# Metabolic Gadolinium Labeling of Clostridium novyi-Nontoxic for Magnetic Resonance Imaging Visualization of Spores and Germinated Bacteria

## TL;DR

This paper introduces a new method to track Clostridium novyi-NT spores and bacteria in tumors using MRI by labeling them with gadolinium.

## Contribution

The novel use of metabolic gadolinium labeling enables dynamic MRI tracking of spore germination and bacterial activity in tumors.

## Key findings

- Gadolinium-labeled spores showed strong T2 contrast and did not affect viability or germination.
- MRI signal transitioned from T2 to T1 contrast as bacteria germinated and proliferated.
- The method enabled noninvasive monitoring of bacterial behavior in an orthotopic liver tumor model.

## Abstract

Bacterial cancer therapy using Clostridium novyi-nontoxic (C. novyi-NT) has emerged as a promising strategy to selectively eradicate hypoxic and necrotic tumor regions. However, noninvasive visualization of injected spores and germinated bacteria remains a major barrier to clinical translation. Here, we developed metabolically responsive gadolinium-labeled C. novyi-NT (Gd-C. novyi-NT) to enable magnetic resonance imaging (MRI)-based tracking of spore localization and bacterial germination. Gd ions were electrostatically condensed onto the negatively charged spore surface, forming Gd clusters that produced strong T2-weighted contrast without affecting spore viability or germination efficiency. Under anaerobic conditions, these Gd aggregates were progressively diluted and redistributed along the bacterial cell wall through metal-chelating metabolites, converting the MRI signal from T2-dominant to T1-weighted contrast. In an orthotopic liver tumor model, intra-arterial infusion of Gd-spores led to selective intratumoral accumulation, with early T2 contrast followed by sustained T1 enhancement persisting for up to 9 days post-administration. This metabolically driven T2-to-T1 contrast transition provides a dynamic, noninvasive imaging signature that correlates with bacterial germination and proliferation. The Gd-C. novyi-NT platform therefore offers a powerful tool for real-time monitoring of bacterial therapeutic behavior in vivo, advancing precision imaging and therapeutic control of bacterial-based oncologic therapies.