Shiwu Zhang

Disulfide-crosslinked chitosan hydrogel for cell viability and controlled protein release

Synthetic hydrogel mimics of the extracellular matrix (ECM) were prepared by cross-linking a thiol-modified chitosan (CS). CS was chemically modified using N-acetyl-l-cysteine (NAC). To minimize interference with biological function, the degree of substitution of thiol groups was kept below 50%. Solution of thiolated CS was prepared in pH 7.4 phosphate buffered saline (PBS) and crosslinked by disulfide bond formation in air. The gelation mainly depended on the content of thiol groups on thiolated CS, concentration of thiolated CS and the molecular weight of CS. Thermogravimetric analysis showed the thermal stabilities of CSS-S hydrogels. Results from SEM observation showed a porous 3D hydrogel structure with pores ranging from 5 to 30microm. In vitro release showed that insulin and BSA release could be controlled by choosing the composition, loading and disulfide bond contents. In vitro cell compatibility of the hydrogels on NIH 3T3 cells was evaluated, indicating that the hydrogels were biocompatible and the cells could migrate into the hydrogels. Moreover, cells were viable and preserved 3D cell morphology inside the hydrogels. These results demonstrate that disulfide-crosslinked CS hydrogels, a new type of macroporous, biocompatible, synthetic polymers, are promising applications in tissue engineering, drug delivery, and cell culture.

Role and mechanism of vasculogenic mimicry in gastrointestinal stromal tumors

Vasculogenic mimicry (VM) is the formation of fluid-conducting channels by highly invasive and genetically dysregulated tumor cells. In this study, we collected specimens of 84 human gastrointestinal stromal tumors (GISTs) along with clinicopathologic data and another 42 GISTs with fresh tissue that was used for gelatin zymography. VM was found in 21 of the 84 GISTs using CD31/periodic acid-Schiff double staining and CD117 and CD31 immunohistochemical staining. There was a significant difference in the VM-positive rate between the lesions with a mitotic rate > or =5/50 high-power fields and those with a lower mitotic rate (P = .000) and between the cases with and without liver metastasis (P = .008). There was a significant difference in the VM-positive rate between the high-risk group (5.9%) and the very low/low-risk group (12.5%) (P = .010) or the intermediate-risk group (39.5%) (P = .020). Kaplan-Meier survival analysis showed VM indicated a poor prognosis (P = .0000). Cox proportional hazards model indicated that the presence of VM, tumor size 10 cm or greater, and hemorrhage were independent predictors of a poor prognosis (P = .000, .005, .032, respectively). The staining indexes of matrix metalloproteinase (MMP)-2 and MMP-9 were higher in the VM-positive than in the VM-negative group (P = .024 and .037, respectively). Gelatin zymography showed that the activity of MMP-2 and MMP-9 was significantly higher in the VM-positive lesions (P = .013 and .033, respectively). We conclude that VM in GISTs is an unfavorable prognostic sign and that patients with VM-positive tumors are prone to suffer liver metastasis. Both MMP-2 and MMP-9 play an important role in VM formation in GISTs.

Chemokine CXCL12 and its receptor CXCR4 expression are associated with perineural invasion of prostate cancer

ObjectiveTo identify the roles of CXCL12 and CXCR4 and the associated mechanism involved in perineural invasion of prostate cancer.MethodsThe distribution and expression of CXCL12, CXCR4, MMP-2 and MMP-9 in human prostate cancer and in tumor cells invading nerve tissue were studied with immunohistochemical staining. The effects of exogenous CXCL12 and CXCR4 antagonist AMD3100 on PC3 prostate cancer cells invasiveness were assessed in vitro and in vivo.ResultsThe expression of CXCL12, CXCR4, MMP-2, and MMP-9 in human prostate cancer were higher than those in hyperplastic prostate tissues (P < 0.05). In vitro CXCL12 could stimulate the PC3 cells invasiveness (P < 0.05) while AMD3100 could inhibit invasiveness. In vivo, the number of nerves around the tumor tissue in the group treated with CXCL12 was significantly higher than that found in the control group (P < 0.05). Both the control group and the CXCL12-treated group had more nerves number near the tumor tissue than it found in the AMD3100-treated group. The positive cell number of CXCL12, CXCR4, MMP-2, MMP-9, and NGF expression ranked from highest to lowest, were the CXCL12-treated, the control, and the AMD3100-treated group(P < 0.05).ConclusionCXCL12 and its receptor CXCR4 along with MMP-2 and MMP-9 are related with prostate cancer perineural invasion.

Thalidomide influences growth and vasculogenic mimicry channel formation in melanoma

AimsTo observe the effects of thalidomide on melanoma tumor growth and blood supply patterns in C57 mice.MethodsThirty mice inoculated subcutaneously with B16F10 cells were randomly divided into the treatment group and the control group. Thalidomide was administered once a day at a dose of 200 mg/kg for the treatment group starting on the fifth day after inoculation, and an equivalent volume of 0.5% carboxylmethyl cellulose was administered similarly in the control group. The diameter of the tumors was measured daily after inoculation until the mice were sacrificed on the 19th day. The different blood supply patterns were counted after immunohistochemical and PAS histochemical double-Staining. VEGF, NF-κB, PCNA, MMP-2 and MMP-9 expression in tumor tissue was also assessed.ResultsThe tumor volume(P = 0.019) and the number of vasculogenic mimicry(P = 0.03) and mosaic vessels(P = 0.004) in the treatment group were significantly decreased compared with the control group. VEGF(P = 0.004), NF-κB(P = 0.009), PCNA(P = 0.002), MMP-2 (P = 0.000), MMP-9(P = 0.002) protein expression and MMP-2(P = 0.000) and MMP-9(P = 0.000) mRNA in the treatment group were significantly lower than those in the control groups.ConclusionThalidomide inhibits vasculogenic mimicry channel and mosaic vessels formation in melanoma through the regulation of vasculogenic factors, and it can induce necrosis of melanoma cells, which may be related with the NF-κB signaling pathway.

Vasculogenic Mimicry: a New Prognostic Sign of Gastric Adenocarcinoma

Vasculogenic mimicry (VM) has been generally recognized as a new pattern of tumor neovascularization. It presents in many human malignancies. Till now, there is no report about VM in gastric adenocarcinoma (GAC). In this study, we collected 173 paraffin-embedded human GAC samples, with detailed follow-up and clinicopathologic data. CD31/ periodic acid-Schiff (PAS) double staining, immunohistochemical staining of CK8 & 18 and laminin were performed to validate the existence of VM in GAC. Microvascular density (MVD) and vasulogenic mimicry density (VMD) were counted respectively. VM was observed in 40 of the 173 GAC patients, especially in poorly differentiated GAC (P = 0.014). Patients with VM were prone to hematogenous metastasis and distant recurrence compared with patients without VM (P = 0.020, 0.029). Higher VMD values was also associated with hematogenous metastasis (P = 0.003). Immunohistochemical staining index (SI) of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9 were compared between the VM and non-VM group. The SI of four factors were all higher in the VM group than those of non-VM group (P = 0.000, 0.000, 0.004, 0.009, respectively). The Kaplan-Meier survival analysis showed that the VM group has shorter life span compared with non-VM group (P = 0.022). Cox proportional hazards model indicated that the presence of VM and TNM stage were independent predictors of poor prognosis (P = 0.039 and 0.004) for GAC. In conclusion, VM exists in GAC, especially in poorly differentiated GAC. Additionally, it is an unfavorable prognostic indictor for GAC. Hypoxia may play a role in VM formation in GAC.

The diagnostic value of SYT-SSX detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) for synovial sarcoma: A review and prospective study of 255 cases

This study aimed to evaluate the diagnostic value of SYT‐SSX detected by reverse transcriptase–polymerase chain reaction (RT‐PCR) and fluorescence in situ hybridization (FISH) for synovial sarcoma (SS) in known and potential cases. SYT‐SSX was analyzed in formalin‐fixed, paraffin‐embedded tissues of 62 known SS, 60 non‐SS and 133 potential SS by RT‐PCR and FISH. FISH was mainly performed on a tissue microarray with some modifications. SYT‐SSX was detected in 94.7% (54/57) of known SS and 70.5% (86/122) of potential SS by RT‐PCR and in 96.7% (58/60) of known SS and 78.1% (100/128) of potential SS by FISH. Moreover, SYT‐SSX was negative in 100% (58/58) of non‐SS by RT‐PCR and in 100% (59/59) of non‐SS by FISH. Accordingly, SYT‐SSX was detected in 106 potential SS by RT‐PCR or FISH, including 80 cases manifested by both methods, 20 specimens verified only by FISH and 6 samples confirmed only by RT‐PCR. Clinical findings and immunohistochemistry data were analyzed in potential SS with final molecular diagnosis. The positive ratio of cytokeratin (CK) and epithelial membrane antigen (EMA) in finally diagnosed SS was 51.9% (55/106) and 61.3% (65/106), respectively. Except EMA, clinical parameters (age, sex, tumor size, tumor sites) and other immunohistochemistry indexes (CK, S‐100, neurone specific enolase (NSE), CD99, myoglobin, smooth muscle actin (SMA), cluster of differentiation (CD) 68 and mesothelial cell) had no significant difference between finally diagnosed SS and non‐SS. It is indicated that the efficiency of FISH is comparable to or even higher than that of RT‐PCR for SYT‐SSX detection. The detection of SYT‐SSX by RT‐PCR or FISH is very useful for the final diagnosis of potential synovial sarcomas. (Cancer Sci 2008; 99: 1355–1361)

Hypoxia influences linearly patterned programmed cell necrosis and tumor blood supply patterns formation in melanoma

To investigate the possibility that tumor cells undergoing linearly patterned programmed cell necrosis (LPPCN) establish a spatial foundation for vasculogenic mimicry (VM) and to reveal that hypoxia influences LPPCN formation as well as Endo G and DNase 1 expression, 78 C57 mice were divided evenly into two groups and engrafted with B16 melanoma. Starting 9 days after inoculation, subgroups of mice were killed every 2 days. LPPCN and the tumor blood supply vessel types were counted and Endo G and DNase 1 mRNA expression were measured. Additionally, 124 cases of human melanoma samples were collected to assess the clinical significance of LPPCN and VM. The data revealed that regions of LPPCN were positive for caspase-3, caspase-9 and Bax, and negative for TUNEL staining. Electron microscopy images indicated that these cells took on the morphologic changes of necrosis. There was more DNase I mRNA expression in the hypoxic group than in the control group (P<0.05) in vitro, and the expression of Endo G mRNA in the hypoxic groups was significantly higher than that in the control groups both in vitro and in vivo (P<0.05). VM and LPPCN cell numbers in the ischemic group were higher than those in the control group in the early stage of tumor growth. Finally, the survival time for patients whose samples showed LPPCN and VM was significantly shorter than that of patients with one or neither of those factors. We speculated that under hypoxic conditions, some melanoma cells might undergo LPPCN, thus providing a spatial foundation for VM channel formation.

Prognostic implication of SYT-SSX fusion type and clinicopathological parameters for tumor-related death, recurrence, and metastasis in synovial sarcoma.

The present study aimed to describe the distribution and features of the SYT–SSX fusion gene in Chinese patients with synovial sarcoma (SS), and to analyze the prognostic value of SYT–SSX fusion type and clinicopathological parameters for tumor‐related death, recurrence, and metastasis in SS. SYT–SSX1 and SYT–SSX2 fusion transcripts were tested by reverse transcription–polymerase chain reaction in 141 formalin‐fixed, paraffin‐embedded SS. The prognostic implication of SYT–SSX fusion type and clinicopathological parameters were analyzed by univariate and multivariate survival analyses. SYT–SSX1 and SYT–SSX2 were detected in 50 (34.5%) and 91 (64.5%) tumors, respectively. SYT–SSX1 (risk ratio [RR] = 2.032, P = 0.004), larger tumor size (RR = 1.859, P = 0.008), and aggressive Fédération Nationale des Centers de Lutte Contre le Cancer grade (RR = 2.094, P = 0.001) were adverse predictors for disease‐specific survival. However, SYT–SSX fusion type was not associated with local recurrence‐free survival (P = 0.216). Patients with larger tumors (RR = 2.071, P = 0.005) and those who received marginal excision (RR = 2.556, P = 0.005) had poor local recurrence‐free survival. Besides, SYT–SSX1 (RR = 1.859, P = 0.037), older age (RR = 1.799, P = 0.040), and aggressive International Union Against Cancer stage (RR = 3.690, P < 0.001) proved to be adverse prognostic factors for metastasis‐free survival. In conclusion, compared to SYT–SSX1, SYT–SSX2 was more frequent in Chinese patients with SS. Moreover, SYT–SSX1 was an adverse predictor for disease‐specific survival and metastasis‐free survival, but had no relation to local recurrence‐free survival. In addition, histological grade and tumor size were also important prognostic factors for SS. (Cancer Sci 2009; 100: 1018–1025)

Differential expression of decorin, EGFR and cyclin D1 during mammary gland carcinogenesis in TA2 mice with spontaneous breast cancer.

BackgroundThe Tientsin Albino 2 (TA2) mouse is an inbred strain originating from the Kunming strain. It has a high incidence of spontaneous breast cancer without the need for external inducers or carcinogens. Until now, the mechanism of carcinogenesis has remained unclear. In this study, we investigate differential gene expression, especially the expression of decorin, EGFR and cyclin D1, during mammary gland epithelial cell carcinogenesis in TA2 mice.MethodsGene expression profiles of spontaneous breast cancer and matched normal mammary gland tissues in TA2 mice were ascertained using an Affymetrix Mouse 430 2.0 array. Twelve mammary tissue samples from five month-old female TA2 mice (Group A), as well as 28 samples from mammary (Group B) and cancer tissues (Group C) of spontaneous breast cancer-bearing TA2 mice, were subsequently used to detect the expression of decorin, EGFR and cyclin D1 by real-time PCR and immunohistochemical methods.ResultsSeveral imprinted genes, oncogenes and tumor suppressor genes were differentially expressed between normal mammary gland tissues and breast cancer tissues of TA2 mice. The imprinted gene decorin and the oncogene EGFR were down-regulated in tumor tissues, while the oncogene cyclin D1 was up-regulated. Immunohistochemistry showed that samples in Group A showed high decorin expression more frequently than those in Group B (P < 0.05). More tissue samples in Group B than Group A were positive for nuclear EGFR, and tissue samples in Group B more frequently showed high nuclear EGFR expression than those in Group A or Group C (P < 0.05). The labeling index for cyclin D1 in Group C was significantly higher than in Group B. Mammary tissues of Group A expressed the highest level of decorin mRNA (P < 0.05), and mammary tissues of Group B expressed the highest level of EGFR mRNA (P < 0.05), while cancer tissues expressed the highest level of cyclin D1 mRNA (P < 0.05).ConclusionsThe expression of decorin, EGFR and cyclin D1 in mammary epithelial cells changes with increasing age. The abnormal expression of them may partly contribute to the genesis of spontaneous breast cancer in TA2 mice.

Clusterin Expression and Univariate Analysis of Overall Survival in Human Breast Cancer

The aim of this research is to investigate the significance of clusterin (CLU) expression as a risk factor for breast cancer through tissue microarray technology and univariate analysis of overall survival. Formalin-fixed, paraffin-embedded tissues from 158 cases of breast cancer and 31 cases of normal adjacent tissues assembled into a tissue microarray. Cytoplasmic CLU expression in tumor tissues was measured by immunochemistry. Survival analysis was used to investigate the relationship between CLU expression and prognosis, tumor volume, pathological classification, and recurrence. Survival time of patients with CLU expression, lymph node metastasis, and limited post-surgery chemotherapy (<6 cycles of treatment) was significantly shorter than that of patients with no detectable CLU expression (P=0.000), without lymph node metastasis (P=0.000) and more comprehensive post-surgery chemotherapy (≥6 cycles of treatment) (P=0.035). CLU expression in tumor cells was higher than in normal adjacent breast epithelial cells (P=0.03). The CLU expression staining coefficient of cancer tissues with lymph node metastasis was higher than those without lymph node metastasis (P=0.000). Cytoplasmic CLU expression was found to be a prognostic factor for human breast cancer.