Hsiao-Ching Chen

Stathmin overexpression cooperates with p53 mutation and osteopontin overexpression, and is associated with tumour progression, early recurrence, and poor prognosis in hepatocellular carcinoma.

Stathmin, a major microtubule‐depolymerizing protein, is involved in cell cycle progression and cell motility. This study aimed to elucidate its role in the progression, early tumour recurrence (ETR), and prognosis of hepatocellular carcinoma (HCC). Stathmin mRNA was overexpressed in 88/156 (56%) resected, unifocal, primary HCCs, while p53 mutation was present in 72 (46%) and osteopontin mRNA overexpression in 79 (51%). Stathmin mRNA expression exhibited high concordance (93%) with protein expression in 107 cases examined by immunohistochemistry. Stathmin overexpression correlated with high α‐fetoprotein (>200 ng/ml, p = 0.02), larger tumour size (>5 cm, p = 0.012), high tumour grade (p < 0.0002), high tumour stage (stage IIIA–IV) with vascular invasion and various degrees of intrahepatic metastasis (p < 1 × 10−8), ETR (p = 0.003), and lower 5‐year survival (p = 0.0007). Stathmin protein expression was often more intense in the peripheral regions of tumour trabeculae, tumour borders, and portal vein tumour thrombi. Stathmin overexpression correlated with p53 mutation (p = 0.017) and osteopontin overexpression (p = 1 × 10−8), both of which were associated with vascular invasion (both p < 0.0001) and poorer prognosis (p < 0.0004 and p = 0.0004, respectively). Regardless of the status of p53 mutation or osteopontin expression, stathmin overexpression was associated with higher vascular invasion (all p < 0.0001). Approximately 90% of HCCs harbouring stathmin overexpression with concomitant p53 mutation or osteopontin overexpression exhibited vascular invasion, and hence the lowest 5‐year survival, p = 0.00018 and p = 0.0009, respectively. However, we did not find that stathmin overexpression exerted prognostic impact independent of tumour stage. In conclusion, stathmin expression correlates with metastatic potential, is an important prognostic factor for HCC, and may serve as a useful marker to predict ETR. Copyright

Visualization of hepatobiliary excretory function by intravital multiphoton microscopy

Intravital imaging of hepatobiliary excretion is vital for elucidating liver metabolism. In this work, we describe a novel method to observe the intravital dynamics of the uptake, processing, and excretion of an organic anion, 6-carboxyfluorescein diacetate (6-CFDA) in the hepatobiliary system. This is achieved by the use of multiphoton microscopy and an intravital hepatic imaging chamber. The high-quality images show sequential uptake and processing of 6-CFDA from the hepatocytes and the subsequent excretion into bile canaliculi within approximately 50 min. This is a promising technique to study intravital hepatic physiology and metabolism.

Optical biopsy of liver fibrosis by use of multiphoton microscopy.

We demonstrate the application of multiphoton microscopy in diagnosing toxin- (CCl4-) induced liver fibrosis in mice. Although hepatocyte autofluorescence does not vary significantly, different degrees of necrosis and stellate cell proliferation at necrotic sites in livers with fibrosis (ex vivo) can be detected easily from multiphoton-induced autofluorescence images by use of 780-nm excitation. Our result suggests that multiphoton microscopy can be developed into an effective technique for the detection and diagnosis of liver fibrosis in vivo.

Ex vivo imaging and quantification of liver fibrosis using second-harmonic generation microscopy

Conventionally, liver fibrosis is diagnosed using histopathological techniques. The traditional method is time-consuming in that the specimen preparation procedure requires sample fixation, slicing, and labeling. Our goal is to apply multiphoton microscopy to efficiently image and quantitatively analyze liver fibrosis specimens bypassing steps required in histological preparation. In this work, the combined imaging modality of multiphoton autofluorescence (MAF) and second-harmonic generation (SHG) was used for the qualitative imaging of liver fibrosis of different METAVIR grades under label-free, ex vivo conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, it is the spectrally distinct SHG signal that allows the characterization of the extent of fibrosis. We found that qualitative SHG imaging can be used for the effective identification of the associated features of liver fibrosis specimens graded METAVIR 0 to 4. In addition, we attempted to associate quantitative SHG signal to the different METAVIR grades and found that an objective determination of the extent of disease progression can be made. Our approach demonstrates the potential of using multiphoton imaging in rapid classification of ex vivo liver fibrosis in the clinical setting and investigation of liver fibrosis-associated physiopathology in animal models in vivo.

Label-free diagnosis of human hepatocellular carcinoma by multiphoton autofluorescence microscopy

Conventional diagnosis for hepatocellular carcinoma (HCC) is time consuming in sample preparation and deficient in quantitative examination. In this work, we developed multiphoton autofluorescence (MAF) microscopy as an effective and efficient tool in HCC diagnosis with qualitative imaging and quantitative measurement. We found that MAF imaging effectively identifies cellular architecture in the liver specimens. Our results also demonstrate the capability of using tissue quantitative parameters of multiphoton autofluorescence intensity ratio, the nuclear number density, and nuclear-cytoplasmic ratio for tumor discrimination. This approach has the potential in clinical diagnosis of HCC and the in vivo investigation of liver tumor development in animal models.

Imaging the bone marrow stem cells morphogenesis in PGA scaffold by multiphoton autofluorescence and second harmonic (SHG) imaging

The ability to image tissue engineering products without damaging histological procedures is important for the understanding of the dynamics of tissue reorganization and formation. In this work, we test the ability of multiphoton autofluorescence and second harmonic generation microscopy to image engineered tissues following chrondrogenic induction. The system we used is human bone marrow stem cells seeded in the scaffold polyglycolic acid (PGA). Our results show that autofluorescence can be used to image cells while second harmonic generation signal can be used to visualize the synthesis of extracellular matrix. This approach demonstrates the ability of multiphoton imaging in the study of tissue engineering products.

Optical biopsy of liver fibrosis using multiphoton microscopy

In this work, we apply multiphoton microscopy in diagnosing toxin (CCl/sub 4/) induced liver fibrosis in mice. We found that multiphoton induced spectroscopic signatures may be used to identify stellate cell proliferation and necrotic sites. Our study suggests that multiphoton imaging may be developed into an effective diagnosis tool for liver fibrosis in vivo.

Human Hepatocellular Carcinoma Diagnosis by Multiphoton Autofluorescence Microscopy

Conventionally, the diagnosis of hepatocellular carcinoma (HCC) is performed by qualitative examination of histopathological specimens, which takes times for sample preparation in fixation, section and stain. Our objective is to demonstrate an effective and efficient approach to apply multiphoton microscopy imaging the HCC specimens, with the advantages of being optical section, label-free, subcellular resolution, minimal invasiveness, and the acquisition of quantitative information at the same time. The imaging modality of multiphoton autofluorescence (MAF) was used for the qualitative imaging and quantitative analysis of HCC of different grades under ex-vivo, label-free conditions. We found that while MAF is effective in identifying cellular architecture in the liver specimens, and obtained quantitative parameters in characterizing the disease. Our results demonstrates the capability of using tissue quantitative parameters of multiphoton autofluorescence (MAF), the nuclear number density (NND), and nuclear-cytoplasmic ratio (NCR) for tumor discrimination and that this technology has the potential in clinical diagnosis of HCC and the in-vivo investigation of liver tumor development in animal models.

Multiphoton analysis of normal and diseased livers

Diseases associated with the liver, a major internal organ, can lead to serious health problems. In this work, we present multiphoton images of normal and diseased liver specimens and we will characterize the changes to pathological liver specimens. In particular, we will focus on the physiological changes associated with liver fibrosis. Our results show that multiphoton microscopy is a useful technique for distinguishing normal and diseased liver tissues and that it has potential applications for in vivo diagnosis of liver diseases.

Multiphoton characterization of tissue engineering scaffolds

The use of seeding scaffolds has facilitated advances in tissue engineering. We found that multiphoton microscopy is useful in imaging commonly used tissue engineering scaffolds such as polyglycolic acid (PGA).