S100a8/S100a9-Emmprin-Vegfa Axis Initiated By Tet2-Deficient Immune Cells Exacerbates Lung Cancer Progression through Promotion of Angiogenesis

Introductions:

Loss-of-function TET2 mutations are frequent in clonal hematopoiesis in patients with solid cancers as well as that in healthy individuals. It remains to be elucidated whether and how TET2-mutated immune cells affect cancer progression in patients with TET2-mutated clonal hematopoiesis. Here, we assessed activity of Tet2-deficient immune cells using a mouse lung cancer model.

Methods:

Lewis Lung Carcinoma (LLC) cells were subcutaneously transplanted into blood-specific Mx-Cre or myeloid-specific LysM-Cre x Tet2 ^f/fmice (Tet2 ^-/-or Tet2 ^mye-) or control mice (CT). Single-cell RNA sequencing (scRNA-seq) was performed to determine the immune-cell profiles and mediators in tumors of Tet2 ^-/-mice (Tet2 ^-/-tumors). Whole transcriptome analysis (WTA) was also performed for granulocytic myeloid-derived cells (GMD), monocytic myeloid-derived cells (MMD), and tumor associated macrophages (TAM), as well as LLC cells sorted from Tet2 ^-/-tumors and CT tumors.

Results:

We found that tumor growth was enhanced in both Tet2 ^-/-and Tet2 ^mye-comparing to CT. Unsupervised clustering of scRNA-seq data identified 14 cell clusters: GMD into 3 (GMD1, GMD2, and GMD3), MMD into 5 (MMD1, MMD2, MMD3, MMD4, and MMD5), TAMs into 4 (TAM1, TAM2, TAM3, and TAM4), and DCs into 2 (DC1 and DC2). Notably, among all subclusters, the proportions of GMD1, GMD3, TAM3 and TAM4 were markedly expanded in Tet2 ^-/-tumors comparing to CT. Differentially expressed gene (DEG) analysis of scRNA-seq data found that S100a8 and S100a9 were highly expressed in Tet2-deficient GMD1 compared to CT. Furthermore, S100a8/S100a9 proteins were elevated in plasmas of Tet2 ^-/-comparing to those of CT. Pathway analysis using DEGs (p < 0.05) from WTA of GMD determined interleukin 1b (Il1b) signaling as upstream of S100a8/S100a9 activity. Gene set enrichment analysis (GSEA) also showed that 6 pathways related to Il1b were enriched in Tet2-deficient group compared to CT group. Gene ontology analysis (GO) for DEGs of GMD, MMD, and TAMs by WTA as well as 13 subclusters by scRNA-seq revealed that the "cellular response to IL-1" pathway was enriched in Tet2-deficient group compared to CT group. To define the downstream effectors in LLC cells, we performed WTA for LLC cells sorted from Tet2 ^-/-and CT tumors. We found that Vegfa, encoding a mediator for angiogenesis was highly upregulated in LLC cells sorted from Tet2 ^-/-tumors comparing to CT tumors. GSEA for WTA further identified that multiple Vegfa-related pathways as well as MAPK cascade were enriched in LLC cells from Tet2 ^-/-tumors comparing to those from CT tumors . Furthermore, S100a8/S100a9 induced Vegfa secretion from LLC cells in vitro. Remarkably, the area of blood vessels was increased in Tet2 ^-/-tumors comparing to CT tumors. Immunostaining exhibited that the number of Ly6g ⁺GMD foci (>1000 px ²) expressing S100a8/S100a9 was increased in Tet2 ^-/-tumors comparing to CT tumors. Furthermore, LLC cells surrounding GMD foci highly expressed Vegfa in Tet2 ^-/-tumors. Finally, administration of an antibody against Emmprin, a receptor for S100a8/S100a9 inhibited the tumor growth in Tet2 ^-/-. Notably, the area of blood vessels in Tet2 ^-/-tumors with anti-Emmprin group was decreased at 2-fold compared to that seen in isotype group (p < 0.05). Consistently, S100A8/S100A9 induced VEGFA production in human lung cancer cells in vitro.

Conclusions:

Tet2-deificient immune cells promote lung cancer progression through S100a8/S100a9-Emmprin-Vegfa axis. Our study suggests a novel role of TET2-mutated clonal hematopoiesis in cancer progression and even provides a novel therapeutic target.