Gene Expression Profiling of Lymph Node Sub-Capsular Sinus Macrophages in Cancer

Abstract

Lymph nodes are key lymphoid organs collecting lymph fluid and migratory cells from the tissue area they survey. When cancerous cells arise within a tissue, the sentinel lymph node is the first immunological organ to mount an immune response. Sub-capsular sinus macrophages (SSMs) are specialized macrophages residing in the lymph nodes that play important roles as gatekeepers against particulate antigenic material. In the context of cancer, SSMs capture tumor-derived extracellular vesicles (tEVs), a form of particulate antigen released in high amounts by tumor cells. We and others have recently demonstrated that SSMs possess anti-tumor activity because in their absence tumors progress faster. A comprehensive profiling of SSMs represents an important first step to identify the cellular and molecular mechanisms responsible for SSM anti-tumor activity. Unfortunately, the isolation of SSMs for molecular analyses is very challenging. Here, we combined an optimized dissociation protocol, careful marker selection and stringent gating strategies to highly purify SSMs. We provide evidence of decreased T and B cell contamination, which allowed us to reveal the gene expression profile of this elusive macrophage subset. Squamous cell carcinomas induced an increase in the expression of Fc receptors, lysosomal and proteasomal enzymes in SSMs. Imaging of mouse and patient lymph nodes confirmed the presence of the top differentially expressed genes. These results suggest that SSMs respond to tumor formation by upregulating the machinery necessary for presentation of tumor particulate antigens to B cells.

Open-ST: High-resolution spatial transcriptomics in 3D

“…Upon closer inspection, our 3D reconstruction revealed its connection to the metastatic mass in the later sections, demonstrating continuity across imaging and transcriptomic modalities (Figure 6C). Similarly, we could follow the continuity of the invading tumor and its stroma within the lymph node context via the markers SPP1, which marks CAMs, ACTA2, which highlights CAFs surrounding the tumor and is proximal to lymph node populations, and LYZ, which marks macrophages and is a transcriptomic feature located at the lymph sinus, as well on invaded tissue 48 . This structure is in general found in close proximity to afferent lymphatic vessels, and sinus macrophages are the first to be exposed to the metastasis 49 .…”

Subcapsular sinus macrophage sensing of extracellular matrix rigidity alters membrane topography and immune complex mobility

“…plexes. SSMs do not express FDC-M1 but do express high levels of Fcγ receptors (FcγRs) (22). We therefore postulated that SSMs were enriched during the cell isolation procedure due to interactions between their FcγRs and the Fc domain of either the rat IgG2c, κ anti-mouse FDC-M1 primary antibody or the biotinylated mouse IgG2a anti-rat Igκ secondary antibody used for cell enrichment.…”

A case for the study of native extracellular vesicles

“…These issues are compounded by the fact that EVs are extremely small [most EV subsets are sub-micron size in diameter ( 1 )], often below the diffraction limit of conventional microscopy ( 9 ), and thus, they can carry limited amounts of fluorescent reporters. As a consequence, only recipient cells that bind nEVs in high numbers can be detected and isolated for profiling studies ( 15 , 20 ). Remote EV-cell communications are much harder to identify as the amount of nEVs exponentially decreases with distance from EV donor cells ( 21 ).…”