Research ArticleHEALTH AND MEDICINE

Bioinorganic hybrid bacteriophage for modulation of intestinal microbiota to remodel tumor-immune microenvironment against colorectal cancer

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Science Advances  15 May 2020:
Vol. 6, no. 20, eaba1590
DOI: 10.1126/sciadv.aba1590
  • Fig. 1 Schematic illustration of phage-based bio/abiotic hybrid system (M13@Ag) to regulate gut microbes for cancer-specific immune therapy.

  • Fig. 2 The screen of protumor gut microbiome in CRC.

    (A) Differential gene expression heat map of identified genes about immunity-associated human CRC gene in the TCGA CRC dataset. (B) Bacteria levels of Fusobacterium colonization in fecal samples of patients with CRC from the NCBI bio-project (PRJEB10878). Significant difference was assessed by using t test. (C) Fluorescence images of microchip from patients with CRC within tumor tissues [Fn visualized with fluorescence in situ hybridization assay is green, tumor cell nucleoid is stained with 4′,6-diamidino-2-phenylindole (DAPI) with blue fluorescence, and Gr-1 antibody–labeled MDSC cell infiltration is red). (D) In vivo bioluminescence imaging of orthotopic CT26-luc tumor-bearing mice with different treatments at 15 days (n = 5). (E) Flow cytometry analysis of the number of CD3+ CD8+ T cells and CD11b+ Gr-1+ MDSC cells at the tumor site after receiving treatment at 10 days (n = 3). Representative dot plots as indicated. (F) Typical images of fluorescence in situ hybridization assay for measuring the Fn level in tumor tissues treated with Fn colonization or antibiotic cocktail. Scale bar, 200 μm. Significant difference was assessed in (B) by using t test. The mean values and SD are presented.

  • Fig. 3 Characterization of bioinorganic hybridization phages.

    (A) In vitro biopanning of specifically Fn-binding M13 phages by Ph.D.-12 peptide phage display library. Eight kinds of clones were examined by ELISA. (B) Screening of a variety of antibacterial nanoparticles (n = 6). (C) Assessment bioactivity of M13 phages after directly assembling with AgNP (n = 3). TEM images of M13 phages (D) and M13@Ag (E), in which M13 phages were native stained. Scale bars, 100 nm. Analysis of element distribution on the surface of M13 phages (F) and M13@Ag (G) detected with EDX. (H) Cell viability of cancerous CT26 cells and normal 3T3 cells after coculture with M13 phages (n = 6). (I) Antibacterial activity of M13@Ag by incubating with Fn for 8 hours (n = 6). (J) Genus-level analysis in the feces from the Fn-colonization CRC murine model with significant increase in antitumor bacteria of Butyricicoccus after PBS, M13, and M13@Ag treatment (n = 3). (K) Venn diagram of identified fecal bacterial strains in Fn-colonization CRC murine model with different treatments (n = 3). Significant differences were assessed in (C) and (J) using one-way ANOVA and t test and in (B), (H), and (I) using t test. The mean values and SD are presented. n.s., not significant; OD, optical density; a.u., arbitrary units.

  • Fig. 4 In vitro activation of anticancer immune responses by M13@Ag.

    (A and B) TEM images of Fn (A-i), M13 phages targeting Fn (A-ii), and M13@Ag targeting Fn (B-i and B-ii). The filamentous M13 phages were indicated with white arrows. Scale bars, 1 μm. (C) Confocal fluorescence images of phage-binding assays of M13 phages and M13@Ag toward different species of bacteria. Phages were marked with red fluorescent Cy5, and bacteria were labeled with green fluorescent FITC, respectively. Scale bar, 30 μm. (D) Flow cytometry for quantification of the targeting capacity of M13@Ag after coincubating with Fn and M13@Ag for 30 min. Phages were stained with green fluorescent FITC (n = 3). (E) Representative flow cytometry images for evaluating the DC maturation and TAMs toward M1 phenotype. BMDCs and M2 macrophages were incubated with M13 phages or M13@Ag for 24 hours (n = 3). Efficacy of immune responses for CTL-mediated cancer cell–specific lysis (F) and cytokine production levels of TNF-α (G) measured by incubating M13 phage–stimulated splenocytes with CT26 tumor cells for 24 hours. The supernatant was collected to detect the LDH leakage level and TNF-α level, with cytotoxic lymphocytes toward CT26 cells with an effector/target ratio of 20:1 (n = 4). (H) In vitro anticancer effects of M13 phage–polarized M1 macrophages measured by LDH assay after coculture with CT26 cells at the ratio of 20:1 (n = 6). Significant differences were assessed in (F), (G), and (H) using one-way ANOVA and t test and in (D) using t test. The mean values and SD are presented. MFI, mean fluorescence intensity.

  • Fig. 5 In vivo M13@Ag mediated tumor targeting and anticancer effect.

    (A) Representative IVIS images for the capacity of phage accumulation in orthotopic CT26-luc tumors after treatment with wild phages (without Fn-binding ability), M13 phages (with Fn affinity), and M13@Ag (n = 3). h, hours. (B) Ex vivo fluorescence imaging of the major organs as well as tumors for measuring the accumulation of M13 phages and M13@Ag after the intravenous injection at 24 hours (n = 3). The tumors were pointed by white circles. (C) Schematic of building orthotopic CRC model and therapeutics-based M13@Ag treatment plan. (D) In vivo bioluminescence imaging of orthotopic CT26-luc tumor-bearing mice after receiving PBS, M13 phages, AgNP, M13@Ag, and M13@Ag combined with α-PD1 or FOLFIRI treatments (n = 5). Mice body weight (E) and survival curves (F) after receiving various treatments (n = 5). (G) Representative fluorescence images of orthotopic tumor-bearing mice showing the number of Fn and Ki67 staining after treatments. (H) Tumor growth curves after different treatments in subcutaneous CT26 tumor model (n = 5). Significant differences were assessed in (F) using one-way ANOVA and in (E) and (H) using t test. The mean values and SD are presented.

  • Fig. 6 M13@Ag reversed immunosuppressive TME for activation of antitumor immune response.

    Orthotopic CT26-luc tumors were harvested from mice at 14 days after different treatments. Representative flow cytometric analysis images (left) and relative quantification (right) of infiltrating immune cells within tumor tissues as indicated. G1, PBS; G2, M13; G3, AgNP; G4, M13@Ag; G5, M13@Ag + α-PD1; G6, M13@Ag + FOLFIRI. (A) The mature of double-positive CD80+CD86+ dendritic cell gating on CD11c+ cells (n = 3). (B) TAM of classical activation M1 phenotype highly expressed CD86 (gated on CD11b+ cells) (n = 3). (C) The level of tumor infiltration CD8+ cytotoxic T cells gating on CD3+ cells (n = 3). (D) Percentage of CD11b+Gr-1+ MDSCs in the total of CD45+ lymphocytes (n = 3). (E) Immunohistochemistry staining images for arginase-1 (Arg-1) expression with immunosuppressive cells in different groups. (F) Relative quantification of MDSCs in spleen. (G and H) Secretion levels of IL-10 and IFN-γ in different treated groups (n = 3). Significant differences were assessed in (A) to (D) and (F) to (H) using one-way ANOVA and t test. The mean values and SD are presented.

Supplementary Materials

  • Supplementary Materials

    Bioinorganic hybrid bacteriophage for modulation of intestinal microbiota to remodel tumor-immune microenvironment against colorectal cancer

    Xue Dong, Pei Pan, Di-Wei Zheng, Peng Bao, Xuan Zeng, Xian-Zheng Zhang

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