Smoking‐Induced M2‐TAMs, via circEML4 in EVs, Promote the Progression of NSCLC through ALKBH5‐Regulated m6A Modification of SOCS2 in NSCLC Cells

Abstract Lung cancer is a commonly diagnosed disease worldwide, with non‐small cell lung cancers (NSCLCs) accounting for ≈ 85% of cases. Cigarette smoke is an environmental exposure promoting progression of NSCLC, but its role is poorly understood. This study reports that smoking‐induced accumulation of M2‐type tumor‐associated macrophages (M2‐TAMs) surrounding NSCLC tissues promotes malignancy. Specifically, extracellular vesicles (EVs) from cigarette smoke extract (CSE)‐induced M2 macrophages promoted malignancy of NSCLC cells in vitro and in vivo. circEML4 in EVs from CSE‐induced M2 macrophages is transported to NSCLC cells, where it reduced the distribution of ALKBH5 in the nucleus by interacting with Human AlkB homolog H5 (ALKBH5), resulting in elevated N6‐methyladenosine (m6A) modifications. m6A‐seq and RNA‐seq revealed suppressor of cytokine signaling 2 (SOCS2)‐mediated activation of the Janus kinase‐signal transducer and activator of transcription (JAK‐STAT) pathway by regulating m6A modification of SOCS2 via ALKBH5. Down‐regulation of circEML4 in EVs from CSE‐induced M2 macrophages reversed EVs‐enhanced tumorigenicity and metastasis in NSCLC cells. Furthermore, this study found that smoking patients showed an increase in circEML4‐positive M2‐TAMs. These results indicate that smoking‐induced M2‐TAMs via circEML4 in EVs promote the NSCLC progression through ALKBH5‐regulated m6A modification of SOCS2. This study also reveals that circEML4 in EVs from TAMs acts as a diagnostic biomarker for NSCLC, especially for patients with smoking history.


Figure S6. Role of inhibition of secretion of EVs from CSE-induced M2 macrophages on proliferation, migration, and invasion of NSCLC cells.
THP-M were exposed to 4% CSE for 48 h. The proliferation of A549 and H1703 cells was measured by (A) CCK-8 assays. (B) EdU assays and (C) EdU-positive rates were determined. (D) Colony formation assays were performed, and (E) colony formation assays were conducted for A549 and H1703 cells. (F) Representative images of Transwell assays, and (G) the migration and invasion were assessed for A549 and H1703 cells. Three independent experiments were conducted. All data represent means ± SD. *P < 0.05; **P < 0.01. THP-M were exposed to siRNA con or to circEML4 siRNA for 24 h. With extracts from A549 and H1703 cells, RIP experiments were performed with an antibody against AGO2. Three independent experiments were conducted. All data represent means ± SD. ns, not significant. *P < 0.05; **P < 0.01. THP-M were exposed to 4% CSE for 48 h. A549 and H1703 cells were exposed to  THP-M were exposed to 4% CSE for 48 h. A549 and H1703 cells were exposed to THP-M-EVs or CSE-THP-M-EVs for 24 h. RNA fluorescence in situ hybridization for circEML4 (red) in A549 and H1703 cells. Nuclei were stained with DAPI (blue). Scale bar, 25 μm.    (C) Expression of SOCS2 for smoking or never-smoking lung cancer patients based on the GEO dataset. Outliers were eliminated during the analysis. Z-score was used for identifying outliers and calculated as follows: , where was the value to be normalized in the data, represented the mean of the data, and was the standard deviation of the data. Data were considered as an outlier if the Z-score was greater than or less than 2 or -2, respectively. All data represent means ± SD. *P < 0.05; **P < 0.01.

Figure S15. Release of EVs derived from CSE-induced M2 macrophages increases m6A levels for SOCS2 in NSCLC cells.
THP-M were exposed to 4% CSE for 48 h. A549 and H1703 cells were exposed to THP-M-EVs or CSE-THP-M-EVs for 24 h. MeRIP-qPCR determinations for m6A enrichment on SOCS2 in A549 and H1703 cells. Three independent experiments were conducted. All data represent mean ± SD. *P < 0.05; **P < 0.01.

Figure S16. Schematic depiction of mutations of the m6A site in SOCS2.
The WT or m6A consensus sequence mutant SOCS2 5'UTR was fused with a firefly luciferase reporter. The m6A mutant sequence was constructed by replacing A with G. Figure S17. Regulation of SOCS2 protein levels by ALKBH5.
A549 and H1703 cells were treated with ALKBH5 siRNA or siRNA con for 24 h. (A) Western blots were performed, and (B) relative protein levels of ALKBH5 and SOCS2 were determined. Three independent experiments were conducted. All data represent means ± SD. *P < 0.05; **P < 0.01.

Figure S18. circEML4 in EVs from CSE-induced M2 macrophages regulates the SOCS2/JAK-STAT pathway in NSCLC cells.
THP-M were treated with circEML4 siRNA or siRNA con for 24 h, and then exposed to CSE (4%) for 48 h. A549 and H1703 cells were exposed to THP-M-EVs or CSE-THP-M-EVs for 24 h. (A) Western blots were performed, and (B) relative protein levels of p-JAK2, p-STAT5, and SOCS2 were determined. Three independent experiments were conducted. All data represent means ± SD. *P < 0.05; **P < 0.01.