We investigated the mechanism of cancer-associated fibroblasts (CAFs) in promoting the invasion and metastasis of pancreatic cancer cells in a non-vascular manner. We verified the original generation of isolated cultured CAFs and normal fibroblasts (NFs) based on the expression of α-SMA and vimentin, and we examined the cell glycolysis level through glucose consumption and lactate production experiments. The mRNA and protein expression of CAF glycolytic enzymes, lactate dehydrogenase and pyruvate kinase m2, were examined by RT-PCR and western blotting, respectively. In vitro culture first-generation pancreatic CAFs were collected and cultured together with pancreas cancer BxPc-3 and Panc-1 cells. Cell invasion and migration were assessed using a Transwell assay and scratch test, respectively. Mitochondrial activity was assessed by experimentally determining oxidative phosphorylation (OP) activity. The aerobic oxidation index of cancer cells was also examined. Succinate dehydrogenase, fumarate hydratase (FH), and monocarboxylate transporter 1 (MCT1) expression were examined using an MCT1-specific inhibitor to remove ‘tumor-stromal’ metabolic coupling to observe the influence of cell interstices on pancreas cancer progression. First-generation isolated cultured CAFs and NFs both grew well, and showed active proliferation. Glucose absorption and lactate production were significantly enhanced in CAFs compared with that in NFs. PCR and western blotting showed that the lactate dehydrogenase and pyruvate kinase m2 mRNA and protein expression levels were increased in the CAFs. After indirect co-culture, OP was increased in the BxPc-3 and Panc-1 cells; correspondingly, succinate dehydrogenase, FH and MCT expression were increased. After the MCT1-specific inhibitor removed ‘tumor-stromal’ metabolic coupling, the migration and invasion abilities of the pancreatic cancer cells were decreased. Pancreatic CAFs can alter metabolism as well as communicate with and respond to cancer cell migration and invasion. This may be an important mechanism for promoting tumor progression in a non-vascular manner in the tumor microenvironment. The mechanism by which CAFs reshape metabolic transition requires further analysis.

Norepinephrine and epinephrine, catecholamine hormones that are major mediators for chronic stress-induced cancers, are implicated in the progression of a number of cancer cells, including gastric adenocarcinoma. However, the underlying mechanisms of these hormones have not been well elucidated. Epithelial–mesenchymal transition (EMT) is a crucial event responsible for cancer cell invasion and metastasis. The hypothesis regarding whether the promotive effects of norepinephrine (NE) on cancer are in part due to its ability to induce an EMT program has not been proven. In this study, we show that NE does not only obviously induce EMT alterations in the morphological characteristics of gastric adenocarcinoma cells, but also increases the markers of EMT, including vimentin expression, and decreases E-cadherin expression, further resulting in cell motility and invasiveness. We also reveal that these actions are mainly mediated through the activation of β2-AR–HIF-1α–Snail signaling pathways. In summary, this study implies that NE induces EMT in gastric adenocarcinoma through the regulation of β2-AR–HIF-1α–Snail activity. The data provide a new perspective on chronic stress in a negative social and psychological state, which may be a risk factor for cancer development and progression.EMT: a novel regulatory program for stress hormone norepinephrine. EMT and gastric adenocarcinoma.

The aim of the study was to investigate the effects of lactic acid on the phenotypic polarization and immune function of macrophages. The human monocyte/macrophage cell line, THP-1, was selected and treated with lactic acid. Immunofluorescence staining, laser confocal microscopy, reverse-transcription polymerase chain reaction (RT-PCR), western blot, siRNA, and ELISA analyses were used to observe changes in the levels of cluster of differentiation (CD)68, CD163, hypoxia inducible factor (HIF)-1α, and programmed death ligand-1 (PD-L1) as well as those of cytokines, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-12, and IL-10. THP-1 macrophages and T cells were co-cultured in vitro to observe the changes in proliferation and apoptosis of T cells. The results showed that, lactic acid (15 mmol/l) significantly upregulated the expression of the macrophage M 2 marker CD163 (P<0.05), cytokines, IFN-γ and IL-10, secreted by M 2-tumor-associated macrophages (TAM, P<0.05), and HIF-1α and PD-L1 (P<0.05), and downregulated the expression of cytokines, TNF-α and IL-12, secreted by M 1-TAM (P<0.05). Redistribution of M 2-TAM subsets and PD-L1 expression was reversed after further transfection of THP-1 cells with HIF-1α siRNA (P<0.05). After co-culturing, T-cell proliferation was inhibited and apoptosis was promoted. In summary, modulation of lactic acid level can redistribute M 2-TAM subsets and upregulate PD-L1 to assist tumor immune escape. The HIF-1α signaling pathway may participate in this process, revealing that macrophages, as 'checkpoints' in organisms, are links that connect the immune status and tumor evolution, and can be used as a target in tumor treatment.

Tumor metastasis is accompanied by a two-stage process of epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). Currently, the exact mechanisms underlying EMT-MET conversion are unclear. In the present study, the mechanisms by which primary sites (hypoxic) and homing sites (normoxic or hyperoxic) participate in EMT-MET conversion were evaluated. Pancreatic cancer cells were grown under different oxygenation conditions. Cell morphology and epithelial (E)-cadherin and vimentin expression were examined. Transwell chambers were used to examine tumor invasiveness, and scratch assays were performed to examine cell migration. Reverse transcription-polymerase chain reaction and western blot analysis were used to quantitate the mRNA and protein expression of E-cadherin, vimentin, Snail and hypoxia-inducible factor (HIF)-1α. BxPc-3 and Panc-1 cells grown under hypoxic conditions demonstrated increased partial EMT, reduced E-cadherin expression, and increased vimentin expression, compared with cells grown under normoxic or hyperoxic conditions. Cells grown under hypoxic conditions also indicated increased migration and invasiveness. HIF-1α mRNA and protein expression was increased in cells grown under hypoxic conditions. These changes were reversed when a specific inhibitor of the HIF-1α receptor was used to block HIF-1α signaling. Differences in oxygen concentration at primary sites and homing sites are important in the EMT-MET process, and the underlying mechanism may involve HIF-1α-Snail signaling.

Abstract. The aquaporins (AQPs) are water channel proteins that exhibit several properties related to tumor development. However, the expression and clinical significance of AQP5 in colorectal cancer, particularly the correlation with circulating tumor cells (CTCs), has not been elucidated. The aim of the present study was to determine whether or not the expression of AQP5 is a strong prognostic biomarker for colorectal cancer. The results showed that of 45 tumor specimens, 14 (31.1%) had high levels of expression of AQP5, 29 (64.4%) exhibited a moderate (intermediate) level of staining, and 2 (4.4%) had an absence of AQP5 staining. AQP5 was only occasionally detected in para-neoplastic [3/45 (6.67%)] and normal tissues [3/45 (6.67%)]. AQP5 protein overexpression frequently accompanied gene amplification detection with fluorescence in situ hybridization (FISH). Moreover, AQP5 expression in colorectal cancer cells was upregulated compared to normal colon cells. AQP5 overexpression was also associated with TNM stage (P=0.002), lymph node metastasis (P=0.016), and distant metastasis (P=0.000). The relationships between age, gender, histologic grade and tumor size with expression of AQP5 were not significant (P>0.05). A positive correlation between the number of CTCs and AQP5 expression (P<0.05) was demonstrated. In addition, patients who did not express AQP5 had a superior cumulative survival rate compared to patients with AQP5 positivity. AQP5 may be used as a novel biomarker for colorectal cancer aggressiveness and metastasis, but it does not reflect drug resistance.