Bi-specific T1 positive-contrast-enhanced magnetic resonance imaging molecular probe for hepatocellular carcinoma in an orthotopic mouse model.

BACKGROUND: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. HCC-targeted magnetic resonance imaging (MRI) is an effective noninvasive diagnostic method that involves targeting clinically-related HCC biomarkers, such as alpha-fetoprotein (AFP) or glypican-3 (GPC3), with iron oxide nanoparticles. However, in vivo studies of HCC-targeted MRI utilize single-target iron oxide nanoprobes as negative (T2) contrast agents, which might weaken their future clinical applications due to tumor heterogeneity and negative MRI contrast. Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles (approximately 5 nm) are potential optimal positive (T1) contrast agents. We previously verified the efficiency of AFP/GPC3-double-antibody-labeled iron oxide MR molecular probe in vitro. AIM: To validate the effectiveness of a bi-specific probe in vivo for enhancing T1-weighted positive contrast to diagnose the early-stage HCC. METHODS: The single- and double-antibody-conjugated 5-nm USPIO probes, including anti-AFP-USPIO (UA), anti-GPC3-USPIO (UG), and anti-AFP-USPIO-anti-GPC3 (UAG), were synthesized. T1- and T2-weighted MRI were performed on day 10 after establishment of the orthotopic HCC mouse model. Following intravenous injection of U, UA, UG, and UAG probes, T1- and T2-weighted images were obtained at 12, 12, and 32 h post-injection. At the end of scanning, mice were euthanized, and a histologic analysis was performed on tumor samples. RESULTS: T1- and T2-weighted MRI showed that absolute tumor-to-background ratios in UAG-treated HCC mice peaked at 24 h post-injection, with the T1- and T2-weighted signals increasing by 46.7% and decreasing by 11.1%, respectively, relative to pre-injection levels. Additionally, T1-weighted contrast in the UAG-treated group at 24 h post-injection was enhanced 1.52-, 2.64-, and 4.38-fold compared to those observed for single-targeted anti-GPC3-USPIO, anti-AFP-USPIO, and non-targeted USPIO probes, respectively. Comparison of U-, UA-, UG-, and UAG-treated tumor sections revealed that UAG-treated mice exhibited increased stained regions compared to those observed in UG- or UA-treated mice. CONCLUSION: The bi-specific T1-positive contrast-enhanced MRI probe (UAG) for HCC demonstrated increased specificity and sensitivity to diagnose early-stage HCC irrespective of tumor size and/or heterogeneity.

[Establishment of a syngeneic mouse model of liver tumor stably expressing hepatitis B virus antigens].

OBJECTIVE: To establish a syngeneic mouse model of liver tumor stably expressing hepatitis B virus (HBV) antigens. METHODS: Melanoma cell line B16 cells were transfected with pLXSN-2HBV. Cells (named B16/HBV) stably and persistently expressing HBV surface (HBsAg) and core (HBcAg) antigens were identified. The cells were injected into the hepatic subcapsular space of fifteen C57BL/6J mice. The mice were divided into 3 groups, receiving 100, 1000 or 5000 cells in a total volume of 5 µl per mouse, respectively, five mice in each group. Two weeks after the tumor cell inoculation, serum samples from the mice were collected weekly and the serum concentration of HBsAg and anti-HBs was quantified by ELISA. The tumor growth in the mouse liver was monitored by a high-resolution ultrasound system. Expression of HBsAg and HBcAg in the tumor tissues was determined by immunohistochemistry. RESULTS: Liver tumors were formed in all the mice receiving 1000 and 5000 B16/HBV cells per mouse, and in 80% of the mice receiving 100 B16/HBV cells. HBsAg and anti-HBs were detectable in their sera from 2 weeks after tumor cell inoculation. The mice receiving 100 cells per mouse began to die 4 weeks, those receiving 1000 cells per mouse began to die 3 - 4 weeks and those receiving 5000 cells began to die 2 - 3 weeks after the cell inoculation. All the tumor cells expressed HBsAg and HBcAg. CONCLUSIONS: The B16/HBV cells stably and persistently express HBV antigens both in vitro and in vivo. A mouse model of transplanted liver tumor stably expressing HBV antigens has been successfully established by inoculation of those cells into the hepatic subcapsular space.

[Estimation and prediction of incidence, mortality and prevalence on liver cancer, in 2008, China].

OBJECTIVE: To estimate the incidence, mortality and 5-year prevalence rates of liver cancer in 2008, China. METHODS: Data from both 36 cancer registries and the Third National Death Survey in China (2004 - 2005) were used to estimate the incidence, mortality and 5-year prevalence of liver cancer in 2008 in the country by using the mathematical models to predict the liver cancer incidence and mortality in the next 20 years. RESULTS: In 2008, the incident cases of liver cancer was 402 208 (14.3% of the total cancers) and the number of deaths from liver cancer was 372 079 (19.0% of the total cancers). The incidence rate was 25.7/100 000, ranking the third among all cancers. The mortality rate was 23.7/100 000, ranking the second among all the cancers. The 5-year prevalence of liver cancer was 296 082 (6.4% of the total cancers) with the proportion as 27.7/ 100 000, ranking the sixth among all the cancers. 72.8% of the liver cancer cases appeared in men and the sex ratio of male to female was 2.7:1. In terms of deaths due to liver cancer, 74.3% of them occurred in men, with sex ratio of male to female as 2.9:1. At any age group, the incidence and mortality of liver cancer among males were higher than those of females. Liver cancer happened more frequently among people older than 40 years of age, particularly among males. Data under our prediction showed that the incidence and mortality of liver cancer in China would gradually increase in the next 20 years. CONCLUSION: Liver cancer is one of the most important public health issues in China. Both incidence and mortality of liver cancer have been increasing in China. The key populations for liver cancer prevention and control programs should be those who were older than 40-year-old, particularly on men.

[Progressive increase of serum circulating immune complexes and its significance in patients during the progression from chronic hepatitis B to hepatocellular carcinoma].

OBJECTIVE: To investigate the significance of increasing circulating immune complex (CIC) in patients during the progression from chronic hepatitis B to hepatocellular carcinoma (HCC). METHODS: Serum levels of CIC from 20 hospitalized patients diagnosed by pathology with primary HCC, and 13 with hepatic hemangioma, and from 45 subjects with chronic HBV infection who finally developed into HCC (45 cases), and age- and gender-matched 45 subjects who kept the chronic HBV infection after consecutively followed up for 10 - 13 years by June of 2009 were quantified by ELISA. The serum levels of anti liver-kidney microsomal (anti LKM-1) antibodies were also measured by ELISA, and that of HBV-DNA were quantified by Taqman probe-based real time PCR in the followed up chronic HBV infection subjects. In the 45 chronic HBV subjects who finally developed into HCC and the 45 controls, serum samples were collected and determined at 3 time points: the baseline when the subjects were recruited, the middle point during the follow-up, and the end of follow-up. RESULTS: The serum level of CIC was significantly higher in the 20 HCC patients than that in the 13 hemangioma cases (P < 0.001). When HCC was diagnosed, the CIC concentration was significantly higher than that in the baselines (P < 0.001) in the 45 chronic HBV subjects who finally developed into HCC after the consecutively follow-up for 5 - 13 years. Of them, 36 patients (80.0%) showed progressively increased CIC during the follow-up (P < 0.001). In the controls, the CIC levels were kept relatively stable during the follow-up. Among them, 17 patients (37.8%) showed CIC slightly increased (P = 0.046). Kaplan-Meier survival analysis indicated that elevated serum CIC during the follow-up increased cumulative HCC incidence (HR = 2.77, 95%CI 1.47 - 5.22). In addition, the serum levels of anti-LKM-1 and HBV-DNA were also significantly higher in the patients who finally progressed into HCC than that in the controls and maintained at a high level during the follow-up tested at all the 3 time points. Further analysis indicated that the serum level of CIC was correlated with that of serum HBV-DNA only when HCC was diagnosed (r = 0.344, P = 0.026). CONCLUSION: Progressive increase of serum CIC level may be one of risk factors reflecting HCC development from chronic HBV infection.

Diagnostic value of glypican-3 in serum and liver for primary hepatocellular carcinoma.

AIM: To evaluate the diagnostic value of glypican-3 (GPC3) in serum and liver for primary hepatocellular carcinoma (HCC). METHODS: Serum levels of GPC3 and α-fetoprotein (AFP) were measured in 75 patients with primary HCC and 32 patients with liver cirrhosis. Expression of GPC3 and AFP in 58 HCC and 12 cirrhotic specimens was detected with immunohistochemical staining. RESULTS: When the cut-off value of serum GPC3 was set at 300 ng/L, its sensitivity and specificity for HCC were 47.0% and 93.5%, respectively. Among the 14 patients with HCC at stage according to the Barcelona Clinic Liver Cancer staging system, the serum GPC3 level was higher than 300 ng/L in 50% (7/14) patients, the serum AFP level was not ≥ 400 µg/L in any patient. Combined serum AFP and GPC3 significantly increased the sensitivity to the diagnosis of HCC. The GPC3 expression was detected in cytoplasm of HCC cells but not in hepatocytes and bile ducts of benign tumors. Among the 58 HCC patients, the GPC3 was expressed in 100% (28/28) patients with their serum AFP level ≥ 400 µg/L, and in 90% (27/30) patients with their AFP level < 400 µg/L, respectively. The GPC3 was weakly or negatively expressed in all paracarcinomatous and cirrhotic tissue samples. AFP positive HCC cells were only found in 1 out of the 58 HCC patients. CONCLUSION: GPC3 protein is a sensitive and specific serum marker for diagnosis of early HCC. Its expression in liver tissues can be used to discriminate tumor cells from benign hepatic cells.

[Mast cell degranulation enhances exogenous dendritic cells homing to draining lymph nodes].

AIM: To establish a method of enhancing exogenous bone marrow derived dendritic cells (BM-DC) homing to draining lymph nodes by induction of local mast cell degranulation. METHODS: Compound 48/80 (c48/80) was injected into C57BL/6 scapular skin to induce local mast cell degranulation. BM-DC generated from bone marrow of syngenic mice were labeled with YG-Microspheres and injected into c48/80 or normal saline treated scapular skin. Cells derived from draining lymph nodes (DLN) were pooled together 48 h after BM-DC injection and stained with conjugated anti-CD11c. The efficiency of BM-DC homing to lymph nodes was analyzed by FCM. RESULTS: Mast cell degranulation was locally boosted by c48/80 injection and caused enhancement of the homing of exogenous BM-DC to DLN, with an increase of 67%+/-43%. The total cells in lymph nodes also increased significantly. The fold of increase was 55%+/-43%. CONCLUSION: Exogenous BM-DC homing to DLN can be boosted by inducing local skin mast cell degranulation.

Exogenous dendritic cell homing to draining lymph nodes can be boosted by mast cell degranulation.

Dendritic cells (DCs), as potent antigen presenting cells, are increasingly used for immunotherapeutic approaches, predominantly in oncology. Low efficiency of injected Ag-pulsed DC homing to draining lymph nodes (DLNs) is one of the factors that affect the efficacy of therapy. As Langerhans cell emigration was enhanced after skin mast cell degranulation, we investigated the effect of local mast cell activation on exogenous bone marrow-derived DCs (BM-DCs) homing to DLNs. Product of activated MC/9 mast cells enhanced chemotaxis of BM-DCs to CCL21 in vitro. Intradermal injection of compound 48/80 (c48/80) induced local skin mast cell obvious degranulation and boosted exogenous BM-DC homing to DLNs. Both Ag-specific lymphocyte proliferation and TH1/TH2 cytokine production increased after HBsAg-pulsed BM-DC was injected into c48/80 pretreated mice. These results suggest that transferred DC homing to DLNs promoted by local mast cell degranulation may have potential application to improve DC-based immunotherapy.