Abstract
The lung tumor microenvironment plays a critical role in the tumorigenesis and metastasis of lung cancer, resulting from the crosstalk between cancer cells and microenvironmental cells. Therefore, comprehensive identification and characterization of cell populations in the complex lung structure is crucial for development of novel targeted anti-cancer therapies. Here, a hierarchical clustering approach with multispectral flow cytometry was established to delineate the cellular landscape of murine lungs under steady-state and cancer conditions. Fluorochromes were used multiple times to be able to measure 24 cell surface markers with only 13 detectors, yielding a broad picture for whole-lung phenotyping. Primary and metastatic murine lung tumor models were included to detect major cell populations in the lung, and to identify alterations to the distribution patterns in these models. In the primary tumor models, major altered populations included CD324+ epithelial cells, alveolar macrophages, dendritic cells, and blood and lymph endothelial cells. The number of fibroblasts, vascular smooth muscle cells, monocytes (Ly6C+ and Ly6C–) and neutrophils were elevated in metastatic models of lung cancer. Thus, the proposed clustering approach is a promising method to resolve cell populations from complex organs in detail even with basic flow cytometers.
Highlights
Lung cancer continues to be a leading cause of cancer-associated mortality worldwide, accounting for about 1.8 million deaths in 2020 [1]
To determine whether flow cytometry can detect major cell subsets in control lungs, a multispectral antibody panel (Table 1) was designed that employed hierarchical clustering and individual fluorochromes that will allow more than one marker for one particular cell subset that was previously separated by other broad markers of cell subsets (Supplementary Figure S1)
CD146 is expressed by blood endothelial cells (BECs) and vascular smooth muscle cells (VSMCs), which can be separated by differences in the expression patterns of platelet endothelial cell adhesion molecule (PECAM-1 or CD31), which is highly expressed on the surfaces of endothelial cells, while Siglec-H is expressed exclusively by plasmacytoid DCs and glucocorticoid-induced tumor necrosis factor receptor (GITR) is expressed at high levels by regulatory T cells (Tregs)
Summary
Lung cancer continues to be a leading cause of cancer-associated mortality worldwide, accounting for about 1.8 million deaths in 2020 [1]. Current therapies aim to target the components of the lung tumor microenvironment (TME) to interfere with cancer progression. Multispectral Analysis of Lung Tumor Heterogenety cancer cells and stromal cells sustains the oncogenic microenvironment, the quantity and distribution of cells in the TME are likely associated with the pathogenesis of lung cancer. Tumor-associated macrophages (TAMs) are among the most abundant cell types in the lung TME, and TAM infiltration is reportedly correlated to the stage and metastasis potential of lung cancer [3, 4]. Since the involvement of microenvironmental cells in disease progression is multifaceted, profiling the TME is critical to elucidate cellular activity, interactions and functions to establish more specific targeted approaches to inhibit lung tumorigenesis. There are currently various techniques available to unravel the cellular composition of the TME such as basic immunohistochemistry, multiplex staining, flow cytometry, mass cytometry, transcriptomic and bioinformatics approaches
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