A Novel Cartesian Plot Analysis for Fixed Monolayers That Relates Cell Phenotype to Transfer of Contents between Fibroblasts and Cancer Cells by Cell-Projection Pumping.

We recently described cell-projection pumping as a mechanism transferring cytoplasm between cells. The uptake of fibroblast cytoplasm by co-cultured SAOS-2 osteosarcoma cells changes SAOS-2 morphology and increases cell migration and proliferation, as seen by single-cell tracking and in FACS separated SAOS-2 from co-cultures. Morphological changes in SAOS-2 seen by single cell tracking are consistent with previous observations in fixed monolayers of SAOS-2 co-cultures.

CSF1/CSF1R Signaling Inhibitor Pexidartinib (PLX3397) Reprograms Tumor-Associated Macrophages and Stimulates T-cell Infiltration in the Sarcoma Microenvironment.

Colony-stimulating factor 1 (CSF1) is a primary regulator of the survival, proliferation, and differentiation of monocyte/macrophage that sustains the protumorigenic functions of tumor-associated macrophages (TAMs). Considering current advances in understanding the role of the inflammatory tumor microenvironment, targeting the components of the sarcoma microenvironment, such as TAMs, is a viable strategy. Here, we investigated the effect of PLX3397 (pexidartinib) as a potent inhibitor of the CSF1 receptor (CSF1R).

Extracellular vesicles from human airway basal cells respond to cigarette smoke extract and affect vascular endothelial cells.

The human airway epithelium lining the bronchial tree contains basal cells that proliferate, differentiate, and communicate with other components of their microenvironment. One method that cells use for intercellular communication involves the secretion of exosomes and other extracellular vesicles (EVs). We isolated exosome-enriched EVs that were produced from an immortalized human airway basal cell line (BCi-NS1.

Potential Hydrodynamic Cytoplasmic Transfer between Mammalian Cells: Cell-Projection Pumping.

We earlier reported cytoplasmic fluorescence exchange between cultured human fibroblasts (Fibs) and malignant cells (MCs). Others report similar transfer via either tunneling nanotubes (TNTs) or shed membrane vesicles, and this changes the phenotype of recipient cells. Our time-lapse microscopy showed most exchange was from Fibs into MCs, with less in the reverse direction.

Increased cell size, structural complexity and migration of cancer cells acquiring fibroblast organelles by cell-projection pumping.

We recently described a hydrodynamic mechanism for cytoplasmic transfer between cells, termed cell-projection pumping (CPP). Earlier image analysis related altered SAOS-2 osteosarcoma cell morphology, to what we now recognize as CPP uptake of fibroblast cytoplasm. We here examine SAOS-2 phenotype following co-culture with human dermal fibroblasts (HDF) in which organelles were pre-labelled with a fluorescent lipophilic marker.

Imaging Tunneling Membrane Tubes Elucidates Cell Communication in Tumors.

Intercellular communication is a vital yet underdeveloped aspect of cancer pathobiology. This Opinion article reviews the importance and challenges of microscopic imaging of tunneling nanotubes (TNTs) in the complex tumor microenvironment. The use of advanced microscopy to characterize TNTs in vitro and ex vivo, and related extensions called tumor microtubes (TMs) reported in gliomas in vivo, has propelled this field forward.

Recombinant TAT-BMI-1 fusion protein induces ex vivo expansion of human umbilical cord blood-derived hematopoietic stem cells.

Transplantation of hematopoietic stem cells (HSCs) is a well-established therapeutic approach for numerous disorders. HSCs are typically derived from bone marrow or peripheral blood after cytokine-induced mobilization. Umbilical cord blood (CB) represents an appealing alternative HSC source, but the small amounts of the individual CB units have limited its applications.

Recapitulating the Size and Cargo of Tumor Exosomes in a Tissue-Engineered Model.

There is a growing interest in the pivotal role of exosomes in cancer and in their use as biomarkers. However, despite the importance of the microenvironment for cancer initiation and progression, monolayer cultures of tumor cells still represent the main in vitro source of exosomes. As a result, their environmental regulation remains largely unknown.

BO-1055, a novel DNA cross-linking agent with remarkable low myelotoxicity shows potent activity in sarcoma models.

DNA damaging agents cause rapid shrinkage of tumors and form the basis of chemotherapy for sarcomas despite significant toxicities. Drugs having superior efficacy and wider therapeutic windows are needed to improve patient outcomes. We used cell proliferation and apoptosis assays in sarcoma cell lines and benign cells; γ-H2AX expression, comet assay, immunoblot analyses and drug combination studies in vitro and in patient derived xenograft (PDX) models.

Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides.

Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and nonglycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy.

A novel cell-stiffness-fingerprinting analysis by scanning atomic force microscopy: comparison of fibroblasts and diverse cancer cell lines.

Differing stimuli affect cell stiffness while cancer metastasis is associated with reduced cell stiffness. Cell stiffness determined by atomic force microscopy has been limited by measurement over nuclei to avoid spurious substratum effects in thin cytoplasmic domains, and we sought to develop a more complete approach including cytoplasmic areas. Ninety μm square fields were recorded from ten separate sites of cultured human dermal fibroblasts (HDF) and three sites each for melanoma (MM39, WM175, and MeIRMu), osteosarcoma (SAOS-2 and U2OS), and ovarian carcinoma (COLO316 and PEO4) cell lines, each site providing 1024 measurements as 32 × 32 square grids.

High affinity and covalent-binding microtubule stabilizing agents show activity in chemotherapy-resistant acute myeloid leukemia cells.

Treatment failure in acute myeloid leukemia (AML) is frequently due to the persistence of a cell population resistant to chemotherapy through different mechanisms, in which drug efflux via ATP-binding cassette (ABC) proteins, specifically P-glycoprotein, is one of the most recognized. However, disappointing results from clinical trials employing inhibitors for these transporters have demonstrated the need to adopt different strategies. We hypothesized that microtubule targeting compounds presenting high affinity or covalent binding could overcome the effect of ABC transporters.

Evaluation of Initial Telomere Length and Changes after Transplantation in Adult Double-Unit Cord Blood Transplant Recipients.

Cord blood (CB) leukocytes have inherent telomere length (TL) variation, and CB hematopoietic stem cells (HSC) can maintain high telomerase levels preventing telomere attrition in vitro. We evaluated TL changes in 13 adult double-unit CB transplant (CBT) recipients. In the 26 units, we observed a marked variation in CB TL at thaw (median, 9.

Total synthesis of glycosylated proteins.

Glycoproteins are an important class of naturally occurring biomolecules which play a pivotal role in many biological processes. They are biosynthesized as complex mixtures of glycoforms through post-translational protein glycosylation. This fact, together with the challenges associated with producing them in homogeneous form, has hampered detailed structure-function studies of glycoproteins as well as their full exploitation as potential therapeutic agents.

CDK4/6 Inhibitor PD 0332991 Sensitizes Acute Myeloid Leukemia to Cytarabine-Mediated Cytotoxicity.

Cyclin-dependent kinase (CDK)4 and CDK6 are frequently overexpressed or hyperactivated in human cancers. Targeting CDK4/CDK6 in combination with cytotoxic killing therefore represents a rational approach to cancer therapy. By selective inhibition of CDK4/CDK6 with PD 0332991, which leads to early G1 arrest and synchronous S-phase entry upon release of the G1 block, we have developed a novel strategy to prime acute myeloid leukemia (AML) cells for cytotoxic killing by cytarabine (Ara-C).

Niche-Based Screening in Multiple Myeloma Identifies a Kinesin-5 Inhibitor with Improved Selectivity over Hematopoietic Progenitors.

Novel therapeutic approaches are urgently required for multiple myeloma (MM). We used a phenotypic screening approach using co-cultures of MM cells with bone marrow stromal cells to identify compounds that overcome stromal resistance. One such compound, BRD9876, displayed selectivity over normal hematopoietic progenitors and was discovered to be an unusual ATP non-competitive kinesin-5 (Eg5) inhibitor.

Arecoline increases basic fibroblast growth factor but reduces expression of IL-1, IL-6, G-CSF and GM-CSF in human umbilical vein endothelium.

Background: Areca nut chewing is associated with oral submucous fibrosis (OSF). Raised vascular basic fibroblast growth factor may induce fibrosis. Arecoline is a muscarinic alkaloid in areca nut, which we earlier reported causes injury and necrosis of human endothelium.

UMG Lenti: novel lentiviral vectors for efficient transgene- and reporter gene expression in human early hematopoietic progenitors.

Lentiviral vectors are widely used to investigate the biological properties of regulatory proteins and/or of leukaemia-associated oncogenes by stably enforcing their expression in hematopoietic stem and progenitor cells. In these studies it is critical to be able to monitor and/or sort the infected cells, typically via fluorescent proteins encoded by the modified viral genome. The most popular strategy to ensure co-expression of transgene and reporter gene is to insert between these cDNAs an IRES element, thus generating bi-cistronic mRNAs whose transcription is driven by a single promoter.

Intercellular communication in malignant pleural mesothelioma: properties of tunneling nanotubes.

Malignant pleural mesothelioma is a particularly aggressive and locally invasive malignancy with a poor prognosis despite advances in understanding of cancer cell biology and development of new therapies. At the cellular level, cultured mesothelioma cells present a mesenchymal appearance and a strong capacity for local cellular invasion. One important but underexplored area of mesothelioma cell biology is intercellular communication.

Smoking accelerates aging of the small airway epithelium.

Background: Aging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals.

AAV-mediated persistent bevacizumab therapy suppresses tumor growth of ovarian cancer.

Rationale: Anti-angiogenesis therapies such as bevacizumab, the monoclonal antibody to vascular endothelial growth factor (VEGF), have been used against ovarian cancer, but transient and low peritoneal drug levels are likely a factor in treatment failure. We hypothesized that a single administration of adeno-associated virus (AAV)-mediated intraperitoneal expression of bevacizumab would direct persistent expression and suppress growth and metastasis of ovarian cancer.

Methods: AAVrh.

SAOS-2 osteosarcoma cells bind fibroblasts via ICAM-1 and this is increased by tumour necrosis factor-α.

We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF) without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of "cellular sipping" changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding.

Synthesis of granulocyte-macrophage colony-stimulating factor as homogeneous glycoforms and early comparisons with yeast cell-derived material.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a medicinally important glycoprotein, used as an immunostimulant following bone-marrow transplant. On the basis of reports of its potential utility as an anticancer vaccine adjuvant, we undertook to develop a synthetic route toward single-glycoform GM-CSF. We describe herein a convergent total synthesis of GM-CSF aglycone and two homogeneous glycoforms.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells.

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation.