| Background & objectivesHepatocellular carcinoma (HCC) is the world’s third leading cause of tumor death. In China, up to 130 thousand patients die from HCC-related illness every year. Surgery remains the most effective treatment currently. However, due to its insidious onset and rapid development, HCC is usually diagnosed at an advanced stage, which is ineligible for surgical resection. Therefore, more and more patients have to receive non-surgical treatments such as transcatheter arterial chemoembolization (TACE). Licartin is a 131I-labeld monoclonal antibody targeting CD 147, a protein which is expressed on the surface of many kinds of tumor cells including HCC cells. After delivered to tumor site by interventional methods, Licartin can specifically binds to CD 147 expressed on liver tumor cells and blocks CD147-related signaling pathways. Also, the embedded 131I can radiate β-rays to directly damage liver cancer cells. The combination of TACE with licartin was demonstrated to be better than TACE monotherapy in the treatment of HCC as reported in a series of clinical trials. However, as a molecule targeted drug, no laboratory or clinical screening techniques were established to predict the efficacy of Licartin and guide the individualized application. Currently, tumor tissues obtained by an excisional or needle biopsy are used for detection of drug targets. However, needle aspiration often fails to locate measurable objectives or obtain sufficient tumor samples, and only a small proportion of patients are eligible for surgical excision at diagnosis. Furthermore, invasive sampling is potentially harmful and expensive, and cannot be performed repeatedly. The collection of circulating tumor cells (CTCs) provides a viable alternative. CTCs are cancer cells shed from either the primary tumor or its metastases that circulate in the peripheral blood, which thus are available noninvasively and can be obtained repeatedly for a readily accessible real-time "liquid biopsy" of tumors.We have previously established and modified the isolation and identification technique of HCC CTCs. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes by magnetic activated cell separation (MACS) strategy following Ficoll density gradient centrifugation. This will reduce the impact from heterogeneity of tumor cells on the efficiency of positive selection. Then the isolated HCC CTCs were identified by a combination of antibodies against Asialoglycoprotein receptor (ASGPR) and carbamoyl phosphate synthetase 1 (CPS1), which improves the sensitivity of identification. In the establishment and verification of the feasibility of the multicolor immunofluorescence staining, we select CD147, the target of Licartin, and also the epithelial cell adhesion molecule (EpCAM), which is considered to possibly be the marker of stem cell-like property of HCC. We then applied this multicolor immunofluorescence staining system in the detection of CD147 in CTCs isolated from patients with HCC and treated with Licartin. By analyzing the correlation with clinical characteristics and evaluation of treatment efficacy, we aimed to investigate the potential value of characterization of CD 147 in HCC CTCs in the direction of individualized therapy of Licartin.MethodsPart one:Establishment of the multicolor immunofluorescence staining system in the detection of CD147 or EpCAM in CTCs from HCC patients and study of the correlation of CD147 expression between tissue specimens and CTCs.(1) HCC cell lines were spiked with peripheral blood from healthy volunteers and then enriched by extracting CD45-expressing leukocytes by MACS following Ficoll density gradient centrifugation.(2) Establishment of the multicolor immunofluorescence staining model of cell-spiking experiment utilizing ASGPR+CPS1 and CD45 as identifying markers and co-staining with DAPI and simultaneously detecting the expression of CD147 or EpCAM.(3) Expression of CD147 or EpCAM were detected in CTCs isolated from 32 patients with HCC.(4) Expression of CD147 was also examined by immunohistochemistry in tissue specimens from the 32 HCC patients, and comparison was performed to analyze the correlation of the detection results between CTCs and tissue specimens.Part two:Detection of the expression of CD147 in CTCs from 92 patients with HCC and treated with TACE+Licartin or TACE alone.Five milliliters of blood samples were collected from 92 HCC patients before and 4 weeks after treatment. Numbers of CTCs were measured and expressions of CD147 were determined. Also, clinical data were collected and survival data were followed up for efficacy evaluation.Results1. ASGPR+CPS1/CD45 can specifically identify HCC CTCs from lymphocytes, and CD 147 or EpCAM can be accurately detected in CTCs by the multicolor immunofluorescence staining. In 32 patients with HCC,91%(n=29) were CTCs positive. The number of CTCs per 5 mL peripheral blood averaged 28 ± 16, ranging from 4 to 62. No CTCs were detected in 15 healthy volunteers and 12 with other malignant tumors. CD147 positive CTCs were detected in 69% (20/29) patients, and EpCAM positive CTCs were detected in 17% (5/29). In patients with CD147+ or EpCAM+ CTCs, also CD147- or EpCAM- CTCs were detected.2. In 29 patients with CTCs detected,76% (n= 22) were CD 147+ determined by immunohistochemistry in tissue specimens.93.1% (27/29) of patients had a molecular classification of tissues concordant with that of CTCs, among which 20 were both CD147+ and 7 were both CD 147-. No significant difference was observed between the two detection methods (P> 0.05).3. Detection of CTCs and molecular characterization of CD 147 were performed in 52 HCC patients treated with TACE+ Licartin and 40 with TACE alone. In all 92 patients, 93% (n= 86) were CTCs positive. In TACE group,95% (38/40) were CTCs positive, and the number of CTCs averaged 38 ± 15, ranging from 5 to 76. In TACE+ Licartin group, 92.3% (48/52) were CTCs positive, and the number of CTCs averaged 36 ± 13, ranging from 2 to 78. Statistical analysis showed no significant differences of the positivity rates and numbers of CTCs between the two groups (P> 0.05).4. In 86 patients with CTCs detected,67% (58/86) were CD147+. In the TACE+ Licartin group of patients,69% (33/48) were CD147+, while in the TACE group this number was 66% (25/38). The expression of CD 147 showed great heterogeneity among different patients and even among different CTCs from the same patients.In patients classified CD147+, the proportions of CD147+ CTCs were different and CD147- CTCs were also found.5. The numbers of CTCs from 86 HCC patients suffered different degrees of decrease after one month of the treatment. In the TACE+ Licartin group (n= 48), CTCs decreased significantly from 36 ± 13 to 21 ± 11 (P< 0.001). Among them, CD147+ patients (n= 33) showed a significant decrease of CTCs numbers before and after treatment (38 ± 12 vs.19 ± 9, P< 0.001), while in CD147- patients (n= 15) no significant decrease was observed (30 ±11 vs.26 ± 13, P= 0.343). Comparison analysis revealed significant difference of the decrease of CTCs numbers between CD147+ and CD147-patients (19 ± 8 vs.4 ± 8, P< 0.001), indicating that CD147+ patients were more sensitive to Licartin. In the TACE group (n= 38), CTCs also showed significant decrease from 38 ± 15 to 29 ± 16 (P= 0.018). However, no significant difference was observed between the decrease of CTCs numbers from CD147+ patients (n= 25) and CD 147-patients (n= 13) (10 ± 8 vs.6 ± 10, P= 0.252) among this group.6. Although there was no significant difference between the numbers of CTCs in patients of the TACE+ Licartin group (n= 48) and TACE group (n= 38) before treatment (36 ± 13 vs.38 ± 15, P= 0.524), the decrease of CTCs numbers after treatment showed significant difference (14 ± 11 vs.9 ± 9, P= 0.009). Similarly, the CD147+ patients from different groups (33 from TACE+ Licartin group and 25 from TACE group) showed no significant difference between the numbers of CTCs before treatment (38 ± 12 vs.42 ± 14, P= 0.247), but suffered significantly different decrease of CTCs numbers (19 ± 8 vs.10 ± 8, P< 0.001). While the decrease of CTCs numbers showed no significant difference between CD 147" patients from TACE+ Licartin group (n= 15) and TACE group (n= 13) (4 ± 8 vs.6 ± 10, P= 0.549).ConclusionsThe multicolor immunofluorescence staining method we established in this study was convenient and applicable, and can be used in the molecular characterization of CD 147 or EpCAM in CTCs from patients with HCC. CTCs could replace tumor tissue biopsies for characterization of CD147 expression. The expression of CD147 showed heterogeneity among individuals and even CTCs from the same individual. The molecular characterization of CD 147 in HCC CTCs may be helpful in the direction of individualized therapy of Licartin, and in dynamic monitoring and real-time evaluation of its efficacy. For CD147+ HCC patients, Licartin would be suggested to be applied, while for CD147- patients would not.
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