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BackgroundMast cells are essential initiators and regulators in both innate and adaptive immunity in addition to being mediators of Type I hypersensitivity. Their role in cancer pathogenesis has been challenging to assess, given they contain both pro- and anti-tumorigenic mediators. Thus, to elucidate the functions of mast cell-derived gene products in cancer pathogenesis and mast cell differentiation, it is critical to study them by comparing their functional responses between those with selected gene knockdown and those without. However, primary human mast cells are known as hard-to-transfect cells and a fast and efficient method for transient transfection of primary human adipose mast cells has not been reported.MethodsWe investigated different approaches to transfect primary human adipose-derived mast cells from 10 donors (24 - 55 yrs.). Three cationic lipid nanoparticles (Metafectene and Metafectene-Pro, Biontex Laboratories, and Lipofectamine 3000, Thermo Fisher), one lipid/histone-based nanoparticle (Turbofectin 8.0, OriGene), and two non-liposomal polymeric reagents (TransIT-LT1 and TransIT-2020, MirusBio) were evaluated in conjunction with green fluorescent protein (GFP)-tagged genes (e.g., tryptase) at different ratios of DNA plasmid to the reagent. The functional and phenotypic responses of the mast cells were compared to non-transfected mast cells.ResultsWe found that transfection of primary human adipose mast cells is highly donor-dependent. Parameters that affect transfection efficiency include donor age, stem cell isolation procedure, in vitro age of mast cells at the time of transfection, the type and dosage of the transfection reagent, incubation time, presence of antibiotics, and serum content. The transfection rate efficiencies of human mast cells using lipid synthetic nanoparticles was higher than the lipid/histone-based and non-liposomal polymeric reagents. The maximum Median Fluorescence Intensity (MFI) fold change compared to the non-transfected mast cells was about 17 for the youngest donor that was transfected 7 to 10 weeks after isolation using Metafectene-Pro.ConclusionsIn general, older donor mast cells and mast cell progenitors were less susceptible to transfection. Also, a higher transfection rate is accompanied by increased cytotoxicity, so the analysis of various transfection conditions limiting cell death was the primary goal. Metafectene-Pro significantly increased GFP reporter gene expression compared to the other reagents. The transfected mast cells were phenotypically and functionally similar to non-transfected cells and were viable compared to control mast cells from the same donors. These studies will aid in determining if mast cell mediators have pro- or anti-tumor cell effects to help define the role these cells have in cancer pathogenesis.Ethics ApprovalThis study was approved by UNCG IRB, approval number 12–099, and under the NIH-defined ”exempt” status for human subjects research.

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.

The purpose of this study was to test the hypothesis that human mast cells (MC) have anti-tumor mediators that can be targeted to tumor specific antigens, induced to release these mediators, and cause cancer cell apoptosis. Breast cancer (BC) continues to be a significant cause of mortality for women in the United States. The oncogene HER2/neu is overexpressed in BC and is validated target for cancer therapeutics. New immunotherapy strategies and combinations of immune agents with non-redundant mechanisms of action are needed. The role of human tissue mast cells (MC) in the pathology of many cancers is not known. Nonetheless, high densities of MC are associated with favorable prognosis in certain types of cancer such as BC suggesting an anti-tumor effect. We previously reported that human adipose cells can be differentiated into autologous, fully functional MC (ADMC) capable of releasing mediators upon FcϵRI stimulation that induce BC cell death of cell suspensions of SK-BR3. Further data is presented demonstrating the anti-tumor effects of ADMC using BC tumor masses. The ADMC, sensitized with breast cancer-specific IgE anti-HER2/neu, bound to and were activated by, HER2/neu-positive human BC cells. The anti-HER2/neu IgE-sensitized ADMC bound to, infiltrated, and induced apoptosis of HER2/neu-positive BT-474 BC tumor masses as determined by time-lapse, live cell confocal imaging. Specific, HER2/neu IgE-mediated binding was also verified in vivo using immunocompromised xenograft models. Further studies are warranted to determine if tumor-targeted, autologous MC may be a new way to treat solid tumors. Citation Format: Chris Kepley, Mohammad Ferrydouni, Mona Motaghed, Kristen Dellinger. IgE receptor-activated human mast cells have potent anti-cancer properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1494.

Background Autologous immune cell-based therapies to re-activate the immune system have been developed to treat patients with cancer. For example, chimeric antigen receptor T cell (CAR T) therapy is an FDA approved strategy that uses autologous T cells as a cancer treatment. Mast cells (MC) are important immune sentinels and widely recognized for their role as mediators of Type I hypersensitivity. However, they also secrete anti-cancer mediators such as tumor necrosis factor alpha (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF) which are being investigated in clinical trials for cancer treatment. Methods We generated adipose-derived mast cells (ADMC) and sensitized with anti-CD20 IgE antibodies and challenged with B-lymphoma cells. In addition, immunocompromised mice were examined for the maximal tolerated dose using ADMC. The ability of the ADMC to be transduced with lentiviral vectors carrying green fluorescence protein was examined. Results It is shown that CD20 IgE-sensitized ADMC bind to and are activated by CD20 positive B-lymphoma cells. The ADMC injected into mice do not display signs of anaphylaxis by the systemic injection of up to 1 × 106 ADMC/mouse. It is also shown for the first time that ADMC and their adipose-derived stem cell precursors can be transduced with a lentiviral vector carrying green fluorescence protein without affecting function. Conclusion This discovery indicates the ADMC could be transduced with other tumor killing molecules to create a “super killing” cell with potent and diverse anti-tumor activity. Using tumor targeting IgE’s, autologous ADMC could be used as a “Trojan Horse” to deliver these anti-tumor mediators to tumors and serve as a new cancer immunotherapy platform.