Jun Yan

IL-17 induces AKT-dependent IL-6/JAK2/STAT3 activation and tumor progression in hepatocellular carcinoma

BackgroundThe Th17 subset and IL-17 have been found in increased frequencies within certain tumors. However, their relevance in cancer biology remains controversial. This study aimed to clarify the biological action of IL-17 on hepatocellular carcinoma (HCC).MethodsEffects and underlying molecular mechanisms of IL-17 on human HCC were explored in vitro using exogenous IL-17 stimulation and in nude mice by implanting IL-17 overexpressed HCC cells. The clinical significance of IL-17 was investigated in tissue microarrays containing HCC tissues from 323 patients following hepatectomy using immunohistochemistry.ResultsAlthough exogenous IL-17 showed no direct effect on the growth rate of HCC cells in vitro, PCR and ELISA showed that IL-17 selectively augmented the secretion of diverse proinvasive factors and transwell showed a direct promotion of invasion of HCC cells by IL-17. Furthermore, transfection of IL-17 into HCC cells significantly promoted neoangiogenesis, neutrophil recruitment and tumor growth in vivo. Using siRNA mediated knockdown of AKT and STAT3, we suggested that the effects of IL-17 were operated through activation of the AKT signaling in HCC, which resulted in IL-6 production. Then, IL-6 in turn activated JAK2/STAT3 signaling and subsequently up-regulated its downstream targets IL-8, MMP2, and VEGF. Supporting these findings, in human HCC tissues, immunostaining indicated that IL-17 expression was significantly and positively associated with STAT3 phosphorylation, neutrophil infiltration and increased tumor vascularity. The clinical significance of IL-17 was authenticated by revealing that the combination of intratumoral IL-17+ cells and phospho-STAT3 served as a better prognosticator for postoperative tumor recurrence than either marker alone.ConclusionsIL-17 mediated tumor-promoting role involves a direct effect on HCC cells through IL-6/JAK2/STAT3 induction by activating the AKT pathway.

Real-Time Monitoring of Rare Circulating Hepatocellular Carcinoma Cells in an Orthotopic Model by In Vivo Flow Cytometry Assesses Resection on Metastasis

The fate of circulating tumor cells (CTC) is an important determinant of metastasis and recurrence, which leads to most deaths in hepatocellular carcinoma (HCC). Therefore, quantification of CTCs proves to be an emerging tool for diagnosing, stratifying, and monitoring patients with metastatic diseases. In vivo flow cytometry has the capability to monitor the dynamics of fluorescently labeled CTCs continuously and noninvasively. Here, we combine in vivo flow cytometry technique and a GFP-transfected HCC orthotopic metastatic tumor model to monitor CTC dynamics. Our in vivo flow cytometry has approximately 1.8-fold higher sensitivity than whole blood analysis by conventional flow cytometry. We found a significant difference in CTC dynamics between orthotopic and subcutaneous tumor models. We also investigated whether liver resection promotes or restricts hematogenous metastasis in advanced HCC. Our results show that the number of CTCs and early metastases decreases significantly after the resection. The resection prominently restricts hematogenous metastasis and distant metastases. CTC dynamics is correlated with tumor growth in our orthotopic tumor model. The number and size of distant metastases correspond to CTC dynamics. The novel in vivo flow cytometry technique combined with orthotopic tumor models might provide insights to tumor hematogenous metastasis and guidance to cancer therapy.

Label-free monitoring of colonic cancer progression using multiphoton microscopy

Abstract: Real-time histology or virtual biopsy for the diagnosis of colonic cancer is of great medical significance. In this work, we show that label-free multiphoton imaging is feasible and effective in monitoring colonic cancer progression by providing cellular and subcellular details in fresh, unfixed, unstained colonic specimens. Our results also demonstrate the capability of using tissue quantitative analysis of the redox ratio for quantifying colonic cancer progression. These results suggest that multiphoton microscopy has potential to become an in situ histological tool, which is free from the labeling requirement of conventional methods, for the early diagnosis and detection of malignant lesions in the colon.

Circulating tumor cells are correlated with disease progression and treatment response in an orthotopic hepatocellular carcinoma model.

Hepatocellular carcinoma (HCC) is a highly malignant tumor characterized by rapid progression, poor prognosis, and frequent hematogenous metastasis. A minimally invasive diagnostic biomarker that can predict disease progression and treatment response would be of extraordinary benefit. Therefore, we have investigated whether the number of circulating tumor cells (CTCs) is correlated with disease progression and treatment response in HCC. Here we report that the number of CTCs, monitored by in vivo flow cytometry (IVFC), is strongly correlated with disease progression and treatment response in a highly metastatic orthotopic nude mouse model of green fluorescent protein (GFP)‐labeled HCC. Sorafenib treatment reduces the number of CTCs significantly. The decreased number of CTCs is consistent with low lung metastasis. This study has demonstrated a considerable clinical value of CTCs as a biomarker in predicting disease progression and monitoring therapeutic efficacy in patients with HCC.

Preclinical study of using multiphoton microscopy to diagnose liver cancer and differentiate benign and malignant liver lesions.

Recently, the miniaturized multiphoton microscopy (MPM) and multiphoton probe allow the clinical use of multiphoton endoscopy for diagnosing cancer via optical biopsy. The purpose of this study was to establish MPM optical diagnostic features for liver cancer and evaluate the sensitivity, specificity, and accuracy of MPM optical diagnosis. Firstly, we performed a pilot study to establish the MPM diagnostic features by investigating 60 surgical specimens, and found that high-resolution MPM images clearly demonstrated apparent differences between benign and malignant liver lesions in terms of their tissue architecture and cell morphology. Cancer cells, characterized by irregular size and shape, enlarged nuclei, and increased nuclear-cytoplasmic ratio, were identified by MPM images, which were comparable to hematoxylin-eosin staining images. Secondly, we performed a blinded study to evaluate the sensitivity, specificity, and accuracy of MPM optical diagnosis by investigating another 164 specimens, and found that the sensitivity, specificity, and accuracy of MPM diagnosis was 96.32%, 96.43%, and 96.34%, respectively. In conclusion, it is feasible to use MPM to diagnose liver cancer and differentiate benign and malignant liver lesions. This preclinical study provides the groundwork for further using multiphoton endoscopy to perform real-time noninvasive optical biopsy for liver lesions in the near future.

Multiphoton microscopic imaging of normal human rectum tissue.

In this paper, multiphoton microscopy (MPM), based on two-photon excited fluorescence and second harmonic generation signals, was used to image microstructures of human rectal mucosa and submucosa. The morphology and distribution of the main components in mucosa layer, goblet cells, intestinal glands, and a little collagen fibers have been clearly monitored, and the content and distribution of collagen, elastic fibers, and blood vessels in submucosa layer have also been distinctly obtained. The variation of these components is very relevant to the pathology in gastrointestinal system, especially early rectal cancer. Our results indicate that the MPM technique has the potential application in vivo in the clinical diagnosis and monitoring of early rectal cancer.

Sorafenib delays recurrence and metastasis after liver transplantation in a rat model of hepatocellular carcinoma with high expression of phosphorylated extracellular signal‐regulated kinase

Liver transplantation (LT) is one of the curative treatments for hepatocellular carcinoma (HCC). However, cancer recurrence and metastasis after LT are common in some HCC patients with high‐risk factors (even in those within the Milan criteria). It remains unclear whether adjuvant therapy with sorafenib inhibits HCC recurrence and metastasis after LT. Therefore, we performed orthotopic LT in an August Irish Copenhagen (ACI) rat model of HCC. Because LT involves immune rejection and tolerance and it is unknown whether sorafenib influences the immune response, we also investigated the effects of sorafenib on immune balance. In this study, we established an allogeneic rat LT model in which liver grafts were taken from Lewis rats and transplanted into ACI rats with orthotopic HCC, and they were administered cyclosporine A to prevent acute allograft rejection. From day 7 after LT, sorafenib was administrated at 30 mg/kg/day for 3 weeks. Our results showed that the serum levels of vascular endothelial growth factor and hepatocyte growth factor significantly increased after LT, and the T helper 1 (Th1)/T helper 2 (Th2) immune balance was shifted toward a Th2 response after immunosuppressant administration. In comparison with controls, the rats in the sorafenib group showed significantly inhibited extracellular signal‐regulated kinase phosphorylation and improved progression‐free survival and overall survival. The tumor proliferation rate and angiogenesis in posttransplant recurrent tumor tissues decreased in the sorafenib group, and the tumor apoptosis rate increased. There was no significant difference in the Th1/Th2 immune balance between the sorafenib and control groups. In conclusion, adjuvant therapy with sorafenib is highly effective at inhibiting cancer recurrence and metastasis without influencing the immune balance after LT for HCC with high expression of phosphorylated extracellular signal‐regulated kinase. This study suggests that sorafenib may have potential, particularly as part of a stratified medicine approach to HCC treatment after LT. Liver Transpl 19:507–520, 2013.

An X-chromosomal association study identifies a susceptibility locus at Xq22.1 for hepatitis B virus-related hepatocellular carcinoma

BACKGROUND AND OBJECTIVEnGenetic epidemiological data in hepatocellular carcinoma (HCC) pedigrees indicate a pattern of X-linked recessive inheritance of HCC susceptibility genes. This study is designed to test the hypothesis that there are genes conferring susceptibility to HCC located on the X-chromosome.nnnMETHODSnAn X-chromosomal association study was conducted among Chinese men recruited from an area with a high prevalence of HCC. The candidate gene was further investigated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).nnnRESULTSnBy analyzing 5454 X-chromosome single nucleotide polymorphisms (SNPs) in 50 HCC patients and 50 controls, we found two promising regions in which the associated SNPs clustered, located at Xq22.1 and Xq26.2. We further selected 35 tag SNPs (tSNPs) from these two regions for additional genotyping analysis in another independent set of 290 cases and 242 controls. Notably, SNP rs5945919 at Xq22.1 exhibited a significant association with HBV-related HCC (odds ratio [OR]=2.22, 95% confidence interval [CI]=1.15-4.30, P=0.016). The expressions of the three genes near the rs5945919 locus, RAB40AL, BEX1, and NXF3, were analyzed by qRT-PCR between another 24 HCC tissues and paired peritumoral liver tissues. The results indicated that NXF3, rather than RAB40AL and BEX1, mRNA level was found to be more abundant in HCC tissue than in peritumoral liver tissue.nnnCONCLUSIONSnOur findings implicated Xq22.1 as a novel susceptibility locus for HCC and NXF3 as a candidate risk factor for relevant HCC.

Use of multiphoton microscopy to diagnose liver cancer and lung metastasis in an orthotopic rat model.

Liver or lung biopsy for suspicious lesions has several disadvantages such as bleeding, bile leak or pneumothorax, needle track seeding, and time-consuming histopathological procedure. The ability to directly observe cellular and subcellular details and then perform optical biopsy is a major goal in the development of new interventional techniques. Multiphoton microscopy (MPM) enables real-time noninvasive visualization of tissue architecture and cell morphology in live tissue. We performed a study to evaluate whether MPMcan make real-time optical diagnosis for liver cancer and lung metastasis using an orthotopic rat model with Morris hepatoma. We found that real-time high-resolution MPMimaging could clearly show tissue architecture and cell morphology. In the normal liver tissue, MPMimaging clearly revealed the blood-filled sinusoids and cords of hepatocytes. In the cancerous tissue, MPMimaging clearly illustrated that cancer cells displayed marked cellular and nuclear pleomorphism. MPMimages were comparable to golden standard hematoxylin-eosin staining images. Moreover, MPMimaging had deep penetration with the capability of optical sectioning. In short, MPMcan make real-time optical diagnosis for liver cancer and lung metastasis. This study provides the groundwork for further using multiphoton endoscopy to perform real-time noninvasive optical biopsy for liver cancer and lung metastasis in the near future.

Expression of hemopexin in acute rejection of rat liver allograft identified by serum proteomic analysis

ABSTRACT Acute rejection (AR) and acceptance of allograft after liver transplantation (LTx) remain critical issues that need addressing to improve prognosis. We therefore performed rat orthotopic LTx and proteomic analyses to screen for immune response–related biomarkers in sera. Markers identified were validated at the mRNA and/or protein levels, and the molecules of interest were functionally explored. Compared with syngeneic controls, signs of AR as well as spontaneous acceptance were observed in hematoxylin and eosin–stained sections of liver allografts. In accordance with the severity of AR, 30 protein spots displaying significant changes in abundance were identified using two-dimensional differential gel electrophoresis. Ultimately, 14 serum proteins were sequenced and five spots of interest were identified as hemopexin (HPX). Expression of HPX was significantly and inversely associated with the severity of AR at both the mRNA and protein levels. In vitro, Mt-1, Ho-1, Fth, Ifn-&ggr;, and Il-17 transcripts were significantly upregulated in lysates of lymphocytes stimulated with HPX, whereas Il-10 markedly was remarkably downregulated. Interferon-&ggr;, IL-10, and IL-17 proteins in the supernatant of HPX-stimulated lymphocytes were significantly altered in keeping with the mRNA level. Our data facilitated the generation of a proteomic profile to enhance the understanding of rat liver AR. In view of finding that the HPX serum level is negatively associated with the severity of AR of rat liver allograft, we propose that in vitro treatment with HPX regulates cytokine expression in rat lymphocytes.