An Engineered Probiotic Platform for Cancer Epitope-Independent Targeted Radionuclide Therapy of Solid Tumors

Abstract

Targeted radionuclide therapy (TRT) is an emerging therapeutic modality for the treatment of various solid cancers. Current approaches rely on the presence of cancer-specific epitopes and receptors against which a radiolabeled ligand is systemically administered to specifically deliver cytotoxic doses of α and β particles to tumors. In this proof-of-concept study, tumor-colonizing Escherichia coli Nissle 1917 (EcN) is utilized to deliver a bacteria-specific radiopharmaceutical to solid tumors in a cancer-epitope independent manner. In this microbe-based pretargeted approach, the siderophore-mediated metal uptake pathway is leveraged to selectively concentrate copper radioisotopes, ⁶⁴ Cu and ⁶⁷ Cu, complexed to yersiniabactin (YbT) in the genetically modified bacteria. ⁶⁴ Cu-YbT facilitates positron emission tomography (PET) imaging of the intratumoral bacteria, whereas ⁶⁷ Cu-YbT delivers a cytotoxic dose to the surrounding cancer cells. PET imaging with ⁶⁴ Cu-YbT reveals persistence and sustained growth of the bioengineered microbes in the tumor microenvironment. Survival studies with ⁶⁷ Cu-YbT reveals significant attenuation of tumor growth and extends survival of both MC38 and 4T1 tumor-bearing mice harboring the microbes. Tumor response to this pretargeted approach correlates with promising anti-tumor immunity, with noticeable CD8⁺ T:T_reg cell ratio. Their strategy offers a pathway to target and ablate multiple solid tumors independent of their epitope and receptor phenotype.

Keywords: cancer theranostics; engineered bacteria; positron emission tomography imaging; pretargeting; siderophore.

Figure 1

Engineered EcN bind and retain Cu‐YbT specifically via FyuA. A) Reaction mechanism depicting complexation of ^64/67Cu by yersiniabactin (YbT). B) Schematic highlighting FyuA‐specific delivery of Cu‐YbT radiopharmaceuticals by EcN‐based pretargeted cancer theranostic platform. C) Immunoblot confirming FyuA expression by engineered EcN. D) Schematic of EcN constructs used for E) quantitative (regression curve) assessment of FyuA overexpression. F) Dissociation curve of ⁶⁴Cu‐YbT from EcN‐fyuA↑Data in (E) and (F) presented as mean ± s.d. (n = 3); data in (E) (10⁹ cfu) analyzed by one‐way ANOVA and Dunnett's T3 multiple comparison test with Brown–Forsythe and Welch's correction, alpha = 0.05. *p < 0.05, **p < 0.01. Source Data: Unprocessed Western Blot for (C) is shown in Figure S6, Supporting Information.

Figure 2

Engineered EcN localize and persist in tumor microenvironment. A) BLI, PET/CT, and ex vivo BioD analyses depicting EcN‐fyuA↑ localization in MC38 tumors and FyuA‐specific retention of ⁶⁴Cu‐YbT 1‐day post intratumoral administration of bacteria. B) General biodistribution of ⁶⁴Cu‐YbT. C) qRT‐PCR assay confirming presence of EcN‐fyuA↑ in MC38 tumors 1‐ and 7‐days post intratumoral administration of bacteria. D) BLI, ex vivo BioD, and PET/CT demonstrating presence of bacteria in MC38 tumors 18‐days after intratumoral injection. Data of bar‐chart in (A) presented as mean ± s.d. (n = 3–4) analyzed by Welch's t‐test. **p < 0.01.

Figure 3

EcN traps ⁶⁷Cu‐YbT in solid tumors to elicit anti‐tumor effects. A) Schematic of the in vivo therapeutic plan. Tumor progression during the initial stages of therapy and survival curves of B) MC38 tumor‐bearing C57BL6/J mice (n = 4–8) and C) 4T1 tumor‐bearing BALB/cJ mice (n = 4) in the four treatment regimens. Data in tumor growth curves presented as mean ± s.d.; the final tumor volumes were analyzed by one‐way ANOVA and Dunnett's T3 multiple comparison test with Brown–Forsythe and Welch's correction, alpha = 0.05. Survival curves were analyzed by Kaplan–Meier with log‐rank (Mantel–Cox) test by comparing two groups at a time and presenting the p‐value at which ⁶⁷Cu‐YbT+EcN‐fyuA↑ is significantly different from the other three treatment strategies. ns = not significant, *p < 0.05, **p < 0.01.

Figure 4

⁶⁷Cu‐YbT+EcN‐fyuA↑ induce changes in tumor immune infiltrate. Flow cytometry analyses of tumor immune cell infiltrates A) total immune cells (CD45⁺), B) total T cells, C) CD4⁺ T cells, D) T_regs (CD4⁺CD25⁺FOXP3⁺) cells, E) CD8⁺ T cells as a percent of total live cells, and F) CD8⁺ T:T_reg ratio 7 days after ⁶⁷Cu‐YbT administration in MC38 tumor‐bearing mice with or without probiotic administration. All data presented as mean ± s.d. (n = 3); data in (F) analyzed by one‐way ANOVA and Dunnett's T3 multiple comparison test with Brown–Forsythe and Welch's correction, alpha = 0.05. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

⁶⁷Cu‐YbT exhibits good biosafety profile. A) Liver and B) kidney function analyses of serum obtained from ⁶⁷Cu‐YbT treated and untreated C57BL6/J mice. C) Weight of mice monitored every 2 days following administration of ⁶⁷Cu‐YbT. Data presented as mean ± s.d. (n = 3).