| Liver cancer is one of the most common cancers in the world. The mortality of liver cancer took the third place among all the malignant tumors and the second place in China. Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is a highly malignant tumor characterized by rapid progression, poor prognosis, and frequent tumor recurrence and metastasis. Hepatocellular carcinoma (HCC), even resectable, carries a poor prognosis with a high risk of mortality for patients because of cancer recurrence and metastasis. Despite significantly improved diagnosis and surgical treatment for HCC in recent years, the long-term therapeutic effect after surgery is compromised by frequent cancer recurrence and metastasis. There are a lot of work need to be done to improve diagnosis and treatment.In terms of diagnosis, pathology currently is still the golden standard for diagnosis of liver cancer or suspicious lung metastasis. Despite great improvements in diagnostic imaging procedures and serological testing, liver and lung biopsies are still performed when the clinical behavior and the test results are not typical. However, liver biopsy and lung biopsy has several disadvantages such as bleeding, bile leak or pneumothorax, needle track seeding, and time-consuming histopathological procedure. Moreover, pathological diagnosis is turning to pathological-biological diagnosis, which not only diagnose cancer but also predict prognosis. Multiphoton microscopy (MPM), based on the advancement of the field of non-linear optics and femtosecond lasers, can provide real-time detailed information about tissue architecture and cell morphology in live tissue using a combination of autofluorescence from cells and second harmonic generation (SHG) signal from collagen. Natural intrinsic fluorophores abundantly present in the most cells include reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotides (FAD). MPM could not only present tissue architecture and cell morphology, but also reveal cell metabolism via NADH and FAD redox ratio in live tissue without the administration of exogenous contrast agents. Recently, the miniaturized MPM and multiphoton probe allow the clinical use of multiphoton endoscopy for diagnosing cancer. Therefore, establishing MPM optical diagnostic features is essential and significant for developing multiphoton endoscopy to diagnose liver cancer and differentiate benign and malignant liver lesions. Until now, these MPM diagnostic features have not been clearly described.Regarding HCC therapy, no effective systemic treatment has been available to inhibit cancer recurrence and metastasis for patients with resectable high-risk HCC, such as large (>5cm) tumor, multinodular disease, and/or vascular invasion. Chemotherapy, chemoembolization, ablation, and radiotherapy, remain disappointing as a neoadjuvant or adjuvant therapy to improve overall survival and progression-free survival. Liver transplantation is a curative therapy of hepatocellular carcinoma (HCC) with underlying cirrhosis, but due to HCC recurrence and metastasis, its benefit is limited to a small population who meet the strict selection criteria. Commonly used selection criteria of liver transplantation for HCC are Milan criteria (single nodule<or=5cm, or<or=3nodules and<or=3cm each, without major vessel invasion and metastasis). Unfortunately, in clinic, most patients with HCC fail to meet Milan criteria. Expanding selection criteria beyond Milan resulted in a high incidence of recurrence and metastasis in patients with HCC. Although some researchers have attempted to extend the limited criteria for liver transplantation, no well-accepted drug or effective treatment has been reported so far to effectively decrease tumor recurrence and metastasis after transplantation. Sorafenib, a multikinase inhibitor which blocks the vascular endothelial growth factor (VEGF) and RAF/MEK/ERK pathways, has been approved for the treatment of advanced HCC by the US Food and Drug Administration (FDA) since2007, which represented a milestone in the treatment of HCC. However, it remains unclear whether sorafenib inhibits HCC recurrence and metastasis after liver transplantation. Therefore, we perform a study to investigate whether sorafenib adjuvant therapy after liver transplantation inhibits cancer recurrence and metastasis and then improves progression-free survival and overall survival via using an orthotopic rat model of HCC.There are three sections in this study as follows:Section1Real-time Optical Diagnosis for Liver Cancer Using Multiphoton Imaging[Background and study aims]:In contrast to liver and lung biopsies which contain several disadvantages such as bleeding, bile leak or pneumothorax, needle track seeding, and time-consuming histopathological procedure, multiphoton imaging enables direct noninvasive visualization of tissue architecture and cell morphology in live tissue without the administration of exogenous contrast agents. The aim of this study was to evaluate the feasibility of using multiphoton imaging to make real-time noninvasive optical diagnosis for liver cancer.[Methods]:Firstly, an animal study was perform to test the feasibility of multiphoton imaging for liver cancer and lung metastasis using an orthotopic rat model with Morris Hepatoma. Six fresh, unfixed, and unstained intact rat livers with1-2cm single Morris hepatoma and six fresh, unfixed, and unstained intact rat lungs with1-3mm metastatic lesions were underwent multiphoton imaging and then went through routine histopathological procedure, which included10%buffered formalin processing, paraffin embedding, sectioning at5μn, and then hematoxylin-eosin (H-E) staining. MPM images and H-E staining images were analyzed and compared by two attending pathologist. Secondly, a pilot study was performed to establish the optical diagnostic features of multiphoton imaging for benign and malignant liver tumor. Sixty fresh human liver lesions, including primary and secondary liver cancers and benign conditions, underwent multiphoton imaging and routine histopathological procedure. Finally, a blinded study was conducted to test the sensitivity, specificity, and accuracy of MPM optical diagnosis of164consecutive specimens from liver tissue bank.[Results]:Multiphoton images were obtained by two channels:broadband autofluorescence from cells and second harmonic generation signal from tissue collagen. In the animal study, real-time high-resolution multiphoton imaging could clearly show tissue architecture and cell morphology. In the normal liver tissue, multiphoton imaging clearly revealed the blood-filled sinusoids and cords of hepatocytes. In the cancerous tissue, multiphoton imaging clearly illustrated that cancer cells displayed marked cellular and nuclear pleomorphism. MPM images were comparable to golden standard hematoxylin-eosin staining images. Moreover, multiphoton imaging had deep penetration with the capability of optical sectioning. In the pilot study, real-time high-resolution multiphoton imaging clearly demonstrated apparent differences between benign and malignant liver tumor 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 multiphoton images, which were comparable to hematoxylin-eosin stained images. In the blinded study, the sensitivity, specificity, and accuracy of optical diagnosis of multiphoton imaging were96.32%,96.43%, and96.34%, respectively.[Conclusions]:It is feasible to make real-time optical diagnosis for liver cancer via using multiphoton imaging. With miniaturization and integration of endoscopy, multiphoton imaging has the potential to provide real-time noninvasive "optical biopsy" for suspicious liver lesions. Section2Sorafenib Decreases the Number of Circulating Tumor Cells and Inhibits Hematogenous Metastasis for Hepatocellular Carcinoma[Background and study aims]:Currently, there are two different opinions about antiangiogenic therapy. One is that antiangiogenic therapy may increase tumor invasiveness and metastasis. The other is that antiangiogenic therapy reduces metastasis. Sorafenib, a multikinase inhibitor blocking the RAF/MEK/ERK pathway and the VEGF pathway, has been approved for the treatment of advanced hepatocellular carcinoma (HCC) by the US Food and Drug Administration since2007. However, it remains unclear whether sorafenib inhibits HCC hematogenous metastasis. Therefore, we performed an animal study to investigate the role of sorafenib in hematogenous metastasis.[Methods]:Firstly, we established a highly metastatic orthotopic nude mouse model of green fluorescent protein (GFP)-labeled human HCC and used in vivo flow cytometry to quantitatively detect circulating tumor cells (CTCs). This model exhibited100%metastatic ability and mice died at the ninth week after orthotopic implantation. Then, ten nude mice implanted orthotopically with highly metastatic GFP-labeled human HCC were randomized into the sorafenib group (n=5) and the control group (n=5) according to body weight. The sorafenib group underwent sorafenib oral administration once daily for4weeks at dose levels of30mg/kg body weight starting on day28after tumor implantation, whereas the control group received solvent oral administration. Both groups received in vivo flow cytometer examination and ultrasound imaging per week. Nude mice were sacrificed on day56after tumor implantation. The primary outcome was number of CTCs detected quantitatively by in vivo flow cytometry. The secondary outcomes include lung metastatic rate, number and size of lung metastasis, primary liver tumor volume, fluorescent intensity of liver tumor and lung metastasis, mice weight, tumor necrosis, apoptosis, proliferation and angiogenesis. [Results]:There were significant differences in the number of CTCs between the sorafenib group and the control group on day35(1.40±0.55versus3.60±1.14, P=0.005), day42(2.00±1.00versus11.60±4.56, P=0.002), day49(2.40±1.95versus22.80±7.33, P<0.001), day56(3.80±2.77versus42.20±11.63, P=0.001) after orthotopic tumor implantation. In addition, there were significant differences between the sorafenib group and the control group on day56after orthotopic tumor implantation in terms of liver tumor volume (529.40±189.23mm3versus2795.60±722.75mm3, P=0,002), fluorescent intensity of lung metastases (169.36±90.21photons/μm2·s versus395.51±86.50Photons/μ·m2s, P<0.001), lung metastatic rate (20%versus100%, P=0.01), number and size of lung metastases (87.50±29.86μm versus404.55±155.01μm, P=0.001), tumor necrosis rate (74.20%±5.12%versus14.40%±7.02%, P<0.001), apoptosis rate (12.40%±3.65%versus4.80%±2.59%, P=0.005), proliferation (Ki-67positive rate:6.40%±2.70%versus78.80%±5.17%, P<0.001), and angiogenesis (microvessel density:65.60±11.26number/mm2versus208.00±19.24number/mm2, P<0.001).[Conclusions]:sorafenib significantly decreases the number of circulating tumor cells and inhibits hematogenous metastasis by inducing tumor necrosis and apoptosis, and suppressing tumor proliferation and angiogenesis. This study provides the groundwork for further using sorafenib as a perioperative therapy for patients with high-risk hepatocellular carcinoma in clinic.Section3Sorafenib Adjuvant Therapy after Liver Transplantation Inhibits Cancer Recurrence and Metastasis in a Rat Model of Hepatocellular Carcinoma[Background and study aims]:It remains unclear whether sorafenib adjuvant therapy after liver transplantation inhibits cancer recurrence and metastasis for hepatocellular carcinoma (HCC). Therefore, we perform an animal study to assess the efficacy and safety of sorafenib adjuvant therapy after liver transplantation in an orthotopic rat model of hepatocellular carcinoma.[Methods]:Firstly, we established an orthotopic rat model of highly metastatic Morris hepatoma and performed allogeneic liver transplantation from Lewis rat to ACI rat with liver rejection. Secondly, twelve ACI rats with Morris hepatoma were randomized into a sorafenib adjuvant therapy group or a control group by body weight after allogeneic liver transplantation. Both groups received subcutaneous cyclosporine A (CsA) injections to prevent liver rejection. The sorafenib group underwent sorafenib oral administration once daily at dose levels of30mg/kg body weight from day3after allogeneic liver transplantation, whereas the control group received solvent oral administration. Cancer recurrence and metastasis were detected by positron emission tomography/computed tomography (PET/CT). Finally, we further explore the underlying mechanisms by investigating whether sorafenib inhibits tumor proliferation, invasion and metastasis under the conditions of ischemia-reperfusion injury and immunosuppression after allogeneic liver transplantation. Liver function marked by the serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were examined by biochemical analysis. Serum levels of VEGF, interferon-gamma (IFN-y), and interleukin (IL)-4were measured by enzyme-linked immunosorbent assay (ELISA).[Results]:Sorafenib adjuvant therapy after liver transplantation, compared with control, significantly inhibited cancer recurrence and metastasis, and then improved progression-free survival (29.17±2.86days versus18.67±3.61days, P=0.001) and overall survival (59.67±7.00days versus35.50±6.60days, P=0.001). The serum levels of VEGF and HGF significantly increased and the Th1/Th2immune balance was shifted towards Th2after allogeneic liver transplantation. Under these conditions of increased growth factors and decreased recipient immunity, sorafenib adjuvant therapy after liver transplantation, compared with control, significantly suppressed tumor proliferation (Ki67-positive rate:14.50%±3.83%versus68.67%±7.29%, P<0.001), inhibited tumor angiogenesis (microvessel density:46.67±8.16number/mm2versus189.33±19.58number/mm2, P<0.001), and induced tumor apoptosis (apoptosis rate:12.40%±3.65%versus4.80%±2.59%, P=0.005).[Conclusions]:Sorafenib adjuvant therapy after liver transplantation is effective in reducing cancer recurrence and metastasis after liver transplantation. This study provides experimental evidence showing the considerable clinical value of sorafenib as an adjuvant therapy after liver transplantation for patients with high-risk HCC such as poor differentiation, microvascular invasion, and/or exceeding Milan criteria. |
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