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.
Techniques to analyze and sort single cells based on functional outputs, such as secreted products, have the potential to transform our understanding of cellular biology as well as accelerate the ...development of next-generation cell and antibody therapies. However, secreted molecules rapidly diffuse away from cells, and analysis of these products requires specialized equipment and expertise to compartmentalize individual cells and capture their secretions. Herein, we describe methods to fabricate hydrogel-based chemically functionalized microcontainers, which we call nanovials, and demonstrate their use for sorting single viable cells based on their secreted products at high-throughput using only commonly accessible laboratory infrastructure. These nanovials act as solid supports that facilitate attachment of a variety of adherent and suspension cell types, partition uniform aqueous compartments, and capture secreted proteins. Solutions can be exchanged around nanovials to perform fluorescence immunoassays on secreted proteins. Using this platform and commercial flow sorters, we demonstrate high-throughput screening of stably and transiently transfected producer cells based on relative IgG production. Chinese hamster ovary cells sorted based on IgG production regrew and maintained a high secretion phenotype over at least a week, yielding >40% increase in bulk IgG production rates. We also sorted hybridomas and B lymphocytes based on antigen-specific antibody production. Hybridoma cells secreting an antihen egg lysozyme antibody were recovered from background cells, enriching a population of ∼4% prevalence to >90% following sorting. Leveraging the high-speed sorting capabilities of standard sorters, we sorted >1 million events in <1 h. IgG secreting mouse B cells were also sorted and enriched based on antigen-specific binding. Successful sorting of antibody-secreting B cells combined with the ability to perform single-cell RT-PCR to recover sequence information suggests the potential to perform antibody discovery workflows. The reported nanovials can be easily stored and distributed among researchers, democratizing access to high-throughput functional cell screening.
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.
Renal cell carcinoma (RCC) is a deadly malignancy due to its tendency to metastasize and resistance to chemotherapy. Stem-like tumor cells often confer these aggressive behaviors. We discovered an ...endoglin (CD105)-expressing subpopulation in human RCC xenografts and patient samples with a greater capability to form spheres in vitro and tumors in mice at low dilutions than parental cells. Knockdown of CD105 by short hairpin RNA and CRISPR/cas9 reduced stemness markers and sphere-formation ability while accelerating senescence in vitro. Importantly, downregulation of CD105 significantly decreased the tumorigenicity and gemcitabine resistance. This loss of stem-like properties can be rescued by CDA, MYC, or NANOG, and CDA might act as a demethylase maintaining MYC and NANOG. In this study, we showed that Endoglin (CD105) expression not only demarcates a cancer stem cell subpopulation but also confers self-renewal ability and contributes to chemoresistance in RCC.
•CD105+ cells sorted from kidney cancer cell line xenografts are stem-like cancer cells•CD105 demarcates a chemotherapy-resistant population in clear cell kidney cancer•CD105 mediates the self-renewal potential via CDA, MYC, and NANOG in ccRCC•CD105 knockdown abrogates chemoresistance in CD105+ ccRCC stem cells
In this article, Xu, Wu, and colleagues show that CD105 not only demarcates the stem-like cancer subpopulation but also confers the self-renewal and gemcitabine chemoresistance through CDA, MYC, and NANOG in clear cell renal cell carcinoma (ccRCC). Downregulation of CD105 might serve as a ccRCC targeted therapy toward the stem-like cancer cell subpopulation.
FSH Directly Regulates Bone Mass Sun, Li; Peng, Yuanzhen; Sharrow, Allison C. ...
Cell,
04/2006, Letnik:
125, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Postmenopausal osteoporosis, a global public health problem, has for decades been attributed solely to declining estrogen levels. Although FSH levels rise sharply in parallel, a direct effect of FSH ...on the skeleton has never been explored. We show that FSH is required for hypogonadal bone loss. Neither FSHβ nor FSH receptor (FSHR) null mice have bone loss despite severe hypogonadism. Bone mass is increased and osteoclastic resorption is decreased in haploinsufficient FSHβ
+/− mice with normal ovarian function, suggesting that the skeletal action of FSH is estrogen independent. Osteoclasts and their precursors possess G
i2α-coupled FSHRs that activate MEK/Erk, NF-κB, and Akt to result in enhanced osteoclast formation and function. We suggest that high circulating FSH causes hypogonadal bone loss.
Abstract Objective The cancer stem cell (CSC) paradigm hypothesizes that successful clinical eradication of CSCs may lead to durable remission for patients with ovarian cancer. Despite mounting ...evidence in support of ovarian CSCs, their phenotype and clinical relevance remain unclear. We and others have found high aldehyde dehydrogenase 1 (ALDHhigh ) expression in a variety of normal and malignant stem cells, and sought to better characterize ALDHhigh cells in ovarian cancer. Methods We compared ALDHhigh to ALDHlow cells in two ovarian cancer models representing distinct subtypes: FNAR-C1 cells, derived from a spontaneous rat endometrioid carcinoma, and the human SKOV3 cell line (described as both serous and clear cell subtypes). We assessed these populations for stem cell features then analyzed expression by microarray and qPCR. Results ALDHhigh cells displayed CSC properties, including: smaller size, quiescence, regenerating the phenotypic diversity of the cell lines in vitro, lack of contact inhibition, nonadherent growth, multi-drug resistance, and in vivo tumorigenicity. Microarray and qPCR analysis of the expression of markers reported by others to enrich for ovarian CSCs revealed that ALDHhigh cells of both models showed downregulation of CD24, but inconsistent expression of CD44, KIT and CD133. However, the following druggable targets were consistently expressed in the ALDHhigh cells from both models: mTOR signaling, her-2/neu, CD47 and FGF18/FGFR3. Conclusions Based on functional characterization, ALDHhigh ovarian cancer cells represent an ovarian CSC population. Differential gene expression identified druggable targets that have the potential for therapeutic efficacy against ovarian CSCs from multiple subtypes.
Cells of the monocyte series respond to follicle stimulating hormone (FSH) by poorly characterized mechanisms. We studied FSH-receptors (FSH-R) and FSH response in nontransformed human monocytes and ...in osteoclasts differentiated from these cells. Western blot and PCR confirmed FSH-R expression on monocytes or osteoclasts, although at low levels relative to ovarian controls. Monocyte and osteoclast FSH-Rs differed from FSH-R from ovarian cells, reflecting variable splicing in exons 8–10. Monocytes produced no cAMP, the major signal in ovarian cells, in response to FSH. However, monocytes and osteoclasts transcribed TNFα in response to the FSH. No relation of expression of osteoclast FSH-R to the sex of cell donors or to exposure to sex hormones was apparent. Controls for FSH purity and endotoxin contamination were negative. Unamplified cRNA screening in adherent CD14 cells after 2h in 25ng/ml FSH showed increased transcription of RANKL signalling proteins. Transcription of key proteins that stimulate bone turnover, TNFα and TSG-6, increased 2- to 3-fold after FSH treatment. Smaller but significant changes occurred in transcripts of selected signalling, adhesion, and cytoskeletal proteins. We conclude that monocyte and osteoclast FSH response diverges from that of ovarian cells, reflecting, at least in part, varying FSH-R isoforms.
The CRISPR-based technology has revolutionized genome editing in recent years. This technique allows for gene knockout and evaluation of function in cell lines in a manner that is far easier and more ...accessible than anything previously available. Unfortunately, the ability to extend these studies to
syngeneic murine cell line implantation is limited by an immune response against cells transduced to stably express Cas9. In this study, we demonstrate that a non-integrating lentiviral vector approach can overcome this immune rejection and allow for the growth of transduced cells in an immunocompetent host. This technique enables the establishment of a von Hippel-Lindau (
) gene knockout RENCA cell line in BALB/c mice, generating an improved model of immunocompetent, metastatic renal cell carcinoma (RCC).
Autosomal recessive osteopetrosis (ARO) is a paradigm for genetic diseases that cause severe, often irreversible, defects before birth. In ARO, osteoclasts cannot remove mineralized cartilage, bone ...marrow is severely reduced, and bone cannot be remodeled for growth. More than 50% of the patients show defects in the osteoclastic vacuolar-proton-pump subunit, ATP6a3. We treated ATP6a3-deficient mice by in utero heterologous hematopoietic stem cell (HSC) transplant from outbred GFP transgenic mice. Dramatic phenotype rescue by GFP osteoclasts was obtained with engraftment, which was observed in most cases. Engraftment survived for variable periods. Recipients were not immunosuppressed, and graft-versus-host disease was not observed in all pups born after in utero treatment. Thus, differentiation of unmatched HSC transplanted in utero is sufficient to prevent fatal defects in ARO and may prevent complications of ARO unresponsive to conventional bone marrow transplantation. The presence of defective cells is not a barrier to the rescue of the phenotype by donor HSC.
The osteoclast degrades bone in cycles; between cycles, the cell is motile. Resorption occurs by acid transport into an extracellular compartment defined by an alphav{szligbeta}3 integrin ring. NO ...has been implicated in the regulation of bone turnover due to stretch or via estrogen signals, but a specific mechanism linking NO to osteoclastic activity has not been described. NO stimulates osteoclast motility, and at high concentrations NO causes detachment and terminates resorption. Here we demonstrate that NO regulates attachment through the cGMP-dependent protein kinase I (PKG I) via phosphorylation of the intermediate protein VASP. VASP colocalized with the alphav{szligbeta}3 ring in stationary cells, but alternating bands of VASP and alphav{szligbeta}3 occurred when motility was induced by NO donors or cGMP. Redistribution of VASP correlated with its phosphorylation. Dependency of NO-induced motility on PKG I and on VASP was shown by siRNA knockdown of each protein. VASP knockdown also altered distribution of alphav{szligbeta}3 at the attachment site. We conclude that PKG I and VASP are essential for reorganization of attachment and cytoplasmic proteins in motility induced by NO or by cGMP.
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