Junctional adhesion molecule-A is dispensable for myeloid cell recruitment and diversification in the tumor microenvironment

Abstract

Junctional adhesion molecule-A (JAM-A), expressed on the surface of myeloid cells, is required for extravasation at sites of inflammation and may also modulate myeloid cell activation. Infiltration of myeloid cells is a common feature of tumors that drives disease progression, but the function of JAM-A in this phenomenon and its impact on tumor-infiltrating myeloid cells is little understood. Here we show that systemic cancer-associated inflammation in mice enhanced JAM-A expression selectively on circulating monocytes in an IL1β-dependent manner. Using myeloid-specific JAM-A-deficient mice, we found that JAM-A was dispensable for recruitment of monocytes and other myeloid cells to tumors, in contrast to its reported role in inflammation. Single-cell RNA sequencing revealed that loss of JAM-A did not influence the transcriptional reprogramming of myeloid cells in the tumor microenvironment. Overall, our results support the notion that cancer-associated inflammation can modulate the phenotype of circulating immune cells, and we demonstrate that tumors can bypass the requirement of JAM-A for myeloid cell recruitment and reprogramming.

Keywords: F11R; JAM-1; JAM-A; extravasation; interleukin-1; junctional adhesion molecule-A; monocyte; tumor-associated macrophage.

Figure 1

Cancer-associated inflammation promotes upregulation of JAM-A on peripheral monocytes in an IL-1β-dependent manner. (A) Frequency of JAM-A⁺ cells within the most abundant immune cell populations in the blood of mice with sc. LLC tumors (21 days post-engraftment, 1678 ± 212mm³ tumor volume). (B) Same as (A) in orthothopic Py8119 breast tumors (32 days post-engraftment, 1199 ± 114mm³ tumor volume). (C) Frequency of JAM-A⁺ cells within the most abundant immune cell populations in the blood of MMTV-PyMT mice at early stage (no palpable tumor) and late stage (tumor volume reached 1500 mm³) of tumor progression. Dots connected with line denote values from the same animal. (D) Representative flow cytometry dot plots of JAM-A expression in Ly6C^hi monocytes from the blood. (E) Changes in the frequency of JAM-A⁺ cells within Ly6C^hi monocytes during tumor progression in the blood of mice with orthotopic Py8119 tumors or sc. LLC tumors (corresponding tumor volumes are shown in Supplementary Figure 2 ). (F) Frequency of JAM-A⁺ cells within Ly6C^hi monocytes in the spleen and bone marrow (BM) of mice with orthotopic Py8119 tumors or sc. LLC tumors (time post-engraftment and tumor volume as in panels A and B). (G) JAM-A expression on GFP⁺ Ly6C^hi bone marrow-derived monocytes adoptively transferred into LLC tumor-bearing mice on day 18 of tumor development. (H) Fold change of JAM-A⁺ cells within Ly6C^hi monocytes in the blood of Il1b ^+/+ or Il1b ^-/- mice with sc. LLC tumors (day 19 post-engraftment) or orthotopic Py8119 tumors (day 35 post-engraftment) compared to naive Il1b ^+/+ mice (tumor burden is shown in Supplementary Figure 3 ). Cell type frequencies were determined using flow cytometry. Graphs show mean and SEM. Panel (C) paired two-tailed t-test; All other panels: unpaired two-tailed t-test; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

JAM-A is expressed on mononuclear phagocytes infiltrating human and mouse tumors. (A) JAM-A expression on endothelial cells and immune cells in LLC tumors (n=4-5). 21 days post-engraftment, 1678 ± 212mm³ tumor volume. (B) Same as (A) in Py8119 tumors (n=4-6). 32 days post-engraftment, 1199 ± 114mm³ tumor volume. (C) Representative histogram of JAM-A expression on blood monocytes, tumor-infiltrating monocytes and tumor-associated macrophages in LLC and Py8119 tumors. (D) Fold change of JAM-A expression on macrophages, monocytes and endothelial cells in Py8119 tumors of Il1b ^-/- mice compared to Il1b ^+/+ mice (n=6). 35 days post-engraftment, tumor burden is shown in Supplementary Figure 3 . (E) JAM-A expression on endothelial cells and immune cells in human non-small cell lung cancer (NSCLC) (n=3). JAM-A expression was determined using flow cytometry. ΔMFI: Delta Median Fluorescent Intensity (MFI) = MFI of anti-JAM-A-stained sample minus MFI of isotype control-stained sample. Bar graphs show mean and SEM.

Figure 3

JAM-A is dispensable for myeloid cell accumulation in the primary tumor and in the pulmonary metastatic site. (A) JAM-A expression in myeloid cell populations and endothelial cells in LLC tumors of F11r ^flox/flox LysM-Cre⁺ mice normalized to F11r ^flox/flox LysM-Cre^- mice. ND = not detected. Day 17 post-engraftment, tumor volume shown in panel (B). (B) LLC (n=16-17) and Py8119-eGFP (n=14-15) tumor growth in F11r ^flox/flox LysM-Cre^- and F11r ^flox/flox LysM-Cre⁺ mice. (C) Frequency of indicated myeloid and lymphoid cell populations in LLC tumors (n=9-10). Day 17 post-engraftment, tumor volume is shown in panel (B). (D) Same as (C) in Py8119-eGFP tumors (n=12-13). Day 31 post-engraftment, tumor volume is shown in panel (B). (E) Frequency of monocytes and neutrophils in the lungs of tumor-bearing mice with Py8119-eGFP tumors (n=14-15). Panel E-G: Day 31 post-engraftment. (F) Proportion of mice with eGFP⁺ metastatic Py8119 cancer cells in the lung detected via flow cytometry. (G) Frequency of eGFP⁺ metastatic cancer cells in the lungs of Py8119-eGFP tumor-bearing mice (n=14-15). (H–J) Same as (E–G) for LLC-eGFP tumors (n=15). Day 17 post-engraftment. JAM-A expression and cell frequencies were determined using flow cytometry. All graphs show mean and SEM.

Figure 4

JAM-A does not affect myeloid cell diversification in the tumor microenvironment. (A) UMAP plot showing CD45⁺ immune cells from LLC tumors of both F11r ^flox/flox LysM-Cre^- and F11r ^flox/flox LysM-Cre⁺ mice color-coded by clusters defined based on single-cell RNA-seq data. (B) Heatmap showing the top 50 enriched genes in each myeloid cell cluster. The top 5 enriched genes per cluster are highlighted. See Supplementary Table 1 for list of top 50 enriched genes for all clusters shown in panel (A). (C) Heatmap showing F11r (encoding JAM-A) expression in tumors of F11r ^flox/flox LysM-Cre^- and F11r ^flox/flox LysM-Cre⁺ mice based on single-cell RNA-seq data. (D) UMAP plots showing CD45⁺ immune cells from LLC tumors of F11r ^flox/flox LysM-Cre^- or F11r ^flox/flox LysM-Cre⁺ mice color-coded by clusters shown in panel (A). (E) Number of differentially expressed (DE) genes (adjusted P value < 0.05, fold change (FC) > 2) between F11r ^flox/flox LysM-Cre⁺ and F11r ^flox/flox LysM-Cre^- mice within each myeloid cell cluster. See Supplementary Table 2 for full list of DE genes.