| ObjectiveTo observe the effect of5-ALA Photodynamic therapy (5-ALA-PDT) on UV-induced cutaneous squamous cell carcinoma (SCC) of SKH-1hairless mice. To investigate the mechanism of action of ALA-PDT from cell death, tumor microvascular injury and part of the immunological effects systemly, which might offer a experimental basis for clinical therapy of cutaneous SCC.Methods1. Built multiple skin squamous cell carcinomas of SKH-1hairless mice by UV radiation. When tumors diameter grew to1mm-5mm, we chose55mice with tumors and randomly divided into four groups included ALA-PDT group(15), ALAgroup(15), red light group(15) and tumors control group(10)without any treatment, which were marked with group A, B, C and D respectively. Monitored PpIX fluorescence intensity of5%,8%,16%concentrations of ALA with Curalux fluorescence detector for12h and at24h after ALA-PDT to determine the best dressing concentration and the optimal duration of treatment. Treated group A mice with the optimal concentration of ALA and the appropriate dressing time. Then gave them632.8nm red light (20mw/cm2,30J/cm2) radiation. Mice in group B were given the same concentration of ALA as group A but without red light irradiation. The group C mice were given the same dose red light irradiation as group A but without ALA. The group D had no any treatment. Mice in each group were treated weakly for4weeks. The length and short diameters of tumors were measured before every treatment while counting the number of tumors, and measured the body weight of mice until2weeks after the end of treatment. Recorded the time of death of the mice or how long the tumor volume reached500mm3in each group from the beginning of treatment to the end of the study, and made statistics on the average survival rate of tumor-bearing mice in each group.2. Selected another21mice with tumors of1mm～5mm in diameter, there were three mice in each time point, mice were sacrificed by cervical dislocation. Tumors were excised and minced using two samples at various time (before and lh,3h,6h,12h,24h and7d after ALA-PDT), half of the tumor was preserved with2.5%glutaraldehyde to be made as electron microscopy specimens, the other half of the tumors were saved in4%paraformaldehyde for the routine pathological examination. Observed and recorded tumor tissue changes before and after ALA-PDT with light microscope and electron microscope.3. The tumor tissue sections of different time except that of7d after ALA-PDT were detected by TUNEL to assay the tumor cell apoptosis and all above tissue sections were immunohistochemical stained of CD34, LC3, CD1a, CD4, CD8antibodies in order to detect the changes of microvessel density, cell autophagy and local immunity of tumors, which might explain the mechanism of the action of ALA-PDT in treatment of squamous cell carcinoma.Results1. Two weeks after the last treatment, the group A that was ALA-PDT group had a significant role in inhibition of tumor growth, The tumor volumes were significantly reduced compared with that before treatment (P<0.05), tumors of1-4mm in diameter and/or lower than2.5mm in depth could disappear completely and tumors of4-6mm in diameter and/or2.5-3mm in depth could partially disappeared or eased. However, tumor volumes of B, C, D three groups were greater, and were statistically different compared with that before treatment (P<0.05). The tumor volumes of group A mice were significantly lower than other groups (P<0.01), but there were no difference among B, C and D groups(P>0.05).The tumor number of group A mice gradually decreased with the increase of treatments.1-2weeks after the end of treatment, the number of the tumor remained unchanged. But tumor numbers of the other three groups gradually increased, there were about dozen of tumors on the back of mice, some of which grown together and distributed densely like a carpet.2. There were4or5mice whose tumor volumes were over than500mm3in groups of B,C and D, which were anesthetized and executed by cervical dislocation. The remaining mice survived till the end of the experiment, except one mouse of group D died from an accident. The survival rate of tumor-bearing mice of each group had no significant difference (χ=8.374, p=0.039<0.05). The body weight of all experimental mice did not differ significantly (P>0.05).3. The result of conventional pathology examination showed that tumor cells distributed densely, the nucleus were big and dyed dark, some of which were apparent atypical. There were a lot of blood capillaries in tumor storm. At1h after ALA-PDT tumor vessels became vasocongestion with a few inflammatory cells aggregation. There a large of inflammatory cells in blood vessels, they adhered and passed through the vessel walls at3h after ALA-PDT. Intercellular edema occurred., the tumor tissue were diffusely bleeding at6h after PDT, vascular showed thrombosis and occlusion, tumor cells peripheral of vessels necrotized, showing spotty red dyed, and a large number of inflammatory cells (such as neutrophils) infiltrated. Tumor vessels still bleeded, and there was deposition of red blood cells in interstitial at12h after treatment.24h later, congestion eased, there were a lot of tumor cells necrosis which were stained lightly red, and many inflammatory cells infiltrated. Till the7th day after treatment, there were large areas of necrosis in the tumor, nuclear fragmented and a large of inflammatory cells infiltrated in local tissue.4. Transmission electron microscopy (TEM) showed tumor cells were polygonal before treatment. The rate of nucleus and cytoplasm was big, and there was much microvillus on the surface of tumor cells. We can saw swollen cells, microvillus reduction, and swollen mitochondria which began to appear empty bubble at1h after ALA-PDT, and early apoptotic cells can be seen. At3h after treatment, chromatin marinated like a ring, the cytoplasm began to bud off that was cell apoptosis.6h later, the cytoplasm vacuolated further. A high degree of cytoplasmic vacuolization, and apoptosis and necrosis formatted12h later. At24h after treatment cell necrosis completely and disappeared into a fuzzy sheet.5. TUNEL indicated that tumor cells began to apoptosis at3h after PDT and gradually increased with time going, but there were no significantly difference among each time(P>0.05).6. Immunohistochemistry showed that microvessel density gradually decreased after ALA-PDT, there was a statistically significant difference from6h after treatment compared with that before treatment(P<0.05). Vascular injury was more serious at12h later(P<0.01). There were only1-2small arteries in the subcutaneous connective tissue at the seventh day after treatment. The positive expression of CD la was observed increasing along with time going, but there was significant difference at24h and7d after ALA-PDT compared with the control. The positive expression of CD4and CD8were more than the baseline (before treatment), and there were statistically significant differences at7d.There was positive linear correlation among CD la, CD4and CD8positive cells(r=0.884,0.862; P<0.05of all). CD34were negatively correlated with CD1a, CD4+T and CD8+T cells (r=-0.923,-0.847,-0.855, and P<0.05of all). LC3-positive cell number increased at0.5h,1h,3h,24h and7d after treatment. The difference was statistically significant (P<0.05) compared with pretreatment.Conclusions1. ALA-PDT of4times treatment could effectively remove tumors of lmm-4mm in diameter and1-2.5mm in depth, reduced the size of tumors with diameter of4mm-6mm and2.5mm-3mm in depth, and improved the survival rate of tumor-bearing mice.2. In this study, ALA-PDT can directly damage the squamous cell carcinoma by cell necrosis and apoptosis but not autophagy.3. ALA-PDT can damage the microvascular endothelial cells of squamous cell carcinoma, so that blood deposited and formatted thrombosis, blocking the blood supply of the squamous cell carcinoma, and thus indirectly kill squamous cell carcinoma cells.4. ALA-PDT damaged tumor cells and produced certain immunogenic substances, such as cell debris, heat shock proteins, raising the aggregation of neutrophils, DC, CD4+, CD8+T cells, and enhanced the local immunological effects.5. In this study, we assumed that autophagy may occur in the macrophages, and it might be an antigen presenting ways. |
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