M2 macrophages in the tumor microenvironment are important drivers of cancer metastasis. Exosomes play a critical role in the crosstalk between different cells by delivering microRNAs or other ...cargos. Whether exosomes derived from pro-tumorigenic M2 macrophages (M2-Exos) could modulate the metastatic behavior of renal cell carcinoma (RCC) is unclear. This study found that M2-Exos promotes migration and invasion in RCC cells. Inhibiting miR-21-5p in M2-Exos significantly reversed their pro-metastatic effects on RCC cells in vitro and in the avian embryo chorioallantoic membrane in vivo tumor model. We further found that the pro-metastatic mechanism of miR-21-5p in M2-Exos is by targeting PTEN-3'UTR to regulate PTEN/Akt signaling. Taken together, our results demonstrate that M2-Exos carries miR-21-5p promote metastatic features of RCC cells through PTEN/Akt signaling. Reversing this could serve as a novel approach to control RCC metastasis.
Poly(ADP-ribosyl)ation is a post-translational modification of proteins by transferring poly(ADP-ribose) (PAR) to acceptor proteins by the action of poly(ADP-ribose) polymerase (PARP). Two tankyrase ...(TNKS) isoforms, TNK1 and TNK2 (TNKS1/2), are ubiquitously expressed in mammalian cells and participate in diverse cellular functions, including wnt/β-catenin signaling, telomere maintenance, glucose metabolism and mitosis regulation. For wnt/β-catenin signaling, TNKS1/2 catalyze poly(ADP-ribosyl)ation of Axin, a key component of the β-catenin degradation complex, which allows Axin's ubiquitination and subsequent degradation, thereby activating β-catenin signaling. In the present study, we focused on the functions of TNKS1/2 in neuronal development. In primary hippocampal neurons, TNKS1/2 were detected in the soma and neurites, where they co-localized with PAR signals. Treatment with XAV939, a selective TNKS1/2 inhibitor, suppressed neurite outgrowth and synapse formation. In addition, XAV939 also suppressed norepinephrine uptake in PC12 cells, a rat pheochromocytoma cell line. These effects likely resulted from the inhibition of β-catenin signaling through the stabilization of Axin, which suggests TNKS1/2 enhance Axin degradation by modifying its poly(ADP-ribosyl)ation, thereby stabilizing wnt/β-catenin signaling and, in turn, promoting neurite outgrowth and synapse formation.
Abstract
Cancer of the urological system commonly occurs in the kidney, bladder, and prostate gland. The clear cell subtype of renal cell carcinoma (ccRCC) constitutes the great majority of kidney ...cancer. Metastatic ccRCC portends a very poor outcome with no effective treatment available. Prostate cancer is the most common cancer in males in the US. Despite recent advances in selective kinase inhibitors and immunotherapies, the rate of developing new treatment from bench to bedside is slow. A time-consuming step is at the animal drug testing stage, in which the mouse model is the gold standard. In the pursuit to streamline the in vivo cancer biology research and drug development, we explored the feasibility of the chicken chorioallantoic membrane (CAM) model to establish xenografts. The CAM model greatly shortens the time of tumor growth and lowers the cost comparing to immunocompromised mice. We generated CAM xenografts from ccRCC, bladder and prostate cancer, with established cancer cell lines and freshly isolated patient-derived tissues, either as primary tumor cells or small pieces of tumors. The successful CAM engraftment rate from the different tumor sources is 70% or above. Using our previously established metastatic ccRCC mouse model, we showed that the CAM xenograft maintains the same tumor growth pattern and metastatic behavior as observed in mice. Taken together, CAM can serve as a valuable platform to establish new patient-derived xenografts (PDXs) to study tumor biology, thus accelerating the development of individualized treatment to halt the deadly metastatic stage of cancer.
Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo cancer models. The chicken chorioallantoic ...membrane (CAM) model provides an inexpensive, rapid alternative that permits direct visualization of tumor development and is suitable for in vivo imaging. As such, we sought to develop an optimized protocol for engrafting gynecological and urological tumors into this model, which we present here. Approximately 7 days postfertilization, the air cell is moved to the vascularized side of the egg, where an opening is created in the shell. Tumors from murine and human cell lines and primary tissues can then be engrafted. These are typically seeded in a mixture of extracellular matrix and medium to avoid cellular dispersal and provide nutrient support until the cells recruit a vascular supply. Tumors may then grow for up to an additional 14 days prior to the eggs hatching. By implanting cells stably transduced with firefly luciferase, bioluminescence imaging can be used for the sensitive detection of tumor growth on the membrane and cancer cell spread throughout the embryo. This model can potentially be used to study tumorigenicity, invasion, metastasis, and therapeutic effectiveness. The chicken CAM model requires significantly less time and financial resources compared to traditional murine models. Because the eggs are immunocompromised and immune tolerant, tissues from any organism can potentially be implanted without costly transgenic animals (e.g., mice) required for implantation of human tissues. However, many of the advantages of this model could potentially also be limitations, including the short tumor generation time and immunocompromised/immune tolerant status. Additionally, although all tumor types presented here engraft in the chicken chorioallantoic membrane model, they do so with varying degrees of tumor growth.
Exosomes are extracellular vesicles that modulate essential physiological and pathological signals. Communication between cancer cells that express the
tumor suppressor gene and those that do not is ...instrumental to distant metastasis in renal cell carcinoma (RCC). In a novel metastasis model, VHL(-) cancer cells are the metastatic driver, while VHL(+) cells receive metastatic signals from VHL(-) cells and undergo aggressive transformation. This study investigates whether exosomes could be mediating metastatic crosstalk. Exosomes isolated from paired VHL(+) and VHL(-) cancer cell lines were assessed for physical, biochemical, and biological characteristics. Compared to the VHL(+) cells, VHL(-) cells produce significantly more exosomes that augment epithelial-to-mesenchymal transition (EMT) and migration of VHL(+) cells. Using a Cre-
exosome reporter system, the fluorescent color conversion and migration were correlated with dose-dependent delivery of VHL(-) exosomes. VHL(-) exosomes even induced a complete cascade of distant metastasis when added to VHL(+) tumor xenografts in a duck chorioallantoic membrane (dCAM) model, while
(+) exosomes did not. Therefore, this study supports that exosomes from VHL(-) cells could mediate critical cell-to-cell crosstalk to promote metastasis in RCC.
Loss of function of the von Hippel-Lindau (VHL) tumor suppressor gene is a hallmark of clear cell renal cell carcinoma (ccRCC). The importance of heterogeneity in the loss of this tumor suppressor ...has been under reported. To study the impact of intratumoral VHL heterogeneity observed in human ccRCC, we engineered VHL gene deletion in four RCC models, including a new primary tumor cell line derived from an aggressive metastatic case. The VHL gene-deleted (VHL-KO) cells underwent epithelial-to-mesenchymal transition (EMT) and exhibited increased motility but diminished proliferation and tumorigenicity compared to the parental VHL-expressing (VHL
) cells. Renal tumors with either VHL
or VHL-KO cells alone exhibit minimal metastatic potential. Combined tumors displayed rampant lung metastases, highlighting a novel cooperative metastatic mechanism. The poorly proliferative VHL-KO cells stimulated the proliferation, EMT, and motility of neighboring VHL
cells. Periostin (POSTN), a soluble protein overexpressed and secreted by VHL non-expressing (VHL
) cells, promoted metastasis by enhancing the motility of VHL-WT cells and facilitating tumor cell vascular escape. Genetic deletion or antibody blockade of POSTN dramatically suppressed lung metastases in our preclinical models. This work supports a new strategy to halt the progression of ccRCC by disrupting the critical metastatic crosstalk between heterogeneous cell populations within a tumor.
Woody biomass comprising cellulose, hemicellulose, and lignin has been the focus of considerable attention as an alternative energy source to fossil fuel for various applications. However, lignin has ...a complex structure, which is difficult to degrade. Typically, lignin degradation is studied using β-O-4 lignin model compounds as lignin contains a large number of β-O-4 bonds. In this study, we investigated the degradation of the following lignin model compounds
organic electrolysis: 2-(2-methoxyphenoxy)-1-(4-methoxyphenyl)ethanol 1a, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol 2a, and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol 3a. The electrolysis was conducted for 2.5 h at a constant current of 0.2 A using a carbon electrode. Various degradation products such as 1-phenylethane-1,2-diol, vanillin, and guaiacol were identified upon separation
silica-gel column chromatography. The degradation reaction mechanisms were elucidated using electrochemical results as well as density functional theory calculations. The results suggest that the organic electrolytic reaction can be used for the degradation reaction of a lignin model with β-O-4 bonds.
Woody biomass comprising cellulose, hemicellulose, and lignin has been the focus of considerable attention as an alternative energy source to fossil fuel for various applications. However, lignin has ...a complex structure, which is difficult to degrade. Typically, lignin degradation is studied using β-O-4 lignin model compounds as lignin contains a large number of β-O-4 bonds. In this study, we investigated the degradation of the following lignin model compounds
via
organic electrolysis: 2-(2-methoxyphenoxy)-1-(4-methoxyphenyl)ethanol
1a
, 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol
2a
, and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol
3a
. The electrolysis was conducted for 2.5 h at a constant current of 0.2 A using a carbon electrode. Various degradation products such as 1-phenylethane-1,2-diol, vanillin, and guaiacol were identified upon separation
via
silica-gel column chromatography. The degradation reaction mechanisms were elucidated using electrochemical results as well as density functional theory calculations. The results suggest that the organic electrolytic reaction can be used for the degradation reaction of a lignin model with β-O-4 bonds.
Woody biomass comprising cellulose, hemicellulose, and lignin has been the focus of considerable attention as an alternative energy source to fossil fuel for various applications.
Abstract
INTRODUCTION
Immunotherapy represents an exciting new strategy to treat patients with malignant gliomas; however, we and others have previously shown that the presence of other immune cell ...populations in the tumor microenvironment can reduce the effectiveness of immunotherapy. To date, a comprehensive and quantitative examination of immune cell composition in the glioma microenvironment has not been performed.
METHODS
We purified CD45+ cells from freshly digested tumor samples of 35 malignant glioma patients undergoing surgical resection at UCLA and performed CyTOF mass cytometry. We then analyzed the lineage and functional marker expression of these populations. Next, to pursue an unbiased analysis of the multi-dimensional data, we used a computational algorithm that clusters cells into phenotypically distinct populations in an unsupervised manner.
RESULTS
CD11b+ myelo-monocytic cells (1542.13 ± 306.82 cells/mg of tumor; 51.33 ± 6.43% of all cells) represented the dominant population and were more abundant than CD3+ T lymphocytes (233.65 ± 59.53 cell/mg of tumor; 8.41 ± 1.47% of all cells; p < 0.001). Meanwhile, our unsupervised clustering algorithm generated 12 distinct phenotypic clusters. Amongst the immune cells, 76.81% were grouped into 6 clusters that derived from populations of a monocytic lineage including a classical monocytic population, an inflammatory monocyte population, and a differentiated M2 macrophage population. 12.37% of the tumor-infiltrating immune cells grouped into 2 clusters of a NK lineage, 9.06% grouped into 2 clusters of a T cell lineage, 0.3% grouped into 1 cluster of a B cell lineage, and 1.46% cells in 1 cluster of an unable to be classified cell lineage.
CONCLUSIONS
Cells of monocytic origin represented the dominant fraction of cells within the tumor-infiltrating immune cell population. Understanding the immune cell composition in the tumor microenvironment may help improve current and future immunotherapies against gliomas.
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