PMC-306: A Novel Murine Mast Cell Line to Study Mast Cell Function and Transformation

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Mast cells (MCs) are immune cells derived from the myeloid lineage, found throughout various tissues in the body. They are a diverse group, varying in their protease content stored in granules and receptor expression profiles. To explore MC biology, researchers often use primary cells isolated from human or mouse tissues or rely on established cell lines such as human HMC-1.1 and HMC-1.2, or rodent lines like RBL-2H3 and L138.8A. However, current cellular models have limitations in replicating all aspects of MC function.

To address this, we developed a new murine mast cell line named PMC-306, derived from primary peritoneal mast cells (PMCs) that underwent spontaneous transformation. We examined this line’s surface receptors, signaling responses, and effector functions, and found that PMC-306 cells behaved comparably to primary wild-type PMCs. Importantly, these cells responded robustly to ligands of the MAS-related G-protein-coupled receptor B2 (MRGPRB2).

PMC-306 also demonstrated accelerated cell cycle progression while still requiring IL-3 and stem cell factor (SCF) for proliferation. Morphologically, the cells resembled immature connective tissue-type MCs. Notably, their transformation was associated with a loss of Cdkn2a and Arf expression, key genes involved in cell cycle regulation. Interestingly, this gene loss could be replicated in bone marrow-derived mast cells (BMMCs) by prolonged SCF treatment, suggesting a role of KIT signaling in suppressing these cell cycle regulators. Thus, PMC-306 offers a valuable model for studying MC biology and potential mechanisms underlying mast cell leukemia.

Introduction

Mast cells are resident immune cells of myeloid origin, widely recognized for their role in type I hypersensitivity reactions. Beyond allergic responses, they also participate in tumor progression by promoting angiogenesis, extracellular matrix remodeling, and epithelial-to-mesenchymal transition. Their strategic location at environmental interfaces allows them to serve as first responders during viral and bacterial infections, alerting the immune system to mobilize to affected tissues. Additionally, MCs play a role in tissue repair and resolution of inflammation by releasing factors such as IL-10 and supporting extracellular matrix remodeling.

Despite their importance, studying MC biology in vitro is challenging due to the heterogeneity of MC subtypes. There is no single "typical" mast cell; different subtypes exist based on receptor expression, granule composition, and tissue localization. Consequently, no in vitro model perfectly represents all MC phenotypes. Moreover, the difficulty in isolating mature tissue-derived MCs has led researchers to use human and rodent MC lines like HMC-1, LUVA, LAD2, RBL-2H3, and L138.8A. However, these lines often carry oncogenic mutations or lack essential receptors, such as the high-affinity IgE receptor (FcεR1), limiting their usefulness for studying key MC signaling pathways.

Mast Cell Subtypes and In Vitro Models

In rodents, mast cells are broadly classified into two categories based on histological staining and granule content:

Connective Tissue Mast Cells (CTMCs): Rich in tryptase, chymases, carboxypeptidase A3 (CPA3), and heparin.

Mucosal Mast Cells (MMCs): Lacking tryptase, with granules containing chondroitin sulfate instead of heparin.

Murine mucosal-like MCs can be cultured from bone marrow precursors using IL-3 and optionally SCF, generating bone marrow-derived MCs (BMMCs). However, these cells often remain incompletely differentiated compared to tissue-resident MCs.

Connective tissue-type MCs, in contrast, can be directly isolated from the peritoneum (PMCs) and expanded in vitro with IL-3 and SCF. Still, the yield from peritoneal lavage is low, and the culture lifespan of PMCs is limited, requiring large numbers of animals for in-depth studies—raising concerns regarding ethical animal use and compliance with the 3R principles (Replacement, Reduction, Refinement).

Establishment and Characterization of PMC-306

To overcome these limitations, we developed PMC-306, a murine MC line originating from peritoneal mast cells that spontaneously transformed in culture. We characterized this line in comparison to wild-type PMCs, assessing morphology, surface markers, protease expression, and responses to different stimuli.

PMC-306 cells preserved key MC features, including functional FcεR1 and MRGPRB2 receptors, allowing them to be used for IgE-related and MRGPRB2-mediated signaling studies. They also showed IL-3 and SCF dependency for growth, consistent with primary MCs. Chromosomal analysis revealed no major structural abnormalities except for a heterozygous deletion in chromosome 4 (qC4-qC7 region), which includes Cdkn2a (p16/INK4A) and Arf (p19), both of which are vital for cell cycle control through the p53 and Rb pathways.

Despite the heterozygous DNA loss, mRNA expression of Cdkn2a and Arf was completely absent, contributing to enhanced proliferation and suggesting these deletions played a role in the spontaneous transformation of PMC-306. Interestingly, continuous SCF exposure in BMMCs also downregulated these genes, implying that sustained KIT signaling may suppress their expression and contribute to transformation.

Conclusion

The PMC-306 cell line represents a significant advancement for mast cell research. It retains critical mast cell characteristics, responds to relevant stimuli, and offers a genetically defined system to investigate the mechanisms behind mast cell transformation and function. This model serves as a complementary tool to existing MC lines, primary cells, and animal models, especially valuable for studying tumorigenic processes such as mast cell leukemia and for refining research in line with ethical standards.