Changes Of Immunity In Rabbits With VX2 Liver Tumors After The Treatment Of High-intensity Focused Ultrasound Combined With HSV-TK/GCV

Background and objectiveAs hepatocellular carcinoma (HCC) is characterized by local invasion and distant metastasis, most of the patients have already lost the chance of operation when hospitalized, for whom, the local adjuvant therapy has become an important treatment. High intensity focused ultrasound (HIFU), a non-invasive local treatment for cancer, has been widely used in a variety of clinical cancer treatments, including HCC. As applied to HCC, HIFU has the advantage of more precise focus on the tumor and less impact on the surrounding tissues, and it is little affected by the patients'general condition, liver function, etc. But because of the energy attenuation, costal arch occlusion, liver movement with respiration shift and other factors, residual cancer cells are often left at the tumor edge after HIFU treatment. Therefore, on the basis of HIFU, combination with other treatments to kill residual cancer cells as much as possible will be the key point to improve the efficacy of HIFU treatment. Suicide gene is one of the most effective schemes in cancer gene therapy. In addition to killing tumor cells directly, bystander effect helps to enlarge the kill effect. Herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) system is the most commonly used therapy system for HCC, but, as with other gene therapy, this system lacks targeting ability and has poor transfection rates. Recently, the emergence of a new gene targeted transfer technology: low-frequency ultrasound targeted-microbubble destruction (UTMD) could improve the transfection and expression of gene in the targeted tissues.CD4+CD25+regulatory T cells (regulatory T cell Treg) are a class of T lymphocyte subsets with immunomodulatory function. They play important roles not only in maintaining self-tolerance, but also in tumor immune. Research shows that there are significantly increases of Treg cells in peripheral blood, ascites and tumor tissue of patients with HCC, indicating that the body is in a state of immune suppression. While killing the tumor cells, HIFU and HSV-TK/GCV can improve immune function and enhance anti-tumor immunity separately.Obviously, HIFU combined with microbubbles carrying HSV-TK targeted therapy for the treatment of HCC provides a new way of thinking. Whether the combination therapy could synergistically enhance tumor killing effect, or improve anti-tumor immunity are worth exploring. In this study, rabbit with VX2 liver tumor was used as research object. We focused on the changes of CD4+CD25+Treg cells and anti-tumor immunity after the treatment of HIFU combined with HSV-TK/GCV, to provide preliminary study of immunological mechanism for the combination treatment in terms of tumor immune tolerance and specific anti-tumor immunity .Methods1. The preparation of ultrasound microbubbles carrying HSV-TK: After the large amplification of DH5a E.coli transfected with HSV-TK gene, the plasmids were extracted and purified in large quantities according to OMEGA kit instructions, identified by PCR and double digestion electrophoresis, and ultraviolet spectrophotometer was used to detect the concentration and purity of the plasmids; The combination of microbubbles with HSV-TK gene and Poly-L-Lysine (PLL) were prepared according to the layer-by-layer (LBL) method, and then detected by optical microscopy.2. The establishment of rabbit models with liver tumor: 42 healthy New Zealand white rabbits, weighing 2-2.5 kg, either sex, 2-3 months old, were inoculated with VX2 tumor cells by laparotomy.Each 3 rabbits were sacrificed at 3th, 4th, 5th week after tumor inoculation to observe tumor morphology and metastasis, and a portion of tumor tissues were stained with HE staining.3. Treatment: While 10 healthy New Zealand white rabbits as the control group, 30 tumor-bearing rabbits were randomly divided into 3 groups: tumor-bearing group, HIFU-treat group, combined-treat group (n=10), and the remaining 3 tumor-bearing rabbits were used for the observation of HIFU treatment. Treatment site was 3th week after tumor inoculation. HIFU parameters: power of 180W, two-layer spacing 3mm, two-line spacing 5mm, scanning speed of 3mm/s.3 rabbits were sacrificed immediately after HIFU treatment to observe the tumor morphology, and parts of the tumor tissue were taken for HE staining and TTC staining. HIFU-treat group were given HIFU treatment separately, and sham treatment was operated in tumor group. 24h after HIFU treatment, combined-treat group were injected with HSV-TK gene(0.2mg/kg) through the ear vein and then transfected by low-frequency ultrasound for 20 minutes with transfection parameters: 300kHz, 2W/cm2. 24h after that, intraperitoneal injection of GCV(50mg/kg) was performed for 10d consecutively. HIFU-treat group and tumor group were injected with saline equally, without low-frequency ultrasound.4. Detection of relevant indicators: Twelve days after treatments, the prevalence of CD4+CD25+Treg cells in peripheral blood of each rabbit was detected by flow cytometry. Elisa kits were used to detect the levels of IL-10 and TGF-βin plasmas. Splenic lymphocytes were also collected to evaluate the cytotoxic activity against VX2 tumor cells by MTT.5. Statistical methods: All of the data were under normal test by SPSS13.0 software, and the data showing a normal distribution were represented with x±s. SPSS13.0 software was used for analysis of variance, with the testing standard a=0.05.Results1. HSV-TK plasmids were successfully extracted, which were confirmed by PCR and double digestion electrophoresis, and the the concentration was adjusted to 1 mg/ml, OD ratio [D(260)/D(280)] to 1.9; Ultrasound microbubbles carrying HSV-TK gene and PLL were also successfully prepared, and the concentration was adjusted to 0.1μg/μl. Under optical microscope, the physical properties of microbubbles were qualified.2 weeks after inoculation, the mean size of tumor was 1.5±0.31cm, and the tumor was isolated, with no visible internal or external metastasis. 3 weeks after inoculation, tumor size was 2.3±0.17. Liquefaction necrosis in the center of tumor was found in one of tumor-bearing rabbits. 4 weeks after inoculation, tumor size was 2.9±0.43cm. Intrahepatic, omental and pulmonary metastasis were observed in all of the 3 rabbits. Macroscopic observation showed that the tumor were round, gray white in color with hard texture, and located in the liver parenchyma with no obvious capsule. The section of the tumor was fish-like tissue, with caseous liquefaction necrosis in the center. Under light microscope, the tumor cells were relatively large with heteromorphism, basophilic cytoplasm and disordered arrangement. Nuclei showed pleomorphism, a lot of which were in split phase, and the nuclear/cytoplasm ratio was increased.3. The gray value of targeted region was significantly increased than that before immediately after HIFU treatment, but no visible changes were observed. TTC staining showed that tumor tissue with HIFU irradiation was pale, indicating that the tissue had been necrotic. HE staining showed the tumor cells were of edema. Although the structure of cells was intact, some cells were deep-stained in nucleus, and the intercellular gap was widened.4. Compared with control group, there were dramatic elevation of levels of Treg (8.5±0.69%), IL-10 (67.82±4.517pg/ml) and TGF-β(6.55±0.451μg/ml) in tumor-bearing group (P<0.01). Significant decreases of both Treg level (7.0±0.56%) and IL-10 level (60.32±4.700pg/ml) were observed in HIFU-treat group in contrast with that in tumor-bearing group (P<0.01), and the decrease tendency was also observed in TGF-βlevel (6.22±0.198μg/ml) (P<0.05). The levels of Treg (6.4±0.72%) and TGF-β(5.86±0.305μg/ml) were significant lower in combined-treat group than those in HIFU-treat group (P<0.05), but there were no significant changes of IL-10 (58.79±4.416 pg/ml) level in these two groups. A significant increase of splenic lymphocyte cytotoxicity to VX2 tumor cells was observed in both HIFU-treat group (31.62±3.09%) (P<0.01) and combined-treat group (29.48±2.68%) (P<0.01) when compared with that of the other two groups (13.94±2.45%, 12.00±2.96%), but there were no significant changes between these two groups. Conclusion1. With the method of extraction in large quantities, HSV-TK plasmids of ideal concentration and purity can be obtained. The concentraion and size of ultrasound microbubbles carrying gene prepared by LBL method can meet the requirements.2. In addition to pathological morphology, the growth and metastasis situation of rabbit VX2 liver tumor is similar with that of HCC in human. Rabbit with VX2 liver tumor is an ideal animal model of hepatic tumor.3. After HIFU treatment, the gray value of targeted region was significantly increased than that before, and simultaneously, coagulation necrosis was occurred in targeted area, indicating that HIFU is effective.4. The combined treatment of HIFU and HSV-TK/GCV could reverse the tumor immunological tolerance and improve the anti-tumor immune activity of the hosts.