Prognostic value and predictive biomarkers of phenotypes of tumour-associated macrophages in colorectal cancer.

BACKGROUND: The roles of different subtypes of tumour-associated macrophages (TAMs) in predicting the prognosis of colorectal cancer (CRC) remain controversial. In this study, different subtypes of TAMs were investigated as prognostic and predictive biomarkers for CRC. METHODS: Expressions of CD68, CD86 and CD163 were investigated by immunohistochemistry (IHC) and immunofluorescence (IF), and the correlation between the expression of CD86 and CD163 was calculated in colorectal cancer tissues from 64 CRC patients. RESULTS: The results showed that high expressions of CD86+ and CD68+ CD86+ TAMs as well as low expression of CD163+ and CD68+ CD163+ TAMs were significantly associated with favourable overall survival (OS). The level of CD86 protein expression showed a negative correlation with CD163 protein expression. In addition, CD86 protein expression remarkably negatively correlated with tumour differentiation and tumour node metastasis (TNM) stage, while CD163 protein expression significantly positively correlated with tumour differentiation and tumour size. As an independent risk factor, high expression of CD86 TAMs had prominently favourable prognostic efficacy, while high expression of CD68+ CD163+ TAMs had significantly poor prognostic efficacy. CONCLUSIONS: These results indicate that CD86+ and CD68+ CD163+ TAMs as prognostic and predictive biomarkers for CRC.

Role of histone deacetylase inhibitors in the aging of human umbilical cord mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are self-renewing cells that exhibit differentiation capacity and immune regulation ability. These versatile cells have a wide range of potential applications. However, the spontaneous differentiation and aging of MSCs during long-term culturing restrict the amount of cells available for therapies and tissue engineering. Thus, maintaining the biological characteristics of MSCs during long-term culturing is crucial. Chromatic modification via epigenetic regulatory mechanisms (e.g., histone acetylation, deacetylation, and methylation) is crucial in stem cell pluripotency. We investigated the effects of largazole or trichostatin A (TSA), a novel histone deacetylase inhibitor (HDACi), against human umbilical cord (hUC)-MSCs aging. Results show that low concentrations of largazole or TSA can significantly improve hUC-MSCs proliferation and delay hUC-MSCs aging. Largazole can better improve MSCs proliferation than TSA. HDAC is modulate histone H3 acetylation and methylation in the telomerase reverse-transcriptase, octamer-binding transcription factor 4, Nanog, C-X-C chemokine receptor 4, alkaline phosphatase, and osteopontin genes. HDACis can promote hUC-MSCs proliferation and suppress hUC-MSCs spontaneous osteogenic differentiation. HDACis can affect histone H3 lysine 9 or 14 acetylation and histone H3 lysine 4 dimethylation, thus increasing the mRNA expression of pluripotent and proliferative genes and suppressing the spontaneous differentiation of hUC-MSCs.

The effect of mesenchymal stromal cells on doxorubicin-induced nephropathy in rats.

BACKGROUND AIMS: The potential protective effects of mesenchymal stromal cells (MSCs) on some kidney diseases has been reported. However, the effect of MSCs on doxorubicin-induced nephropathy is still poorly understood. METHODS: Rats with doxorubicin-induced kidney injuries were treated with human cord-derived MSCs. Human MSCs were first labeled with 5-bromo-2'-deoxyuridine to track their homing in kidneys after infusion. RESULTS: Alleviation of proteinuria, decreased serum albumin, alleviation of lipid disorders and histologic alterations were found in rats 4 weeks after treatment with MSCs, particularly in rats that were given repeat doses. Decreases in serum levels of interleukin-6, tumor necrosis factor-α and prostaglandin E2 and decreases in messenger RNA levels of kidney tissue cylooxygenase-2 and EP4 were found in MSC-treated rats. MSC-treated rats also displayed an increase in serum interleukin-10 levels. CONCLUSIONS: These results indicate that MSCs ameliorate doxorubicin-induced kidney injuries and inflammation, suggesting a potential clinical treatment for inflammatory kidney diseases.

CDX2 as a Predictive Biomarker Involved in Immunotherapy Response Suppresses Metastasis through EMT in Colorectal Cancer.

Background: Studies have confirmed that Caudal Type Homeobox 2 (CDX2) plays a tumor suppressor role in colorectal cancer (CRC) and as a prognostic and predictive marker for colorectal cancer. The epithelial to mesenchymal transition (EMT) is a transdifferentiation process, providing migratory and invasive properties to cancer cells during tumor progression. However, the role of CDX2 during the activation of EMT in CRC maintains controversial. Aim: To investigate whether CDX2 is associated with EMT in CRC. Methods: Forty-six CRC patients were included in the study. Expressions of CDX2, E-cadherin, and N-cadherin in all CRC patients were detected by IHC. ROC assays were applied to detect cut-off points for IHC scores to distinguish high and low expressions of CDX2 in 46 CRC samples. The prognostic value of CDX2 was statistically analyzed. MTT, Western blot, invasion, and migration assays in vitro were employed to explore the function of CDX2. Results: We observed that high expressions of CDX2 and E-cadherin as well as low expressions of N-cadherin were significantly correlated with favorable prognosis. The levels of CDX2 protein exhibited a positive associated with E-cadherin while negative correlation with N-cadherin. Then, the low expression of CDX2 and high expression of CA199 in combination are positively related with poor prognosis. Overexpression of CDX2 reduced expression of MMP-2 and diminished cell proliferation, invasion, and migration, while knockdown CDX2 enhanced MMP-2 expression and increased cell proliferation, invasion, and migration in HCT-116 cells. CDX2 was correlated with expression of EMT markers. Overexpression of CDX2 suppressed the EMT markers indicating that CDX2 suppresses CRC cell viability, invasion, and metastasis through inhibiting EMT. Finally, we found that the expression of CDX2 was negatively associated with Th1 cells, macrophages, Th2 cells, cytotoxic cells, T cells, and T helper cells. Conclusions: These results indicated CDX2 as prognostic biomarkers involved in immunotherapy response for CRC. CDX2 loss promotes metastasis in CRC through a CDX2-dependent mechanism.

Methylation of CDX2 gene promoter in the prediction of treatment efficacy in colorectal cancer.

The aim of the present study was to examine the diagnosis of methylation of CDX2 gene promoter in colorectal cancer (CRC) and assessed its value in the prediction of treatment efficacy. Sixty patients who were diagnosed as CRCs for the first time, 60 patients with hyperplastic polyps (HPs) and adenomas, and 60 patients with inflammatory lesions or healthy patients (control group) were included in the present study. The methylation levels of CDX2 gene promoter were detected by methylation-specific polymerase chain reaction (MSP), and the expression levels of CDX2 mRNA were detected by fluorescence quantitative PCR. Treatment options, such as surgery, radiotherapy and chemotherapy, were chosen on the basis of TNM staging of CRC patients. The tumor-free survival, relapse rate and mortality were also recorded. The methylation rate was 71.67% (43/60) and significantly higher in the CRC group as compared to the HP/adenoma and control groups, P<0.05. Moreover, they showed further increase with higher degree of TNM staging. The expression levels of CDX2 mRNA was significantly lower in the CRC group in comparison to HP/adenoma and control groups, P<0.05, and showed a further decrease with a higher degree of TNM staging. The tumor-free survival was shorter, and the relapse rate and mortality were higher in patients with positive methylation in the CRC group, P<0.05. Multivariate logistic regression analysis demonstrated that TNM staging and positive methylation were independent risk factors of mortality. In conclusion, higher methylation degree of CDX2 gene promoter resulted in decreased expression of CDX2 gene, and was closely associated with TNM staging and prognosis. TNM staging and positive methylation were independent risk factors of mortality for CRC patients.

Methylation of promoter region of CDX2 gene in colorectal cancer.

The incidence of colorectal cancer is on the increase owing to changes in daily diet. In the present study, the methylation status of caudal type homeobox transcription factor 2 (CDX2) gene in lesion tissue of colorectal cancer (CRC) was investigated. Additionally, the correlation between the promoter methylation of CDX2 gene, CRC and gene expression in patients with CRC and normal population was examined. Between April 2014 and May 2015 78 cases with CRC were enrolled in the study. Using methylation-specific polymerase chain reaction (PCR), the promoter methylation of CDX2 in normal tissues and colorectal tissues was examinned. Through the fluorescence quantitative PCR technique, the expression levels of CDX2 gene were determined in a normal population and lesion tissue of patients with CRC. At the same time, we evaluated the levels of the CDX2 gene product in the normal population and lesion tissue of patients with CRC. The results showed that the methylation rate of the promoter region of CDX2 gene in normal colorectal tissue was 43.5%, whereas that in the lesion tissue of CRC was 78.5%. The result was statistically significant (P<0.05). The quantity of mRNA and protein expression of CDX2 gene in colorectal and normal tissue was significantly different (P<0.05). In conclusion, the methylation of the CDX2 gene promoter region was associated with risk of CRC, i.e., methylation of the promoter region of CDX2 gene favors the occurrence of CRC.

[Differentiation of bone marrow mesenchymal stem cells into insulin producing cells induced by rat injured pancreatic tissue extract].

OBJECTIVE: To investigate the effects and mechanisms of differentiation of bone marrow mesenchymal stem cells (BMSCs) into insulin producing cells (IPCs) induced by injured pancreatic tissue extract of rat. METHODS: Eighty 6-week-old Sprague Dawley rats were selected. Forty rats underwent removal of 60% pancreas and the injured pancreas tissue was obtained after 48 hours to prepare the injured pancreatic tissue extract; and normal pancreatic tissue extract was prepared from the other 40 rats. The BMSCs were isolated from the tibia and femur of 4-week-old Sprague Dawley rats. BMSCs at passage 3 were co-cultured with rat injured pancreatic tissue extract as experimental group, with rat normal pancreatic tissue extract as normal control group, and with cell culture medium as blank control group for 14 days. The expressions of pancreas development related genes and proteins were detected, and cell morphological changes were observed. Then the C peptide positive cell rate was detected by flow cytometry analysis and insulin secretion levels were detected by glucose stimulation experiment at 14 days. RESULTS: Injured pancreatic tissue extract can induced BMSCs differentiating into IPCs. The pancreatic development related genes of pancreatic duodenal homeobox 1 (PDX-1), islet 1, Nkx6.1, glucose transporter type 2, proprotein convertase 2, neurogenin 3, and somatostatin were expressed sequentially in the differentiation process of experimental group; mature pancreatic proteins of PDX-1, insulin, C peptide, Nkx6.1 also were expressed. But there was no morphological changes and expression of pancreatic development related genes and proteins in normal control and blank control groups. The C peptide positive cell rate of experimental group (13.8% +/- 1.8%) were significantly higher than those of normal control and blank control groups (1.6% +/- 0.4%) (P < 0.05). The insulin secretion of experimental group was significantly higher than that of normal control and blank control groups (P < 0.05), but it was 1/40 and 1147 of natural islet cells (P < 0.05). CONCLUSION: After pancreatic injury, injured pancreas would secrete transcription proteins related to development, differentiation, and repair of pancreas, which can promote the differentiation of BMSCs into IPCs.

Role of injured pancreatic extract promotes bone marrow-derived mesenchymal stem cells efficiently differentiate into insulin-producing cells.

Mesenchymal stem cells (MSCs) can be successfully induced to differentiate into insulin-producing cells (IPCs) by a variety of small molecules and cytokines in vitro. However, problems remain, such as low transdifferentiation efficiency and poor maturity of trans-differentiated cells. The damaged pancreatic cells secreted a large amount of soluble proteins, which were able to promote pancreative islet regeneration and MSCs differentiation. In this study, we utilized the rat injured pancreatic tissue extract to modulate rat bone marrow-derived MSCs differentiation into IPCs by the traditional two-step induction. Our results showed that injured pancreatic tissue extract could effectively promote the trans-differentiation efficiency and maturity of IPCs by the traditional induction. Moreover, IPCs were able to release more insulin in a glucose-dependent manner and ameliorate better the diabetic conditions of streptozotocin (STZ)-treated rats. Our study provides a new strategy to induce an efficient and directional differentiation of MSCs into IPCs.

[Role of INGAP-pp in the differentiation of hUCMSCs into insulin producing cells].

OBJECTIVE: To find out a protocol for the efficient and directional differentiation from human umbilical cord mesenchymal stem cells (hUCMSCs) into insulin-producing cells (IPCs). METHODS: HUCMSCs were induced to differentiate into IPCs by an improved protocol based on a traditional protocol with addition of islet neogenesis-associated protein-pp (INGAP-pp), and the IPCs were identified by morphological observation, flow cytometry, real-time quantitative PCR, immunofluorescence and ELISA. RESULTS: Compared with the traditional protocol, the improved protocol resulted in the higher rate of C peptide positive cells (P<0.05) and the increased mRNA expressions of pancreas development related transcription factor PDX-1, Ngn3, NeuroD1, MafA, insulin, Glut-2 (P<0.05). Meanwhile, by the improved protocol the protein expressions of C peptide, PDX-1 and Nkx6.1 were positive, and insulin and C-peptide release were promoted (P<0.05) as demonstrated by glucose challenge test. CONCLUSION: INGAP-pp may facilitate the differentiation of hUCMSCs into IPCs. However, the IPCs are not as mature as normal human islet ß cells.