of 32
Nature Biotechnology
| Volume 41 | July
2023 | 919–931
919
nature biotechnology
https://doi.org/10.1038/s41587-022-01581-y
Article
Genomically mined acoustic reporter genes
for real-time in vivo monitoring of tumors
and tumor-homing bacteria
Robert C. Hurt
1,5
, Marjorie T. Buss
2,5
, Mengtong Duan
1,5
, Katie Wong
1
,
Mei Yi You
1
, Daniel P. Sawyer
1
, Margaret B. Swift
3
, Przemysław Dutka
1,2
,
Pierina Barturen-Larrea
2
, David R. Mittelstein
4
, Zhiyang Jin
4
,
Mohamad H. Abedi
1
, Arash Farhadi
1
, Ramya Deshpande
2
&
Mikhail G. Shapiro
2,3
Ultrasound allows imaging at a much greater depth than optical methods,
but existing genetically encoded acoustic reporters for in vivo cellular
imaging have been limited by poor sensitivity, specificity and in vivo
expression. Here we describe two acoustic reporter genes (ARGs)—one
for use in bacteria and one for use in mammalian cells—identified through
a phylogenetic screen of candidate gas vesicle gene clusters from diverse
bacteria and archaea that provide stronger ultrasound contrast, produce
non-linear signals distinguishable from background tissue and have stable
long-term expression. Compared to their first-generation counterparts,
these improved bacterial and mammalian ARGs produce 9-fold and 38-fold
stronger non-linear contrast, respectively. Using these new ARGs, we
non-invasively imaged in situ tumor colonization and gene expression in
tumor-homing therapeutic bacteria, tracked the progression of tumor gene
expression and growth in a mouse model of breast cancer, and performed
gene-expression-guided needle biopsies of a genetically mosaic tumor,
demonstrating non-invasive access to dynamic biological processes at
centimeter depth.
Basic biological research, in vivo synthetic biology and the development
of cell-based medicine require methods to visualize the functions of
specific cells deep inside intact organisms. In this context, optical tech
-
niques based on fluorescent and luminescent proteins are limited by the
scattering and absorption of light by tissue
1
. Ultrasound is a widely used
technique for deep tissue imaging, providing sub-100-μm spatial resolu
-
tion and penetrating several centimeters into tissue
2
. Super-resolution
methods
3
,
4
have pushed its spatial resolution below 10 μm. Recently, the
first genetically encodable reporters for ultrasound
5
7
were introduced
based on gas vesicles (GVs)—air-filled protein nanostructures encoded
by clusters of 8–20+ genes, which evolved as flotation devices in diverse,
mostly aquatic bacteria and archaea
8
,
9
. The low density and high com-
pressibility of their air-filled interiors compared to surrounding tissues
allow GVs to scatter sound waves and, thereby, produce ultrasound
contrast when heterologously expressed as acoustic reporter genes
(ARGs) in genetically engineered bacteria
6
or mammalian cells
7
.
Despite the promise of first-generation ARGs, their utility for
monitoring bacterial or mammalian gene expression in vivo is limited.
Received: 21 February 2022
Accepted: 20 October 2022
Published online: 2 January 2023
Check for updates
1
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA.
2
Division of Chemistry and Chemical Engineering,
California Institute of Technology, Pasadena, CA, USA.
3
Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.
4
Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA, USA.
5
These authors contributed equally: Robert C. Hurt,
Marjorie T. Buss, Mengtong Duan.
e-mail:
mikhail@caltech.edu