High-intensity Focused Ultrasound Ablation For Comprehensive Therapy In Primary Liver Cancer Patients And Low-intensity Ultrasound Exposure For CD133Positive Population In SMMC-7721Cell Line With Multidrug Resistance

Background: Primary liver cancer is one carcinoma with high grademalignancy and poor prognosis. Comprehensive treatment of combinedseveral approaches reasonably and sequentially is becoming a tendency ofclinical treatment for patients with primary liver cancer. However, standardcombination treatment method remains to be determined. Althoughhigh-intensity focused ultrasound ablation (HIFU) combined withtransarterial chemoembolization (TACE) had been used for treatingunresectable patients with mid-late stage primary liver cancer, the role of thiscombination method in the treatment of mid-late stage primary liver cancerremains to be determined for present studies without long-term follow-upresults and detailed clinical analysis. Radiotherapy is also one effectivemethod for treating unresectable patients with primary liver cancer. It hasbeen demonstrated that radiotherapy combined with thermotherapy for treating cancer patients has synergistic effects. Similarly, it suspectstheoretically that a combination therapy method of radiotherapy and HIFUablation with main mechanism of heat effect in the treatment of cancerpatients has synergistic effects too,showing that this combination methodmaybe probably improve the efficacy for treating patients with primary livercancer, but fewer relative studies has been reported.It demonstrated that both resection and liver transplantation caneffectively prong the survival in patients with small primary liver cancer. Inchina, liver transplantation for treating small primary liver cancer is difficultto develop widespreadly because of absent liver donor and high expense.Present clinical trials showed the survival of small primary liver cancerpatients treated by radiofrequency ablation with the main mechanism ofhyperthermia effect is similar as compared to resection. The role ofradiofrequency ablation for small primary liver cancer is becomingimportant more and more for its advantages of mini-invasive feature andpreserving residual liver function as far as possible. As compared toradiofrequency ablation, HIFU ablation with the same main mechanism ofhyperthermia effect has advantages of non-invasive feature, the wholeprocedure guided and monitored really by real ultrasound images and thecandidate tumors without tumor location and size limits, indicating thatHIFU ablation may be a new optional method for treating small primaryliver cancer. However, fewer relative researches have been reported. Cancer stem cells (CSC) with the features of drugs and radiationresistance are highly correlated with cancer occurrence, development,recurrence and metastasis, which become hot in tumor studies. It is a newidea of inhibit CSC targetedly to obstruct cancer occurrence, development,recurrence and metastasis in order to probably improve treatment effects incancer patients. Liver CSC may be existed in liver cancer mass, which arehighly relevant to cancer occurrence, development, recurrence andmetastasis. Low-intensity ultrasound may increase the permeability of cellmembrane and influence the activities of signal pathway in cells by themechanisms of cavitation, sonochemistry and mechanical effects, which aredifferent to the radiobiological effects induced by radiation. No relativeresearches of the influence of low-intensity ultrasound exposure different toradiation on CSC with the features of multidrug resistance, radiationresistance and self proliferation and renewal have been reported. Once theresults show that low-intensity ultrasound exposure may change thebiological features of CSC and inhibit the cell proliferation, differentiationand resistance to chemoradiation in liver cancer stem cells, it is possible thatCSC in the liver tumor mass may be inhibited targetedly by the targetedirradiation of tumor mass using low-intensity ultrasound in order to improvethe treatment effects in liver cancer patients. Moreover, low-intensityultrasound exposure can effectively avoid the side effects and limitationsinduced by HIFU ablation because of its lower acoustic power, indicating that it be probably used to really treat tumor safely and noninvasively. Inaddition, this treatment method may provide the new direction of ultrasoundtherapy and improve treatment effects of HIFU ablation as a supplementapproach.Objective:1. To investigate the safety and long-term effect forassessing the feasibility of high-intensity focused ultrasound ablationcombined with transarterial embolization (TAE) for patients with smallprimary liver cancer in order to evaluate the role of this combinationapproach in the treatment of these clinically.2. To investigate the safety and long-term effect of HIFU ablationcombined with transarterial chemoembolization for treating patients withunresectable mid-late stage of primary liver cancer and find majorprognostic factors by clinical analysis in order to evaluate the role of thiscombination approach in the treatment of these patients clinically.3. To investigate the safety and efficacy of HIFU combined withradiotherapy for treating patients with primary liver cancer in order to assessthe feasibility of this combination method in the treatment of these patientsclinically.4. To investigate the influence of low-intensity ultrasound exposure onthe multidrug resistance (MDR) and cell proliferation in liver cancerstem-like cells in order to assess the effect of low-intensity ultrasoundexposure on liver cancer stem cells. Methods:1.30unresectable small primary liver cancer patients (30liver tumors) with cirrhosis accepted HIFU ablation combined with TAE. Allpatients received mid-long term follow-up. The changes of liver function,the level of tumor markers and response of enhanced CT or MRI scanningbefore and after HIFU ablation were observed. Survival analysis wasassessed by Kaplan-Meier method.2. A total of73patients with unresectable HCC (III A25, ⅣA48;TNM classification,5th edition) were enrolled in this study. Seriouscomplications of both short and long term were observed. All patientsreceived follow up after HIFU ablation combined with TACE. Survivalanalysis was evaluated by Kaplan-Meier method. The Variables of gender,age, AFP level, liver function, tumor location, tumor number, tumor size,TNM staging system, portal vein invasion, ultrasonic pathway of HIFU (ribresection), TACE session and ablation response were evaluated byunivariate analysis. The variables with significant difference resulted fromunivariate analysis were assessed by multivariate analysis using Coxregression method.3.25liver tumors in20liver cancer patients (16primary liver cancer,4liver cholangiocellular carcinoma) after3dimension conformal radiotherapyor stereotactic radiotherapy accepted HIFU ablation. All patients receivedfollow-up. The changes of skin situation, liver function and peripheral bloodof blood cells counts before and after HIFU ablation were observed. Ablation responses2weeks after HIFU were evaluated by enhanced MRI.Survival analysis was assessed by Kaplan-Meier method.4. Liver cancer cell line of SMMC-7721with multidrug resistance(MDR) was established by gradually increasing the concentration ofadriamycin (ADM) during cell culture. MDR of the new establishedsubgroup was identified by evaluating the resistance index. The CD133positive population of MDR cell line was sorted by magnetic activated cellssorting (MACS). The rate of CD133positive was assessed by FACS. BothCD133positive and negative population were irradiated by low-intensityultrasound. The mRNA expression of MDR1, cyclinD1and β-catenin indifferent subgroups were evaluated by reverse transcription polymerasechain reaction (RT-PCR). The protein expression of MDR and β-cateninwere measured by Western blot method.Results:1. The mean period of follow up was49.7±28.6months (range,12-120months). The median survival time was82.3months (95%CI:75.8-108.3months), and overall survival rates of1,3,5-year were96.7%,73.0%and63.8%,respectively. The tumors in29patients acquired completeablation were observed by enhanced CT or MRI scanning2weeks afterHIFU. The rate of complete ablation was96.7%(29/30). The rates ofcumulative recurrence and metastasis in the liver at1,2,3and4-year afterHIFU+TAE were11.1%,22.3%,41.7and52.3%, respectively.No serious complications including gastric-intestine tract perforation were observed after HIFU+TAE.2ribs inside the region of ultrasoundpathway in2patients were fractured induced by HIFU ablation duringmid-long term follow-up. No significant difference in peripheral blood totalbilirubin, albumin and prothrombin activity between before and after HIFUwas found (t test, all P>0.05). The level of α-fetoprotein (AFP) in22AFPpositive patients was decreased from742.1±1120.9ng/ml before HIFU to319.9±405.8ng/ml1week after HIFU. The significant difference in the AFPlevel between before and1week after HIFU was found (t test, P<0.01).2. The mean period of follow up was11.7±11.1months (range,1-60months). At the end of follow-up,51patients (69.9%) died from tumorprogression (27patients), liver function failure (18patients), hemorrhage ofupper digestive tract (3patients) and infection (3patients). The mediansurvival time was12months (95%CI:9-15months), and overall survivalrates of1,2,3-year were49.1%,18.8%and8.4%,respectively.33patients(45.2%) received complete ablation.1patient with liver abscess,2withserious skin burns and2with rib fracture were observed after HIFU ablation.On univariable analysis, age (P=0.017), tumor size (P=0.000), tumornumber (P=0.039), TNM staging system (P=0.023), portal vein invasion(P=0.02) and ablation response (P=0.000) had significant difference. Onmultivariable analysis, ablation response (P=0.001) and tumor size (P=0.013)were major prognostic predictors.3. The mean follow-up time was12.6±8.0months (range,1-27months). The median survival time and1-year survival rate were22months and87.5%respectively. At the end of follow-up,3patients died. No serious skinburns were observed in all patients. No serious complication includinggastric-intestine perforation was found after HIFU ablation. There were nosignificant differences in the levels of albumin, alanine transarninase,aspartate transarninase, total bilirubin, direct bilirubin, blood red cell counts,blood white cell counts and blood platelet counts between before and1weekafter HIFU (paired t test, P=0.156,0.356,0.203,0.659,0.531,0.519,0.310,0.346, respectively). Among25liver tumors, sixteen with completeablation and six with ablation volume of≥80%were observed by enhancedMRI at2weeks after HIFU.4. The new established subgroup of SMMC-7721induced by ADM hadresistance to ADM, vincristine (VCR) and cisplatin (DDP), indicating thatit had MDR characteristics. The rates of CD133positive population inSMMC-7721line and MDR line induced by ADM were1%and10.26%respectively assessed by flow cytometric analysis cell scan (FACS). Therates of CD133positive population in CD133positive and negative groupsorted by MACS were0.005%and45.16%respectively assessed by FACS.The mRNA expression of MDR1, cyclinD1and β-catenin were the highestin CD133positive population of MDR line assessed by RT-PCR. Ascompared to the groups before low-intensity ultrasound exposure, themRNA expression of MDR1, cyclinD1and β-catenin in the groups irradiated by low-intensity ultrasound were decreased variably. Although the proteinexpression of MDR and β-catenin in all groups were found by Western blot,it was expressed highest in CD133positive MDR cells. As compared to thegroups before low-intensity ultrasound exposure, the protein expression ofMDR and β-catenin in the groups irradiated by low-intensity ultrasoundwere decreased variably.Conclusions:1. A combination approach of HIFU ablation and TAEis a safe and effective method for treating unresectable small primary livercancer patients with cirrhosis. This comprehensive therapy method may beone of selective and effective approaches in the treatment of small primaryliver cancer patients who are unresectable or unwilling to accept operation.HIFU ablation combined with TAE could acquire high rate of tumorcomplete ablation in the treatment of small primary liver patients, indicatingthat this combination approach may be one of effective methods for treatingsmall primary liver cancer patients planned to receive liver transplantation inorder to reduce the probability of missing liver transplantation or influencethe efficacy of liver transplantation because of tumor progression.2. As a safe and effective combination approach, HIFU combined withTACE is one of selective comprehensive therapy methods in the treatment ofunresectable patients with mid-late stage liver cancer. As major prognosticpredictors, ablation response and tumor size could help us predict thesurvival and select eligible patients clinically. Meanwhile, ablation response and tumor size may be used for prognostic evaluation in these patients.3. HIFU could ablate viable residual tumor safely and effectively inliver patients after radiotherapy to reduce the probability of tumor recurrenceand may improved the survival, showing that HIFU combined withradiotherapy is a effective comprehensive therapy method for treating livercancer and a safe, effective and feasible new approach for treating livercancer.4. SMMC-7721cell line with MDR can be established by graduallyincreasing the concentration of ADM during cell culture. Chemotherapydrugs including ADM may increase the amount of liver cancer stem-likecells to enrich liver cancer stem-like cells. Low-intensity ultrasoundexposure might effectively invert MDR in liver cancer stem-like cells, whichmight be a physical approach for effectively improving MDR in liver cancerstem cells. Low-intensity ultrasound exposure may inhibit the transductionof Wnt/β-catenin signal pathway to inhibit cell proliferation in liver cancerstem-like cells, which might be a physical method for inhibiting cancer stemcells.