inversely proportional to the temperatures and the time durations of hyperthermia despite the kind of cancer cell line, although there was variability in cell viability.
Reculture After Thermal Treatment
The fatal conditions to which H460 cultured cells were exposed in this study were as follows: thermal treatment at 55°C for 5 minutes, 53°C for 10 minutes, and 51°C for 15 minutes. Representational microscopic findings of therapeutic response to thermal treatment are shown in Figure 4. Recultured cells after thermal treatment at 53°C or less for 5 minutes were propagated. However, the number of recultured cells after thermal treatment at ≥55°C for 5 minutes was reduced and almost all cells were detached from the bottom of the flasks. The results of the A549 and MGH-7 cell lines were the same as those of the H460 cell line.
In Vivo Study
In all 3 mice in the laser therapy with saline group, tumor sizes were significantly increased and the tumor diameter reached 1 cm within 24 days after treatment (Fig. 5). However, in all 3 mice in the laser therapy within ICG group, tumor sizes were gradually reduced. In 2 of the 3 mice, tumors had disappeared macroscopically. A significant difference was found between the survival rates of ICG/laser and saline/laser groups (P<0.05). One mouse treated by laser with ICG suffered a burn, and its tumor could not be measured because of scarring until 24 days after treatment. The burn eventually healed without treatment and the tumor had completely disappeared.
In the present study, we clarified the required conditions for photothermal ablation therapy of human lung cancer using relatively low-output power laser to apply this technique for future bronchoscopic treatment. We confirmed the efficacy of the low-power laser ablation therapy with a topical injection of 0.5 g/L ICG as a photosensitizer to compensate for the low-output power laser. To clarify the required conditions for the photothermal treatment, human lung cancer cells were used for the in vitro study and the level of cytotoxicity at each temperature was demonstrated. On the basis of the result of the in vitro study, we confirmed the efficacy of the treatment using a nude mouse subcutaneous human lung cancer xenograft in vivo model. For the in vivo setting, we needed to take several factors into consideration. For example, normal tissue located between the skin and tumor may absorb light and decrease photothermal effect. This is called “heat sink effect” caused by cooling effect of surrounding vascular structures,”19,20 and normal tissue that is located between the skin and the tumor that may absorb the laser light and decrease the photothermal effect. Therefore, we showed the efficacy of the in vivo study using a 500 mW output laser instead of a 250 mW output laser used in the in vitro study. As a result, the surface temperature of the tumor, which had a diameter of 5 mm, was heated to the temperature of 48°C by the photothermal ablation therapy with 500 mW for 10 minutes and in 2 of the 3 mice, the tumor had completely disappeared during the 60-day follow-up. Although the in vivo study was carried out on only 3 animals in each group, the difference between the survival rates of ICG/laser and saline/laser was statistically significant (P<0.05). From the obvious result of the study and from the point of view of the animal’s welfare, we decided not to carry out an additional study. The 500 mW output power laser is a significantly lower powered laser when compared with currently used lasers in the clinic and is classified as class 3B.21 For example, the output power of a continuous laser for an endobronchial tumor is 20 to 100 W,22 that of a pulse laser for an endobronchial tumor is 25 to 80 W,23 and that of a laser for varicose vein is 10 to 14 W.24 This significantly lower powered laser compared with these currently used clinical lasers may potentially be useful for photothermal ablation therapy.
We clarified that 0.5 g/L was an appropriate concentration of ICG for the thermal treatment in the in vitro study of “photothermal enhancement of ICG.” The relation between the temperature and cytotoxicity using human lung cancer cells was also clarified in an in vitro study of “hyperthermia on human lung cancer cultured cells.” To start, we attempted an preliminary in vitro experiment using the actual ICG/laser. However, it was difficult to control the local temperature by laser irradiation because of heat accumulation and the increasing local temperature during laser irradiation. Hence, we carried out the in vitro study using a constant-temperature water bath as a heat source instead of the actual ICG/laser. This was done to gain more accurate control of the local temperature. The result of the reculture study showed that the fatal conditions to the human lung cancer cells were as follows: thermal treatment at 55°C for 5 minutes, 53°C for 10 minutes, and 51°C for 15 minutes. The MTS assay study suggested that thermal treatment at 59°C for 5 minutes and 57°C for 20 minutes yielded a severe cytotoxic effect. The discrepancy in the results of the 2 assays was probably because of a difference in the method of evaluation. The enzyme activity was evaluated in the MTS assay and the total cell viability was evaluated in reculturing. To maintain total cell viability, both the enzyme activity and the other intracellular activities have to be preserved. Therefore the reculturing study showed a severe cytotoxic effect with lower temperature settings compared with the MTS assay study. When considering the results of the reculture study and the MTS assay study, if the cell viability measured by the MTS assay is <0.5, it may potentially indicate “cell death.”
Feng et al25 reported in vitro data from an experiment conducted with human prostate cancer cells. In that study, it was shown that thermal treatment at 54°C for 4 minutes represented a fatal condition. The method of our reculture study is relatively similar to that of Feng and colleagues and the relation between temperature and cytotoxicity was shown. They started the thermal treatment immediately after exchanging the medium set at the intended temperature to accurately evaluate the cytotoxic effect. In contrast, the method in our study was different from Feng's study where we started heating the medium from 37°C. Although we cannot perform a simple comparison between these results because of the difference in the methodology, the results suggest that even though the types of cancer cells are different, there was no significant difference in the cytotoxicity against thermal treatment.
A laser at 500 mW with a 10 mm spot (0.75 cm2) was irradiated to the tumor in the nude mouse subcutaneous human lung cancer xenograft model. We confirmed that laser ablation therapy at 500 mW/0.75 cm2=666.6 mW/cm2 with a topical injection of 0.5 g/L ICG was effective for treatment of the mouse subcutaneous tumor with human lung cancer cells.
Our current method shows cellular cytotoxicity by heat energy. However, PDT is being utilized to treat NSCLC by exposing light of a specific wavelength; thus, photosensitizers produce singlet oxygen instead of heat that mediates cellular cytotoxicity. Although PDT has been readily available for the treatment of centrally located lung cancer, it has not been easily adopted in clinical practice. Because of the side effects of photosensitizers, post-PDT patients must adopt sun-protection measures, with avoidance of full-spectrum light for 6 weeks. In contrast, using ICG as a photosensitizer during PDT can avoid light sensitivity and will not limit patients to sun exposure even immediately after the treatment.
There are several limitations to this study. First, in the current study, ICG was administered using a topical injection through the skin into the tumor site. The mouse subcutaneous tumor was then irradiated with the laser from outside the body. To translate this into the clinical treatment of lung cancer, we need to consider how to approach the target lesion. In terms of central lung cancer, we can easily do this using a bronchoscopy and can topically administer ICG under bronchoscopy guidance. This technique, using endoscopy, may potentially be applied to malignant lesions in other organs, such as the esophagus, stomach, and colon. To translate this into the treatment of peripheral lung cancer, one will require precise access to the peripheral lung tumor, which can be achieved using recently developed navigational bronchoscopy.26,27 The second limitation is the limited tissue penetration of NIR light. Kim et al28 have described that NIR light penetrates human tissues to a depth of 10 mm. Tanaka et al29 have also reported that 10 mm is the maximum limit of depth for detecting NIR light. However, taking the thermal conductivity in the ICG-injected tumor into consideration, tumors larger than 1 cm may be treated with this approach. The third limitation is the procedure time. In our experiments, we used a minimally thin optical fiber (300 μm) that could fit in the accessory channel of the smallest ultrathin bronchoscope (1.2 mm ID); thus, it took 10 to 20 minutes. If we increase the diameter of the fiber, we can potentially decrease the duration of photothermal ablation treatment.
In conclusion, we clarified the efficacy and feasibility of photothermal ablation therapy by a low-power NIR laser and a topical injection of ICG using a mouse subcutaneous human lung cancer xenograft model. This system may potentially be applied for transbronchial laser ablation of peripheral lung cancers.
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