Soft tissue sarcoma (STS) develops in a variety of mesenchymal tissues [20]. In general, local recurrence is common following conservative excision. Therefore, other therapies including radiation therapy and chemotherapy are used to prevent recurrence after performing a wide first excision [13,21,22,26,27]. Radiation therapy plays a particularly important role in the management of STS [9]. However, this type of therapy can only be performed in restricted facilities. Some patients cannot undergo radiation therapy due to financial difficulties or because the number of available facilities is limited. Therefore, it is necessary to develop novel and effective techniques to treat STS.

Indocyanine green (ICG) induces heat generation in response to light at a wavelength of 808 nm [5,6,7] and oxygen radicals upon exposure to light at wavelengths of 600~800 nm [18]. Based on these evidences, we developed a novel cancer therapy using the properties of ICG and a broadband light source instead of a diode laser to establish a combination of photodynamic therapy and hyperthermia. This method is known as photodynamic hyperthermia (PHT) and can be combined with local chemotherapy to create a triple therapy strategy (photodynamic therapy, hyperthermia therapy, and chemotherapy) called photodynamic hyperthermal chemotherapy (PHCT). To date, no studies on the application of PHT and PHCT for the treatment of STS have been published. This is the first report of the use of PHCT to treat STS.

Table 2 presents a summary of the study results. PHCT was performed three to 21 times. The treatment frequency depended on the wishes of the owner. No severe side effects, including severe skin burns, necrosis, or rupture of skin sutures, were observed in any of the animals although skin redness and minor skin burns occurred. The overall canine survival time (ST) except for 1 case of amputation (case C06) ranged from 225 to 1,901 days (median survival time: 767 days). In seven out of 10 dogs (70.0%), no recurrence was observed during the follow-up periods ranging from 238 to 1,901 days. In five of these seven dogs, no recurrence was observed after more than 2 years. The remaining three dogs (two with undifferentiated soft tissue sarcomas and one with liposarcoma) experienced recurrence or metastasis over intervals ranging from 72 to 162 days after surgery. One case with liposarcoma (case C06) experienced local recurrence occurring within 1 month after the first surgery. In this animal, local recurrence was also observed 72 days after the first round of PHCT following the second surgery. The disease-free time (DFT) was prolonged by the use of PHCT. However, amputation was performed in accordance with the owner's desire, and the dog is currently alive. One dog (case C08) of the remaining three dogs died due to tumor progression. In case 08, metastasis to the sublumbar lymph nodes was observed. In that animal, no local recurrence was found. In case C09, local recurrence was observed 162 days after the first round of PHCT. PHCT was repeated for this animal five times at intervals of 1 to 2 weeks. Thereafter, the dog was only followed up. Sixty-seven days after the fifth round of PHCT, local recurrence was observed. The dog eventually died of cardiac failure 225 days after the first round of PHCT.

The overall feline ST, excluding two cases of amputation (cases F04 and F06), ranged from 383 to 1,521 days (median survival time: 1,344 days). In three out of the six cats (50.0%), no recurrence was observed over the follow-up periods ranging from 1,344 to 1,521 days. The remaining three cats (one case of fibrosarcoma, one case of rhabdomyosarcoma, and one case of undifferentiated STS) experienced recurrence over intervals ranging from 20 to 175 days after surgery. In two (cases F04 and F06) out of the remaining three cases, amputation was performed on one cat (case F04) that is currently alive and one cat (case F06) that died due to unknown cause. The remaining cat died due to tumor progression. In case F02 for which no surgery was performed, we periodically assessed tumor status using fine needle aspiration (FNA). No living tumor cells were observed by FNA during the eighth round of PHCT. We diagnosed the tumor as degenerative following PHCT, and the degenerative tumor tissue was removed using an ultrasonic aspiration device (Sono Cure; Tokyo Iken, Japan). Thereafter, PHCT was performed for 3 months. This cat had no recurrence for about 5 years.

Malignant STSs rarely metastasize; however, they are locally invasive [3]. If the surgical margin is insufficient, the rate of recurrence is 10 times of that when the surgical margin is sufficient [20]. Therefore, complete resection with a sufficient surgical margin is necessary. In general, adjuvant therapies including radiation therapy and chemotherapy are administered to cases of insufficient surgical margins [13,21,22,27]. In particular, radiation therapy plays an important role in the management of STS. In the present study, the surgical margins were insufficient in all cases. However, 70.0% (7/10) of the dogs and 50.0% (3/6) of the cats did not experience recurrence over follow-up periods ranging from 238 to 1,901 days. These results suggest that PHCT decreases the risk of recurrence.

As an alternative to radiation therapy, we developed a new therapy, PHT, by combining Photodynamic therapy (PDT) and hyperthermia with ICG using a broadband light source. Furthermore, we administered PHT in combination with local chemotherapy and designated this technique "PHCT". No previous studies have demonstrated the efficacy of the combination of ICG and use of a broadband light source in cancer therapy.

It has been suggested that fibrosarcomas, malignant schwannomas, and hemangiopericytomas are sensitive to PHCT considering the relationship between tumor type and PHCT efficacy. The local control rate of fibrosarcoma, malignant schwannoma, and hemangiopericytoma was 100% in the present study. However, PHCT is not effective for other tumor types including liposarcoma, rhabdomyosarcoma, and undifferentiated STSs occurring in the limbs. In these cases, the tumors recurred ranging from 20 to 175 days after the first PHCT. These results suggest that the effects of PHCT might differ depending on tumor type. Further investigation into this possibility is necessary. In the present study, no relationship between treatment frequency and recurrence was observed. In one case (case C09), local recurrence was observed 63 days after the final round of PHCT. This phenomenon suggests that it is important to periodically continue treatment with PHCT. Additionally, no relationship was observed between the use of anti-cancer drugs and outcomes.

In two out of three canine and two out of three feline recurrence cases, the tumor cells had infiltrated deeply. We speculate that the light used for PHCT did not reach the tumor cells located in deep tissues. In a preliminary experiment, we found that the temperature at a depth of 2 cm from the skin surface did not reach 40℃ (data not shown), indicating that PHCT is not effective for treating tissues beyond this depth. Additionally, the ICG solution was not equally distributed. These drawbacks will be investigated in the future.

Hyperthermia is not a common treatment in veterinary medicine because the required device is expensive and few fundamental data related to the efficacy of hyperthermia are available. On the other hand, there are reports of PDT being used to treat spontaneous tumors in canines and felines, and the efficiency of this technique has been recognized [11,28,29,30,33,34,35]. However, the photosensitizer for PDT is very expensive and a special diode laser is needed. Therefore, PDT is also rarely administered by veterinarians. ICG was developed as a drug to promote liver and bile duct function, and it has been used medically since 1956. This drug is safe for both human and animals [8]. In particular, ICG is widely used in ophthalmology for hyperthermia therapy with an 808-nm diode laser to treat chorioretinopathy [12]. ICG is also used for sentinel biopsies [17,19].

Some reports have suggested that ICG induces the formation of oxygen radicals [1,2,4,10,23]. Most of these findings are from in vitro studies of different human cell lines. Bozkulak et al. [4] reported that ICG with near-infrared light (809 nm, 60 mW/cm², 24 J/cm²) is very effective for eliminating human breast cancer cells. In this study, the temperature of the medium was controlled to ensure that it remained at 37℃. The authors concluded that ICG acts as a new photosensitizer. Hirano et al. [18] was the first to demonstrate that ICG induces the formation of oxygen radicals in response to irradiation with light at a wavelength of 600~800 nm. The concentration of oxygen radicals produced by ICG dissolved in ethanol is similar to that generated by other photosensitizers. Oxygen radicals are formed even if ICG is dissolved in water although the concentration is lower. The total volume of oxygen radicals produced is thought to result from the accumulation of oxygen radicals produced with each wavelength. Therefore, a broadband light emitting light over a range of 600~800 nm would induce the formation of more oxygen radicals than a diode laser. Additionally, a broadband light that has an emission range of 600~800 nm would stimulate ICG to simultaneously induce heat and oxygen radical production in tumor tissues.

We previously investigated the effects of PHT on B16F10 murine melanoma cells in vitro [31,32]. Our results demonstrated that PHT induces early morphological changes in tumor cells that promote more cell death than hyperthermia alone. Furthermore, PHT induces early apoptosis and cell arrest. These in vitro data support the present in vivo results.

Tumor cells are more sensitive to heat under acidic conditions [14]. Therefore, saline with a pH adjusted to 5.0 with acetic acid was used as a solvent for ICG. In a preliminary experiment, we found that saline with a pH of 4.0 induced tissue inflammation (data not shown).

The effects of anti-tumor drugs are enhanced by heat [15,16,24,25]. Furthermore, tumor tissues have a tendency to be acidic [36]. Therefore, we selected bleomycin as an anti-tumor drug because it is effective in acidic environments [15]. Platinum-based drugs such as cisplatin and carboplatin are also useful anti-tumor reagents. In the present study, we were concerned that PHCT would affect the stability of the anti-tumor drug. However, our preliminary data showed that the anti-tumor effects of PHCT were enhanced by the addition of an anti-tumor drug (data not shown). This means that PHCT does not only affect the stability of the anti-tumor drug but also enhances its effects.

In conclusion, PHCT is a simple procedure, is not associated with any severe side effects, and requires no special facilities. However, further investigation is necessary to establish PHCT as a therapeutic technique due to some associated problems involving treatment times and intervals, and selection of anti-cancer drugs. Nevertheless, this modality is expected to become a useful alternative to radiation therapy for treating superficial tumors such as STS in companion animals.