| BackgroundPhotodynamic therapy (PDT) is clinically adopted for the treatment of tumors in1970s. Now it has become a routine tumor treatment modality approved in America, England, France, German and Japan. PDT is currently successfully applied to the treatment of bladder cancer, prostate cancer, esophageal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, lung cancer, malignant pleural mesothelioma, head and neck tumors and brain tumors. In the study of PDT, researchers further expanded its application to nonmalignant disease such as port wine stain, age-related macular degeneration, mucosal vascular lesions on the digestive tract, verruca acuminate, acne and periodontal disease.PDT consists of three essential components:photosensitizer (PS), light and oxygen. PDT is a two stage procedure. After the administration of a light-sensitive PS, tumor locis are irradiated with a light of appropriate wavelength. The PS administered locally or systemically can accumulate selectively in the tumor tissues. This therapy results in a sequence of photochemical and photobiologic processes that cause irreversible photodamage to tumor tissues. The necrosis and apoptosis of tumor cells was derived from three interconnected approaches:the direct cell toxicity of singlet oxygen and other reactive oxygen species; shut down of tumor microvasculature and consequently the deprivation of tumor oxygen and nutrition; the stimulation of the host immune system. The high selectivity of PDT is derived from both the ability of useful PSs to localize in neoplastic lesions and the precise delivery of light to the treated site. And that makes PDT an excellent tumor treatment modality. PDT is a minimally invasive treatment with low toxicity and good repeatability. It is capable of protecting feature and organ function.But as a local treatment, PDT has its own limitations such as the penetration is usually not enough, the requirement of protection from sun and white light to avoid photosensitivity caused by PSs, and insufficient activation of the host immune system as well as tumor recurrence.For tumors at early stage, radical resection is capable of curing them. But large amount of patients are diagnosed at a late stage who have lost chance for surgical resection. Radiotherapy and chemotherapy turned out to be the choice on this occasion, but radiotherapy and chemotherapy would damage normal cells especially those fast growing cells such as skin, mucosa and bone marrow. What’s more, radiotherapy and chemotherapy usually function as a palliative treatment for late stage patients. Metastasis is the most frequent cause of treatment failure and death in cancer patients. As a local treatment, PDT is useless for metastatic lesions. An ideal tumor treatment modality should not only eradicate the treated primary tumors but also control untreated metastases at remote sites.PDT induces an acute inflammation and the infiltration of neutrophils, mast cells and monocytes as well as the secretion of various cytokines. Tumor cells swell and break into pieces after PDT, allowing the release of antigens. Antigen presenting cells, particularly dendritic cells can capture these antigens and migrate to lymph nodes and finally lead to the generation of a tumor specific immune response. But the immune system of cancer patients is often compromised, and tumor debris may not be sufficient enough in inducing a potent antitumor response. Thus, additional immunological stimulations are required to achieve a protective immune response against residual tumor cells.In the exploration of photodynamic therapy, researchers explored the combination of PDT with other approaches for a better antitumor effect. The combination of PDT with immunotherapy was one of them. And researchers defined this combination photodynamic immunotherapy (PDIT). These immune-therapies include immunoadjuvant, monoclonal antibody, cytokines, complement activators and immune cells. Results revealed that the combination could improve the antitumor effect. Laser immunotherapy is among these combinations. It is consisted of PDT and immunoadjuvant. The tumor destruction mechanism is that the laser-photosensitizer interaction induces direct tumor destruction and an acute inflammation response, while the immunoadjuvant administered intratumorally or peritumorally help strengthen the immunity and finally the induction of a tumor specific immune response. The tumor specific immunity is capable of clearing residual tumors in the irradiated site as well as metastases. Furthermore, the immune memory help resist tumor recurrence. These characteristics make laser immunotherapy a cancer treatment of wonderful prospect.ObjectiveThis study was aimed to evaluate the long and systemic antitumor tumor effect of laser immunotherapy on mouse H22tumor and explore its antitumor mechanism for further clinical application.Methods (1) Analysis of the antitumor effect with different light doses:Kunming mice aged4-6weeks and weighed18～22gram were chosen as the subject. Mice with the hair on their lower back depilated were anesthetized with1%pentobarbital sodium at a dose of60milligram per kilogram of body weight (60mg/kg), and then injected subcutaneously with2×106H22cells suspended in100μL of phosphate-buffered saline into the right hip. Tumors reached a size of7to9mm in diameter5to7days after transplantation. They were randomized to a control group and5treatment groups (30J/cm2,60J/cm2,90J/cm2,120J/cm2and150J/cm2). The control group was observed without any treatment. Mice in the5treatment groups were administered Photofrin5mg/kg24h before laser irradiation, after a24h drug-light interval, they were irradiated with the designed energy respectively. Then the appearance of the tumor and treatment region were observed. Tumor growth was assessed3times a week and the orthogonal tumor dimensions (a and b) were measured with a Vernier caliper. The tumor volume was calculated according to the formula, V=axb2×1/2.(2) Long term antitumor effect of laser immunotherapy:30mice with tumors growing in their right lower back were randomized to control group, PDT group and LIT group. The control group didn’t receive any treatment. Mice in the PDT and LIT group were injected with Photofrin intravenously.24hours later, the lesions were illuminated by a diode laser (630PDT laser, Diomed) through a microlen fiber at a dose of120J/cm2(power300mW, duration12minutes, spot diameter1.5cm2) Mice in the LIT group were further injected with0.2mL of1%GC intratumorally immediately after the illumination. Mice were considered cured when they were healthy and tumor free90days after treatment. Cured mice were rechallenged with H22tumor cells at the same dose (2×106). Tumor occurrence and growth were observed and recorded.(3) Systemic anti-tumor effect of laser immunotherapy:Kunming mice aged4-6 weeks and weighed18～22gram were transplanted subcutaneously with H22tumor cells on bilateral hips and then randomized to PDT group and LIT group. Only the tumors on the right side were treated, the tumors on the left side were not treated. The tumor size on both sides were recorded and controlled with the control group of the unilateral tumor model.(4) Statistics:SPSS13.0software package was applied to statistical analysis in this study. The measurement dates were recorded as (mean±standard deviation). One-way ANOVA was performed to compare tumor volume at different times. Repeated measures was performed to analyze the growth curve of different groups.Results(1) Analysis of the antitumor effect with different light doses:There is no significant difference in tumor size before treatment (P=0.995). The tumor size of the control group increased gradually. The skin above the tumor in the30J/cm2and60J/cm2became brown3-4days after laser irradiation, and there is no obvious scabs in these two groups. Tumor growth in these two groups has no significant difference compared to the control group. While tumor growth was significantly inhibited in the90J/cm2,120J/cm2and150J/cm2(P<0.01). No mice were cured in the90J/cm2group. One mouse in the120J/cm2was cured and showed no tumor recurrence. The tumor of three mice in the150J/cm2group disappeared after treatment but one of them experienced tumor recurrence.(2) Long term antitumor effect of laser immunotherapy:Tumor growth speed was significantly lower in the PDT and LIT group (P<0.01), and the tumors in the LIT group grow at an even slower speed than the PDT group (P=0.016). Tumors in the control group couldn’t disappear automatically. There are two mice cured in the PDT group and seven cures in the LIT group. Cured mice were given a second challenge with the same dose H22tumor cell. The two cured mice in the PDT group failed to resist the second challenge, but tumors grow at a lower speed compared to the first challenge. Five out of seven cured mice in the LIT group were resistant to the second challenge. The tumor occurrence was delayed and tumor growth slowed down in the two mice that failed the rechallenge.(3) Systemic antitumor effect of laser immunotherapy:In the bilateral tumor model with only the tumor on the right side treated, PDT inhibited the growth of the treated side, but showed no effect on the non-treated side. The growth of the left tumor showed no significant difference when compared with the blank control group. However, both sides of tumors in the LIT group were significantly inhibited (P<0.01), tumors on the non-treated left side in the LIT group grow at a comparable speed of the treated right side tumors in the PDT group (P=0.56). Furthermore, three mice were cured with tumors on both sides disappeared, and there was no tumor recurrence after a long time follow-up.ConclusionsWe concluded that photodynamic therapy is curable to H22tumor under certain light dose. The combination of GC and photodynamic therapy can improve the antitumor effect of PDT on H22tumor. Our findings indicate that laser immunotherapy can induce a long-term and systemic antitumor effect. It’s capable of eradicating both treated primary tumors and untreated metastases at remote sites. |
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