Comprehensive Mutation Profiling of Colorectal Cancer Patients With Lung or Liver Metastasis by Targeted Next-Generation Sequencing.

OBJECTIVES: Primary tumor tissue is often analyzed to search for predictive biomarkers and DNA-guided personalized therapies, but there is an incomplete understanding of the discrepancies in the genomic profiles between primary tumors and metastases, such as liver and lung metastases. METHODS: We performed in-depth targeted next-generation sequencing of 520 key cancer-associated genes for 47 matched primary and metastatic tumor samples which were retrospectively collected. RESULTS: A total of 699 mutations were detected in the 47 samples. The coincidence rate of primary tumors and metastases was 51.8% (n = 362), and compared to patients with liver metastases, patients with lung metastases had a significantly greater coincidence rate (P = .021). The number of specific mutations for the primary tumors and liver and lung metastases was 186 (26.6%), 122 (17.5%), and 29 (4.1%), respectively. Analysis of a patient with all three occurrences, including a primary tumor, liver metastasis, and lung metastasis, indicated a possible polyclonal seeding mechanism for liver metastases. Remarkably, multiple samples from patients with primary and metastatic tumors supported a mechanism of synchronous parallel dissemination from primary tumors to metastatic tumors that were not mediated through pre-metastatic tumors. We also found that the PI3K-Akt signaling pathway significantly altered lung metastases compared to matched primary tumors (P = .001). In addition, patients with mutations in CTCF, PIK3CA, or TP53 and LRP1B, AURKA, FGFR1, ATRX, DNMT3B, or GNAS had larger primary tumor sizes and metastases, especially patients with both LRP1B and AURKA mutations. Interestingly, CRC patients with TP53-disruptive mutations were more likely to have liver metastases (P = .016). CONCLUSION: In this study, we demonstrate significant differences in the genomic landscapes of colorectal cancer patients based on the site of metastasis. Notably, we observe a larger genomic variation between primary tumors and liver metastasis compared to primary tumors and lung metastasis. These findings can be used for tailoring treatments based on the specific metastatic site.

Quantitative proteomics profiling reveals the inhibition of trastuzumab antitumor efficacy by phosphorylated RPS6 in gastric carcinoma.

BACKGROUND: The anti-HER2 antibody trastuzumab is a standard treatment for gastric carcinoma with HER2 overexpression, but not all patients benefit from treatment with HER2-targeted therapies due to intrinsic and acquired resistance. Thus, more precise predictors for selecting patients to receive trastuzumab therapy are urgently needed. METHODS: We applied mass spectrometry-based proteomic analysis to 38 HER2-positive gastric tumor biopsies from 19 patients pretreated with trastuzumab (responders n = 10; nonresponders, n = 9) to identify factors that may influence innate sensitivity or resistance to trastuzumab therapy and validated the results in tumor cells and patient samples. RESULTS: Statistical analyses revealed significantly lower phosphorylated ribosomal S6 (p-RPS6) levels in responders than nonresponders, and this downregulation was associated with a durable response and better overall survival after anti-HER2 therapy. High p-RPS6 levels could trigger AKT/mTOR/RPS6 signaling and inhibit trastuzumab antitumor efficacy in nonresponders. We demonstrated that RPS6 phosphorylation inhibitors in combination with trastuzumab effectively suppressed HER2-positive GC cell survival through the inhibition of the AKT/mTOR/RPS6 axis. CONCLUSIONS: Our findings provide for the first time a detailed proteomics profile of current protein alterations in patients before anti-HER2 therapy and present a novel and optimal predictor for the response to trastuzumab treatment. HER2-positive GC patients with low expression of p-RPS6 are more likely to benefit from trastuzumab therapy than those with high expression. However, those with high expression of p-RPS6 may benefit from trastuzumab in combination with RPS6 phosphorylation inhibitors.

High dissolved oxygen exacerbates ammonia toxicity with sex-dependent manner in zebrafish.

Ammonia nitrogen is one of the important environmental factors, and causes negative effects for fish health in ecosystem and aquaculture. The toxic effects and mechanisms of ammonia in fish deserve further investigation. In the present study, we exposed female and male zebrafish (Danio rerio) to ammonia (50 mg/L NH4Cl) with oxygenated (7.5-7.8 mg/L) or non­oxygenated (3.8-4.5 mg/L) water, to identify the combined effects of dissolved oxygen and ammonia on fish with gender difference. The results showed that oxygenated ammonia exposure increased fish mortality, gill secondary lamellas damage and gill tissue spaces, gene expressions of proinflammatory interleukin 1 beta (il-1ß) and apoptotic caspase8 as compared with non­oxygenated ammonia. Besides, oxygenated ammonia elevated plasma ammonia contents, and decreased the discharge of body ammonia through gills by depressing the enzyme activity of Na+/K+-ATPase. Notably, when zebrafish were subjected to ammonia stress, more severe mortality, gill damage and tissue inflammatory response were observed in males than females. This is the first study to clarify the gender-dependent impacts of ammonia toxicity, and the adverse effects of oxygenation on ammonia resistance in zebrafish.

PIAS3 suppresses damage in an Alzheimer's disease cell model by inducing the STAT3-associated STAT3/Nestin/Nrf2/HO-1 pathway.

BACKGROUND: Alzheimer's disease (AD), the most common form of dementia, is caused by the degeneration of the central nervous system (CNS). A previous study reported that signal transducer and activator of transcription 3 (STAT3) is activated during AD development; nonetheless, the related mechanism remains unknown. Thus, this study used a cell model to explore whether and how the protein inhibitor of activated STAT3 (PIAS3) is involved in AD development. METHODS: Cerebrospinal fluid (CSF) specimens of 30 patients with AD and 10 normal participants were included in this study. SH-SY5Y cells were used to constructed AD model. Relevant indices were then detected and analyzed. RESULTS: The results showed that compared with the control group, PIAS3 expression was substantially decreased in patients with AD and amyloid beta (Aß)-treated SH-SY5Y cells. PIAS3 overexpression was able to reverse the detrimental effects of Aß treatment on cell survival and growth. Further, it could also ameliorate apoptosis and oxidative stress in Aß-treated SH-SY5Y cells. Additionally, PIAS3 was shown to reduce the activated form of STAT3 and increase the activity of the downstream Nestin/nuclear factor erythroid 2-related factor/heme oxygenase-1 pathway. CONCLUSIONS: STAT3 reactivation by colivelin treatment negated the influence of PIAS3 on the survival, growth, apoptosis, and oxidative stress of Aß-treated SH-SY5Y cells.

miR-29c-3p regulates TET2 expression and inhibits autophagy process in Parkinson's disease models.

Autophagy in dopamine (DA) neurons is concerned to be associated with Parkinson's disease (PD), but the detailed mechanism remains unknown. Herein, we aimed to investigate the function of microRNA (miR)-29c-3p in autophagy in PD models. Intraperitoneal injection of MPTP (20 mg/kg) was given to C57BL/6 mice to establish PD mouse model. SH-SY5Y cells were treated with MPP+ (1 mmol/L) to establish in vitro PD model. The results indicated that in the substantia nigra pars compacta (SNpc) DA neurons of PD mice, autophagy was activated accompanied by down-regulated miR-29c-3p and up-regulated ten-eleven translocation 2 (TET2) expression. Up-regulation of miR-29c-3p inhibited TET2 expression and SNpc (including DA neurons) autophagy in PD mice. In vitro PD model confirmed that MPP+ treatment markedly down-regulated miR-29c-3p expression and up-regulated TET2 expression in SH-SY5Y cells in a dose/time-dependent manner. Moreover, miR-29c-3p up-regulation also inhibited autophagy and TET2 expression in vitro. Additionally, TET2 was proved to be targeted and down-regulated by miR-29c-3p. TET2 knockdown inhibited MPP+ -induced autophagy, whereas TET2 over-expression reversed the effects of miR-29c-3p over-expression on SH-SY5Y cell autophagy. Overall, miR-29c-3p over-expression inhibits autophagy in PD models, which may be mediated by TET2. Our finding may provide new insights for regulating autophagy to improve PD progression.

DDX54 Plays a Cancerous Role Through Activating P65 and AKT Signaling Pathway in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most malignant cancers, and its incidence is still steadily increasing. The DDX RNA helicase family members have been found to play a role in various cancers; however, the role of DDX54 in colorectal cancer is still unclear and needed to be defined. Here, we found DDX54 was overexpressed in CRC tissues by the label-free mass spectrum, which was also verified in tissue microarray of colon cancer, as well as the CRC cell lines and TCGA database. High DDX54 level was correlated with tumor stage and distant metastasis, which always indicated a poor prognosis to the CRC patients. DDX54 could promote the proliferation and mobility of CRC cells through increasing the phosphorylation level p65 and AKT leading to the tumorigenesis. Here, we have preliminarily studied the function of DDX54 in CRC, which would improve our understanding of the underlying biology of CRC and provide the new insight that could be translated into novel therapeutic approaches.

Inhibition of Long Noncoding RNA SNHG15 Ameliorates Hypoxia/Ischemia-Induced Neuronal Damage by Regulating miR-302a-3p/STAT1/NF-κB Axis.

PURPOSE: Ischemic brain injury results in high mortality and serious neurologic morbidity. Here, we explored the role of SNHG15 in modulating neuronal damage and microglial inflammation after ischemia stroke. MATERIALS AND METHODS: The hypoxia/ischemia models were induced by middle cerebral artery occlusion in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Quantitative real-time PCR (qRT-PCR) and Western blot were conducted to determine the levels of SNHG15, miR-302a-3p, and STAT1/NF-κB. Moreover, gain- or loss-of functional assays of SNHG15 and miR-302a-3p were conducted. MTT assay was used to evaluate the viability of HT22 cells, and the apoptotic level was determined by flow cytometry. Furthermore, enzyme-linked immunosorbent assay was performed to detect oxidative stress and inflammatory mediators in the ischemia cortex and OGD/R-treated BV2 microglia. RESULTS: The SNHG15 and STAT1/NF-κB pathways were both distinctly up-regulated, while miR-302a-3p was notably down-regulated in the ischemia cortex. Additionally, overexpressing SNHG15 dramatically enhanced OGD/R-mediated neuronal apoptosis as well as the expression of oxidative stress and inflammation factors from microglia. In contrast, knocking down SNHG15 or overexpressing miR-302a-3p relieved OGD/R-mediated neuronal apoptosis and microglial activation. Moreover, the rescue experiment testified that overexpressing miR-302a-3p also attenuated SNHG15 up-regulation-induced effects. In terms of the mechanisms, SNHG15 sponged miR-302a-3p and activated STAT1/NF-κB as a competitive endogenous RNA, while miR-302a-3p targeted STAT1 and negatively regulated the STAT1/NF-κB pathway. CONCLUSION: SNHG15 was up-regulated in the hypoxia/ischemia mouse or cell model. The inhibition of SNHG15 ameliorates ischemia/hypoxia-induced neuronal damage and microglial inflammation by regulating the miR-302a-3p/STAT1/NF-κB pathway.

The individual and combined effects of hypoxia and high-fat diet feeding on nutrient composition and flesh quality in Nile tilapia (Oreochromis niloticus).

Hypoxia and high-fat diet (HFD) feeding are two factors commonly existing in aquaculture. However, their individual and combined effects on nutrient composition and flesh quality in fish have not been investigated. The present study evaluated the alterations of growth, nutrient composition and flesh quality in Nile tilapia (initially 7.0 ± 0.1 g and 5.6 ± 0.2 cm) fed with normal fat diet (5.95% fat) or HFD (11.8% fat) at two dissolved oxygen levels (1.1 ± 0.1 and 7.2 ± 0.1 mg/L) for 8 weeks. The results showed that hypoxia and HFD had similar effects in inducing lipid deposition, reducing flesh protein and amino acids content, pH values and water holding ability. Hypoxia had additional adverse effects in decreasing meat yield, flesh contents of n-3 PUFA and glycogen, increasing flesh fragmentation and causing liver damages. The combination of hypoxia and HFD significantly decreased feed intake, survival rate and muscle protein content, but didn't affect flesh quality-related parameters.

High tumor mutation burden in a patient with metastatic gastric cancer sensitive to trastuzumab: a case report.

Patients with HER2-positive gastric cancer (GC) can benefit from the addition of trastuzumab. However, not all patients with HER2-positive GC respond to trastuzumab. Biomarkers affecting its efficacy in patients with advanced gastric cancer (AGC) are largely unknown. Therefore, classifying GC into molecularly distinct subtypes to accurately distinguish between GC patients who would and would not benefit from trastuzumab is worthwhile. Tumor mutation burden (TMB) is a notable feature in GC and whether TMB influences trastuzumab efficacy is still unknown. Herein, we report the case of a 61-year-old man who was diagnosed with metastatic HER2-positive gastric adenocarcinoma that had spread to the liver (T4aN0M1, stage IV). Esophagogastroduodenoscopy revealed a circular ulcer in the posterior wall of the stomach. A computed tomography (CT) scan revealed a 2-cm diameter liver metastasis. Immunohistochemical analysis of the endoscopic biopsy tumor revealed 3+ positive expression for HER2. Whole-exome sequencing (WES) of the tumor tissue revealed 3,736 somatic mutations in 2,423 genes and a very high TMB of 50.3 mutations/Mb. Immunohistochemistry revealed that the patient had mismatch repair-proficient (pMMR) GC. The patient received first-line trastuzumab-containing chemotherapy, and after 2 courses of sequential metronomic trastuzumab-containing chemotherapy, restaging CT showed that the liver metastasis had disappeared. Following resection, the patient had no recurrence and no new tumor metastasis after a follow-up of period nearly 7 years. This study is the first to report that pMMR GC with a high TMB has a favorable response to trastuzumab. The combination of HER2 positivity and a high TMB may be sufficiently predictive of sensitivity to trastuzumab in AGC.

Gemfibrozil improves lipid metabolism in Nile tilapia Oreochromis niloticus fed a high-carbohydrate diet through peroxisome proliferator activated receptor-α activation.

High carbohydrate diet (HCD) can induce lipid metabolism disorder, characterized by excessive lipid in farmed fish. Peroxisome proliferator activated receptor-α (PPARα) plays an important role in lipid homeostasis. In this study, we hypothesize that PPARα can improve lipid metabolism in fish fed HCD. Fish (3.03 ± 0.11 g) were fed with three diets: control (30% carbohydrate), HCD (45% carbohydrate) and HCG (HCD supplemented with 200 mg/kg gemfibrozil, an agonist of PPARα) for eight weeks. The fish fed HCG had higher growth rate and protein effiency than those fed the HCD diet, whereas the opposite trend was observed in feed conversion ratio, hepatosomatic index and mesenteric fat index. Additionally, fish fed HCG significantly decreased lipid accumulation in the whole body, liver and adipose tissues compared to those fed the HCD diet. Furthermore, fish in the HCG group significantly increased the mRNA and protein expression and protein dephosphorylation of PPARα. The HCG group also significantly increased the mRNA level of the downstream target genes of PPARα, whereas the opposite trend occured in the mRNA level of lipolysis-related genes compared to the HCD group. Besides, fish in the HCG group remarkably decreased the contents of alanine aminotransferase, aspartate aminotransferase and malondialdehyde, whereas the opposite occured in the activities of antioxidative enzymes and anti-inflammatory cytokine genes compared to the HCD group. This study indicates that gemfibrozil can improve lipid metabolism and maintain high antioxidant and anti-inflammatory capacity through activating PPARα in Nile tilapia fed a high carbohydrate diet.

High-carbohydrate diet promotes the adaptation to acute hypoxia in zebrafish.

Oxygen deprivation (hypoxia) is a common challenge in water environment, which causes lack of energy and oxidative damage in organisms. Many studies have indicated a number of physiological and metabolic changes under hypoxia, but the effects of dietary nutrients on hypoxia tolerance have not been well evaluated. In the present 7-week feeding trial, we fed zebrafish with low-protein diet (LP), high-protein diet (HP), low-fat diet (LF), high-fat diet (HF), low-carbohydrate diet (LC), and high-carbohydrate diet (HC), respectively. Afterward, the resistance to acute hypoxia challenge, growth, body composition, activities of metabolic enzymes, and expressions of energy homeostasis-related genes and six hifαs genes were measured. The results indicated that only the HC diet could significantly improve the resistance to hypoxia challenge. Moreover, the HC diet feeding caused higher glycogen deposition in the liver and muscle, and these glycogens were significantly reduced after 6-h acute hypoxia challenge. Meanwhile, the lactate content in the liver and blood was increased in the HC groups. At hypoxia status, the relative mRNA expressions of the genes related to glycolysis, ATP production, insulin signaling pathway, and hif-3a (hif1al) were all significantly increased in the muscle of the HC diet-fed fish. This study revealed that high-carbohydrate diet could improve the resistance to hypoxia by activating glycolysis and hif/insulin signaling pathway in zebrafish, mainly in the muscle, to efficiently supply energy. Therefore, our results highlight the importance of dietary carbohydrate in resisting hypoxia in fish.

Protective effect of sakuranetin in brain cells of dementia model rats.

Alzheimer's disease (AD) is a high-incidence neurodegenerative disease with complex and diverse pathogenesis. With aging of the population and continuous improvement of living standards, the incidence of AD is on the increase. Therefore, there is need to develop more effective AD drugs in order to improve the quality of life of the elderly. Sakuranetin (SAK) is a dihydroflavonoid compound extracted from plants. It has many physiological properties. In this study, the effect of SAK on spatial discrimination in a rat model of cognitive dysfunction exposed to D-galactose was investigated with respect to its effect on malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels, and on the expressions of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and nuclear factor-κB inhibitory factor-α (IκBα) in hippocampus of rats. The results obtained suggest that SAK may exert protective effects on brain cells through anti-oxidation mechanism. Moreover, the improvement in learning and memory impairment by SAK may also be related to the inhibition of inflammatory mediators in brain tissue. These findings provide scientific evidence that can be exploited for more effective treatment of Alzheimer's disease.

Pyridoxine exerts antioxidant effects in cell model of Alzheimer's disease via the Nrf-2/HO-1 pathway.

Pyridoxine is a water- soluble pyridine derivative. The effect of pyridoxine in cell models of Alzheimer's disease (AD), and the potential mechanisms involved, are not fully understood. In this study, the anti-AD effects of pyridoxine were studied in an AD cell model using a combination of techniques viz MTT assay, western blotting and assays for reactive oxygen species (ROS). Assays were also carried out to determine the mechanism underlying the antioxidant effects of pyridoxine. The results obtained revealed that pyridoxine exerted a protective potential against AD, attenuated ROS levels, decreased the expressions of cytoplasmic Nrf2, and upregulated whole-cell HO-1 expression. These results suggest that the anti-AD effect of pyridoxine may be attributed to its anti-oxidant property elicited via stimulation of the Nrf2/HO-1 pathway.

Gastrin inhibits gastric cancer progression through activating the ERK-P65-miR23a/27a/24 axis.

BACKGROUND: To test the hypothesis that activated extracellular signal-regulated kinase (ERK) regulates P65-miR23a/27a/24 axis in gastric cancer (GC) and the ERK-P65-miR23a/27a/24 axis plays an important role in the development of GC, and to evaluate the role of gastrin in GC progression and ERK-P65-miR23a/27a/24 axis. METHODS: The component levels of the ERK-P65-miR23a/27a/24 axis in four fresh GC tissues, 101 paraffin-embedded GC tissues and four GC cell lines were determined by Western blotting, immunohistochemistry (IHC) or qRT-PCR. The effects of gastrin on GC were first evaluated by measuring gastrin serum levels in 30 healthy and 70 GC patients and performing a correlation analysis between gastrin levels and survival time in 27 GC patients after eight years of follow-up, then evaluated on GC cell lines, GC cell xenograft models, and patient-derived xenografts (PDX) mouse models. The roles of ERK-P65-miR23a/27a/24 axis in GC progression and in the effects of gastrin on GC were examined. RESULTS: ERK- P65-miR23a/27a/24 axis was proved to be present in GC cells. The levels of components of ERK-P65-miR23a/27a/24 axis were decreased in GC tissue samples and PGC cells. The decreased levels of components of ERK-P65-miR23a/27a/24 axis were associated with poor prognosis of GC, and ERK-P65-miR23a/27a/24 axis played a suppressive role in GC progression. Low blood gastrin was correlated with poor prognosis of the GC patients and decreased expression of p-ERK and p-P65 in GC tissues. Gastrin inhibited proliferation of poorly-differentiated GC (PGC) cells through activating the ERK-P65-miR23a/27a/24 axis. Gastrin inhibited GC growth and enhanced the suppression of GC by cisplatin in mice or PGC cell culture models through activating the ERK-P65-miR23a/27a/24 axis or its components. CONCLUSIONS: ERK-P65-miR23a/27a/24 axis is down-regulated, leading to excess GC growth and poor prognosis of GC. Low gastrin promoted excess GC growth and contributed to the poor prognosis of the GC patients by down-regulating ERK-P65-miR23a/27a/24 axis. Gastrin inhibits gastric cancer growth through activating the ERK-P65-miR23a/27a/24 axis.

Prohibitin, relocated to the front ends, can control the migration directionality of colorectal cancer cells.

Directional migration is a cost-effective movement allowing invasion and metastatic spread of cancer cells. Although migration related to cytoskeletal assembly and microenvironmental chemotaxis has been elucidated, little is known about interaction between extracellular and intracellular molecules for controlling the migrational directionality. A polarized expression of prohibitin (PHB) in the front ends of CRC cells favors metastasis and is correlated with poor prognosis for 545 CRC patients. A high level of vascular endothelial growth factor (VEGF) in the interstitial tissue of CRC patients is associated with metastasis. VEGF bound to its receptor, neuropilin-1, can stimulate the activation of cell division cycle 42, which recruits intra-mitochondrial PHB to the front end of a CRC cell. This intracellular relocation of PHB results in the polymerization and reorganization of filament actin extending to the front end of the cell. As a result, the migration directionality of CRC cells is targeted towards VEGF. Together, these findings identify PHB as a key modulator of directional migration of CRC cells and a target for metastasis.

Ethylene Diamine Tetraacetic Acid Etched Quantum Dots as a "Turn-On" Fluorescence Probe for Detection of Trace Zinc in Food.

In the present paper, a simple and rapid "turn-on" fluorescence sensor for Zn2+ based on ethylene diamine tetraacetic acid (EDTA) etched CdTe quantum dots (QDs) was developed. First, the initial bright fluorescence of mercaptopropionic acid (MPA) capped CdTe QDs was effectively quenched by EDTA, and then the presence of Zn2+ could "turn on" the weak fluorescence of QDs quenched by EDTA due to the formation of ZnS passivation shell. The increase of fluorescence intensity of EDTA etched QDs was found to be linear with the concentration of Zn2+ added. Under the optimum conditions, the calibration curve of this method showed good linearity in the concentration range of 9.1-1 09.1 µM of Zn2+ with the correlation coefficient R2 = 0.998. The limit of detection (3σ/K) was 2 µM. The developed QDs-based sensor was successfully applied to detect trace zinc in zinc fortified table salts and energy drinks with satisfactory results.

Genetic analysis of 15 mtDNA SNP loci in Chinese Yi ethnic group using SNaPshot minisequencing.

SNaPshot minisequencing is a rapid and robust methodology based on a single base extension with a labeled ddNTP. The present study detected 15 selected SNPs in the mitochondrial DNA (mtDNA) control and coding regions by minisequencing methodology using SNaPshot for forensic purpose. The samples were collected from 99 unrelated individuals of the Yi ethnic minority group in Yunnan Province. We have predominantly found high-frequency transitions (91.7%) and a significantly lower frequency of transversions (8.3%). The nt152, 489, 8701, 10,398, 16,183, and 16,362 loci were highly polymorphic, while the nt231, 473 and 581 loci were not polymorphic in the studied population. Based on these 15 SNPs, a total of 28 mtDNA haplotypes were defined in 99 individuals with the haplotype diversity of 0.9136. Also, we compared the mtDNA sequences of Yi group and other 9 populations worldwide and drew a Neighbor-Joining tree based on the shared 12 mtDNA SNP loci, which demonstrated a close relationship between Yi and Bai groups. In conclusion, the analysis of the 15 selected SNPs increases considerably the discrimination power of mtDNA. Moreover, the SNaPshot minisequencing method could quickly detect mtDNA SNPs, and is economical and sensitive. The set of selected 15 SNPs is highly informative and is capable for anthropology genetic analysis.

MIF, secreted by human hepatic sinusoidal endothelial cells, promotes chemotaxis and outgrowth of colorectal cancer in liver prometastasis.

Growth and invasion of metastatic colorectal cancer (CRC) cells in the liver depend on microenvironment. Here, we showed that human hepatic sinusoidal endothelial cells (HHSECs) induce chemotaxis and outgrowth of CRC cells. Macrophage migration inhibitory factor (MIF), released by HHSECs, stimulated chemotaxis of CRC cells. MIF secreted by HHSECs, but not by CRC cells themselves, promoted migration and epithelial-mesenchymal transition (EMT) and facilitated proliferation and apoptotic resistance of CRC cells. In orthotopic implantation models in nude mice, exogenous MIF stimulated growth of CRC cells and metastasis. Furthermore, MIF accelerated mobility of CRC cells by suppressing F-actin depolymerization and phosphorylating cofilin. Noteworthy, MIF levels were correlated with the size of hepatic metastases. We suggest that HHSECs and paracrine MIF promote initial migration and proliferation of CRC cells in the hepatic sinusoids to generate liver metastases.

CLIC4, ERp29, and Smac/DIABLO derived from metastatic cancer stem-like cells stratify prognostic risks of colorectal cancer.

PURPOSE: Cancer stem-like cells have been well accepted to be involved in recurrence and metastasis of cancers, but the prognostic potential of biomarkers integrating with metastasis and cancer stem-like cells for colorectal cancer is unclear. EXPERIMENTAL DESIGN: We identified three proteins, CLIC4, ERp29, and Smac/DIABLO, from metastatic cancer stem-like cells of colorectal cancer and verified the proteins' role in metastatic behaviors. The proteins were detected by IHC in colorectal cancer tumors and matched colonic mucosa from patients with colorectal cancer who underwent radical surgery in the training cohort. The associations between proteins expression levels and five-year disease-specific survival (DSS) were evaluated to predict the survival probability in the training cohort of 421 cases and the validation cohort of 228 cases. RESULTS: A three-protein panel including CLIC4, ERp29, and Smac/DIABLO, which was generated from multivariate analysis by excluding clinicopathologic characteristics from the training cohort, distinguished patients with colorectal cancer into very low-, low-, middle-, and high-risk groups with significant differences in five-year DSS probability (88.6%, 63.3%, 30.4%, 11.4%; P < 0.001). The panel is independent from tumor-node-metastasis staging system and histologic grading to predict prognosis, and also enables classification of validation cohort into four risk stratifications (five-year DSS probability is 98.2%, 80.2%, 25.6%, and 2.7%; P < 0.001). CONCLUSIONS: CLIC4, ERp29, and Smac/DIABLO integrated into a novel panel based on cancer stem-like cells in association with metastasis stratify the prognostic risks of colorectal cancer. Prediction of risks with molecular markers will benefit clinicians to make decisions of individual management with postoperative colorectal cancer patients.

[A simple method for isolating and culturing single cancer stem cells].

OBJECTIVE: To develop an effective method for isolating and culturing single cancer stem cells. METHODS: The capillary glass tube was stretched on fire and connected to a sterile plastic tube to prepare the single cell separation apparatus. Single SW480 cell clone spheres in serum-free culture were marked with CD133 and CK7, and the single cancer stem cells were separated and cultivated in 96-well plates or microdrop covered by paraffin. RESULTS: SW480 cell clone formation rate was about 1.04%, and the cell clone spheres highly expressed CD133 with low CK7 expression. The isolation of the single cancer stem cells showed a success rate of 98.99% using the separation device. The cell division profile was comparable between the cell cultures in microdrop and 96-well plates in the initial 2 cell divisions (P>0.05), whereas prolonged cell division occurred afterwards in the microdrop culture as compared to 96-well plate culture. The cell population expansion of the single cancer stem cells was similar between microdrop culture (11.5%, 22/192) and 96-well plate culture (9.2%, 17/184) (P>0.05). CONCLUSIONS: Single SW480 cells can develop into cancer stem cell spheres. Microdrop culture is convenient and stable, and can be the primary choice for single cancer stem cell culture.