Echinacea purpurea-derived homogeneous polysaccharide exerts anti-tumor efficacy via facilitating M1 macrophage polarization

Abstract

Echinacea purpurea modulates tumor progression, but the underlying mechanism is poorly defined. We isolated and purified a novel homogeneous polysaccharide from E. purpurea (EPPA), which was shown to be an arabinogalactan with a mean molecular mass (Mr) of 3.8 × 10⁴ Da and with α- (1 → 5) -L-Arabinan as the backbone and α-L-Araf-(1→, →6)-β-D-Galp-(1→, and →4)-α-D-GalpA-(1→ as the side chains. Interestingly, oral administration of EPPA suppresses tumor progression in vivo and shapes the immune cell profile (e.g., facilitating M1 macrophages) in tumor microenvironment by single-cell RNA sequencing (scRNA-seq) analysis. More importantly, EPPA activates the inflammasome through a phagocytosis-dependent mechanism and rewires transcriptomic and metabolic profile, thereby potentiating M1 macrophage polarization. Collectively, we propose that EPPA supplementation could function as an adjuvant therapeutic strategy for tumor suppression.

Graphical abstract

Figure 1

Extraction, purification, and chemical characterization of homogeneous polysaccharide (EPPA) (A and B) HPGPC (A) and ¹H and ¹³C NMR spectra (B) of homogeneous polysaccharide. (C) The putative structure of EPPA.

Figure 2

Effects of EPPA on tumor growth of colorectal cancer in mice (A) Schematic image for the establishment of colorectal cancer mouse model with or without EPPA treatment. (B–D) The tumor number (C) and colon length (D) of the mice in different groups at 15 weeks post-induction (n = 12). (E) Representative IHC staining image for IFN-γ and F4/80. Scale bar, 100 μm. (F) Positive staining area/total tissue area in colorectal tumor tissue sections (n = 6–9). Data were analyzed using unpaired t tests (C, D, and F) and are represented as mean ± SD. ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.

Figure 3

Transcriptional landscape of macrophages in tumor tissue of colorectal cancer in mice by scRNA-seq analysis (A) Schematic diagram of scRNA-seq process in mice. (B) Immune cell profiles of tumor tissues in different groups of mice. (C) The percentage of cells in control, model, and polysaccharide group. (D–F) Volcano map (D), GO analysis (E), and KEGG analysis (F) of differential gene obtained from macrophages between model group and polysaccharide group. (G–I) Immunofluorescence analysis of F4/80⁺ iNOS⁺ macrophages (G and I) and F4/80⁺ CD206⁺ macrophages (H and I) in tumor tissue of colorectal cancer in mice (n = 5–7). Scale bar, 50 μm. Data were analyzed using an unpaired t test (I) and are represented as mean ± SD. ∗p < 0.05 and ∗∗p < 0.01.

Figure 4

EPPA suppresses M1 macrophage inflammation (A and B) The secretion of IL-1β (A) and TNF-α (B) from M1 macrophages treated with or without EPPA (500 μg/mL) (n = 3). Results represent three independent experiments. (C) Heatmap analysis of DEGs in M1 macrophages treated with or without EPPA (500 μg/mL) (n = 4). (D) Lists of signaling pathways (count ≥ 3) in M1 macrophages treated with or without EPPA (500 μg/mL) according to KEGG analysis (n = 4). (E–G) Heatmap analysis of DEGs related to cytokine-cytokine receptor interaction (E), NF-κB signaling pathway (F), and NOD-like receptor signaling pathway (G) in M1 macrophages treated with or without EPPA (500 μg/mL) (n = 4). (H) Protein abundance of p65, p-p65, NALP1, NLRP3, NLRC4, AIM2, caspase-1, and IL-1β in M1 macrophages treated with or without EPPA (500 μg/mL) (n = 4). Data were analyzed using one-way ANOVA with Bonferroni correction (A, B, and H, right) and are represented as mean ± SD.∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

Figure 5

EPPA activates inflammasome in M1 macrophages via endocytosis (A) The secretion of IL-1β from M1 macrophages treated with EPPA (500 μg/mL) or rhodamine B-labeled EPPA (500 μg/mL) (n = 3). Results represent two independent experiments. (B) Representative images of the uptake of EPPA (500 μg/mL) by M0 and M1 macrophages at different time points (10 min, 30 min, and 1 h) (×100, n = 3). (C) The secretion of IL-1β from CPZ (10 μM), Col (10 μM), NY (50 μg/mL), or AMR (50 μM)-pretreated M1 macrophages with or without EPPA (500 μg/mL) supplementation (n = 3). Results represent two independent experiments. (D and E) Protein abundance of NLRP3, caspase-1, and IL-1β in CPZ (10 μM) or Col (10 μM)-pretreated M1 macrophages with EPPA (500 μg/mL) supplementation (n = 3). (F and G) Representative images (F) and statistical analysis (G) of the uptake of EPPA (500 μg/mL) by Col (10 μM)-pretreated M1 macrophages at different time points (10 min, 30 min, and 1 h) (n = 5). Results represent two independent experiments. Scale bar, 10 μm. (H and I) Representative images (H) and statistical analysis (I) of the uptake of EPPA (500 μg/mL) by AMR (50 μM)-pretreated M1 macrophages at different time points (10 min, 30 min, and 1 h) (n = 6). Results represent two independent experiments. Scale bar, 10 μm. Data were analyzed using one-way ANOVA with Bonferroni correction (A, C, and E) or unpaired t tests (G and I) and are represented as mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Figure 6

EPPA shapes the metabolic profile of M1 macrophages (A) Heatmap analysis of different metabolites in M1 macrophages treated with or without EPPA (500 μg/mL) (n = 6). (B) Fold change of the levels of different metabolites in glucose metabolism of M1 macrophages treated with or without EPPA (500 μg/mL) (n = 6). Blue, decreased; gray, undetermined; black, unchanged. (C and D) The glycolysis, glycolic capacity, and glycolic reserve of M1 macrophages treated with or without EPPA (500 μg/mL) (n = 10). Results represent two independent experiments. Data were analyzed using unpaired t tests (B and D) and are represented as mean ± SD.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001.