Alteration Of Metastatic Potential Of Hepatocellular Carcinoma After Radiofrequency Ablation-an Experimental Study

Hepatocellular carcinoma(HCC) is the third most common cause of death from cancer in the world and the second cancer killer in China.Surgical resection remains the most effective treatment for HCC.Unfortunately,less than 20%of patients with HCC are candidates for resection at the time of diagnosis because of multinodular pattern,vascular invasion and liver cirrhosis.Since early 1990s,radiofrequency ablation(RFA) has been widely used to treat liver tumor and developed rapidly.For small HCC,randomized control trials indicated that RFA yielded similar long-term survival as compared to resection.However,the residual cancer rate and recurrence rate remain high after RFA.Even after curative RFA,residual cancer could be detected in 3%-52%of patients and recurrence rate has been reported up to 53%-81%.Questions raise based on this observation:which one of RFA and resection can promote the invasiveness and metastatic potential of residual cancer? What can be done to improve the outcome of RFA? Our present study was designed to clarify these issues by treating nude mice bearing human HCC orthotopic xenografts with RFA and palliative reseetion.After treatment the invasiveness and metastatic potential of residual HCC was evaluated and possible molecular background was investigated using invasion and metastasis related cDNA microarray(Oligo Tumor Metastasis Microarry,Super Array) and Real-time PCR. Moreover,we try to explore combination therapies to improve the outcome of RFA. We found that RFA inhibits the invasion and metastasis of residual cancer associated with down regulation of MET and up-regulation of CDKN2A,FAT,COL4A2 and TIMP2 in different phases;whereas palliative resection promotes the invasion and metastasis of residual cancer associated with upregulation of MMP-9,MMP-10 and MTA2.Combined Pseudomonas aeruginosa vaccine(PA) can further inhibit the growth and metastasis of residual cancer after RFA associated with down regulation of HGF and up-regulation of KAI1;and may be a novel approach to improve outcome of RFA for HCC. 1.Alteration of the invasiveness of MHCC97L cells after thermotherapy in vitroAlteration of the proliferation and invasiveness of MHCC97L cells after thermotherapy was examined using water bath of 42℃(30min×2).Compared to the control group,the proliferation of MHCC97L cells was significantly increased after thermotherapy(P＜0.01),the augmented ratio of proliferation reached the maximum at 48h after thermotherapy to 27.9%and the doubling time decreased 15%,the plate efficiency of cells at 48h after thermotherapy decreased and increased at 96h(9.73±0.29 Vs 8.87±0.59,9.37±0.26 Vs 11.9±0.70)(P＜0.01),the ratio of G1 phase decreased and S+G₂ phase increased of ceils at 48h and 96h after thermotherapy by flow cytometric analysis(P＜0.01 and P＜0.05).The average amount of invading cells per field in cell invasion assay and motility assay of the same number of cells 48h and 96h after thermotherapy were significantly lower than control group(P＜0.01 and P＜0.05).This decrease in invasiveness was associated with down regulation of expression of MMP-2 and VEGF.The levels of protein for MMP-2 and VEGF were determined by Elisa analysis in supernatant of the same number of cell culture and showed a significant decrease after thermotherapy. The maximum inhibition ratio reached to 37.5%(48h) and 43.7%(72h),respectively.These results suggested that thermotherapy(water bath,42℃) increased the proliferation of MHCC97L cells and inhibited invasiveness of them in vitro.2.Alteration of the metastatic potential of residual HCC after R1FA in vivo2.1.Comparison between RFA and control group:1 and 4 weeksIn order to investigate the metastatic potential of residual HCC after RFA in vivo, Nude mice model with orthotopic implantation of metastatic human HCC(MHCC97L) was employed.The mice were randomized into different groups and were performed a palliative RFA/sham operation(control group) 30 days after implanation.The mice were sacrificed 1 or 4 weeks afer RFA,and tumor volumes,incidences of lung metastasis,lung metastatic nodules were examined.A Part of residual cancers were replanted into the liver of other nude mice and were sacrificed 6 weeks after replantation.Again,tumor volumes,incidences of lung metastasis,lung metastatic nodules were measured.Compared with their controls,the tumor volume of 1 week and 4 weeks-post-RFA groups were 1570±380mm³ vs 1450±460mm³,and 2290±430mm³ Vs 1540±930mm³(P＞0.05);also,statistical difference was not found regarding incidence of lung metastasis of 4 weeks-post-RFA group[85.7%(6/7) vs 85.7%(6/7)](P＞0.05), however,the number of the lung metastatic nodule were markedly decreased(23.57±14.26 vs 9.86±7.43)(P＜0.05).Compared to its replanted control group,the replanted tumor volume of 1 week-post-RFA group were markedly decreased(1240±440mm³ vs 30±30mm³) (P＜0.05),the incidence of lung metastasis and the number of the lung metastatic nodule were also decreased[40%(2/5) vs 0(0/5) and 0.8±1.3 vs 0]but not significant (P＞0.05).Compared to its replanted control group,the replanted tumor volume of 4 weeks-post-RFA group showed no obvious difference(720±510mm³ Vs 800±270mm³)(P＞0.05),the incidence of lung metastasis were decreased[57.1%(4/7) vs 28.6%(2/7)]without significance(P＞0.05),the number of the lung metastatic nodule were markedly decreased(2.00±1.91 Vs 0.29±0.49)(P＜0.05).Invasion and metastasis related cDNA microarray and Real-time PCR was used to analyze changes of gene expression profile after RFA.The results showed that the mRNA of MET,which can promote invasion and metastasis was significantly down regulated in residual cancers of 1 week-post-RFA.While the mRNA of CDKN2A and FAT,two factors that inhibit invasion and metastasis were up-regulated in residual cancers 4 weeks-post-RFA.These results were associated with the suppressed metastatic potential of residual HCC after RFA.These results suggested that compared to control group,RFA suppressed the metastatic potential of short term residual HCC,and were associated with differential expressed genes of MET,CDKN2A and FAT.2.2.Comparison between RFA and palliative resection in vivo:6 weeksIn order to clarify the long-term alteration of metastatic potential of residual HCC after RFA and compared with palliative resection,metastatic nude mice model with human HCC was employed.The mice were performed a palliative RFA/resection/sham operation(control group) 30 days after implanation and were sacrificed 6 weeks later.The tumor volumes,incidences of lung metastasis,lung metastatic nodules were measured.Parts of the residual cancers were replanted into the liver of other nude mice and were sacrificed 6 weeks after replantation.The tumor volumes,incidences of lung metastasis,lung metastatic nodules were examined.Compared to its control group,the tumor volume of 6 weeks-post-RFA group were increased(2430±380mm³ vs 3340±970mm³) without significance(P＞0.05),the incidence of lung metastasis showed no obvious difference[100%(6/6) Vs 100% (6/6)](P＞0.05),the number of the lung metastatic nodule were markedly decreased (18.00±7.54 Vs 9.17±5.64)(P＜0.05).Compared to its control group,the tumor volume of 6 weeks-post-resection were markedly increased(2430±380mm³ Vs 3640±1240mm³)(P＜0.05),the incidence of lung metastasis showed no obvious difference[100%(6/6) Vs 100%(6/6)](P＞0.05),the number of the lung metastatic nodule were markedly increased(18.00±7.54 Vs 30.33±7.40)(P＜0.05).Compared to replanted control group,the replanted tumor volume of 6 weeks-post-RFA group were decreased(2800±1000mm³ Vs 2200±830mm³) without significance(P＞0.05),the incidence of lung metastasis were decreased [83.3%(5/6) vs 50.0%(3/6)]without significance(P＞0.05),the number of the lung metastatic nodule were markedly decreased(4.2±3.3 Vs 0.8±1.1)(P＜0.05). Compared to replanted control group,the replanted tumor volume of 6 weeks-post-rsection group were decreased(2800±1000mm³ vs 2100±720mm³) without significance(P＞0.05),the incidence of lung metastasis were increased [83.3%(5/6) vs 100%(6/6)]without significance(P＞0.05) and the number of the lung metastatic nodule were markedly increased(4.2±3.3 Vs 10.2±4.3)(P＜0.05).Invasion and metastasis related cDNA microarray and Real-time PCR was used to analyze changes of gene expression profile after RFA/palliative resection.The results showed that the mRNA of COL4A2 and TIMP2,which can inhibit invasion and metastasis,were significantly up-regulated in residual cancers after RFA,while the mRNA of MMP-9,MMP-10 and MTA2,which can promote invasion and metastasis, were up-regulated in residual cancers after palliative resection.These results were associated with the alteration of metastatic potential of residual HCC after RFA/palliative resection.The life span of nude mice after RFA(61 d±5d) and palliative resection(65d±8d) was slightly longer than their control group(57d±6d),respectively,but without significance(P＞0.05),which suggested that both palliative radiofrequency and palliative resection could not significantly improve the outcome of HCC in vivo.These results suggested that compared to control group,RFA suppressed the metastatic potential of long term residual HCC,while palliative resection enhanced the metastatic potential of long term residual HCC;which were associated with differential expressed genes of COL4A2,TIMP2 and MMP-9, MMP-10,MTA2,respectively.3.Indomethacin and PA combined with RFA improved outcome of HCC3.1.1ndomethacin and PA inhibited the proliferation and invasiveness of MHCC97L cells in vitroAlteration in the proliferation and invasiveness of MHCC97L cells after using Indomethacin and PA at various concentrations was examined.Compared to its control group,Indomethacin can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose dependent manner.The inhibition ratio increased and plate efficiency decreased gradually when cells treated with 0.1,0.2,0.5mM Indomethacin(P＜0.01).Indomethacin can induce cell cycle arrest at the G1 phase in a dose dependent manner by flow cytometric analysis.The average amount of invading cells per field in cell invasion assay and motility assay of the same number of cells were 2.2±1.3 and 4.4±1.1 after cells treated with 0.2mM Indomethacin for 72h,significantly lower than control group(11.4±1.9 and 12.8±1.8)(P＜0.01).This decrease in invasiveness was associated with down regulation of expression of VEGF and MMP-2.The levels of protein for VEGF and MMP-2 were determined by Elisa analysis in supematant of the same number of cells culture and showed a significant decrease compared to the control group:VEGF, (607.8±22.8) pg/ml vs(869.2±8.6) pg/ml(P＜0.01);MMP2(Diluted 200 times), (46.6±4.1) pg/ml vs(76.1±4.2) pg/ml(P＜0.01).These results suggested that Indomethacin could inhibit invasiveness of HCC cell line MHCC97L,which were associated with the VEGF and MMP2 protein activity.Compared to its control group,PA can inhibit the proliferation and plate efficiency of MHCC97L cell markedly in a dose dependent manner.The IC50 of cells treated with PA for 48h and 72h were 3.1×10⁸/ml and 1.9×10⁸/ml,respectively.The doubling time increased and plate efficiency decreased gradually when cells treated with 0.5×10⁸/ml,1×10⁸/ml,2×10⁸/ml PA(P＜0.01).PA can induce cell cycle arrest at the G1 phase in a dose dependent manner by flow cytometric analysis.The average amount of invading cells per field in cell invasion assay and motility assay of the same number ofcells were 4.8±1.3 and 8.8±2.2 after cells were treated with1×10⁸/ml PA for 72h,significantly lower than control group(8.6±2.1 and 15.6±1.2 )(P＜0.01).Comparison between the same number of cells treated with1×10⁸/ml PA and control group,no remarkable difference was found regarding expression of VEGF and MMP2 in supematant of cell culture.This result suggested that PA could inhibit invasiveness of HCC cell line MHCC97L,which was unrelated to the VEGF and MMP2 protein activity.3.2.Indomethacin and PA Inhibit the growth and metastasis of HCC in vivo In order to investigate the inhibition effects of Indomethacin and PA on HCC in viva,metastatic nude mice model of human HCC was employed and were randomized into 4 groups:control,Indomethacin(3mg/kg·d),PA(4.4×10⁸/d), low-dose Indomethacin(1.5mg/kg·d) and PA(2.2×10⁸/d).The mice were administrated different therapies for 40 days and sacrificed 24h later.The tumor volumes,incidences of lung metastasis,lung metastatic nodules were detected.Compared to control group,the tumor volume of PA group,low-dose Indomethacin and PA group were markedly decreased with inhibition ratio of 36.7%and 62.2%, respectively(P＜0.05 and P＜0.01),the incidence of lung metastasis were markedly decreased,respectively[both are 66.7%(4/6) vs 0(0/6)](P＜0.05).Compared to control group,the tumor volume,incidence of lung metastasis and lung metastatic nodules of Indomethacin group were decreased[1670±370 Vs 1130±610, 66.7%(4/6) vs 50%(3/6),2.34±0.95 vs 1.75±0.71,repectively]without significance (P＞0.0s).In order to investigate the mechanism involved in this process, immunohistochemistry assay(IHA) was used to investigate the expression of COX-2 following different therapies.Compared to control group,the expression of COX-2 slightly down regulated in PA group,and significantly down regulated in low-dose Indomethacin and PA group.These results suggested suggested PA,low-dose Indomethacin and PA suppressed growth and metastasis of HCC in viva,which were associated with down regulation of COX-2.3.3.Indomethacin and PA suppressed thermotherapy augmented proliferation of MHCC97L cells,further inhibited the invasiveness in vitroIndomethacin(0.2mM) or PA(1×10⁸/ml) was added 2h before thermotherapy, Chemicals and thermotherapy were omitted for the thermotherapy and control group, respectively.Compared to the control group,the proliferation of MHCC97L cells were significantly decreased after thermotherapy combined Indomethacin or PA, respectively(P＜0.01),and reached the maximum at 48h after thermotherapy with inhibition ratio up to 53.6%,42.3%and the doubling time increased 2.07,1.68 times, respectively.The plate efficiency of the same number of cells 48h and 96h after thermotherapy combined Indomethacin or PA further decreased,respectively (P＜0.01).Flow cytometric analysis:The ratio of G1 phase increased and S+G2 phase decreased of cells 48h and 96h after thermotherapy combined Indomethacin or PA, respectively,which suggested that Indomethacin and PA could inhibit proliferation and plate efficiency of cells after thermotherapy,which were in part mediated by the cell cycle arrest at the G1 phase.The average amount of invading cells per field in cell invasion assay and motility assay of the same number of cells 48h and 96h after thermotherapy combined Indomethacin or PA were significantly lower than solitary thermotherapy group, respectively(P＜0.01).The decrease in invasiveness was associated with further down regulation of expression of MMP-2 and VEGF after thermotherapy combined Indomethacin,which were determined by Elisa analysis in supematant of the same number of cells culture and showed a significant decrease compared to the solitary thermotherapy group.But comparison between cells added PA 2h before thermotherapy and solitary thermotherapy group,no remarkable difference was found regarding expression of VEGF and MMP2 in supernatant of the same number of cells culture(P＞0.05).These results suggested Indomethacin could further inhibit invasiveness of HCC cell line MHCC97L after thermotherapy,which were associated with the VEGF and MMP2 protein activity;PA could further inhibit invasiveness of HCC cell line MHCC97L after thermotherapy,which were unrelated to the VEGF and MMP2 protein activity.3.4.Combined PA(2.2×10⁸/d) further suppressed the growth and metastasis of residual HCC and prolonged life span after RFA in vivoIn order to investigate combined therapies after RFA in vivo,metastatic animal model of human HCC and palliative RFA same as above and were randomized into 4 groups:control(chemicals and RFA were omitted),RFA(chemicals were omitted), RFA combined Indomethacin(3mg/kg·d),RFA combined PA(2.2×10⁸/d).The mice were performed a palliative RFA/sham operation(control group) 30 days after implanation.The mice were administrated different chemicals(or omitted) 48h later for 40 days and sacrificed 24h later.The tumor volumes,incidences of lung metastasis,lung metastatic nodules were detected.Parts of the residual cancers were replanted into the liver of other nude mice and were sacrificed 6 weeks after replantation.The tumor volumes,incidences of lung metastasis,lung metastatic nodules were detected.Compared to RFA group,the tumor volume of RFA combined Indomethacin group were increased(3340±970mm³ vs 3730±840mm³) without significance(P＞0.05), the incidence of lung metastasis were decreased[100%(6/6) vs 83.3%(5/6)]without significance(P＞0.05),the number of the lung metastatic nodule were increased(9.17±5.64 Vs 13.83±15.72) without significance(P＞0.05);Compared to solitary RFA group,the tumor volume of RFA combined PA group were markedly decreased(3340±970mm³ vs 1940±450mm³)(P=0.01),the incidence of lung metastasis were decreased[100%(6/6) vs 50.0%(3/6)]without significance(P＞0.05),the number of the lung metastatic nodule were markedly decreased(9.17±5.64 vs 2.83±3.49) (P＜0.05).Compared to replanted RFA group,the replanted tumor volume,the incidence of lung metastasis,the number of the lung metastatic nodule of RFA combined Indomethacin group showed no obvious difference[2200±830mm³ vs 2010±430mm³,50.0%(3/6) vs 50.0%(3/6),0.8±1.2 vs 0.7±0.8](P＞0.05).Compared to replanted solitary RFA group,the replanted tumor volume of RFA combined PA group were decreased(2200±830mm³ vs 1800±140mm³) with significance (P＞0.05),the incidence of lung metastasis were decreased[50.0%(3/6) vs 0(0/6)] without significance(P＞0.05),the number of the lung metastatic nodule were markedly decreased(0.8±1.2 vs 0)(P＜0.05).These results suggested that combined PA(2.2×108/d) further suppressed the growth and metastasis of residual HCC after RFA.Invasion and metastasis related cDNA microarray and Real-time PCR was used to analyze changes of gene expression profile after different therapies.The results showed that the mRNA of KAI1 which can inhibit invasion and metastasis,were significantly up-regulated,while the mRNA of HGF which can promote invasion and metastasis were down regulated in residual cancer of RFA combined PA group. Immunohistochemistry assay(IHA) showed that compared to solitary RFA group,the expression of COX-2 in RFA combined PA group significantly down regulated. These results were associated with the further suppressed metastatic potential of residual HCC after RFA combined PA.The life span of nude mice of RFA combined with PA group(86d±10d) was significantly longer than control group(57d±6d) and RFA group(57d±6d), respectively;RFA combined Indomethacin group(50d±3d) showed no obvious difference(P＞0.05).This result suggested that combined administration of PA (2.2×108/d) prolonged life span after RFA.Conclusions1.The metastatic potential of residual HCC was suppressed after RFA,and associated with down regulation of MET and up-regulation of CDKN2A,FAT, COL4A2 and TIMP2 in different phase.2.Palliative resection enhanced the metastatic potential of the residual HCC,and associated with up-regulation of MMP-9,MMP-10 and MTA2.3.Combined administration of PA(2.2×108/d) further suppressed the growth and metastasis of residual HCC and prolonged life span after RFA.These results associated with down regulation of HGF,up-regulation of KAI1.4.Low-dose Indomethacin(1.5mg/kg·d) and PA(2.2×10⁸/d markedly suppressed growth and metastasis of HCC in vivo.These results associated with down regulation of COX-2 expression.The potential application of this work1.Palliative resection but not RFA enhanced the metastatic potential of residual HCC,which will help to select optimal ways to improve the outcome of HCC in clinical.2.Combined PA further suppressed the growth and metastasis of residual HCC and prolonged life span after RFA,thereby provides a new clue to improve the outcome of RFA for HCC.3.Combination with Indomethacin and PA markedly suppressed growth and metastasis of HCC in vivo,thus provides a new approach for treatment of HCC.Originalities of this work1.Clarify the alteration of invasion and metastatic potential of residual HCC after RFA and compared with palliative resection,which will help for treatment choice of HCC patients.2.Demonstration that RFA combined with PA could further suppressed the growth and metastasis of residual HCC and prolonged life span.3.Pointout that combination of Indomethacin and PA markedly suppressed growth and metastasis of HCC.