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Genomically Mined Acoustic Reporter Genes Enable In Vivo Monitoring of Tumors and Tumor-Homing Bacteria

Hurt, Robert C. and Buss, Marjorie T. and Duan, Mengtong and Wong, Katie and You, Mei Yi and Sawyer, Daniel P. and Swift, Margaret B. and Dutka, Przemysław and Mittelstein, David R. and Jin, Zhiyang and Abedi, Mohamad H. and Farhadi, Arash and Deshpande, Ramya and Shapiro, Mikhail G. (2021) Genomically Mined Acoustic Reporter Genes Enable In Vivo Monitoring of Tumors and Tumor-Homing Bacteria. . (Unpublished)

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A major outstanding challenge in the fields of biological research, synthetic biology and cell-based medicine is the difficulty of visualizing the function of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 µm. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging in the in vivo context. Here, we describe second-generation ARGs with greatly improved acoustic properties and expression characteristics, identified through a phylogenetic screen of candidate gene clusters from diverse bacteria and archaea. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue, and stable long-term expression. We demonstrate the utility of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing probiotic bacteria, revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells noninvasively in living subjects.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Hurt, Robert C.0000-0002-4347-6901
Buss, Marjorie T.0000-0002-4266-9197
Sawyer, Daniel P.0000-0003-2926-191X
Swift, Margaret B.0000-0001-9610-0687
Dutka, Przemysław0000-0003-3819-1618
Mittelstein, David R.0000-0001-8747-0483
Jin, Zhiyang0000-0002-4411-6991
Abedi, Mohamad H.0000-0001-9717-6288
Farhadi, Arash0000-0001-9137-8559
Shapiro, Mikhail G.0000-0002-0291-4215
Alternate Title:Genomically Mined Acoustic Reporter Genes Enable On-Demand In Vivo Monitoring of Tumor-Homing Bacteria
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Version 1 - April 27, 2021; Version 2 - October 29, 2021. The authors would like to thank Dianne Newman for a sample of Streptomyces coelicolor A3(2), and Yunfeng Li, Avinoam Bar-Zion and Hongyi (Richard) Li for help with tissue histology. Electron microscopy was performed in the Beckman Institute Resource Center for Transmission Electron Microscopy at Caltech. Mammalian cell sorting was performed at the Analytical Cytometry Core at City of Hope in Duarte, CA. Confocal microscopy was performed in the Beckman Institute Biological Imaging Center. This research was supported by the National Institutes of Health (R01-EB018975 to M.G.S.) and Pew Charitable Trust. R.C.H. was supported by the Caltech Center for Environmental Microbial Interactions. M.T.B. was supported by an NSF GRFP fellowship. Related research in the Shapiro Laboratory is supported by the David and Lucille Packard Foundation, the Burroughs Wellcome Fund and the Heritage Medical Research Institute. Author Contributions: R.C.H., M.T.B., M.D., K.W., M.B.S., P.D., Z.J., M.Y.Y., A.F. and R.D. planned and performed experiments. M.T.B. and M.D. performed all in vivo experiments, with help from M.B.S. P.D. and M.D. performed TEM imaging. D.R.M. built the ultrasound plate-scanning setup, and D.R.M. and D.P.S. wrote the associated MATLAB scripts for controlling it. Z.J. and D.P.S. wrote the MATLAB scripts for ultrasound imaging of EcN in vitro and in vivo. Z.J. performed the calibration of the L22-14v transducer. M.H.A. provided the axe-txe stability cassette, and advised on tumor colonization experiments. R.C.H., M.T.B., M.D., D.P.S., P.D., and Z.J. analyzed data. R.C.H., M.T.B., M.D., and M.G.S. wrote the manuscript with input from all other authors. M.G.S. supervised the research. The authors declare no competing financial interests.
Group:Heritage Medical Research Institute, Caltech Center for Environmental Microbial Interactions (CEMI)
Funding AgencyGrant Number
Pew Charitable TrustUNSPECIFIED
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
David and Lucile Packard FoundationUNSPECIFIED
Burroughs Wellcome FundUNSPECIFIED
Heritage Medical Research InstituteUNSPECIFIED
Record Number:CaltechAUTHORS:20210429-092722876
Persistent URL:
Official Citation:Genomically Mined Acoustic Reporter Genes Enable In Vivo Monitoring of Tumors and Tumor-Homing Bacteria. Robert C. Hurt, Marjorie T. Buss, Mengtong Duan, Katie Wong, Mei Yi You, Daniel P. Sawyer, Margaret B. Swift, Przemysław Dutka, David R. Mittelstein, Zhiyang Jin, Mohamad H. Abedi, Arash Farhadi, Ramya Deshpande, Mikhail G. Shapiro bioRxiv 2021.04.26.441537; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108863
Deposited By: Tony Diaz
Deposited On:29 Apr 2021 18:27
Last Modified:02 Nov 2021 18:08

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