Solid tumors are highly heterogeneous, and will exhibit very different distributions of specific cell types, such as immune system macrophages—which are involved in tumor progression—in the tumor microenvironment. Scientists in Germany have now developed a technology that combines a form of optoacoustic imaging and harmless photosynthetic purple Rhodobacter bacteria to visualize the presence of these tumor-associated macrophages (TAM) directly in breast tumors in experimental mice.

The researchers, at the Helmholtz Zentrum München, the Juelich Research Center, the Technical University of Munich, and the Heinrich Heine University Düsseldorf, suggested that their method could ultimately allow the use of bacteria to detect tumors, identify increased macrophage activity, and monitor response to immunotherapy. Ultimately the technique could help to develop better, personalized anticancer treatment strategies.

“We were able to demonstrate that bacteria of the genus Rhodobacter, which are harmless to humans, are suitable as indirect markers of macrophage presence and activity,” said Andre C. Stiel, PhD, head of the cell engineering group at the Institute of Biological and Medical Imaging (IBMI), Helmholtz Zentrum München.

Tumors exhibit very spatially heterogeneous morphology, physiology, and immunology, including subpopulations of distinct cell types, such as macrophages. While macrophages cells are essential to the functioning of a healthy immune system, they can also trigger inflammation of the tumor tissue, which can lead to further disease progression. Such tumor heterogeneity complicates our understanding of tumor progression and is a challenge to treatment decision making, the authors explained. Current microscopy and radiological-based imaging methods aren’t ideal for studying tumor heterogeneity, either because they have limited field of view and penetration depth, or because they offer limited spatial resolution, or are too expensive. “Understanding spatial heterogeneity necessitates high resolution in vivo imaging of anatomical and pathophysiological tumor information,” the researchers commented.

They turned instead to multispectral optoacoustic (OA) tomography (MSOT), an imaging technique that combines optical contrast with ultrasound resolution, to enable high-resolution, real-time in vivo imaging that goes beyond the penetration depth of current microscopy methods. “Therefore, it is emerging as a particularly interesting alternative imaging method in cancer research,” the researchers commented. However, one drawback of the technique is that when used without labels, MSOT can only highlight a limited number of tumor features.

Several labeling approaches have been tested to extend optoacoustic capacity, but the researchers turned to a novel labeling approach that harnessed photosynthetic bacteria of the genus Rhodobacter, a harmless organism that lives in stagnant and flowing waters, and which produces different pigments to carry out photosynthesis. One of these pigments is bacteriochlorophyll a (Bchl a), which can be used alongside MSOT to localize solid tumors. However, as the authors stated, to date, “ … no study has attempted to use bacteria for in vivo monitoring of pathophysiological processes.”

During an MSOT scan, light is initially converted into sound, and then into visual information, according to the Helmholtz Zentrum München. A weak, pulsating laser beam is directed towards the site of interest on the body, and this causes the cells and molecules to heat up minimally and respond with similarly minimal vibrations, which generate acoustic signals. These signals are detected by sensors, and converted into images. Exactly how the individual cells and molecules respond to the laser depends on their optical properties, and in the reported study on the properties of the Rhodobacter pigment.

The scientists used MSOT in combination with administration of the purple Rhodobacter organisms to image macrophages in the tumor environment in live mice. Macrophages engulfed the bacteria as part of their natural scavenging, which acted to change the environment of the bacteria, and so their absorption of electromagnetic radiation, and this, in turn, changed the optoacoustic signal, which could then be detected. “These properties can be used, e.g., in the case of tumors, to report macrophage accessibility in the tumor microenvironments,” the authors commented. “Remarkably, we found reproducible (n = 3) spatiotemporal variation of the spectral pattern depending on the strain, the tumor model and the localization within the tumor at different times after injection.”

Change of optoacoustic signals of purple bacteria located outside (blue) and inside (red) of macrophages visualized via schematic (first row), microscopic (second row), and MSOT analysis (bottom). The change of MSOT spectra (third row) can be used to differentiate between Rhodobacter cells located inside and outside of macrophages and hence macrophage localization and activity.
[Helmholtz Zentrum München]

They claim their results clearly link the change of the spectral signature to macrophage activity in different areas of the tissue, “which is an important contribution to understanding the tumor microenvironment and the spatially and temporally diverse plasticity, activity, and overall functionality of macrophages … The present work, therefore, provides a starting point for applying OA imaging and Rhodobacter strains as reporters for studying the pathophysiological role of macrophages in preclinical cancer research and can help to develop strategies for bacterial tumor therapy.”

“In further steps, these bacteria will enable novel approaches to noninvasive technologies and so open up entirely new possibilities for innovative diagnostic and therapeutic procedures,” stated Thomas Drepper, PhD, who heads the Bacterial Photobiotechnology Group at Heinrich Heine University Düsseldorf.

“In summary, Rhodobacter species offer multiple properties making them promising candidates for preclinical studies of tumor heterogeneity,” the authors concluded. “Our results now demonstrate the usefulness of this class of photosynthetic bacteria by showing that their BChl a can be very effectively identified via MSOT and that changes in their unique spectral signature are a function of macrophage activity, which can provide insights into tumor biology in vivo.”

The researchers further suggest that the Rhodobacter may represent “a truly versatile smart micro-packaging system,” which could be used to deliver DNA and active substances into tumor-associated macrophages, as well as simultaneously enable MSOT-based monitoring of drug release.

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