Advances in molecular and cell therapy for immunotherapy of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly malignant tumor of the hepatobiliary system that has failed to respond to many traditional therapies to a certain extent, including surgery, chemotherapy and radiotherapy. In recent years, the new therapeutic schemes based on immunology have fundamentally changed the systemic treatment of various malignant tumors to a certain extent. In view of the immunogenicity of CCA, during the occurrence and development of CCA, some immunosuppressive substances are released from cells and immunosuppressive microenvironment is formed to promote the escape immune response of its own cells, thus enhancing the malignancy of the tumor and reducing the sensitivity of the tumor to drugs. Some immunotherapy regimens for cholangiocarcinoma have produced good clinical effects. Immunotherapy has more precise characteristics and less adverse reactions compared with traditional treatment approaches. However, due to the unique immune characteristics of CCA, some patients with CCA may not benefit in the long term or not benefit at all after current immunotherapy. At present, the immunotherapy of CCA that have been clinically studied mainly include molecular therapy and cell therapy. In this article, we generalized and summarized the current status of immunotherapy strategies including molecular therapy and cell therapy in CCA in clinical studies, and we outlined our understanding of how to enhance the clinical application of these immunotherapy strategies.

Research progress of bile biomarkers and their immunoregulatory role in biliary tract cancers.

Biliary tract cancers (BTCs), including cholangiocarcinoma and gallbladder carcinoma, originate from the biliary epithelium and have a poor prognosis. Surgery is the only choice for cure in the early stage of disease. However, most patients are diagnosed in the advanced stage and lose the chance for surgery. Early diagnosis could significantly improve the prognosis of patients. Bile has complex components and is in direct contact with biliary tract tumors. Bile components are closely related to the occurrence and development of biliary tract tumors and may be applied as biomarkers for BTCs. Meanwhile, arising evidence has confirmed the immunoregulatory role of bile components. In this review, we aim to summarize and discuss the relationship between bile components and biliary tract cancers and their ability as biomarkers for BTCs, highlighting the role of bile components in regulating immune response, and their promising application prospects.

Decreased expression of serum semaphorin 3B is associated with poor prognosis of patients with hepatocellular carcinoma.

Semaphorin 3B (SEMA-3B), which belongs to the semaphorin family, has an important role in cell apoptosis and inhibition of angiogenesis. A previous study by our group revealed that SEMA-3B was downregulated in tumor tissues of patients with hepatocellular carcinoma (HCC) and exerts anti-motility and anti-invasion effects on tumor cells. However, the serum levels of SEMA-3B and their clinical significance have remained elusive; therefore, the aim of the present study was to monitor its expression in HCC and investigate its clinical significance. ELISA was used to determine the serum levels of SEMA-3B in 132 patients with HCC and 57 healthy individuals. The association between SEMA-3B and clinicopathological parameters was investigated. Serum SEMA-3B was indicated to be significantly decreased in patients with HCC as compared with that in the controls (P<0.05) and it was negatively associated with tumor size (P=0.039), encapsulation (P=0.002) and TNM stage (P=0.034). The prognosis of patients with low expression of SEMA-3B was poor. In conclusion, the results of the present study revealed that serum SEMA-3B is decreased in HCC and is negatively associated with prognosis; therefore, it may be used as a prognostic marker in HCC.

Downregulation of EVI1 Expression Inhibits Cell Proliferation and Induces Apoptosis in Hilar Cholangiocarcinoma via the PTEN/AKT Signalling Pathway.

Aims: Hilar cholangiocarcinoma (HCCA) is a tumour with high malignancy, low surgical resection potential, and a poor prognosis. Ecotropic Viral Integration site 1 (EVI1) is a transcriptional regulator that has been proven to be associated with tumourigenesis and progression in many human solid tumours. However, the expression of EVI1 and its role in HCCA progression remain unclear. The aim of this study was to clarify the association between EVI1 expression and clinical outcomes in patients with HCCA. Methods: The expression of EVI1 in HCCA tissue samples and cell lines was examined by quantitative real-time PCR (qRT-PCR), Western blotting, and immunohistochemistry (IHC). Kaplan-Meier analysis was used for survival analysis. A log-rank test was performed for univariate analysis of survival, and a Cox regression model was utilized for multivariate analysis of survival. Cell proliferation was measured by cell counting kit-8 (CCK-8), colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) assays. The cell cycle was evaluated by flow cytometry. Cell apoptosis was detected by flow cytometry and a terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling (TUNEL) assay. In vivo tumour growth was observed for xenografts in nude mice. Results: EVI1 expression was upregulated in HCCA tissue samples and correlated with a poor prognosis. In clinical specimens, the expression of EVI1 correlated with tumour histological grade and tumour size. Knocking down EVI1 expression reduced HCCA cell proliferation, blocked cell cycle progression, and promoted apoptosis in vitro and in vivo. Furthermore, we found that EVI1 could regulate the AKT signalling pathway by regulating PTEN levels in HCCA. Conclusion: Our data revealed that EVI1 played important roles in HCCA tumourigenesis and development. Our findings suggest that EVI1 may be a potentially useful therapeutic target in HCCA.

HMGB1 correlates with angiogenesis and poor prognosis of perihilar cholangiocarcinoma via elevating VEGFR2 of vessel endothelium.

Perihilar cholangiocarcinoma (PHCCA) is the most common type of cholangiocarcinoma with low resection rate and high morbidity. The study of PHCCA biomarkers made progresses slowly compared with intrahepatic cholangiocarcinoma because of surgical complexity and low possibility of radical surgery, which resulted in the difficulty of specimen obtainment. To screen and identify new biomarkers in PHCCA, we constructed a retrospective cohort with 121 PHCCA patients and a prospective cohort consisting of 64 PHCCA patients, and screened the candidate biomarkers by immunohistochemistry and quantified PCR. In our study, expression of high mobility group box 1 (HMGB1) was demonstrated to be significantly associated with microvascular density (MVD) and unfavorable prognosis of PHCCA in both retrospective and prospective study. Moreover, HMGB1 concentrations in bile and serum of PHCCA patients and healthy controls were detected and compared. Postoperative serum HMGB1 and reflux cholangitis indicated recurrence and unfavorable prognosis of PHCCA. With experiments in vitro and in vivo, we demonstrated that intracellular HMGB1 could be released from PHCCA cells and induce invasion and angiogenesis with LPS stimulation. VEGFR2 expression in vessel endothelial cells was upregulated by the released HMGB1 from PHCCA, resulting in the ectopic angiogenesis. In conclusion, intracellular HMGB1 could be released from PHCCA cells and promote angiogenesis via elevating VEGFR2 in vessel endothelial cells. High expression of HMGB1 was associated with MVD and poor prognosis in clinical analyzation. Postoperative serum HMGB1 and cholangitis could predict high recurrence and unfavorable prognosis.

Sprouty2 suppresses progression and correlates to favourable prognosis of intrahepatic cholangiocarcinoma via antagonizing FGFR2 signalling.

Fibroblast growth factor receptor 2 (FGFR2) was demonstrated to correlate to the progression and prognosis of intrahepatic cholangiocarcinoma (ICC) by numerous evidences. However, as a well-recognized suppressor of FGFR2 signalling, the clinical significance of Sprouty (SPRY) family of ICC has not been investigated. In our study, the expressions of SPRY1-4 in 20 pairs of fresh tumour tissues were detected with qPCR, and in 108 cases of paraffin-embedded tissues with immunohistochemistry. The prognostic value of SPRY family in ICC was estimated with univariate analysis and multivariate analysis. As a result, SPRY2 was identified as an independent prognostic biomarker predicting favourable prognosis of ICC. High SPRY2 expression was correlated with good differentiation of ICC. With silencing SPRY2 expression, we demonstrated that SPRY2 could suppress FGFR2-induced ERK phosphorylation, migration, invasion and epithelial-mesenchymal transition (EMT) under FGF1 stimulation. By overexpressing SPRY2-wide type or SPRY2-Y55F, the tyrosine-55 of SPRY2 was demonstrated to be essential in suppressing ERK phosphorylation, tumour invasion and EMT of ICC cells. In conclusion, SPRY2 was correlated with favourable prognosis of ICC via suppressing FGFR2-induced ERK phosphorylation, invasion and EMT. The phosphorylation of SPRY2-Y55 was required in this tumour-suppressing function of SPRY2.

Biodistribution and preparation of technetium-99m-labeled D-D3 monoclonal antibody against pro-gastrin-releasing peptide (31₋98) in mice.

BACKGROUND: We previously reported that iodine-131((131)I)-labeled anti-pro-gastrin-releasing peptide (ProGRP(31-98)) monoclonal antibody D-D3 could selectively accumulate in the tumor sites of nude mice bearing small cell lung cancer (SCLC) xenografts. However, (131)I-D-D3 was cleared slowly from the body, and the best radioimmunoimaging time for SCLC was 72 - 96 hours after injection. The aims of this study were to radiolabel anti-ProGRP(31-98) D-D3 monoclonal antibody with technetium-99m ((99m)Tc) and to investigate the biodistribution of this antibody in healthy ICR mice. METHODS: D-D3 was labeled with (99m)Tc via the 2-mercaptoethanol reduction method. (99m)Tc-D-D3 was purified by the gel column separation method. The labeling efficiency and radiochemical purity were measured by thin-layer chromatography. The immunological activity of (99m)Tc-D-D3 was determined with cell conjugation assays. (99m)Tc-D-D3 was injected into healthy ICR mice via a tail vein, and all the healthy ICR mice were sacrificed by cervical dislocation at a designated time. Then, the blood and major organs were removed and weighed, and counted in a gamma scintillation counter to determine the percentage of the injected dose per gram (%ID/g). RESULTS: The labeling rate and the radiochemical purity of (99m)Tc-D-D3 were (73.87 ± 2.89)% and (94.13 ± 4.49)%, respectively. The immunobinding rates of (99m)Tc-D-D3 to the human small cell lung cancer NCI-H446 cell line and lung adenocarcinoma A549 cell line were (81.2 ± 2.37)% and (24.3 ± 1.46)%, respectively. The distribution data of normal ICR mice demonstrated that (99m)Tc-D-D3 was mainly distributed in the liver, kidney and lung, and less in the brain tissue and muscle. CONCLUSIONS: (99m)Tc-D-D3 antibody not only had high radiochemical purity, but also had good stability both in vitro and in vivo, and maintained good immunological activity. (99m)Tc-D-D3 was metabolized mainly in the kidney and liver, and the blood radioactivity decreased rapidly. Thus, (99m)Tc-D-D3 is conducive to the radioimmunoimaging of SCLC.

[Values of (99m)TcO(-)(4)thyroid imaging plus ultrasonography in the differential diagnoses of thyroid nodules].

OBJECTIVE: To evaluate the diagnostic values of (99m)TcO(-)(4) thyroid imaging plus ultrasonography in the differentiation of benign and malignant thyroid nodules. METHODS: (99m)TcO(-)(4) thyroid imaging and ultrasonography were performed for 114 patients with 125 thyroid nodules. And the examination results were compared with the post-operative pathological findings. RESULTS: (1) Among 125 thyroid nodules, there were thyroid adenoma (n = 64, 51.2%), thyroid cancer (n = 30, 24.0%) and other thyroid diseases (n = 31, 24.8%). On thyroid images, 73.6% of them were of cold nodules. And among these cold nodules, 25.0% were of thyroid cancer. Among the ultrasonic results, 125 nodules were predominantly of solid nodules and mixed solid and cystic nodules while the malignant rate of solid nodules was the highest. (2) The malignant rate of solid cool or cold nodules was greater than those of mixed solid and cystic cool or cold nodules (P < 0.005). (3)In solid cool or cold nodules with a diameter of ≥ 2.0 cm, thyroid cancer accounted for 62.5%. CONCLUSION: The combination of (99m)TcO(-)(4) thyroid imaging and ultrasonography may help to evaluate the (99m)TcO(-)(4) uptaking functions of thyroid nodules so that it plays an important role in the differentiation of benign and malignant thyroid nodules.

Adenovirus-mediated and tumor-specific transgene expression of the sodium-iodide symporter from the human telomerase reverse transcriptase promoter enhances killing of lung cancer cell line in vitro.

BACKGROUND: The sodium-iodide symporter (NIS) protein can mediate the active radioiodine uptake. The human telomerase reverse transcriptase (hTERT) promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting. We constructed a recombinant adenovirus containing the human sodium iodide symporter (hNIS) gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of (131)I in a lung cancer cell line in vitro. METHODS: The hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL*-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack. The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system. A positive control adenovirus Ad-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly. A549 cells were transduced with recombinant adenoviruses. (125)I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function. Toxic effects of (131)I on tumor cells were studied by in vitro clonogenic assay. RESULTS: We first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter. When infected with recombinant adenovirus constructs expressing hNIS directed by hTERT- and CMV-promoters (Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively), the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30-fold compared to the control parental cells, respectively. The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate (NaClO4). The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% (Ad-CMV-hNIS) and 30% (Ad-hTERT-hNIS) of the control group after receiving radioiodide for 7 hours (P < 0.001). CONCLUSION: Our preliminary study indicates that an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter has the potential to become an effective wide-spectrum yet highly specific anti-cancer strategy.

Potential biochemical therapy of glioma cancer.

Glioma is a highly invasive, rapidly spreading form of brain cancer that is resistant to surgical and medical treatment. The recent progresses made in intracellular and ion channels of glioma cells provide a potential new approach for biochemical therapy of brain tumor. In this paper, we reviewed clinical data on chemotherapy by temozolomide and results from new studies on voltage-gated potassium channels, large-conductance Ca(2+)-activated K(+) channels, volume-activated chloride channels, glioma-specific chloride channel and their modulators. These new findings may represent future directions for brain tumor studies and treatment.