Targeting polarized phenotype of microglia via IL6/JAK2/STAT3 signaling to reduce NSCLC brain metastasis

Abstract

Tumor-associated macrophages have emerged as crucial factors for metastases. Microglia are indispensable components of the brain microenvironment and play vital roles in brain metastasis (BM). However, the underlying mechanism of how activated microglia promote brain metastasis of non-small cell lung cancer (NSCLC) remains elusive. Here, we purified cell lines with brain-metastatic tropism and employed a co-culture system to reveal their communication with microglia. By single-cell RNA-sequencing and transcriptome difference analysis, we identified IL6 as the key regulator in brain-metastatic cells (A549-F3) to induce anti-inflammatory microglia via JAK2/STAT3 signaling, which in turn promoted the colonization process in metastatic A549-F3 cells. In our clinical samples, patients with higher levels of IL6 in serum showed higher propensity for brain metastasis. Additionally, the TCGA (The Cancer Genome Atlas) data revealed that NSCLC patients with a lower level of IL6 had a longer overall survival time compared to those with a higher level of IL6. Overall, our data indicate that the targeting of IL6/JAK2/STAT3 signaling in activated microglia may be a promising new approach for inhibiting brain metastasis in NSCLC patients.

Fig. 1

Human NSCLC A549 cells alter phenotype of microglia. a Evaluating the ability of invasion in different cell lines by Transwell invasion assay (magnification, 400×). b Evaluating the ability of migration in different cell lines by Transwell migration assay (magnification, 400×). c Representative images of IHC staining with IBA-1 (brown) and nuclear counterstaining with hematoxylin (blue) were depicted. Left image was from a control mouse and the right image was brain specimens of a mouse with brain metastasis. Top panels: magnification was 200×. Bottom panels were with higher magnification (800×). d Human specimens of NSCLC-BM showing metastasis free and positive areas, metastatic zone was surrounded with activated IBA-1⁺ microglia cells. Top panels: magnification was 200×. Bottom panels were with higher magnification (800×). e Morphologic changes of HMO6 cells treated with ACM for 0 h, 12 h, 24 h, 48 h were evaluated by phase-contrast microscopy (magnification: 400×). f The relative expression of M1-markers (iNOS and CD86) and M2-markers (Arg1 and CD206) mRNA in HMO6 cells treated with ACM for 0 h, 12 h, 24 h, 48 h are detected by quantitative RT-PCR. β-Actin was used to normalize gene expression. g Western blot and the corresponding gray value analysis were used to investigate the expression iNOS and CD206 in HMO6 cells cultured in ACM for 0, 12, 24, 48 h. h Representative images of immunofluorescence staining with DAPI (blue) for nuclei and Arg1(red) for M2-marker were depicted (magnification: 20×). ACM: A549 cells-conditioned media. Data are mean ± SD. P > 0.05 no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 2

M2-Microglia CM promotes NSCLC cells colonization. a, b E-cadherin (magnification: 400×) and vimentin (magnification: 200×) expression of A549 and H292 cells were presented by immunofluorescence staining. The blue signal represents the DAPI-stained nuclei. c qRT-PCR analysis of E-cadherin and vimentin for A549 cells cultured with different treatment. d qRT-PCR analysis of E-cadherin and vimentin for H292 cells cultured with different treatment. e Western blot for E-cadherin and vimentin of A549 cells cultured under different conditions. The right panels were gray value analysis. f Western blot for E-cadherin and vimentin of H292 cells cultured under different conditions. The right panels were gray value analysis. control: normal A549 cells; TGF-β1: A549 cells treated with TGF-β1 (2.5 ng/ml) for 24 h; co-HCM and co-CCM: A549 cells pretreated with TGF-β1 (2.5 ng/ml) and incubated with the co-cultured CM for 24 h. co-HCM/co-CCM: CM from A549 and HMO6/CHME5 cells co-cultured for 48 h. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

Single-Cell RNA-Seq Reveals activated microglia in metastatic lesions. a Schematic representation of the in vivo selection process. Parent A549 cells were inoculated into the left cardiac ventricle of nude mice. Tumor cells were isolated from brain lesions and reinoculated after expansion in culture. Cells isolated from the second round of metastases were expanded in culture and reinoculated to get the third-generation cells. b Histogram showed incidence of brain metastases in different cell lines (parental: 10.0%; A549-F1: 28.6%; A549-F2: 50.0%; A549-F3: 63.6%). c Schematic overview of the experimental design for the single-cell RNA-seq analyses was generated from the online tool (BioRender: https://biorender.com/). d UMAP analyzed cells reveal the existence of 20 clusters within the three experimental groups (untreated mouse brain, brain metastases from A549 and A549-F3 cells). e UMAP plots indicated distribution of cell clusters in different groups. f The numbers of different cell types in each group. g The numbers and percentages of anti-inflammatory and pro-inflammatory microglia in different groups. h The top 20 signal pathways with significant difference showed in KEGG

Fig. 4

IL6 emerged as for a mediator of A549-F3 and M2 microglia communication in BM. a Heat map showed different gene expression profiles of A549-F3 populations and parental cells. b Heat map showed different gene expression profiles of A549 co-cultured with HMO6 for 48 h and parental cells. c Venn diagram of genes positively correlated with brain metastasis analyzed through transcriptome microarrays between parent A549 cells and A549-F3 or co-cultured A549 cells. d Comparison of mRNA microarray data showed top 30 changed genes (up or down) with the same expressed pattern between parent A549 cells and A549-F3 or co-cultured A549 cells. e Heat map of the most prominently upregulated cytokine in A549-F3 cells and co-cultured system. f qRT-PCR analysis showed expression of IL6 in different cell lines. g Western blot showed expression of IL6 in different cell lines. h ELISA assay of IL6 in culture supernatants from A549 and A549-F3 cells. i Brain metastases were determined by bioluminescence imaging. j Histogram showed incidence of brain metastases in different cell lines. k Photon flux detected from brain-metastatic focus in the different groups. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

IL6/JAK2/STAT3 signaling mediates M2 polarization of microglia. a Western blot analysis of M2-marks (CD206 and Arg1) in HMO6 and CHME5 cells with control media or F3-CM alone or F3-CM treated with Tocilizumab (2.5 μg/ml for 24 h) or with IL6 (200 ng/ml for 24 h) alone or IL6-supplemented with Tocilizumab. b Western blot analysis of p-JAK2, JAK2, p-STAT3, and STAT3 in HMO6 and CHME5 cells with control media or F3-CM alone or F3-CM treated with Tocilizumab (2.5 μg/ml) or with IL6 (200 ng/ml) alone or IL6-supplemented with Tocilizumab. c Western blot analysis of CD206, Arg1, p-JAK2, JAK2, p-STAT3, and STAT3 in HMO6 and CHME5 cells with control media or F3-CM alone or F3-CM treated with fedratinib (20 μm for 24 h) or plus IL6(200 ng/ml) alone or IL6-supplemented with fedratinib. d The speculative binding sites in Arg1 for STAT3 were showed. e The dual-luciferase reporter experiment was applied to detect the luciferase activity of Arg1 stimulated by STAT3. f ChIP analysis was performed using a negative control immunoglobulin G (IgG) or anti-STAT3 antibody in CHME5 cells. g qPT-PCR of ChIP analysis was conducted to show binding site of STAT3 in ARG1. F3-CM: conditioned media of A549-F3 cells. Vector: pGL3-vector; STAT3: STAT3-vector. WT: wild type (#1); MUT: mutant type. M0:CHME5 cells; M2: F3-CM induced M2-CHME5 cells. Data are mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 6

Targeting IL6/JAK2/STAT3 signaling for suppression of A549-F3 metastasis in vivo. a Brain metastases were determined by bioluminescence imaging. b Histogram showed incidence of brain metastasis in the different groups. c Photon flux detected from brain-metastatic focus in the different groups. d Brain metastases were determined by bioluminescence imaging. e Histogram showed incidence of brain metastasis in the different groups. f Photon flux detected from brain-metastatic focus in the different groups. g Kaplan–Meier estimates of the cumulative probability of brain metastasis among. NSCLC patients according to the level of IL6 in serum. h Kaplan–Meier survival curves of NSCLC patients with IL6 high expression and IL6 low expression. i Proposed model for IL6/JAK2/STAT3 activation in M2-microglia to promote colonization of brain-metastatic cells. Data are mean ± SD. *P < 0.05; **P < 0.01