The Impact And Its Mechanisms On Circulating Tumor Cells After Transcatheter Arterial Embolization To Hepatocellular Carcinoma

Part IEstablishment of stable enhanced green fluorescent protein expressing (EGFP) hepatocellular carcinoma (HCC) model in Buffalo ratsPurpose:To establish a transplanted EGFP expressing HCC model in Buffalo rats and evaluate its characteristics of tumor growth, metastasis, circulating tumor cells (CTCs) and to study imaging findings of the model.Materials and Methods:McA-RH7777/EGFP cells were subcutaneously injected into the Buffalo rats’ right thighs. Pieces of tumor tissue were implanted into the left lobes of the livers in 30 rats and right thighs in 10 rats. The subsequent growth, invasion, metastasis of the transplanted tumors were observed and evaluated with doppler ultrasonography, CT, MRI, DSA, PET-CT. The stability and advantage of EGFP expression in tumor cells were observed and evaluated with fluorescent microscopy. The number of CTCs of the orthotopic tumor model and the subcutaneous tumor model was calculated by flow cytometry. The different rates of lung metastasis were observed after animals were sacrificed on day 28.Results:Lung metastasis was detected in orthotopic implantation rats in their advanced-stage. EGFP expression in tumor cells was stable during their growth in vivo. The invasion and metastasis of tumor were readily observed and accurately evaluated by whole-body fluorescence optical imaging system and fluorescent microscopy. Doppler ultrasound showed hyperechoic nodular or oval mass with clear boundary. The tumor demonstrated well-demarcated hypo-intensity signal on T1WI and hyper-intensity signal on T2WI and DWI. Heterogeneous signal was showed with the enlargement of the lesions. The liver tumors were manifested by CT as low density foci. Lung metastases occured in peripheral lung with rounded shape and clear boundary. Some metastases collected together as irregular regiment mass. DSA demonstrated a mass with nodular or circular tumor staining, and with enlarged or twisted feeding artery. Iodized oil deposited in the lesions after iodized oil infusion. Portography demonstrated portal vein was not the tumor-feeding vessel. PET-CT scan revealed that the tumor lesions had higher levels of glucose metabolism. Small metastases in lung could be readily observed and tumor-feeding artery could be clearly showed with fluorescent microscopy. Futhermore, the change of GFP intensity gradually weakened of the lesions during microwave ablation treatment could be dynamically observed with fluorescent microscopy. Compared with subcutaneous implantation rats, orthotopic implantation rats had more CTCs, which increased with the enlargement of the lesions and higher rate of lung metastasis.Conclusion:The EGFP tagged HCC models in Buffalo rats are superior for the detection and studying relevant characteristics of HCC invasion and metastasis in vivo. These rat models have the similar imaging and pathological patterns to that of human HCC. These rat models with ideal implantation and tumor growth rate and high rate of lung metastasis are suitable for the researches about tumor metastasis and interventional therapy of human HCC. Portography demonstrated portal vein was not the tumor-feeding vessel. PET-CT scan revealed that the tumor lesions had higher levels of glucose metabolism. Small metastases in lung could be readily observed and tumor-feeding artery could be clearly showed with fluorescent microscopy. Futhermore, the change of GFP intensity gradually weakened of the lesions during microwave ablation treatment could be dynamically observed with fluorescent microscopy. Compared with subcutaneous implantation rats, orthotopic implantation rats had more CTCs, which increased with the enlargement of the lesions and higher rate of lung metastasis.Conclusion:The EGFP tagged HCC models in Buffalo rats are superior for the detection and studying relevant characteristics of HCC invasion and metastasis in vivo. These rat models have the similar imaging and pathological patterns to that of human HCC. These rat models with ideal implantation and tumor growth rate and high rate of lung metastasis are suitable for the researches about tumor metastasis and interventional therapy of human HCC.Part ⅡTranscatheter arterial embolization (TAE) facilitated lung metastasis by increasing the number of circulating tumor cells in transplanted hepatocellular carcinoma (HCC) Buffalo ratsPurpose:To evaluate whether transcatheter arterial embolization (TAE) can facilitate lung metastasis by increasing the number of circulating tumor cells (CTCs) in transplanted hepatocellular carcinoma (HCC) Buffalo rats.Materials and Methods:McA-RH7777/EGFP cells were subcutaneously implanted into the Buffalo rats’ right thighs. Pieces of tumor tissue were transplanted into the left lobes of the livers in 42 rats. On day 14 after tumors implanted, tumor-bearing rats were randomly divided into two groups (n=21):group Control (saline,0.6ml/kg), group TAE (low dose iodized oil,0.2ml/kg). Body weights and tumor volumes were evaluated.15 rats in each group were sacrificed to detect liver and lung metastasis. The influence of TAE on invasiveness was observed and evaluated by tumor tissue with HE and transmission electron microscopy (TEM). The number of CTCs of two groups at different points was calculated by flow cytometry.Results:Body weights in two groups were similar before treatment. The mean body weights and 217.30 ± 10.59 g in Control group and 233.40 ± 6.13 g in TAE group respectively on day 21 after treatment, P<0.05. TAE obviously inhibited liver tumor growth on day 7 and 14 after treatment. The liver and lung metastasis rates were no significant difference between two groups on day 14 after treatment, but the number of lung metastasis foci was higher in TAE groups than that in Control group. Pathological examination with HE revealed the liver tumors were unclearly boundary with vessels involved and tumor thrombus in portal vein in TAE group. The chief characteristics of liver tumors in TEM were:the perivascular erosion, tumor cells invasion, the incomplete basement membrane, etc.There were no significant difference in the number of CTCs between two groups on day 0,1 and 3 after treatment. The average level of CTCs was significantly increased by TAE treatment from day 7 to day 21 (day 7:51.80 vs 72.93/106 PBMC cells; day 14:92.73 vs 189.20/106 PBMC cells; day 21:285.33 vs 592.50/106 PBMC cells, P<0.05 for all)Conclusion:TAE can inhibit liver tumor growth but facilitate lung metastasis by increasing the number of CTCs in transplanted HCC Buffalo rats. iodized oil,0.2ml/kg). Body weights and tumor volumes were evaluated.15 rats in each group were sacrificed to detect liver and lung metastasis. The influence of TAE on invasiveness was observed and evaluated by tumor tissue with HE and transmission electron microscopy (TEM). The number of CTCs of two groups at different points was calculated by flow cytometry.Results:Body weights in two groups were similar before treatment. The mean body weights and 217.30 ± 10.59 g in Control group and 233.40 ± 6.13 g in TAE group respectively on day 21 after treatment, P<0.05. TAE obviously inhibited liver tumor growth on day 7 and 14 after treatment. The liver and lung metastasis rates were no significant difference between two groups on day 14 after treatment, but the number of lung metastasis foci was higher in TAE groups than that in Control group. Pathological examination with HE revealed the liver tumors were unclearly boundary with vessels involved and tumor thrombus in portal vein in TAE group. The chief characteristics of liver tumors in TEM were:the perivascular erosion, tumor cells invasion, the incomplete basement membrane, etc.There were no significant difference in the number of CTCs between two groups on day 0,1 and 3 after treatment. The average level of CTCs was significantly increased by TAE treatment from day 7 to day 21 (day 7:51.80 vs 72.93/106 PBMC cells; day 14:92.73 vs 189.20/106 PBMC cells; day 21:285.33 vs 592.50/106 PBMC cells, P<0.05 for all)Conclusion:TAE can inhibit liver tumor growth but facilitate lung metastasis by increasing the number of CTCs in transplanted HCC Buffalo rats.Part ⅢThe significance of epithelial-mesenchymal transiton (EMT) on increasing CTCs level after transcatheter arterial embolization (TAE)Purpose:To investigate the molecular mechanisms on increasing circulating tumor cells (CTCs) level after transcatheter arterial embolization (TAE).Materials and Methods;McA-RH7777/EGFP cells were cultured with 100umol/L CoCl2 to induce hypoxia in vitro. Morphological change was observed by optical microscope. Invasiveness capability of the tumor cells was evaluated by transwell assay. E-cadherin, N-cadherin, Vimentin of the tumor cells were detected by real time PCR and immunofluorescence. And those of the tumor tissues were dectected by Western-blot and immunohistochemistry. The different expression of E-cadherin, N-cadherin and Vimentin between CTCs and McA-RH7777/EGFP cells was evaluated by flow cytometry.Results:McA-RH7777/EGFP cells displayed Interstitial morphological change and enhanced the ability of invasion 24h after cultured with CoCl2.31.4±6.8 in CoCl2-McA-RH777/EGFP cells group and 19.3±3.3 in McA-RH7777/EGFP cells group were evaluated by transwell assay, P<0.05. Imunofluorescence examination demonstrated down-regulation expression of E-cadherin protein and up-regulation expression of Vimentin, N-cadherin protein in CoCl2-McA-RH777/EGFP cells compared to McA-RH777/EGFP cells. Real time PCR examination showed the level of mRNA of McA-RH777/EGFP cells and CoCl2-McA-RH777/EGFP cells, respectively:0.40 ± 0.04 vs 1.64 ± 0.06 (HIF-la,P<0.05),0.92 ± 0.10 vs 0.52 ± 0.07 (E-cadherin, P<0.05),0.55 ± 0.28 vs 1.21 ± 0.30 (N-cadherin, P<0.05) and 0.87 ± 0.14 vs 1.25 ± 0.29 (Vimentin,P<0.05).Western-blot analysis demonstrated that the relative expression of E-cadherin, N-cadherin, Vimentin, and HIF-1α in the control group and the TAE group was 0.74 ± 0.21 vs 0.39 ± 0.17,0.20 ± 0.08 vs 0.35 ± 0.10,0.49 ± 0.10 vs 0.75 ± 0.12 and 0.21 ± 0.11 vs 0.79 ± 0.14, respectively (P<0.05 for all). The results of immunostaining showed that E-cadherin expression was markedly reduced, while the mesenchymal markers N-cadherin and Vimentin were significantly upregulated in the TAE group. Nuclear expression of HIF-1α was also significantly increased in the TAE-treated tumors compared with the control group. Moreover, the mean percentages of E-cadherin-, N-cadherin-, Vimentin-, and HIF-1α-positive cells in the control group and the TAE group were 33.33 ± 7.24 vs 13.00 ± 7.27,42.00 ± 8.62 vs 71.33 ± 9.16, 54.00 ± 9.86 vs 82.00 ± 9.41, and 43.33 ± 11.13 vs 81.33 ± 9.72, respectively (P< 0.05 for all).The results of expression of E-cadherin, N-cadherin and Vimentin between CTCs and McA-RH7777/EGFP cells evaluated by flow cytometry were 36/156 (23.1%) vs 127/215 (59.1%),86/199 (43.1%) vs 14/179 (7.8%) and 75/137 (52.6%) vs 21/207 (10.1%), respectively (P<0.05 for all).Conclusion:Epithelial-mesenchymal transiton (EMT) induced by hypoxia plays an important role in increasing CTCs level after TAE in the transplanted HCC rat models.The results of expression of E-cadherin, N-cadherin and Vimentin between CTCs and McA-RH7777/EGFP cells evaluated by flow cytometry were 36/156 (23.1%) vs 127/215 (59.1%),86/199 (43.1%) vs 14/179 (7.8%) and 75/137 (52.6%) vs 21/207 (10.1%), respectively (P<0.05 for all).Conclusion:Epithelial-mesenchymal transiton (EMT) induced by hypoxia plays an important role in increasing CTCs level after TAE in the transplanted HCC rat models.Part ⅣCirculating epithelial cell adhesion molecule-positive tumor cells in the central and peripheral venous compartment---assessing hematogenous dissemination after transarterial chemoembolization (TACE) and the prognostic significance of advanced hepatocellular carcinoma (HCC)Purpose:To assess the effect of transarterial chemoembolization (TACE) on circulating tumor cells (CTCs) in peripheral blood (PB) and right atrium blood (RAB) of hepatocellular carcinoma (HCC) patients, to compare the difference between the CTCs at these two locations, and to evaluate whether CTCs perioperatively shed. The prognostic significance was investigated further.Materials and Methods:Before and after TACE, PB and RAB samples (7.5 ml) were collected for 42 HCC patients who were initially treated. CTCs in PB and RAB were isolated and enriched using epithelial cell adhesion molecule (EpCAM) antibody-conjugated nano-magnetic bead, and investigated using immunochemical staining. The effects of TACE on CTCs in PB and RAB of HCC patients were evaluated by comparing the number of CTCs at these two locations before and after the treatment ROC curve analysis was used to determine the predictive value of the parameters, and the differences in the area under the curve (AUC) were detected. Logistic regression analysis was used to screen predictive risk factors for early progress (EP). Univariate and multivariate analyses to screen independent prognostic factors for time to progress (TTP) were based on the Cox proportional hazard regression model. Kaplan-Meier survival curves and a log-rank test were used to analyze the TTPs of the patients.Results:Among the 42 HCC patients,22 (52.4%) had CTCs before TACE and 27 (64.3%) had CTCs after TACE in their PB (P=0.267); 31 (73.8%) had CTCs before TACE and 35 (83.3%) had CTCs after TACE in their RAB (P=0.219). The CTC-positive rates were higher in RAB than that in PB before (P=0.012) and after (P=0.039) TACE treatment. The number of CTCs was between 0 and 30, and between 0 and 54 in peripheral blood before and after TACE, respectively (Z=-1.387, P=0.166). The number of CTCs was between 0 and 65, and between 0 and 98 in right atria before and after TACE, respectively (Z=-1.633, P=0.102). The number of CTCs were significantly different between peripheral blood and right atrial blood both before (P= 0.007) and after (P=0.021) TACE treatment. In EP group and in No EP group, respectively, the mean number of CTCs was 9.8 and 2.2 (P=0.028, pre-TACE),13.1 and 2.9 (P=0.002, post-TACE) in PB and 25.0 and 7.6 (P=0.003, pre-TACE),27.2 and 9.6 (P=0.015, post-TACE) in RAB. Multivariate logistic regression analysis showed that CTC7.5 of >9 pre-TACE in RAB was a strong novel predictor for EP (OR 33.13,95% CI 1.02～1072.45, P=0.049). The AUC was 0.838, with a sensitivity of 88.9% and specificity of 78.8%. In univariate and multivariate analyses, a CTC7.5 of >9 pre-TACE in RAB was the strongest independent prognostic factor for TTP (HR 5.124,95% CI 1.185-22.162; P=0.029).Conclusion:There were more CTCs in RAB than in PB. The numbers of CTCs in PB and RAB were unchanged after TACE treatment. A preoperative CTC75 of>9 in RAB is a strong predictor for EP and a novel independent dangerous factor for TTP in HCC patients treated with TACE.