Optimized flow cytometry panel for the detection and analysis of human tumor-induced memory-like NK cells.

Recent studies indicate that under certain conditions such as viral infection or exposure to pro-inflammatory cytokines, NK cells may acquire features of adaptive immune cells. In this context, various forms of adaptive NK cells have been described, i.e. "liver-resident" memory-like NK cells, cytomegalovirus (CMV)-induced memory NK cells and interleukin (IL)12/15/18 cytokine-induced memory-like (CIML)-NK cells. We recently provided evidence that upon a 7-day co-culture with irradiated leukemia specimens NK cells can exhibit a memory-like phenotype with substantial anti-leukemic functionality. Here, we propose an antibody panel that allows the identification of subtle changes in the activation status and maturation during memory cell conversion of these so-called tumor-induced memory-like (TIML)-NK cells but also the comparison of those with other forms of memory NK cells. As tremendous efforts are currently undertaken to evaluate the clinical benefit of adoptive cell transfer of various forms of NK cells, we here delineate the process of our panel design in detail to provide future researchers with the means to optimize the flow cytometric analysis of various forms of memory NK cells within their clinical trial protocols.

Metabolic alteration--Overcoming therapy resistance in gastric cancer via PGK-1 inhibition in a combined therapy with standard chemotherapeutics.

BACKGROUND AND OBJECTIVES: It can be assumed that PGK1 is involved in metastatic spread of gastric carcinomas. Furthermore PGK1 has a proven influence on the characteristics of tumor stem cells. The presence of malignant stem cells, regarding treatment resistance and recurrence, is of considerable importance. We hypothesized that inhibition of PGK1 makes these cells more sensitive to chemotherapeutic agents and therefore mediates an overcome of the existing therapy resistance. METHODS: All investigations were performed with human gastric adenocarcinoma cell lines. Small hairpin RNA knockdown of PGK1 via adenovirus-shPGK1 was used for PGK1-inhibition. Chemotherapeutic agents were 5-FU and mitomycin. FACS, qRT-PCR, and xCELLigence were performed. RESULTS: Using the medium-sole-control indicating the highest cell viability and Triton indicating the lowest, mitomycin and 5-FU alone showed a significant decrease in cell viability. The treatment with AdvshPGK1 alone already showed a better decrease. The simultaneous application of chemotherapeutics and adenovirus showed the strongest effect and is comparable to the effect of Triton. CONCLUSIONS: We showed a significant decrease in cell viability after the simultaneous application of chemotherapeutics and adenovirus. These results suggest that PGK1-inhibition is able to increase the vulnerability of gastric cancer cells and tumor stem cells to overcome the chemotherapeutic therapy resistance.

Phosphoglycerate kinase 1 as a promoter of metastasis in colon cancer.

Oxidative stress due to intratumoral hypoxia in solid cancer has been shown to be associated with increased mortality. Phosphoglycerate kinase 1 (PGK1) is an enzyme of the glycolytic pathway, which is regulated by hypoxia-inducible factor-1α (HIF-1α) and has been described for its role in tumor progression and metastasis in several malignancies. We investigated whether the expression of PGK1 varies between metastatic and non-metastatic colon cancer. We compared PGK1 expression in colon cancer patients either with or without metastasis via polymerase chain reaction (PCR) and immunohistochemistry. Microarray analysis was performed to test altered gene expression after PGK1 silencing, using isolates from HCT116 cell lines. PCR results showed an increased expression of PGK1 in colon cancer tissue from metastatic patients in comparison to patients with no metastasis (fold change 2.6, p<0.001). Immunohistochemical staining of PGK1 showed stronger staining in metastatic tissue in comparison to non-metastatic cancer tissue according to a semi-quantitative evaluation. Microarray and subsequent pathway analysis provided 4 genes of interest (CYR61, FOS, JUN and EGR1) used for pathway proposal. The results indicate that increased expression of PGK1 in colon cancer tissue is associated with metastasis. Furthermore, we propose several genes induced by PGK1 that could account for cell migration, mainly EGR1 and CYR61 together with the transcription factors FOS and JUN.

Induction of tumor stem cell differentiation--novel strategy to overcome therapy resistance in gastric cancer.

PURPOSE: Metastases are a frequent finding in gastric cancer and are associated with poor prognosis. A recently discovered link between metabolic changes, differentiation, and therapy resistance due to tumor stem cells could depict a novel approach in cancer research and therapy. Phosphoglycerate kinase 1 (PGK1) is a metabolic enzyme and is known to be involved in enabling gastric cancer cells to be invasive and to disseminate. In this study, we investigated if PGK1 is a promising candidate in inducing stem cell differentiation in gastric cancer. MATERIALS AND METHODS: MKN45 gastric cancer cells were used due to their known cancer stem cell population, which is defined by the surface marker CD44. MKN45 cells were separated between CD44+ and CD44- cells and, in equal parts, incubated with shRNA anti-PGK1 using fluorescence-activated cell sorting (FACS) analysis; they were then injected into nude mice to evaluate their tumor growth behavior in vivo. Further, the invasive potential of gastric cancer cells was evaluated in vitro using the xCelligence analyzing system. RESULTS: CD44+ gastric cancer cells treated with and without shRNA anti-PGK1 were capable to cause tumor growth in vivo, whereas tumor growth in CD44+ cells treated with shRNA anti-PGK1 was considerably smaller in comparison with that in CD44+ cells without treatment. CD44- cells did not show any noticeable tumor growth in vivo. By targeting PGK1, the invasive potential of gastric cancer cells was impressively reduced in vitro. In all our cells, which were targeted with shRNA anti-PGK1, we did not find any change that is in accordance with the phenotype of the cells using FACS analysis. CONCLUSIONS: Our findings suggest that targeting the key metabolic enzyme PGK1 in gastric cancer cells may open a new chapter in cancer treatment, which is well worth for further exploration in combination with recent chemotherapy, and might be a promising possibility to overcome therapy resistance in gastric cancer.

Lactate influences the gene expression profile of human mesenchymal stem cells (hMSC) in a dose dependant manner.

BACKGROUND/AIMS: Wounds, especially non-healing wounds are characterized by elevated tissue lactate concentrations. Lactate is known for being able to stimulate collagen synthesis and vessel growth. Lately it has been shown that lactate, in vivo, plays an important role in homing of stem cells. With this work we aimed to show the influence of lactate on the gene expressionprofile of human mesenchymal stem cells (hMSC). MATERIALS AND METHODS: hMSCs were obtained from bone marrow and characterized with fluorescence-activated cell sorting (FACS) analysis. Subsequently the hMSCs were treated with either 0, 5, 10 and 15 mM lactate (pH 7,4) for 24 hours. RNA Isolation from stimulated hMSCs and controls was performed. The Microarray analysis was performed using AffymetrixHuGene 1.0 ST Gene Chip. Selected targets were subsequently analysed using quantitative real time PCR (RTq-PCR). RESULTS: We were able to show that lactate in moderate concentrations of 5 respectively 10 mM leads to an anti-inflammatory, anti-apoptotic but growth and proliferation promoting gene expression after 24 h. In contrast, high lactate concentrations of 15 mM leads to the opposed effect, namely promoting inflammation and apoptosis. Hypoxia induced genes did not show any significant regulation. Contrary to expectation, we were not able to show any significant regulation of candidates associated with glycolysis. CONCLUSION: We were able to show that lactate alters gene expression but does not change the cell phenotype, which might be helpful for further investigations of new treatment strategies for chronic non-healing wounds as well as tumor-therapy and neuronal plasticity.

Phosphoglycerate kinase 1 promoting tumor progression and metastasis in gastric cancer - detected in a tumor mouse model using positron emission tomography/magnetic resonance imaging.

BACKGROUND/AIMS: Tumor dissemination is frequent in gastric cancer and implies a poor prognosis. Cure is only achievable provided an accurate staging is performed at primary diagnosis. In previous studies we were able to show a relevant impact of increased phosphoglycerate kinase 1 expression (PGK1; a glycolytic enzyme) on invasive properties of gastric cancer in-vivo and in-vitro. Thus the aim of the present study was to evaluate the effect of enhanced PGK1 expression in gastric cancer employing magnetic resonance (MR)-imaging combined with positron emission tomography (PET), a recently emerging new high resolution imaging technique in a mouse model. METHODS: A metastatic nude mouse model simulating human gastric cancer behavior by orthotopic tumor implantation was established. Mice were divided into one control group (n=5) and two experimental groups (n=30) divided by half in animals baring tumors from MKN45-cells and MKN45-cells with plasmid-mediated overexpression of PGK1. In the course of tumor growth MR-imaging and PET/MRI fusion was performed. Successively experimental animals were examined macroscopically and histopathologically regarding growth, metastasis and PGK1 expression. RESULTS: Elevated PGK1 expression increased invasive and metastatic behavior of implanted gastric tumors significantly. MR/PET- imaging results in-vivoand subsequent ex-vivo findings concerning tumor growth and metastasis correlated excellently and could be underlined by concordant immuohistochemical PGK1 staining. CONCLUSION: Consistent in-vivo findings suggest that PGK1 might be crucially involved in gastric malignancy regarding growth and metastasis, which was also underlined by novel imaging techniques. Thus, PGK1 may be exploited as a prognostic marker and/or be of potential therapeutic value preventing malignant dissemination.

Phosphoglycerate kinase 1 a promoting enzyme for peritoneal dissemination in gastric cancer.

Peritoneal carcinomatosis is a frequent finding in gastric cancer associated with a poor prognosis. The features that enable gastric tumors to disseminate are poorly understood until now. Previously, we showed elevated mRNA levels of phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate-generating enzyme in the glycolytic pathway, the chemokine receptor 4 (CXCR4), the corresponding chemokine ligand 12 (CXCL12) and beta-catenin in specimens from gastric cancer patients with peritoneal carcinomatosis. In this study, the influence of PGK1 on CXCR4 and beta-catenin was assessed as well as the invasiveness of PGK1 overexpressing cancer cells. In this current study, we found that PGK1 regulates the expression of CXCR4 and beta-catenin at the mRNA and protein levels. On the other hand, CXCR4 regulates the expression of PGK1. Plasmid-mediated overexpression of PGK1 dramatically increased the invasiveness of gastric cancer cells. Interestingly, inhibition of CXCR4 in cells overexpressing PGK1 produced only a moderate reduction of invasiveness suggesting that, PGK1 itself has a critical role in tumor invasiveness. Immunohistochemistry in specimens from diffuse gastric cancer patients also revealed an overexpression of PGK1 in patients with development of peritoneal carcinomatosis. Therefore, PGK1 may be a crucial enzyme in peritoneal dissemination. Together these findings suggest that the enhanced expression of PGK1 and its signaling targets CXCR4 and beta-catenin in gastric cancer cells promote peritoneal carcinomatosis. Thus, PGK1 may serve as prognostic marker and/or be a potential therapeutic target to prevent dissemination of gastric carcinoma cells into the peritoneum.