The quantitative proteomic analysis of E. coli in the rich medium and the tumor microenvironment.

(a) The Venn diagram of the E. coli protein IDs identified in the rich medium and in the TME. (b) The volcano plot of the E. coli protein IDs quantified in the rich medium and in the TME. (c) The GO-term analysis of the protein IDs enriched in the rich medium condition. (d) The GO-term analysis of the protein IDs enriched in the tumor condition. (e) The hierarchical clustering analysis of the protein IDs identified in the rich medium and in the TME. Each column is a biological replicate. (f) Left: the fold changes of individual proteins in the iron ion homeostasis process. These proteins are involved in transporting or processing the iron ions. Right: the fold changes of individual proteins in the enterobactin biosynthesis process. (g) The label-free quantification of LCN2 in the tumors with and without E. coli inoculation.

Combination of iron chelator and DGC-E. coli for cancer therapy.

(a) Toxicity profiles of various iron chelators against MC38 cancer cells and E. coli. (b) Identification of cyclic-di-GMP secretion from DGC-E. coli by LC-MS/MS. The precursor ion and the fragmented product ions correspond to the correct molecule weights of CDG. (c) IFN-β secretion by RAW264.7 cells treated with the supernatants from wild-type E. coli or DGC-E. coli. (d) Schematic illustration of mouse treatments. The DGC-E. coli was intratumorally delivered on Day 0 and Day 9, whereas VLX600 was intravenously administrated every three days from Day 0 to Day 9. (e) Tumor growth curve for various treatment groups. The complete remission was only achieved in the CDG+VLX600 group (CR=2/4). (f) The Kaplan-Meier analysis for different treatment groups. The mouse was considered dead when the tumor volume exceeded 1,500 mm3.

Characterization of IroA-E. coli for anti-tumor activity

(a) E. coli viability in varying concentrations of LCN2 protein. The ΔentE strain, which could not generate enterobactin, was used as a negative control. (b) Iron acquisition ability of E. coli determined by the CAS assay. (c) Cytotoxicity of enterobactin on the MC38 colon cancer cells. The enterobactin was extracted from an equal supernatant volume of the WT-E. coli or the IroA-E. coli culture. The extraction buffer (DMSO) was used as a negative control. (d) Treatment schedule of IroA-E. coli in tumor-bearing mice. Two intratumoral injections were administered on Day 0 and Day 9. (e) Tumor growth curves across various treatment groups. (f) The Kaplan-Meier analysis for the mice in different treatment groups. Mice were considered dead when the tumor volumes exceeded 1,500 mm3.

Quantitative proteomic analysis comparing IroA-E. coli and WT-E. coli in the TME.

(a) Volcano plot analysis between the proteomes of WT-E. coli and IroA-E. coli in the mouse tumors. (b) Fold changes of the proteins involved in the iron ion homeostasis. All the fold changes are > 1. (c) Fold changes of the proteins involved in the enterobactin biosynthetic process. All the fold changes are > 1. (d) Fold changes of transferrin and transferring receptor in the tumor.

IroA-E. coli treatment stimulated the adaptive immune system for anti-tumor activity.

(a) The mice cured by IroA-E. coli were re-challenged with a subcutaneous inoculation of 2.5 × 105 MC38 cells. No tumor formation was observed. The naïve mice were used as controls. (b) The proportions of tumor-infiltrating CD4+ and CD8+ T cells in different treatment groups. (c) The tumor-bearing mice were treated with IroA-E. coli in the presence or absence of the anti-CD8 depletion antibody. (d) Survival curves of mice in different treatment groups. The mice were considered dead when tumor volumes exceeded 1,500 mm3.

Synergistic anti-tumor activity of IroA-E. coli and oxaliplatin.

(a) The scheme of the systemic delivery of IroA-E. coli and oxaliplatin in the tumor-bearing mice. (b) The alteration of mouse weights during the treatment course. (c) Survival curves of the mice in various treatment groups. The mice were considered dead when tumor volumes exceeded 1,500 mm3. (d) The average tumor growth curves of different treatment groups. (e) The tumor growth curves of individual mice in (d).