Concomitant Treatment With Glycated Chitosan Improves Immunological Effect By Photodynamic Therapy

BackgroundAt present, cancer has become one of the diseases that seriously threat human’s health. The latest version of World Cancer Report that released by the International Agency for Research on Cancer (IARC) forecasted that global cancer cases would grow rapidly, from 14 million in 2012 to 19 million in 2025, and in 2035 would reach 24 million people. Cancer seems to have become the strongest human killer in the new century. Therefore, the control of cancer is the focus task of governments around the world, and comprehensive treatment for cancer has become the focus of researchs in the world.Among the methods of cancer therapy, photodynamic therapy (PDT) is a new research field which is under developing. So far, PDT has made remarkable achievements in clinical cancer therapy, and with the advantages of efficiency, safety, few side effects, cooperativity, repeatability, and low cost, it has showed strong vitality in cancer treatments.To precancerous lesions and early cancer, PDT can achieve the purpose of healing. To the advanced cancer patients, especially that cannot (or refuse to) be provided conventional therapy, PDT is a new choice. For decades, PDT has become a very active area of cancer prevention. Many countries have carried out studies of PDT for malignancy, and tens of thousands of different cancer patients have benefited from this therapy. It is not only effective in treating superficial tumors, but also cancers of respiratory tract, gastrointestinal tract, brain, eye, bladder and other parts.Utilizing the various half-life of photosensitizers in different tissue, photosensitizers selective retain in tumor tissue. And then the photosensitizer is activated by light of specific wavelength and reacts with the oxygen within the tumor tissue. The reaction produces chemically active singlet oxygen (1O2) and some other reactive oxygen species (ROS), leading to the oxidative damage of intracellular proteins, nucleic acids and lipids and other biological macromolecules, causing direct cytotoxic effect, causing tumor tissue microvascular damage. Furthermore, local oxidative stress can activate the complement system, prompt the production and the release of various inflammatory and immune factors, induce a variety of inflammatory cells, immune cells rapidly invasing into the local tumor and finally activate the anti-tumor immune systemic. PDT contains three essential components: 1.a photosensitizer applied systematically or topically; 2. a near-infrared laser with absorption wavelength compatible with the photosensitizer; 3. molecular oxygen.However, PDT has its own limitations, such as limited treatment depth of the existing photodynamic technology, high rate of recurrence, limited therapeutic efficacy for metastatic lesions and so on. To overcome these drawbacks, the researchers began to explore on the combination of PDT and immunotherapy, tried to enhance the immunogenicity of the tumor cells and activate of the host’s immune system. They tried to stimulate the body immune system, achieve the healing and prevent tumor recurrence by the means of active immunization.Laser immunotherapy (LIT) was suggested by Chen et al. of Oklahoma State University in 1997. The combination of laser irradiation and photosensitizer causes selective photothermal destruction and the release of tumor antigenic determinants. The immunoadjuvant binding with the antigen stimulates the host immune system to develop a specific immune reaction against both the remaining tumor cells in the primary site and metastases.The key feature of this novel modality is the combination of photothermal, photochemical and immunologic reactions.Object1. Discussion the cell killing characteristics of Photofrin-PDT to EMT6 murine breast cancer cell in vitro to find the ideal parameters of GC-LIT.2. On EMT6 mouse breast cancer model, observe tumor inhibitory effect and the survival of tumor-bearing mice that treated with the various combinations of the three components (photosensitizers, laser and immune adjuvant) of laser immunotherapy. Observe tumor formation of cure mice which were rechallenged with the same number of EMT6 cells to explore the long-term anti-tumor effects of laser immunotherapy.3. Compare the expression of inducible nitric oxide synthase (iNOS), heat shock protein 70 (HSP70), cyclooxygenase-2 (COX-2), Actived-Caspase3 of EMT6 tumors after different treatments. Compare the number of CD3+ T cells, CD8+ T cells and Granzyme B positive lymphocytes that infiltrating into the tumor tissues. Compare the level of oxidative stress, apoptosis and cellular immune effector of different treatment groups.4. On the mouse model of 4T1 metastatic breast cancer, compare the efficiency of laser immunotherapy and photodynamic therapy of killing distant metastases. Compare the spleen coefficient of tumor-bearing mice of each group to confirm the involvement of the immune system.Methods1. Research characteristics of Photofrin absorption by EMT6 cells using fluorescence microscopy and multifunction microplate reader. Detect the contribution of the three PDT efficacy factors (photosensitizer concentration, power density and energy density) to the cell killing power of PDT.2. Establish hip subcutaneous EMT6 breast cancer model of BALB/c mice, these mice were divided into six groups (PDT+ GC group, PDT group, Laser+ GC group, Laser group, GC group and Control group). They were received various combinations of the three components of laser immunotherapy (photosensitizers, laser, immune adjuvant) respectively. And the tumor volume of each group mice were measured every other day and the death time of the mice were recorded. The tumor growth curve and survival curve of tumor-bearing mice were plotted and analyzed. When the local tumor and metastases disappeared and the mouse still survival up to 90 days after treatment, we considered it a cured mice. The cured mice were rechallenged with the same tumor cells at the same dose as the first challenge and were observed whether the tumor emerged and the growth speed of it.3. In the 2nd day after treatment, killed 3 mice of each group, took out the tumor tissues and compared the expression of inducible nitric oxide synthase (iNOS), heat shock protein 70 (HSP70), cyclooxygenase (COX-2), Actived-Caspase3 of different groups by immunohistochemical methods and Western Blotting. Killed 3 mice of each group 2 weeks after treatment and compared the quantity of infiltrating CD3+T cells, CD8+T cells and the immue cells that express Granzyme B of different groups by immunohistochemical methods. The level of oxidative stress, cell apoptosis and cell immune effector strength were compared of each group.4. Establish inguinal subcutaneous 4T1 mouse breast cancer model of BALB/c. These mice were divided into three groups (PDT+ GC group, PDT group and Control group) and were treated accordingly. Three mice of each group were sacrificed 1 week after treatment and the lungs of them were removed aseptically. The single cell suspension of the lung tissue was prepared by mechanical and enzymatic digestion process. The single cell suspension was added into a medium containing 6-thioguanine, using the selectively 6-thioguanine resistant characteristics of 4T1 cells, and was used for cloning experiment. Count the clones of each group 14 days after planking. Three mice of each group were sacrificed 3 weeks after treatment and were measured the body weight. The lungs of which were removed and fixed with Bouin’s solution for 24 hr. Then count the number of lesions of each mouse. Confirming the formation of pulmonary metastases by HE staining. Remove the mouse spleen and measure its weight to calculate the spleen coefficient (The spleen coefficient is the mouse spleen weight divided by the body weight of itself) of each mouse.Results1. When EMT6 cells were incubated with 8μg/ml Photofrin for less than 8h, the cells emit a faint red fluorescence and cell outline is not clear under an inverted fluorescence microscope. During 8h-24h, cell outline more clearly. And strongest fluorescence was observed at 12h. Moreover, the cell morphology did not change significantly and continued to proliferate. By detecting fluorescence values using multifunctional microplate reader, we found that within a certain range, the intracellular content of the photosensitizer was positive correlation with the concentration of the photosensitizer and the incubation time.2. Under the irradiation conditions with the power density of 12.5mW/cm2 and the energy density of 7.5J/cm2, within a certain range, the inhibition rate was positive correlation with the photosensitizer concentration and incubation time. When 8ug/ml Photofrin incubated with EMT6 for 6h, the inhibition rate reached 50%. Under this irradiation conditions with the energy density of 7.5J/cm2, the power density in a certain range had no significant effect on the killing effect (P>0.05), and the laser treatment alone had no significant toxicity (P> 0.05). Under the irradiation conditions with the power density 12.5mW/cm2, the energy density within a certain range, PDT killing effect was enhanced with the increase of energy density, and the laser treatment alone had no significant toxicity (P> 0.05).3. The tumor formation rate of EMT6 mouse breast cancer model was 100%. There was no significant difference of tumor volume before treatment among 6 groups (P=0.594). The tumors of PDT+GC group were observed after treatment and were found the occurrence of edema, black, eschar formation and off, shrinking or disappearing after a slight increase. The tumor volume of PDT group was significantly reduced after treatment. The residual tumor tissue were visible at about nine days after treatment when the eschar off. Due to swelling, the tumor volume of PDT+GC group has increased significantly one day after treatment, but then reduced with edema reducing. To about 9 days after treatment, the tumor volume of PDT+GC group was considerable with PDT group, and then the tumors growed slowly, moreover, some tumors were even still narrowing. Repeated measures analysis showed that there was no significant difference between PDT+GC group and PDT group in the tumor volume (P=0.282). Survival analysis showed that media survival time of mice of PDT+GC group, PDT group, Laser+GC group was longer than the Control group (P=0.000,0.001,0.003 respectively), and PDT+GC group existed a survival advantage when compared with PDT group or Laser+GC group (P=0.036, 0.000 respectively). Cure mice were rechallenged with the same tumor cells at the same dose as the first challenge, but the subcutaneous masses of them appeared later and the tumor growth slower when compared with age-matched control mice. Moreover, the tumor of two cured mice of PDT+GC group disappeared completely at 27d and 35d after inoculation.4.HE staining of tumor tissue showed significant necrosis in PDT+GC group and PDT group, but minor necrosis in Laser+GC group, Laser group, GC group. The infiltration levels of CD3+T cells, CD8+T cells and the expression of cytotoxic marker Granzyme-B were higher in PDT+GC group, PDT group and Laser+GC group when compared with the Control group (P<0.001). There was no significant difference in these three immune cells infiltrate level between the GC group with Control group (P=0.978,0.424,0.430 respectively). The CD8+T cells’ and Granzyme B positive expression immune cells’ infiltrate level is higher in Laser group than Control group (CD8+T cells, P=0.001; Granzyme B+ cell, P=0.025), but there was no significant difference in CD3+T cells (P=1.000). Although there was no significant difference between PDT group with PDT+GC group in CD3+T cells’infiltration level (P=0.712), the signs of activated immune cells, CD8 and Granzyme B, were higher in PDT+GC group than that in PDT group (P=0.030,0.022 respectively). In our study, we investigated iNOS, HSP70, COX-2 and Actived-Caspase3 expression of the treated tumor in situ two days (48hr) after treatment in different group with various combinations of the components in laser immunotherapy. The results showed that the expression of iNOS, HSP70, COX-2 and Actived-Caspase3 in tumor tissue was higher in PDT+GC group, PDT group, Laser+GC group than the Control group (P<0.001). Although there was no significant difference between PDT+GC group with PDT group in tumor COX-2 expression level (P=0.078), but the levels of iNOS (oxygenation index), HSP70 (stress response indicators) and Actived-Caspase3 (apoptosis index) were higher in PDT+GC group (P=0.038,0.039,0.040 respectively). The expression levels of these four proteins were significantly higher in PDT+GC group than Laser+GC group (P=0.000,0.021,0.040,0.021, respectively). There was no significant difference in these proteins expression between GC group and Control group.5. The Western blot results show that the expression of Caspase-3 was decreased in PDT+GC group and the expression of Actived-caspase-3, COX-2 and HSP70 was increased in PDT+GC group when compared with the Control group. PDT+GC group had lower-Caspase-3 expression and higher Actived-caspase-3 expression than PDT group.6. The tumor formation rate of inguinal subcutaneous 4T1 mouse breast cancer model was 100%. One weeks after different treatments (PDT+GC group, PDT group and Control group), the metastatic tumor cells in lungs of PDT+GC group and PDT group were significantly less than that of the Control group (P=0.002,0.042 respectively) through a plat cloning experiment using the selectively 6-thioguanine resistant characteristics of 4T1 cells. The metastatic tumor cells of lungs was less in PDT+GC group than PDT group (P=0.035). Three weeks after different treatments, the number of lung tumor nodules in PDT+GC group and PDT group was significantly less than that of Control group (P=0.001,0.017 respectively), and PDT+GC group was less than PDT group (P=0.028). We confirmed the formation of pulmonary metastases by HE staining.The spleen coefficient of PDT+GC group and PDT group was greater than Control group (P=0.001,0.029 respectively), and PDT+GC group was greater than that of PDT group (P=0.031).Conclusions1. We confirm the absorption kinetics of Photofrin by EMT6 cell and the killing effect of Photofrin-PDT on EMT6 cells in vitro, indicating that finding more ideal parameter about the light dose, photosensitizer concentration and interval between administration and treatment are the key to improving PDT efficacy.2. LIT can significantly inhibit tumor growth and prolong the survival of treated mice. Moreover, the cured mice get specificity anti-tumor immune. LIT had an increased production of NO in situ and expression of HSP70 when compared to PDT. LIT can enhance the local inflammatory response, promote the apoptosis of local tumor cells, attract more actived T cells to the tumor locally when compared with PDT alone.3. Concomitant treatment with Glycated chitosan improves systemic effect of photodynamic therapy with Photofrin, and we confirme that the body’s immune system involves in this system antitumor effect.4.4T1 metastatic breast cancer model is an ideal model for laser immunotherapy. On the basis of 4T1’s resistance to 6-thioguanine, we can be more visually observed the system effect of LIT, and early detect the metastatic tumor cells.