Establishment of an orthotopic transplantation tumor model of hepatocellular carcinoma in mice

AIM:To improve the outcome of orthotopic transplantation in a mouse model,we used an absorbable gelatin sponge(AGS) in nude mice to establish an orthotopic implantation tumor model.METHODS:MHCC-97L hepatocellular carcinoma(HCC)cells stably expressing the luciferase gene were injected into the subcutaneous region of nude mice.One week later,the ectopic tumors were harvested and transplanted into the left liver lobe of nude mice.The AGS was used to establish the nude mouse orthotopic implantation tumor model.The tumor suppressor gene,paired box gene 5(PAX5),which is a tumor suppressor in HCC,was transfected into HCC cells to validate the model.Tumor growth was measured by bioluminescence imaging technology.Semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) and histopathology were used to confirm the tumorigenicity of the implanted tumor from the MHCC-97L cell line.RESULTS:We successfully developed an orthotopic transplantation tumor model in nude mice with the use of an AGS.The success rate of tumor transplantation was improved from 60% in the control group to 100% in the experimental group using AGS.The detection of fluorescent signals showed that tumors grew in all live nude mice.The mice were divided into 3 groups:AGS-,AGS+/PAX5-and AGS+/PAX5 +.Tumor size was significantly smaller in PAX5 transfected nude mice compared to control mice(P < 0.0001).These fluorescent signal results were consistent with observations made during surgery.Pathologic examination further confirmed that the tissues from the ectopic tumor were HCC.Results from RT-PCR proved that the HCC originated from MHCC-97L cells.CONCLUSION:Using an AGS is a convenient and efficient way of establishing an indirect orthotopic liver transplantation tumor model with a high success rate.

Kanglaite combined Gemcitabine inhibits growth of nude mouse subcutaneous transplantation tumor of human PC-3 pancreatic cancer cell

Objective:To study the mechanisms of pancreatic cancer treatment with Kanglaite combined Gemcitabine by investigating the relationship between the apoptosis and the expression of bcl-2, Bax and VEGF in pancreatic cancer cells.Methods:Nude mouse subcutaneous transplantation tumor model of Human PC-3 pancreatic cancer was established; the expressions of bcl-2, Bax and VEGF of transplantation tumor cell were determined; the earlier apoptosis rate of pancreatic cancer cell and the gross tumor volume were determined. Results:Kanglaite combined Gemcitabine remarkably decreased the protein expression of bcl-2,raised the expression of Bax,increased the apoptosis rate of the pancreatic cancer and contract the gross tumor volume. Kanglaite greatly decreased the protein expression of VEGF of the tumor cell. Conclusion:Therapeutic efficacy of Kanglaite combined Gemcitabine is far better than separate use of the two medicines in the pancreatic cancer transplantation tumor treatment.

Liver transplantation for hepatocellular carcinoma on cirrhosis:Strategies to avoid tumor recurrence

Hepatocellular carcinoma(HCC) is one of the most frequent neoplasms worldwide and in most cases it is associated with chronic liver disease.Liver transplantation(LT) is potentially the optimal treatment for those patients with HCC who have a poor functional hepatic reserve due to their underlying chronic liver disease.However,due to the limited availability of donors,only those patients whose oncologic profile is favorable can be considered for LT.Despite the careful selection of candidates based on strict rules,10 to 20%of liver transplant recipients who have HCC in the native cirrhotic liver develop tumor recurrence after transplantation.The selection criteria presently employed to minimize the risk of recurrence are based on gross tumor characteristics defined by imaging techniques;unfortunately,the accuracy of imaging is far from being optimal.Furthermore,microscopic tumor features that are strictly linked with prognosis can not be assessed prior to transplantation.Pre-transplantation tumor downstaging may allow transplantation in patients initially outside the selection criteria and seems to improve the prognosis;it also provides information on tumor biology.Themain peculiarity of the transplantation setting,when this is compared with other modalities of treatment,is the need for pharmacological immunosuppression:this is based on drugs that have been demonstrated to increase the risk of tumor development.As HCC is an aggressive malignancy,immunosuppression has to be handled carefully in patients who have HCC at the time of transplantation and new categories of immunosuppressive agents should be considered.Adjuvant chemotherapy following transplantation has failed to show any significant advantage.The aim of the present study is to review the possible strategies to avoid recurrence of HCC after liver transplantation based on the current clinical evidence and the more recent developments and to discuss possible future directions.

IL-24/MDA-7 Suppressed the Transplantation Tumor in BALB/c Mice

It is well-documented that interleukin-24 （IL-24） can induce apoptosis in a large spectrum of human cancer derived cell lines, but the effect of MDA-7/IL-24 gene transfer on mouse melanoma cells remains unknown. The eukaryotic expressing plasmid of IL-24 （pEGFP-IL-24） was constructed by DNA recombination technique. The recombination plasmid and empty vector were transfected into B16F0 cells and the expressions of IL-24 were determined by LSM, the proliferation of B16F0 cells was measured by MTT assay, and apoptosis rate and cell-cycle distribution of B16F0 cells were measured by FCM. The inhibitory effect of IL-24 gene transfection in mouse solid tumor was observed and measured. Compared with the control, the proliferation of B16F0 cells was inhibited by transfection with pEGFP-IL-24 and the G2/M phase of the transfected cells was also increased. Moreover, the percentage of mice with detectable tumor was decreased after inoculated with B16F0 cells transfected with pEGFP-IL-24. Growth rate of tumor in mouse model was significantly inhibited in IL-24 gene therapy group compared with the control. Proliferation of B16F0 cells was inhibited by pEGFP-IL-24 transfection. The intratumor injection of pEGFP-IL-24 could inhibit the growth of solid tumor in mice remarkably. Cellular ＆ Molecular Immunology.

HSP70 inhibitor combined with cisplatin suppresses the cervical cancer proliferation in vitro and transplanted tumor growth:An experimental study

Objective:To study the regulating effect of HSP70 inhibitor(PES) combined with cisplatin on cervical cancer proliferation in vitro and transplanted tumor growth.Methods:Cervical cancer Hela cell lines were cultured and divided into control group,cisplatin group,PES group and cisplatin+PES group that were treated with serum-free DMEM,cisplatin with final concentration of 10 μmol/L,PES 20 μmol/L and cisplatin 10 μmol/L combined with PES with 20 μmol/L,respectively;animal models with cervical cancer xenografts were established and divided into control group,cisplatin group,PES group and cisplatin+PES group who received intra-tumor injection of normal saline,10 μmol/L cisplatin,20 μmol/L PES as well as 10 μmol/L cisplatin+20 μmol/L PES,respectively.Cell proliferation activity,transplanted tumor volume and mitochondria apoptosis molecule expression were detected.Results:Cell viability value and Bcl-2 mRNA expression in cells of cisplatin group,PES group and cisplatin+PES group were significantly lower than those of control group while Bax,Caspase-3 and Caspase-9 mRNA expression in cells were significantly higher than those of control group;transplanted tumor volume and the Bcl-2 mRNA expression in transplanted tumor tissue of cisplatin group,PES group and cisplatin+PES group were significantly lower than those of control group while Bax,Caspase-3 and Caspase-9 m RNA expression in transplanted tumor tissue were significantly higher than those of control group.Conclusions:HSP70 inhibitor combined with cislatin can inhibit cervical cancer cell proliferation in vitro and transplanted tumor growth through mitochondrial apoptosis pathway.

OB glue paste technique for establishing nude mouse human gastric cancer orthotopic transplantation models

AIM： To establish nude mouse human gastric cancer orthotopic transplantation models using OB glue paste technique. METHODS： Using OB glue paste technique, orthtopic transplantation models were established by implanting SGC-7901 and NKN-45 human gastric cancer cell strains into the gastric wall of nude mice. Biological features, growth of the implanted tumors, the success rate of transplantation and the rate of auto-metastasis of the two models were observed. RESULTS： The success rates of orthotopic transplanration of the two models were 94.20% and 96%. The rates of hepatic metastasis, pulmonary metastasis, peritoneal metastasis, lymphocytic metastasis and splenic metastasis were 42.13% and 94.20%, 48.43% and 57.97%, 30.83% and 36.96%, 67.30% and 84.06%, and 59.75% and 10.53%, respectively. The occurrence of ascites was 47.80% and 36.96%. CONCLUSION： OB glue paste technique is easy to follow. The biological behaviors of the nude mouse human gastric cancer orthotopic transplantation models established with this technique are similar to the natural processes of growth and metastasis of human gastric cancer, and, therefore, can be used as an ideal model for experimental research of proliferative metastasis of tumors.

A three-factor preoperative scoring model predicts risk of recurrence after liver resection or transplantation in hepatocellular carcinoma patients with preserved liver function

BACKGROUND： No staging systems of hepatocellular carcinoma（HCC） are tailored for assessing recurrence risk. We sought to establish a recurrence risk scoring system to predict recurrence of HCC patients receiving surgical curative treatment（liver resection or transplantation）.METHODS： We retrospectively studied 286 HCC patients with preserved liver function receiving liver resection（n=184） or transplantation（n=102）. Independent risk factors were identified to construct the recurrence risk scoring model. The recurrence free survival and discriminatory ability of the model were analyzed. RESULTS： Total tumor volume, HBs Ag status, plasma fibrinogen level were included as independent prognostic factors for recurrence-free survival and used for constructing a 3-factor recurrence risk scoring model. The scoring model was as follows： 0.758×HBs Ag status（negative： 0; positive： 1）＋0.387×plasma fibrinogen level（≤3.24 g/L： 0; 〉3.24 g/L： 1）＋0.633×total tumor volume（≤107.5 cm3： 0; 〉107.5 cm3： 1）. The cutoff value was set to 1.02, and we defined the patients with the score ≤1.02 as a low risk group and those with the score 〉1.02 as a high risk group. The 3-year recurrence-free survival rate was significantly higher in the low risk group compared with that in the high risk group（67.9% vs 41.3%, P〈0.001）. In the subgroup analysis, liver transplantation patients had a better3-year recurrence-free survival rate than the liver resection patients in the low risk group（80.0% vs 64.0%, P〈0.01）. Additionally for patients underwent liver transplantation, we compared the recurrence risk model with the Milan criteria in the prediction of recurrence, and the 3-year recurrence survival rates were similar（80.0% vs 79.3%, P=0.906）.CONCLUSION： Our recurrence risk scoring model is effective in categorizing recurrence risks and in predicting recurrencefree survival of HCC before potential surgical curative treatment.

Effects of Ad-p27mt Gene Transfer on the Expression of Bax,Bcl-2,VEGF and MMP-9 in the Transplanted Liver Tumors in Nude Mice

In this study, the mechanism by which Ad-p27mt inhibits the growth, invasion and metastasis of transplanted liver tumor was studied by examining the effects of Ad-27mt gene transfer on the expression of Bax, Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice.The model of transplanted hepatic tumor was established in nude mice.The mice were then divided into three groups, which were injected with PBS, Ad-LacZ and Ad-p27mt and the growth of the transplanted liver tumor was observed.The expressions of P27, Bax and Bcl-2 proteins were detected by Western blotting and the expressions of VEGF and MMP-9 were immunohistochemically determined.Our result showed that the tumor size, expressions of Bax, Bcl-2 proteins, VEGF and MMP-9 were all lower than those in PBS and Ad-LacZ groups and the differences were statistically significant (P【0.05).Our study suggested that Ad-p27mt could inhibit the growth, invasion and metastasis of hepatic cancer by lowering the expressions of VEGF and MMP-9.

The Influence of Different Single Radiation Dose on Delayed Growth of Transplanted Tumor in Athymic Mouse

OBJECTIVE To reveal the biological effects and effective dosage in radiotherapy model which applies high single-dose irradiation by animal experiment. METHODS We inoculated subcutaneouly human pancreatic carcinoma cell line （MIA PaCa-2） in the lateral of the right lower extremity of the athymic mouse to grow transplantation tumor. While the median diameter of transplantation tumor reached 10 mm approximately, the animals were randomly divided into 7 groups （6 animals per group） and irradiation by different dose in one fixed with consciousness for fraction （0, 2, 5, 10, 17, 25, 35 Gy）. All were kept on to be bred for observation of the change in gross tumor volume, calculation of delayed growth time and delayed growth curve. RESULTS With increased dose per fraction, cutaneous reaction on the neoplasma surface of the animal, which was mainly moist yellow effusion was more and more severe. When dosage is less than 10 Gy, all animals showed similar effects, that＇s the delayed tumor growth was not obvious. Tumors receiving more than 10 Gy in one fraction showed very good biological effect and the delayed tumor growth was obviously related to dosage. The difference in delayed tumor growth between the 2 groups was statistically significant. The delayed tumor growth time in 10, 17, 25 Gy group was respectively 3 weeks, 6 weeks and more. CONCLUSION The biological effect of the model which applies high single-dose irradiation （more than 10 Gy in one fraction） was very good. The effect of delayed tumor growth was obviously related to the dosage after transplantation tumor was radiated. Because of its higher dose per fraction and biological effects, the model of high single-dose irradiation can get better clinical effects.

PERCUTANEOUS INJECTING ANTINEOPLASTIC AGENT INTO TRANSPLANTED TUMORS OF MICE BY CT－GUIDED

Antineoplastic agent and contrast medium were injected into transplanted tumors of mice under guidance with CT, site and range of the intratumoural drug were shown on CT image immediately. It was value of multipoint injections, concentration of 0.1 mg/0.l ml MMC every point, 1 cm interval of injection. After the injections, the tumor size of mice reduced and at last disappeared (ratio of inhibited tumor 59.32% in 0.05 mg MMC group, 43.86% in 0.1 mg MMC group).The pathologic examination showed coagulatic necrosis of the tumor tissues. The higher concentration of antineoplastic agent (0.2 mg MMC) could make the tumors enlarged (ratio of inhibited tumor -15.3%). The tissues and vessels around the tumors were not injured, if MMC overflow out off the tumor.

Combined use of retroperitoneal laparoscopy and bladder resectoscope to treat renal and ureteral tumor occurring at the same side of transplanted kidney (report of 5 cases)

To evaluate the operative characteristics and efficacy of retroperitoneoscopic resection of renal,ureter and partial bladder for the treatment of native renal pelvic and ureteral transitional cell cancer occurring at the same side of transplanted kidney.Methods In 5 cases of renal transplantation,there were 2 cases of right native renal pelvic cancer,1 case of right native renal pelvic and ureter cancer and 2 cases of right ureter cancer respectively.The transplanted kidney was in the same iliac fossa side of the tumor.All 5 patients were subjected to nephroureterectomy and bladder cuff excision by retroperitoneoscopic technique.Results Five operations were completed successfully.The operative time was 180 to 280 min,and the blood loss was 50 to 200 ml.The recovery of intestinal function after operation was 12 to 36 h.The urine output was 1 500 to 4 000 per day.Postoperative serum creatinine was still normal.The mean hospital stay after operation was 4.5 days.Conclusion Retroperitoneal laparoscopic nephroureterectomy and bladder cuff excision is a good method to treat the native renal pelvic and ureteral transitional cell cancer occurring at the same side of transplanted kidney.The procedure is safe and less invasive,which provides a good protection of transplanted kidney.12 refs.

Mechanism of AiTongXiao granule in the treatment of hepatocellular carcinoma based on network pharmacology and rat transplanted liver cancer model

Objective:To investigate the mechanism of action and material basis of AiTongXiao granule in the treatment of hepatocellular carcinoma(HCC)based on network pharmacology and transplanted liver cancer rat model.Methods:TCMSP database was used to screen out effective components and its corresponding potential pharmaceutical targets,and databases including Gene Cards,OMIM,Drugbank and TTD were further used to collect HCC-related drug targets.The intersecting targets were obtained by mapping the drug and disease targets.The component-targets network was constructed and visualized by Cytoscape 3.8.2 software.Protein-protein interaction(PPI)network was built by STRING online platform,and the topological relationship and core targets was analyzed and screened by using CytoNCA software.In addition,Metascape database was used to perform gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis of the core targets.At last,rat liver transplanted liver cancer model was established by using Walker-256 cell line and treated by AiTongXiao granule for 15 days.Western blot was used to further compare the expression levels of AKT,pAKT,p53,p-p53,ERK1/2 and ERK1/2 in the tumor between treatment group and the control group.Results:257 active components were obtained from AiTongXiao granule,corresponding to 294 drug targets.Meanwhile,233 of the 7993 HCC disease targets were screened out between AiTongXiao granule drug and HCC disease targets.11 core targets including AKT1,IL6,TP53,MAPK3,TNF,JUN,CASP3,MAPK1,MYC,PTGS2,MMP9 were further obtained by median screening.GO and KEGG analysis results showed that these core targets enriched to HBV,TNF and cancer related pathways.The rat transplanted liver cancer model results indicated significant down regulation for AKT,p-AKT,pERK1/2,and significant up regulation of p-p53 after AiTongXiao granule treatment(P<0.05).Conclusion:AiTongXiao granule could act to multiple cancer related pathways,and AKT,p53 and ERK1/2 were validated to be regulated by ATXF in rat model.The mechanism may be through the regulation of the above signaling pathways to exert anti-liver cancer effect.

Protective effect of antioxidant on renal damage caused by Doxorubicin chemotherapy in mice with hepatic cancer

Objective:To investigate the protective effects and mechanism of antioxidant TBHQ on renal damage caused by doxorubicin chemotherapy in mice with hepatic cancer.Methods:Cell H22 of mice with hepatic cancer which was subcultured for three times was subcutaneously transplanted to the groin of right lower limb of 45 SPF Kunming mice to establish the transplanted tumor model.The doxorubicin chemotherapy group and antioxidant intervention group received intraperitoneal injection of ADM(1 mg/kg·0.2 mL/2d).The model control group received normal saline(NS) of the same volume at the same time.1%TBHQ was added into the diet of mice of the antioxidant intervention group.Seven weeks later,morning urines and peripheral blood were randomly collected to detect UAIb,UCr,BUN,Scr and UAlb/Cr levels.All mice were beheaded.The renal tissues were made into homogenate,and SOD,T-AOC and MDA content in tissues were detected followed by cell lysis.All data were processed using SPSS 19.0.Results:The UAlb/Cr,BUN.Scr and MDA of doxorubicin chemotherapy group were significantly higher those of model control group and the activities of SOD,T-AOC in doxorubicin chemotherapy group were lower than those of model control group(P<0.01).The UAlb/Cr,BUN,Scr and MDA of antioxidant intervention group were lower than those of doxorubicin chemotherapy group and the activities of SOD,T-AOC of antioxidant intervention group were higher than those of doxorubicin chemotherapy group doxorubicin chemotherapy group(P<0.05).The BUN of model control group was higher than that of blank group,and T-AOC was lower than that of blank group,and difference was statistically significant(P<0.05).Conclusions:Doxorubicin chemotherapy could lead to abnormal antioxidant capacity and renal function of tumor-bearing mice with hepatic cancer.TBHQ antioxidant intervention could effectively improve the antioxidant capacity of renal tissue and reduce the renal damage caused by doxorubicin to some extent.

A Review of Progress in the Construction and Application of Human-Derived Xenograft Models for Bladder Cancer

The patient-derived xenografts (PDX) model is an animal model established by transplanting primary tumors or fresh tumor tissues of patient origin directly into immunodeficient mice, which preserves the heterogeneity and survival microenvironment of the primary tumor and is widely used in preclinical and precision medicine research of tumors. This article reviews the construction of the PDX model of human bladder cancer and the progress of the application of the PDX model in bladder cancer.

Effects of hydroxy safflower Yellow-A on tumor capillary angiogenesis in transplanted human gastric adenocarcinoma BGC-823 tumors in nude mice

OBJECTIVE:To study the effects of hydroxy safflower yellow A(HSYA) on tumor capillary angiogenesis in transplanted human gastric adenocarcinoma BGC-823 tumors in nude mice.METHODS:BGC-823 cells were injected subcutaneously into the right anterior armpit of nude mice to establish an animal model of transplanted tumors.After 24 h,18 nude mice injected with tumor cells were randomized into model,control,and HSYA 0.028 g/L groups,with six mice in each group.Transplanted tumors were excised on day 20.Tumor inhibition ratios were calculated for the transplanted tumors.Pathological changes and capillary angiogenesis in the tumors were observed by light microscopy.RESULTS:Tumors in the model group grew more quickly than those in the control and HSYA groups,with inhibition ratios of 48% and 30%,respectively.The microvessel count in the HSYA group was lower than in the model group(P<0.01),and microvessel density was also lower in the HSYA group(P<0.05).Pathological changes were more obvious in tumors in the model group compared to the HSYA group.CONCLUSION:HSYA inhibits the growth of transplanted BGC-823 tumors,and its effects on tumor capillary angiogenesis may represent one of the mechanisms responsible for this antineoplastic effect.

Activity of Recombinant Human Interleukin-15 against Tumor Recurrence and Metastasis in Mice

Transplantable experimental tumor models were constructed to study the activities of recombinant human interleukin-15 （rhIL-15） against tumor recurrence and metastasis. The results showed that tumor nodule formation was retarded and tumor growth was inhibited in the subcutaneous tumor model of LA795 lung adenocarcinoma after treatment with rhIL-15, and the survival rate of T739 tumor-bearing mice treated with rhIL-15 was much higher than that of mice treated with either saline or with the same dose of rhIL-2. This indicats that rhIL-15 had better antitumor effect than rhIL-2 at the same dose level. In some rhIL-15 treated mice, the tumor cells inoculated subcutaneously were eradicated and there was no tumor formation even 138 days after tumor cell inoculation. The tumor-free mice were rechallenged with live tumor cells and no tumor reoccurred in the following two months in all of these mice, indicating that long-lasting antitumor systemic immunity developed. It was also shown that tumor recurrence and metastasis were inhibited markedly after treatment with rhIL-15, but not with the same dose of rhIL-2, in both subcutaneously and intravenously disseminated tumor models of LA795 lung adenocarcinoma. Simultaneously, the CTL and NK cell activities of the splenocytes obtained from tumor-bearing mice that had been treated with either rhIL-15 or rhIL-2 were both markedly enhanced. However, the enhancement of CTL and NK cell activities was more significant in rhIL-15 treated mice than that in rhIL-2 treated mice. This suggests that the anti-tumor effect of rhIL-15 in vivo was achieved by enhancing the CTL and NK cell activities in tumor immune response.

Effect of Spirulina Platensis Polysaccharide on Hematopoietic Recovery and Related Cytokines in Mice with Transplanted Tumor Treated by Chemotherapy

Objective:To evaluate the effect of Spirulina platensis polysaccharide (SPP) on hematopoietic recovery and related cytokines in mice with transplanted tumor after chemotherapy. Methods:Mouse model of transplanted solid tumor was established and treated with chemotherapy. Peripheral blood cells, bone marrow nucleated cells, and colony forming unit spleen (CFU S) were counted; the content of DNA in bone marrow was inspected by ultraviolet spectrophotometer; serum content of cytokines, interleukin (IL)1, IL 3, granulocyte macrophage colony stimulating factor (GM CSF) and tumor necrosis factor α (TNF α) were determined by double antibody sandwich ELISA.Results:Cyclophosphamide (CTX) could induce evident myelosuppression, manifested as decrease of peripheral blood cells, bone marrow nucleated cell and DNA, and the CFU S number. SPP could significantly ameliorate the myelosuppression induced by CTX without reducing anti tumor effect of CTX. In addition, it could also increase the contents of IL 1, IL 3, GM CSF, TNF α in serum. Conclusion:SPP can probably accelerate the hematopoietic recovery in mice after chemotherapy through promoting endogenous secretion of cytokines.