Research ArticleHEALTH AND MEDICINE

Engineered algae: A novel oxygen-generating system for effective treatment of hypoxic cancer

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Science Advances  20 May 2020:
Vol. 6, no. 21, eaba5996
DOI: 10.1126/sciadv.aba5996
  • Fig. 1 Characterization of the RBCM-Algae biosystem.

    (A) Illustrative description of engineered processes and treatments. (B) Photograph of algae (inset, large-scale preparation of algae). Pseudocolor SEM images of algae (C) and RBCM-coated algae (D). (E) Optical absorption of the algae (inset, photographs). (F) Oxygenation of the RBCM-Algae under white/red light irradiation. (G) Concentration-dependent oxygenation of the RBCM-Algae. Data are means ± SD, n = 3 for each group. a.u., arbitrary units.

  • Fig. 2 Engineered algae could increase oxygen, improve radiotherapeutic effect, release chlorophyll, and induce PDT.

    (A) Confocal fluorescence images showing increased intracellular O2 level after treatment with RBCM-Algae in hypoxia incubation with hypoxia probe [Ru(dpp)3]Cl2. Scale bars, 40 μm. (B) Clonogenic assay of 4T1 cells in vitro showing increased sensibility of RT. More cancer cells were killed by RBCM-Algae–mediated x-ray irradiation. (C) Cell DNA damage evaluated by γ-H2AX foci. (D) The corresponding quantitative analysis of the number of foci per cell. More DNA damages and cancer cell–killing effects were found for the hypoxia cancer cells by the RBCM-Algae under red light irradiation (6000 lux, 2 hours). (E) Live/dead staining of the algae showed that a large number of the dead algae were found after the x-ray treatment. Scale bars, 50 μm. X-ray treatment could kill the algae, and the internal chlorophyll of the algae was released after the treatment. (F) Scheme of the process of the RBCM-Algae after x-ray treatment. (G) Fluorescence images of the 4T1 cancer cells after the incubation of the released chlorophyll, demonstrating the internalization of the chlorophyll in the cancer cells [4′,6-diamidino-2-phenylindole (DAPI), blue; chlorophyll, red]. Scale bars, 5 μm. (H) Representative confocal fluorescence microscopy images of live/dead staining assay of 4T1 cells via calcein-AM (green, viable cells) and PI (red, dead cells) staining. Scale bars, 100 μm. Data are means ± SD, n = 3, Student’s two-tailed t test, not significant (n.s.) P ≥ 0.05; ***P < 0.001. Photo credit for (B): M.Z., Zhejiang University.

  • Fig. 3 Engineered algae could efficiently reach tumor site after intravenous injection and improve oxyhemoglobin.

    In vivo fluorescence imaging (A) of the mice and the quantitative analysis (B), ex vivo imaging (C), and the quantitative analysis (D) of the tumor uptake after intravenous injection of RBCM-Algae in 4T1 tumor-bearing mice. (E) Pseudocolor SEM images of tumor tissues; RBCM-Algae (green) were found in the tumor (red) 2 hours after intravenous injection of RBCM-Algae. (F) CD31 staining for tumor microvessels (green). The red signals are RBCM-Algae. (G) In vivo PA images of oxygen hemoglobin in 4T1 tumor at different time points after intravenous administration of RBCM-Algae and (H) the corresponding sO2 quantitative analysis. Data are means ± SD, n = 3.

  • Fig. 4 Engineered algae-mediated RT, and PDT can inhibit efficiently the tumor growth (4T1 breast cancer model) after intravenous injection.

    (A) Scheme illustrating treatments. (B) Tumor growth curves of individual mice. (C) Average tumor growth curves of all the groups. Photographs (D) and weight (E) of the tumors collected from the 4T1 tumor–bearing mice of each group (day 12). (F and G) Representative H&E, CD31, Ki-67, and cleaved caspase-3 staining of tumor sections from the tumor tissues in different treatment groups (day 12) and corresponding immunohistochemical index quantitative analysis. Treatments: 1, control; 2, laser alone; 3, RBCM-Algae alone; 4, x-ray irradiation (RT) alone; 5, RT + laser; 6, RBCM-Algae + laser; 7, RBCM-Algae + RT; and 8, RBCM-Algae + RT + laser. Data are means ± SD, n = 5, Student’s two-tailed t test, not significant P ≥ 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 5 Mechanism of the treatments, RBCM-Algae decrease the protein level of HIF1α and VEGF and promotes cell apoptosis and necrosis.

    (A) Tumor collection timetable from the process of in vivo anticancer PBS biosystem therapy. (B) Bioluminescence imaging and (C) the corresponding quantitative analysis of tumor tissues that were stained with ROS probe (DCFH). (D) Representative HIF1α, CD31, Ki-67, cleaved caspase-3, and H&E staining of tumor section from in situ 4T1 tumor–bearing mice of RBCM-Algae treated by RBCM-Algae + RT + laser and the corresponding immunohistochemical index quantitative analysis (E). (F) Immunoblotting of HIF1α, VEGF, cleaved caspase-3, proliferating cell nuclear antigen (PCNA), and caspase-3 protein level from 4T1 tumor–bearing mice of the RBCM-Algae + RT + laser group and the corresponding quantitative analysis. (G) Possible mechanism of the RBCM-Algae–mediated combination therapy. Data are means ± SD, n = 3.

Supplementary Materials

  • Supplementary Materials

    Engineered algae: A novel oxygen-generating system for effective treatment of hypoxic cancer

    Yue Qiao, Fei Yang, Tingting Xie, Zhen Du, Danni Zhong, Yuchen Qi, Yangyang Li, Wanlin Li, Zhimin Lu, Jianghong Rao, Yi Sun, Min Zhou

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