ORIGINAL INVESTIGATION
Photothermal Ablation of Human Lung Cancer by Low-power Near-Infrared Laser and Topical Injection of Indocyanine Green Kentaro Hirohashi, MD,*w Takashi Anayama, MD, PhD,*w Hironobu Wada, MD, PhD,* Takahiro Nakajima, MD, PhD,* Tatsuya Kato, MD, PhD,* Shaf Keshavjee, MD, MSc,* Kazumasa Orihashi, MD, PhD,w and Kazuhiro Yasufuku, MD, PhD*
Summary: The present study was designed to evaluate
the efficacy of photothermal ablation therapy for lung cancer by low-power near-infrared laser and topical injection of indocyanine green (ICG). In vitro study 1: an 808 nm laser with 250 mW was irradiated for 10 minutes using different dilutions of ICG and the temporal thermal effect was monitored. ICG (1 mL of 0.5 g/L) was heated to a temperature of >301C from the base temperature by laser irradiation. In vitro study 2: the cytotoxic effect of hyperthermia on human lung cancer cells was examined in different temperature and time settings. Cell viability was quantified by both an MTS assay and reculturing. Fatal conditions evaluated by reculturing were as follows: thermal treatment at 551C for 5 minutes, 531C for 10 minutes, and 511C for 15 minutes. The MTS assay study suggested that thermal treatment at 591C for 5 minutes and 571C for 20 minutes showed a severe cytotoxic effect. In vivo study: nude mouse subcutaneous NCI-H460 human lung cancer xenograft models were used for the study. Saline or 0.5 g/L of ICG was injected topically into the tumor (n = 3/group). Tumors were irradiated with a laser at 500 mW for 10 minutes. Although the tumor diameter reached 1 cm within 24 days after treatment in all 3 mice using saline/laser, tumor sizes were gradually reduced in all 3 mice using the ICG/laser. In 2 of the 3 mice using ICG/laser, tumors had disappeared macroscopically. The efficacy of the photothermal ablation therapy by low-power near-infrared
Received for publication September 1, 2014; accepted February 9, 2015. From the *Division of Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, Toronto, ON, Canada; and wDepartment of Surgery, Kochi University, Kochi, Japan. The care and handling of the animals were performed in accordance with a protocol approved by the University Health Network’s Animal Resource Centre. Disclosure: K.Y. has received an unrestricted grant for CME and research from Olympus Medical Systems Corp. There is no conflict of interest or other disclosures for all remaining authors. Reprints: Kazuhiro Yasufuku, MD, PhD, Division of Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, 200 Elizabeth St, 9N-957, Toronto, ON, Canada M5G 2C4 (e-mail: [email protected]). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.
J Bronchol Intervent Pulmonol
laser and a topical injection of ICG was clarified using a mouse subcutaneous a lung cancer xenograft model. Key Words: photothermal ablation therapy, near-infrared laser, indocyanine green, lung cancer (J Bronchol Intervent Pulmonol 2015;22:99–106)
L
ung cancer is the leading cause of cancerrelated 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
Volume 22, Number 2, April 2015
www.bronchology.com |
99
Hirohashi et al
J Bronchol Intervent Pulmonol
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 mm), the diameter of the laser fiber would need to be 801C was used as a positive control and a conical tube maintained in a 371C incubator was used as a negative control. After the treatment, we immediately added 20 mL per well of MTS/PMS solution (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI) and returned the tubes to the 371C 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-(4,5dimethylthiazol-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 (mQuant; 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
Copyright
r
2015 Wolters Kluwer Health, Inc. All rights reserved.
J Bronchol Intervent Pulmonol
Volume 22, Number 2, April 2015
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 constanttemperature 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 571C 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 371C 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 mL of tumor suspension containing 1106 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 mL 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 mL 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, Copyright
r
Photothermal Ablation of Human Lung Cancer
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 Analysis
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 301C 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 671C in vivo. The terminal temperature increase of 0.02 g/L ICG was 151C. During the irradiation, the surface temperature of saline was almost unchanged.
FIGURE 1. Indocyanine green (ICG) as a photothermal enhancer. The terminal temperature increase of 1 mL of 0.5 g/L ICG, which was irradiated with the 808 nm laser at 250 mW for 10 minutes, was >301C.
2015 Wolters Kluwer Health, Inc. All rights reserved.
www.bronchology.com |
101
Hirohashi et al
J Bronchol Intervent Pulmonol
Volume 22, Number 2, April 2015
In Vitro Study MTS Assay
The temperature-dependent and timedependent 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 571C after 20 minutes of heating, and at 591C and 611C 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.
FIGURE 3. Responses to the hyperthermic treatment of H460, A549, and MGH-7 cancer cells. The cancer cell viability was inversely proportional to the temperatures and the time durations of hyperthermia.
Reculture After Thermal Treatment
The fatal conditions to which H460 cultured cells were exposed in this study were as follows: thermal treatment at 551C for 5 minutes, 531C for 10 minutes, and 511C for 15 minutes. Representational microscopic findings of therapeutic response to thermal treatment are shown in Figure 4. Recultured cells after thermal treatment at 531C or less for 5 minutes were propagated. However, the number of recultured cells after thermal treatment at Z551C 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. DISCUSSION
FIGURE 2. Time-dependent loss of cell viability at each temperature. The viability of H460 human lung cancer cells was lost at 571C after 20 minutes of heating, at 591C after 5 minutes of heating, and at 611C after 5 minutes of heating; at these points, the graphs reached a plateau.
102 | www.bronchology.com
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
Copyright
r
2015 Wolters Kluwer Health, Inc. All rights reserved.
J Bronchol Intervent Pulmonol
Volume 22, Number 2, April 2015
Photothermal Ablation of Human Lung Cancer
FIGURE 4. Hyperthermic treatment and reculturing. The effect of hyperthermia on H460 cancer cells at 371C, 531C, and 551C for 5 minutes; (A) before treatment; (B) immediately after treatment; and (C) 5 days after treatment. The cells remained immediately after treatment at 551C for 5 minutes. Almost all cells were detached from the bottom of the flask after thermal treatment at 5 days after treatment.
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
FIGURE 5. A, Representative case of in vivo photothermal therapy. The mouse treated with ICG/laser suffered a burn. The burn eventually healed and the tumor completely disappeared, whereas the tumor treated with saline/laser reached 1 cm in diameter at 13 days after treatment. B, Tumor size after laser irradiation. Tumors had completely disappeared in 2 of the 3 mice treated with ICG/laser, whereas tumor sizes were significantly increased and the tumor diameter reached 1 cm in all 3 mice treated with saline/laser. The dashed line represents the timepoints at which the tumor could not be measured because of a scar. *The point at which the mice were killed. Copyright
r
2015 Wolters Kluwer Health, Inc. All rights reserved.
www.bronchology.com |
103
Hirohashi et al
J Bronchol Intervent Pulmonol
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 481C 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 551C for
104 | www.bronchology.com
Volume 22, Number 2, April 2015
5 minutes, 531C for 10 minutes, and 511C for 15 minutes. The MTS assay study suggested that thermal treatment at 591C for 5 minutes and 571C 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
Photothermal Ablation of Human Lung Cancer by Low-power Near-Infrared Laser and Topical Injection of Indocyanine Green Kentaro Hirohashi, MD,*w Takashi Anayama, MD, PhD,*w Hironobu Wada, MD, PhD,* Takahiro Nakajima, MD, PhD,* Tatsuya Kato, MD, PhD,* Shaf Keshavjee, MD, MSc,* Kazumasa Orihashi, MD, PhD,w and Kazuhiro Yasufuku, MD, PhD*
Summary: The present study was designed to evaluate
the efficacy of photothermal ablation therapy for lung cancer by low-power near-infrared laser and topical injection of indocyanine green (ICG). In vitro study 1: an 808 nm laser with 250 mW was irradiated for 10 minutes using different dilutions of ICG and the temporal thermal effect was monitored. ICG (1 mL of 0.5 g/L) was heated to a temperature of >301C from the base temperature by laser irradiation. In vitro study 2: the cytotoxic effect of hyperthermia on human lung cancer cells was examined in different temperature and time settings. Cell viability was quantified by both an MTS assay and reculturing. Fatal conditions evaluated by reculturing were as follows: thermal treatment at 551C for 5 minutes, 531C for 10 minutes, and 511C for 15 minutes. The MTS assay study suggested that thermal treatment at 591C for 5 minutes and 571C for 20 minutes showed a severe cytotoxic effect. In vivo study: nude mouse subcutaneous NCI-H460 human lung cancer xenograft models were used for the study. Saline or 0.5 g/L of ICG was injected topically into the tumor (n = 3/group). Tumors were irradiated with a laser at 500 mW for 10 minutes. Although the tumor diameter reached 1 cm within 24 days after treatment in all 3 mice using saline/laser, tumor sizes were gradually reduced in all 3 mice using the ICG/laser. In 2 of the 3 mice using ICG/laser, tumors had disappeared macroscopically. The efficacy of the photothermal ablation therapy by low-power near-infrared
Received for publication September 1, 2014; accepted February 9, 2015. From the *Division of Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, Toronto, ON, Canada; and wDepartment of Surgery, Kochi University, Kochi, Japan. The care and handling of the animals were performed in accordance with a protocol approved by the University Health Network’s Animal Resource Centre. Disclosure: K.Y. has received an unrestricted grant for CME and research from Olympus Medical Systems Corp. There is no conflict of interest or other disclosures for all remaining authors. Reprints: Kazuhiro Yasufuku, MD, PhD, Division of Thoracic Surgery, Toronto General Hospital, University Health Network, University of Toronto, 200 Elizabeth St, 9N-957, Toronto, ON, Canada M5G 2C4 (e-mail: [email protected]). Copyright r 2015 Wolters Kluwer Health, Inc. All rights reserved.
J Bronchol Intervent Pulmonol
laser and a topical injection of ICG was clarified using a mouse subcutaneous a lung cancer xenograft model. Key Words: photothermal ablation therapy, near-infrared laser, indocyanine green, lung cancer (J Bronchol Intervent Pulmonol 2015;22:99–106)
L
ung cancer is the leading cause of cancerrelated 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
Volume 22, Number 2, April 2015
www.bronchology.com |
99
Hirohashi et al
J Bronchol Intervent Pulmonol
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 mm), the diameter of the laser fiber would need to be 801C was used as a positive control and a conical tube maintained in a 371C incubator was used as a negative control. After the treatment, we immediately added 20 mL per well of MTS/PMS solution (CellTiter 96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, WI) and returned the tubes to the 371C 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-(4,5dimethylthiazol-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 (mQuant; 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
Copyright
r
2015 Wolters Kluwer Health, Inc. All rights reserved.
J Bronchol Intervent Pulmonol
Volume 22, Number 2, April 2015
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 constanttemperature 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 571C 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 371C 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 mL of tumor suspension containing 1106 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 mL 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 mL 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, Copyright
r
Photothermal Ablation of Human Lung Cancer
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 Analysis
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 301C 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 671C in vivo. The terminal temperature increase of 0.02 g/L ICG was 151C. During the irradiation, the surface temperature of saline was almost unchanged.
FIGURE 1. Indocyanine green (ICG) as a photothermal enhancer. The terminal temperature increase of 1 mL of 0.5 g/L ICG, which was irradiated with the 808 nm laser at 250 mW for 10 minutes, was >301C.
2015 Wolters Kluwer Health, Inc. All rights reserved.
www.bronchology.com |
101
Hirohashi et al
J Bronchol Intervent Pulmonol
Volume 22, Number 2, April 2015
In Vitro Study MTS Assay
The temperature-dependent and timedependent 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 571C after 20 minutes of heating, and at 591C and 611C 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.
FIGURE 3. Responses to the hyperthermic treatment of H460, A549, and MGH-7 cancer cells. The cancer cell viability was inversely proportional to the temperatures and the time durations of hyperthermia.
Reculture After Thermal Treatment
The fatal conditions to which H460 cultured cells were exposed in this study were as follows: thermal treatment at 551C for 5 minutes, 531C for 10 minutes, and 511C for 15 minutes. Representational microscopic findings of therapeutic response to thermal treatment are shown in Figure 4. Recultured cells after thermal treatment at 531C or less for 5 minutes were propagated. However, the number of recultured cells after thermal treatment at Z551C 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. DISCUSSION
FIGURE 2. Time-dependent loss of cell viability at each temperature. The viability of H460 human lung cancer cells was lost at 571C after 20 minutes of heating, at 591C after 5 minutes of heating, and at 611C after 5 minutes of heating; at these points, the graphs reached a plateau.
102 | www.bronchology.com
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
Copyright
r
2015 Wolters Kluwer Health, Inc. All rights reserved.
J Bronchol Intervent Pulmonol
Volume 22, Number 2, April 2015
Photothermal Ablation of Human Lung Cancer
FIGURE 4. Hyperthermic treatment and reculturing. The effect of hyperthermia on H460 cancer cells at 371C, 531C, and 551C for 5 minutes; (A) before treatment; (B) immediately after treatment; and (C) 5 days after treatment. The cells remained immediately after treatment at 551C for 5 minutes. Almost all cells were detached from the bottom of the flask after thermal treatment at 5 days after treatment.
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
FIGURE 5. A, Representative case of in vivo photothermal therapy. The mouse treated with ICG/laser suffered a burn. The burn eventually healed and the tumor completely disappeared, whereas the tumor treated with saline/laser reached 1 cm in diameter at 13 days after treatment. B, Tumor size after laser irradiation. Tumors had completely disappeared in 2 of the 3 mice treated with ICG/laser, whereas tumor sizes were significantly increased and the tumor diameter reached 1 cm in all 3 mice treated with saline/laser. The dashed line represents the timepoints at which the tumor could not be measured because of a scar. *The point at which the mice were killed. Copyright
r
2015 Wolters Kluwer Health, Inc. All rights reserved.
www.bronchology.com |
103
Hirohashi et al
J Bronchol Intervent Pulmonol
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 481C 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 551C for
104 | www.bronchology.com
Volume 22, Number 2, April 2015
5 minutes, 531C for 10 minutes, and 511C for 15 minutes. The MTS assay study suggested that thermal treatment at 591C for 5 minutes and 571C 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
