Description and Characterization of Three-Dimensional Human Mast Cell Progenitor Spheroids In Vitro.

Human mast cells (MC) are an essential component of the immune system as they uniquely store and release a wide range of soluble mediators through IgE and non-IgE mechanisms. Several tissue sources can be used to differentiate functional MC for in vitro and in vivo studies. Here we describe an improved method for obtaining large numbers of human MC from adipose tissue with advantages over current methods. We analyzed donor parameters (e.g. age, race) on MC-isolation following adipose and skin tissue digestion from healthy donors. Adipose and skin-derived MC were morphologically and immunophenotypically similar in all donors regardless of age. However, donor-dependent variations in MC numbers were observed following tissue digestion. In addition, we identified and characterized three-dimensional structures from which mature MC emerged in vitro using peripheral blood and human tissue sources. MC progenitor spheroids (MCPS) appeared approximately one week following progenitor isolation and were consistently observed to have mature MC attached, emerging, or nearby when cultured in a stem cell factor-containing medium. The overall characteristics of the MCPS were similar from each tissue source. We propose that these MCPS serve as the common source of human MC in vitro.

Identification and Characterization of Tunneling Nanotubes Involved in Human Mast Cell FcεRI-Mediated Apoptosis of Cancer Cells.

Mast cells (MCs) are found in practically all tissues where they participate in innate and adaptive immune responses. They are also found in and around tumors, yet their interactions with cancer cells and the resulting impact on cancer cell growth and metastasis are not well understood. In this study, we examined a novel mechanism of IgE-FcεRI-mediated, intercellular communication between human adipose-derived mast cells (ADMC) and cancer cells. The formation of heterotypic tunneling nanotubes (TnT) and membrane structures between MCs and tumor cells in vitro was examined using microscopy and a diverse array of molecule-specific indicator dyes. We show that several MC-specific structures are dependent on the specific interactions between human tumor IgE-sensitized MCs and antigens on the tumor cell surface. The formation of TnT, membrane blebs and other MC-specific structures paralleled FcεRI-degranulation occurring within 30 min and persisting for up to 24 h. The TnT-specific adhesion of FcεRI-activated MCs to tumor cells was characterized by the transport of the MC granule content into the tumor cells, including tryptase and TNF-α. This interaction led to apoptosis of the tumor cells, which differs from previous studies examining tissue cells within the cancer microenvironment. The formation of heterotypic TnT results in stimulation of an invasive tumor cell phenotype and increased tumor cell invasion and chemoresistance of the cancer cells. These studies describe a heretofore-unrecognized mechanism underlying IgE-mediated interactions and FcεRI-activated MC-mediated killing of tumor cells through the formation of TnT.

Human Tumor Targeted Cytotoxic Mast Cells for Cancer Immunotherapy.

The diversity of autologous cells being used and investigated for cancer therapy continues to increase. Mast cells (MCs) are tissue cells that contain a unique set of anti-cancer mediators and are found in and around tumors. We sought to exploit the anti-tumor mediators in MC granules to selectively target them to tumor cells using tumor specific immunoglobin E (IgE) and controllably trigger release of anti-tumor mediators upon tumor cell engagement. We used a human HER2/neu-specific IgE to arm human MCs through the high affinity IgE receptor (FcεRI). The ability of MCs to bind to and induce apoptosis of HER2/neu-positive cancer cells in vitro and in vivo was assessed. The interactions between MCs and cancer cells were investigated in real time using confocal microscopy. The mechanism of action using cytotoxic MCs was examined using gene array profiling. Genetically manipulating autologous MC to assess the effects of MC-specific mediators have on apoptosis of tumor cells was developed using siRNA. We found that HER2/neu tumor-specific IgE-sensitized MCs bound, penetrated, and killed HER2/neu-positive tumor masses in vitro. Tunneling nanotubes formed between MCs and tumor cells are described that parallel tumor cell apoptosis. In solid tumor, human breast cancer (BC) xenograft mouse models, infusion of HER2/neu IgE-sensitized human MCs co-localized to BC cells, decreased tumor burden, and prolonged overall survival without indications of toxicity. Gene microarray of tumor cells suggests a dependence on TNF and TGFß signaling pathways leading to apoptosis. Knocking down MC-released tryptase did not affect apoptosis of cancer cells. These studies suggest MCs can be polarized from Type I hypersensitivity-mediating cells to cytotoxic cells that selectively target tumor cells and specifically triggered to release anti-tumor mediators. A strategy to investigate which MC mediators are responsible for the observed tumor killing is described so that rational decisions can be made in the future when selecting which mediators to target for deletion or those that could further polarize them to cytotoxic MC by adding other known anti-tumor agents. Using autologous human MC may provide further options for cancer therapeutics that offers a unique anti-cancer mechanism of action using tumor targeted IgE's.

Harnessing the Anti-Tumor Mediators in Mast Cells as a New Strategy for Adoptive Cell Transfer for Cancer.

The emergence of cancer immunotherapies utilizing adoptive cell transfer (ACT) continues to be one of the most promising strategies for cancer treatment. Mast cells (MCs) which occur throughout vascularized tissues, are most commonly associated with Type I hypersensitivity, bind immunoglobin E (IgE) with high affinity, produce anti-cancer mediators such as tumor necrosis factor alpha (TNF-α) and granulocyte macrophage colony-stimulating factor (GM-CSF), and generally populate the tumor microenvironments. Yet, the role of MCs in cancer pathologies remains controversial with evidence for both anti-tumor and pro-tumor effects. Here, we review the studies examining the role of MCs in multiple forms of cancer, provide an alternative, MC-based hypothesis underlying the mechanism of therapeutic tumor IgE efficacy in clinical trials, and propose a novel strategy for using tumor-targeted, IgE-sensitized MCs as a platform for developing new cellular cancer immunotherapies. This autologous MC cancer immunotherapy could have several advantages over current cell-based cancer immunotherapies and provide new mechanistic strategies for cancer therapeutics alone or in combination with current approaches.

Preparation of nano cationic liposome as carrier membrane for polyhexamethylene biguanide chloride through various methods utilizing higher antibacterial activities with low cell toxicity.

This study suggested successful encapsulation of polyhexamethylene biguanide chloride (PHMB) into nano cationic liposome as a biocompatible antibacterial agent with less cytotoxicity and higher activities. Phosphatidylcholine, cholesterol and stearylamine were used to prepare nano cationic liposome using thin film hydration method along with sonication or homogeniser. Sonication was more effective in PHMB loaded nano cationic liposome preparation with smaller size (34 nm). FTIR, 1H NMR and XRD analyses were used to confirm the encapsulation of PHMB into nano cationic liposome. PHMB inclusion in nano cationic liposome was beneficial for increased antibacterial activity against Staphylococcus aureus and Escherichia coli. PHMB-loaded cationic liposome enables to deliver high concentrations of the antibacterial agent into the infectious cell. The cytotoxicity of PHMB entrapped in positively charged liposome was prominently reduced showing no significant visible detrimental effect on normal primary human skin fibroblast cell lines morphology confirming the effective role of cationic liposome encapsulation. Comparing with PHMB alone, encapsulation of PHMB in nano cationic liposome resulted in significant increase in cell viability from 2.4 to 63%.