The Research On Effect Of Photodynamic Therapy With PSD-007 On Osteosarcoma

The Research on Effect of Photodynamic Therapy with PSD-007 on OsteosarcomaMalignant bone and soft tissue tumors are serious diseases threat to human health. Osteosarcoma is one of the most representative of the tumors, characteristic of relatively high morbidity, high degree and early metastasis. Malignant bone and soft tissue tumors typically require surgical procedure, supplemented by radiotherapy and chemotherapy. However, a wide surgical resection often results in poor physical function postoperatively according to the amount of excised normal tissue, including muscle, vessels, and nerves. Preservation of such normal surrounding tissue can lead to better postoperative function for the patient, althoμgh there is a higher risk of local recurrence. Adjuvant treatment may reduce the risk of local recurrence when decreasing the surgical margin. However, most mesenchymal tumors are not sensitive to chemotherapy except for classic osteosarcoma.In addition, chemotherapy have serious side effects.The effect of chemotherapy and radiotherapy remains controversial regarding musculoskeletal tumors.Therefor, we are eager to explore a new adjuvant therapy. Photodynamic therapy is a unique cancer treatment modality based on the dye-sensitized photooxidation of biologic matter in target tissue.An intravenous injection of a light-sensitive agent (the photosensitizer) is retained selectively by tumor cells. The photosensitizer can be focally excited by laser light in the presence of oxygen using light of a wavelength matched to an absorption peak of the photosensitizer, and it transfers energy from photons to oxygen molecules. Direct killing of tumor cells, vascular damage, and inflammatory responses contribute to tumor destruction. It has advantages such as anatomic and functional preservation of adjacent normal tissues, enabling minimally invasive procedures and adjuvant therapy for unresectable cancers. Photodynamic therapy could be a novel adjuvant treatment for bone and soft tissue tumors.Objective:Exploring the possibility that photodynamic therapy (PDT) using PSD-007 could be a new therapy for osteosarcoma. To evaluate the PSD-007-mediated photodynamic effect on human osteosarcoma cell line MG-63 and mouse osteosarcoma cell line LM-8 in vitro. To ask whether PDT using PSD-007 and laser irradiation could cause tumor necrosis and inhibit tumor progression in vivo. We also investigated whether PDT using PSD-007 and laser irradiation after marginal resection of the tumor of C3H mice bearing osteosarcoma LM-8 could reduce the rate of local recurrence of the tumor. Methods:To assess an in vitro cytotoxic effect of PDT, we measured the cell viability of two cell lines (LM-8,MG-63) after PDT using laser irradiation (0-12J/cm2) following incubation with PSD-007 (0-16μg/ml) for 4 hours. We incubated human osteosarcoma cell line MG-63 and mouse osteosarcoma cell line LM-8 and seed the two cells into 96-well microplate (2×103 per well) respectively. The cells were treated with 0,0.5, 1,2,4,8μg/ml PSD-007 for 4 hours. The cells were washed with medium twice and then irradiated with 0,1.5,3,6,9J/cm2 using the 630-nm laser. After additional 24-hour incubation of the cells, cell viability was assessed by an MTT assay. Quantitation then was measured using a multiwall scanning spectrophotometer We prepared five wells for each condition and the inhibiting rate in treated cells was represented as a percentage of MTT value, and compared with that of untreated control cells. The curves of the inhibiting rate of cell after PDT were drawn then. We also observed the morphological changes of the cells after PDT in light microscope and detected the death mechanism of the cells by flow cytometry. Similarly, the cells were incubated into three 96-well microplates(2×103 per well) with 0,1,2,4,8,16μg/ml PSD-007 for 4 hours and then irradiated with laser.The dose of laser was fixed at 6J/cm2.After irradiation the cells were incubated in a humidified atmosphere at 37℃and 5% CO2 for 15min,60min,90min. The OD values were determined then by MTT assay in order to assess the early photodynamic effect. Experments were repeated three times.Fifty 4-week-old female C3H mice were used for implantation of LM-8 cells. We injected single cell suspensions of LM-8 cells (105cells per mouse) subcutaneously into the lumbosacral site of the mice. Growth of the tumors was measured using a slide caliper. When the diameter of the tumor on the back of the nude mice reached 6-8mm, PSD-007 and (5 and 10 mg/kg,) was injected intravenously into the LM-8-bearing mice. HpD was injected in the same way in order to compare their photodynamic effects. We irradiated the tumor site with 630 nm laser light (240J/cm2) 6 hours after injection (seven mice for each group). After PDT, the animals were maintained in a dark room to avoid skin irritation. The length and width of the tumors were measured at Days 0,1,3,5,7 after laser irradiation and then tumor volume was represented by using the equation:[maximum diameter (mm)]+[minimum diameter (mm)] /2. The mice were euthanized by an inhalation of ether on Day 7 after PDT. We measured the size and the weight of the tumors and compared with the controls. Tumor specimens excised from mice were fixed and embedded in paraffin before undergoing microscopic examinations. Slides of the 4um-thick specimens were stained with hematoxylin and eosin.In addition, we also examined the efficacy of PDT as an adjuvant treatment to reduce the rate of tumor local recurrence and the surgical margins, such as for marginal resections of a tumor, without excising surrounding normal tissue. After the diameter of the tumor on the back of the mice was 10-12 mm, a marginal resection of the tumor on mice bearing LM-8 was performed 6 hours after intravenous injection of PSD-007. We divided the mice into three groups:(1) marginal resection without PDT (control), which means resection of the tumor without excision of the surrounding normal tissue; (2) marginal resection followed by PDT with 240 J/cm2 laser irradiation 6 hours after intravenous injection of 5 mg/kg PSD-007; and (3) marginal resection followed by PDT with 360 J/cm2 laser irradiation 6 hours after intravenous injection of 5 mg/kg PSD-007 (n= 10 for each group). The mice were observed until 4 weeks after marginal resection of the tumors. We compared the rate of tumor recurrence after marginal resection of the tumors with or without PDT using Fisher's exact probability test. Taking into account our power analysis, we considered a probability value less than 5% as significant throμghout the study.Results:MTT assay revealed PDT using PSD-007 had a cytotoxic effect on and inhibited the growth of MG-63,LM-8 cells in vitro. The cytotoxic effect of PDT on the cells was dependent on the PSD-007 concentration and the level of laser irradiation. No antitumor effect was observed in the cells treated with laser irradiation without PSD-007 and also with the exposure to PSD-007 without laser irradiation. A higher dose of laser irradiation induced a strong antitumor effect under each concentration of PSD-007. PDT lead to cell death through apoptosis and (or) direct killing. PDT using PSD-007 and laser irradiation inhibited tumor growth of LM-8-bearing mice compared with the untreated control group. Injection of PSD-007 without irradiation or laser irradiation without injection of PSD-007 did not inhibit tumor growth. PSD-007-mediated photodynamic therapy substantially suppressed local recurrence after marginal resection of the tumor. Nine of 10 mice had local recurrence within 4 weeks after marginal resection of the tumor without PDT. Four of 10 mice had local recurrence after marginal resection of the tumor followed by PDT (5 mg/kg PSD-007,240 J/cm2 laser irradiation).Two of 10 mice had local recurrence after marginal resection of the tumor followed by PDT (5 mg/kg PSD-007, 360 J/cm2 laser irradiation). No side effects, such as delayed wound healing or skin defects, were evident with PDT with marginal resection. The rate of local recurrence after marginal resection followed by PDT (5mg/kg PSD-007,360J/cm2 laser irradiation) was lower than that after marginal resection only. Local tumor recurrence occurred in the peripheral area of PDT.Conclusion:Photodynamic therapy with PSD-007 showed an in vitro cytotoxic effect on the cultured LM-8,MG-63 cells. The in vitro effect of PDT depended on the treatment concentration of PSD-007 and the laser dose of irradiation. Photodynamic therapy suppressed the tumor growth in vivo, and high-dose irradiation induced a strong antitumor effect. Photodynamic therapy with PSD-007 performed after marginal resection of the tumor of the mice reduced the rate of local recurrence and reduced the surgical margin and preserve critical anatomic structures such as muscles, nerves, and vessels adjacent to the tumor. Our results indicated PSD-007-mediated PDT may be a potentially useful treatment modality for osteosarcoma.