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. 2022 Jun 30:12:732860.
doi: 10.3389/fonc.2022.732860. eCollection 2022.

Sanguinarine Regulates Tumor-Associated Macrophages to Prevent Lung Cancer Angiogenesis Through the WNT/β-Catenin Pathway

Yajing Cui  1 , Yingbin Luo  1 , Qiaohong Qian  2 , Jianhui Tian  1 , Zhihong Fang  1 , Xi Wang  1 , Yaoying Zeng  1 , Jianchun Wu  1 , Yan Li  1
Affiliations

Sanguinarine Regulates Tumor-Associated Macrophages to Prevent Lung Cancer Angiogenesis Through the WNT/β-Catenin Pathway

Yajing Cui et al. Front Oncol. .

Abstract

Tumor-associated macrophage (TAM)-mediated angiogenesis in the tumor microenvironment is a prerequisite for lung cancer growth and metastasis. Therefore, targeting TAMs, which block angiogenesis, is expected to be a breakthrough in controlling the growth and metastasis of lung cancer. In this study, we found that Sanguinarine (Sang) inhibits tumor growth and tumor angiogenesis of subcutaneously transplanted tumors in Lewis lung cancer mice. Furthermore, Sanguinarine inhibited the proliferation, migration, and lumen formation of HUVECs and the expression of CD31 and VEGF by regulating the polarization of M2 macrophages in vitro. However, the inhibitory effect of Sanguinarine on angiogenesis remained in vivo despite the clearance of macrophages using small molecule drugs. Further high-throughput sequencing suggested that WNT/β-Catenin signaling might represent the underlying mechanism of the beneficial effects of Sanguinarine. Finally, the β-Catenin activator SKL2001 antagonized the effect of Sanguinarine, indicating that Sanguinarine can regulate M2-mediated angiogenesis through the WNT/β-Catenin pathway. In conclusion, this study presents the first findings that Sanguinarine can function as a novel regulator of the WNT/β-Catenin pathway to modulate the M2 macrophage polarization and inhibit angiogenesis, which has potential application value in immunotherapy and antiangiogenic therapy for lung cancer.

Keywords: Wnt/β- catenin; angiogenesis; lung cancer; sanguinarine; tumor associated macrophages.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Sang may inhibit tumor growth and angiogenesis in mice through a TAM-dependent mechanism. A total of 5 × 105 LLCs per mouse were subcutaneously injected into the right flank of C57BL/6 mice, and then mice were intraperitoneally injected with saline (0.1 ml/mouse), Sang (2.5 mg/kg, 5 mg/kg) or cisplatin (2 mg/kg) from day 2. Tumor size was measured every 2 days, and following treatment for 3 weeks, the mice were sacrificed, and tumor tissues were isolated. Data are presented as the mean ± SEM of measurements from five mice per group. *p < 0.05; ***p < 0.001. ns, not statistically significant (p > 0.05). (A) Tumor volume curves; (B) Tumor tissues collected at the end point; and (C) Tumor weights. (D) The expression of CD31, (E) F4/80, and (F) CD206 in the subcutaneous tumor tissue of lung cancer mice were revealed by immunofluorescence staining. Nucleus was stained with DAPI solution. Scale bars: 100, 100, and 100 μm, respectively.
Figure 2
Figure 2
Sang suppresses M2 polarization in macrophages. Bone marrow-derived macrophages (BMDMs) were isolated from mice and treated with IL-4 (20 ng/ml) for 24 h for M2 polarization. (A) Flow cytometry analysis of the expression of the M2-like macrophage surface marker CD206. (B) Western blot analysis of CD206 protein expression in IL-4- stimulated BMDMs. (C) BMDMs were incubated in the presence of Sang at different concentrations (0, 0.2, 0.4, 0.8, 1, 2, and 4 µM) for 24 h, and cell viability was determined by the CCK-8 assay. (D) BMDMs in the presence or absence of IL-4 were treated with different concentrations of Sang (0.1, 0.3, and 1 µM), and the expression of CD68 and CD206 proteins was detected through a western blot assay. (E) Flow cytometry detection of CD206 expression treated with 1 µM Sang Each experiment was reproduced three times. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant (p > 0.05).
Figure 3
Figure 3
Sang inhibits neovascularization by suppressing M2 macrophage polarization. Conditioned medium (CM) was used to determine the effects of Sang pre-treatment on angiogenesis promoted by M2-like macrophages. (A) Representative images of the HUVEC tube formation assay on Matrigel (magnification 20x) and quantification of tubes and branch points. (B) Representative images of the HUVECs wound healing assay at 0 h and 24 h, as well as quantitative analysis of the wound healing area. (C) HUVECs migration and invasion was detected using a Transwell assay. HUVECs were plated in the upper chamber, and the conditioned medium of macrophages after different treatments was collected and placed in the lower chamber. Representative images of HUVECs invasion, as well as quantification of the number of migrated HUVECs (20X). (D) Western blot assay for CD31 and VEGF protein expression in HUVECs that were co-cultured with macrophage-CM, and treated with Sang. Each experiment was reproduced three times. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant (p > 0.05).
Figure 4
Figure 4
Sang inhibits tumor growth and neovascularization in mice by suppressing macrophage M2 polarization. CLP (200 µl/mouse) was intraperitoneally administered on the day before starting tumor inoculation (day -1). A total of 5 × 105 LLCs per mouse were subcutaneously injected into the right flank of C57BL/6 mice (day 0), and then 100 µl of CLP per mouse was administered by intraperitoneal injection on days 4, 8, 12, 15 and 19 and Sang (5 mg/kg) from day 1. Tumor size was measured every 2 days, and following treatment for 3 weeks, the mice were sacrificed and tumor tissues isolated. Data are presented as the mean ± SEM of measurements from five mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant (p > 0.05). (A) Experimental setup for depletion of macrophages in the Lewis lung subcutaneous transplantation mouse model. (B) Tumor volume curves; (C) Tumor tissues collected at the end point; and (D) Tumor weights. (E) The expression of CD31, (F) F4/80 colocalization with CD206, and (G) VEGF in the subcutaneous tumor tissue of lung cancer in macrophage-cleared mice was revealed by immunofluorescence staining. Nucleus was stained with DAPI solution. Scale bars: 100, 100, and 100 μm, respectively. In vivo, Sang reduced the secretion of VEGF by inhibiting M2 polarization, which ultimately resulted in inhibition of angiogenesis and tumor growth in mice.
Figure 5
Figure 5
Sang regulates the M2 phenotype of macrophages by the WNT/β-Catenin pathway. RNA sequencing (RNA-Seq) was used to profile genome-wide gene expression and transcriptome changes in M0 and M2 macrophages. |log2FC| >1 and p-value<0.05 were taken as thresholds to screen differential genes. (A) Volcano plot showing the differentially expressed genes (DEGs) in M0 versus M2, n = 3. FC, fold change. (B) KEGG pathway enrichment analysis of differentially expressed genes (DEGs). Each of these blue entries represents a signaling pathway; broken yellow lines indicate the number of differential genes enriched in the pathway. (C) qRT-PCR was performed to determine Wnt ligand expression in M0, M2, and M2S. (D) Western blot assay for WNT5A and β-Catenin in M0, M2, and M2S, are considered to be key proteins in the non-classical and classic WNT/β-Catenin pathway, respectively. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 6
Figure 6
Sang inhibits M2-like polarization of macrophages and M2-mediated angiogenesis by targeting the WNT/β-Catenin pathway in macrophages. (A) The WNT/β‐Catenin activator SKL2001, alone or in combined with Sang, was utilized to treat BMDMs. Western blotting was performed to detect the protein expression of CD68, CD206, and β‐Catenin, and the results indicated that SKL2001 could upregulate β-Catenin and CD206 expression; however, β-Catenin and CD206 were markedly decreased following the SKL2001/Sang combination. (B–D) HUVECs were cocultured with M2 macrophages’ conditioned medium treated with SKL2001 alone or combined with Sang. (B) Representative images of the HUVEC tube formation assay on Matrigel (magnification 20x) and quantification of tubes and branch points. (C) Representative images of the wound healing assay of HUVECs at 0 h and 24 h, as well as quantitative analysis of the wound healing area. (D) HUVEC migration and invasion was detected using a Transwell assay. HUVECs were plated in the upper chamber, and the conditioned medium of macrophages after different treatments was collected and placed in the lower chamber. Representative images of HUVEC invasion and quantification of the number of migrated HUVECs (20X). Each experiment was reproduced three times. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant (p > 0.05).
Figure 7
Figure 7
Sang targets the WNT/β-Catenin signaling pathway in TAMs, which represses M2 polarization and thus inhibits TAM-mediated neoangiogenesis. M2-like TAMs show upregulation of WNT ligands (1-7a-7b-10b), leading to transcriptional activation of β-Catenin. β-Catenin translocation into the nucleus induces M2 macrophage polarization, which further promotes the secretion of VEGF protein in HUVECs, leading to the initiation of tumor angiogenesis. However, Sang inhibited WNT/β-Catenin signaling in M2 TAMs by downregulating ligands (1-7b) and degrading β-Catenin. β-Catenin could not enter the nucleus to induce M2 macrophage polarization, which thus restrained the secretion of VEGF protein in HUVECs and ultimately suppressed tumor angiogenesis and tumor growth.
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