Genomically mined acoustic reporter genes for real-time in vivo monitoring of tumors and tumor-homing bacteria

Abstract

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.

Biomolecular actuators for genetically selective acoustic manipulation of cells

“…Acoustic patterning of GV-expressing cells has minimal impact on cell viability, as established in the current study, complementing previous findings that cells remain viable after GV expression and culturing in 3D hydrogels ( 16 , 32 , 34 ). These results set the stage for future studies involving continued culturing of acoustically patterned GV-expressing cells in 3D hydrogels for tissue engineering and living material applications.…”

“…We chose a fluorescent protein, EBFP2, as our model cargo protein to allow us to quantify the cargo expression level optically. We expressed this construct alongside the remaining GV genes in mammalian cells via transient transfection ( 34 ), which results in the range of transgene expression levels due to the stochastic incorporation of the transfection complex by the cells. Fluorescence and phase-contrast imaging of the engineered cells revealed colocalized expression of EBFP2 and GVs to the same cells ( Fig.…”

“…The ability of GVs to connect an acoustophoretic phenotype to the output of genetic circuits in both bacterial and mammalian cells will allow their expression to designate specific cells for separation, trapping, and patterning using ultrasound. GV expression is compatible with several methods of genetic modification including chemical transformation, electroporation, transient transfection, and piggyBac integration ( 16 , 32 , 34 ), whereupon both endogenous and engineered promoters can be connected to gene expression, allowing the formation of GVs to indicate a wide variety of cellular states, based on which the cells can now be selectively manipulated, patterned, and sorted. Alternatively, GVs can be used as exogenous cellular labels.…”

“…HEK293T cells expressing GVs and EBPF2 were prepared by transient transfection as described previously ( 34 ). Briefly, a transfection mixture containing 420 fmol of GvpA-IRES-EBFP2 plasmid and 70 fmol of each of the accessory plasmids ( GvpC , GvpF , GvpG , GvpJ , GvpK , GvpN , GvpV , and GvpW ) was mixed with polyethyleneimine (PEImax, Polyscienes) at a 1:2.6 ratio (w/w) by vortexing and added to a 70% confluent culture.…”

“…Having established the ability of GVs to experience strong ARF, we tested the ability of these genetically encodable nanostructures to modify the ARF response of genetically engineered cells. GVs have been successfully expressed as well-tolerated reporter genes for ultrasound imaging in multiple bacterial and mammalian cell types (16,(32)(33)(34). We hypothesized that because intracellular GV expression would reduce the volume-averaged density and increase the volume-averaged compressibility of the cell, it could invert a cell's acoustic contrast factor from positive to negative.…”