Breast cancer can be divided into four subgroups HER2+, Luminal A, Luminal B and triple negative using multiple clinically validated monoclonal antibodies. Each subtype presents a different prognosis and treatment option. It is also increasingly clear that immune subsets within the tumor microenvironment may also have a bearing on prognosis, with certain subsets being associated with increased patient survival. Determining the tumor and immunologic phenotype of initial tumor biopsies both in the composition of cells and their relative spatial distribution is becoming key in selecting optimal treatment. The aim of our research has been to develop a multiplex immunofluorescence technology that can rapidly and simultaneously detect 5+ markers on a single section, allowing multiparametric quantitative analysis of cell phenotype and tissue composition as well as providing important spatial information of cells relative to each other. Our multiplex immunofluorescence (mxIF), modified-hapten based technology allows the simultaneous detection of multiple biomarkers using a standard two-step procedure. The technology is antibody species independent and produces signals 3-4X stronger than direct fluor-labeled secondary antibodies. It can detect multiple co-localized markers and while it has been engineered to detect four biomarkers and DAPI, it has the potential for significantly higher multiplexing. In addition it is modular and can be readily modified for any multiplex panel. The data generated from a staining experiment can be readily exported into any image analysis program. In comparison to existing technologies from GE and PerkinElmer, our technology allows multiple targets to be interrogated rapidly and simultaneously without the need for multiple rounds of stripping and imaging. The breast cancer phenotyping panel (BC-1) provides rapid phenotyping information allowing identification of Luminal A, Luminal B, HER2+ or triple negative cancer. Our immuno-oncology panel (IO-1) identifies CD4 and CD8 T cells, CD20+ B cells and CD68+ macrophages. Application of each panel to one of two sequential sections and then image overlay allows both tumor and immunophenotyping. The modular and non-species specific nature of this technology means that panels can be modified and expanded to characterize multiple different tumors and immune cell subpopulations in a rapid, sensitive and quantitative manner.
Citation Format: Matt Levin, Stephen J. Kron, David Schwartz, Helen Snyder. Rapid 5-marker multiplex phenotyping of breast cancer subtypes & tumor-infiltrating leukocytes “in situ” in FFPE sections. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3949.
Background
Individuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI).
Methods
The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1β (IL-1β), and C–C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM.
Results
Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1β signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1β, and CCL2 neutralization, or ICS treatment.
Conclusions
Collectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC).
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