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) Label-free quantification of LCN2 in the tumors with and without E. coli inoculation. The error bars represent mean ± SD. Statistical analyses were performed by Student’s T test (** p <0.01).

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. The error bars represent mean ± SD. Statistical analyses were performed by Student’s T test (* p <0.05).

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 consuming ability of E. coli determined by the CAS assay reagent. (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. (g) E0771 breast tumor growth curves for the different treatment groups. (h) Survival curves for mice in different treatment groups. (i) B16F10 melanoma tumor growth curves for the different treatment treatment groups. (j) Survival curves for mice in the different treatment groups. (k) The bacterial burden from the blood of mice on day 1, 3, and 7 following intravenous administration with different bacteria. (l) Whole blood cell analyses for the different treatment groups. The error bars represent mean ± SD. Statistical analyses were performed by One-way ANOVA (** p <0.01).

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 error bars represent mean ± SD. Statistical analyses were performed by One-way ANOVA (** p <0.01, *** p <0.001).

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. (d) The average tumor growth curves of different treatment groups. (e) The tumor growth curves of individual mice in (d). The error bars represent mean ± SD. Statistical analyses were performed by One-way ANOVA (*p <0.05, **p <0.01, ****p <0.0001).

Workflow of quantitative proteomics for E. coli cultured in rich medium and E. coli colonized in murine tumors.

The E. coli BL21(DE3) were harvested from rich medium and tumors, respectively, and subject to the LC-MS/MS analysis.

STRING network analysis.

The proteins involving enterobactin biosynthesis and iron ion homeostasis were enriched in the E. coli colonized in tumors. The STRING diagram was drawn for the representative proteins in these two processes. The node size represents the fold change (tumor/rich medium) of the protein determined the label-free quantification method. The different interior colors of the nodes represent various biological processes defined by GO terms. The lines indicate the existence of experimental evidence between the connected nodes.

High concentration of cyclic-di-GMP partially blocks LCN2.

E. coli (1 × 104) were cultured in RPMI medium supplemented with LCN2 at 0.15 µM and Cyclic-di-GMP at 7.5 µM overnight. The number of survival bacteria were determined by plating the overnight culture on the LB agar plates. The survival rate was normalized to the no-treatment group.

Tumor re-challenge experiments in mice.

The mice cured by DGC-E. coli and VLX600 combination therapy were re-challenged with the same cell line (MC38) subcutaneously at the opposite flank. No tumor growth was observed. The naïve mice were used as controls.

The tumor growth curves of individual mice in MC38, E0771, B16F10 tumor models

(a) The tumor growth curves of individual mice in MC38 tumor model (b) The tumor growth curves of individual mice in E0771 tumor model (c) The tumor growth curves of individual mice in B16F10 tumor model

IroA-E. coli treatment resulted in higher bacterial colonization in tumors as compared to WT bacteria.

The bacteria were intratumorally delivered to tumor-bearing mice for fifteen days. The tumors were collected and homogenized for measuring the bacteria number. The number of survival bacteria was determined by plating the overnight culture on the LB agar plates.