Periodic Reporting for period 2 - TheraSonix (Ultrasonic Imaging and Drug Propulsion Into Tumors Using Genetically Encoded Gas Nanostructures)
Période du rapport: 2020-05-01 au 2021-04-30
One of the important limitations of today's anti-cancer therapies is their inefficient and non-specific delivery—the systemic delivery of treatments like chemotherapy results in significant damage to healthy tissue and severe side effects. At the same time, most drugs have a limited penetration depth of only a few cell layers into the tumor. These drugs are concentrated around the heterogeneous vasculature and produce only a local therapeutic effect. In this project, we proposed a method of overcoming these limitations by using focused ultrasound for selective activation of targeted bio-molecules and tumor homing cells. The resulting vibrations can locally release anti-cancer drugs and deliver them deep into the tumor core.
The proposed approach is based on ultrasonic cavitation, a phenomenon in which gas bubbles expand and collapse under the influence of ultrasound waves. This process produces fluid streaming that propels drugs deeper into the tumor mass. The use of ultrasound for drug delivery is attractive due to its availability and affordability. However, the use of this technology is currently limited by the properties of conventional microbubble-based cavitation nuclei: their large size prevents them from penetrating into the tumor, and their short circulation times do not match the pharmacokinetic time constants of many drugs.
To overcome these challenges, we will utilize gas vesicles (GVs), a unique class of genetically encoded, gas-filled protein nanostructures, as cavitation nuclei. In nature, GVs are used by buoyant photosynthetic microbes that use them to regulate their flotation. Unlike microbubbles, GVs are physically stable. Their nanoscale dimensions have the potential to enable them to extravasate into tumors and bind to specific cellular targets. Recently our lab was able to express GVs in tumor-homing bacteria and mammalian cells and use their acoustic signature as a deep-tissue reporter of gene expression. We hypothesized that GVs could act as both imaging agents and cavitation nuclei. This therapeutic approach could provide vastly improved efficacy and selectivity and the potential to combine cavitation-enhanced drug delivery with emerging advancements in cell-based therapeutics.
The proposed approach is based on ultrasonic cavitation, a phenomenon in which gas bubbles expand and collapse under the influence of ultrasound waves. This process produces fluid streaming that propels drugs deeper into the tumor mass. The use of ultrasound for drug delivery is attractive due to its availability and affordability. However, the use of this technology is currently limited by the properties of conventional microbubble-based cavitation nuclei: their large size prevents them from penetrating into the tumor, and their short circulation times do not match the pharmacokinetic time constants of many drugs.
To overcome these challenges, we will utilize gas vesicles (GVs), a unique class of genetically encoded, gas-filled protein nanostructures, as cavitation nuclei. In nature, GVs are used by buoyant photosynthetic microbes that use them to regulate their flotation. Unlike microbubbles, GVs are physically stable. Their nanoscale dimensions have the potential to enable them to extravasate into tumors and bind to specific cellular targets. Recently our lab was able to express GVs in tumor-homing bacteria and mammalian cells and use their acoustic signature as a deep-tissue reporter of gene expression. We hypothesized that GVs could act as both imaging agents and cavitation nuclei. This therapeutic approach could provide vastly improved efficacy and selectivity and the potential to combine cavitation-enhanced drug delivery with emerging advancements in cell-based therapeutics.
Since the beginning of the project, we were able to demonstrate the ability of gas vesicles to seed to seed cavitation activity in vitro, in cellulo, and in vivo. We first demonstrated the ability of purified GVs to nucleate cavitation activity using passive acoustic detection and high frame rate microscopy at 5 million frames per second. We showed that the collapse of GVs under ultrasound pressure releases the air from these vesicles. If the ultrasound pressure is high enough, over several cycles, the nanobubbles are converted into micron-scale bubbles, which can eventually undergo violent inertial cavitation. Passive acoustic measurement also enabled us to study the physical parameters that effects this activity, including the ultrasound pressure, pulse length, frequency, and concentration of GVs.
Then, we demonstrated that molecularly-targeted GVs could serve as ultrasound-triggered disruptors of tumor cells. The outer protein of GVs GvpC was edited to include an RGD peptide, targeting them to the overexpressed surface receptors of U87 glioblastoma cells. These GVs seeded cavitation activity that opened the membranes of nearby U87 tumor cells, enabling propidium iodide dye to enter into these cells. High frame rate microscopy directly showed the formation of bubbles during ultrasound application to cells treated with GVs.
After showing the GVs can nucleate cavitation in free medium and when attached to cells, we investigated them as genetically encoded seeds for cellular inertial cavitation and payload release. We showed that GVs expressed by bacteria and mammalian cells could be used as a remote kill switch for engineered cells. Moreover, we showed that by detonating these GVs, we release co-expressed molecular payload from these cells with spatiotemporal control.
Finally, we performed three proof of concept experiments demonstrating GV-seeded cavitation and tissue disruption in vivo. GVs were directly administered into MC26 hind limb tumors under ultrasound imaging. The acoustic contrast of these GVs disappeared after exposure to focused ultrasound, while passive acoustic measurements showed significantly higher cavitation activity compared to control tumors. The ability of systemically administered GVs to damage surrounding tissue was demonstrated using the natural accumulation of GVs in the liver. Insonation in the presence of GVs was shown to produce selective tissue damage with a high number of hemorrhagic foci surrounded by necrotic regions. Finally, we endeavored to demonstrate the use of genetically encoded cavitation nuclei in the context of in vivo cell-based therapy. Mechanotherapy with GV-expressing tumor-homing cells enhanced cancer immunotherapy with checkpoint inhibitors.
Then, we demonstrated that molecularly-targeted GVs could serve as ultrasound-triggered disruptors of tumor cells. The outer protein of GVs GvpC was edited to include an RGD peptide, targeting them to the overexpressed surface receptors of U87 glioblastoma cells. These GVs seeded cavitation activity that opened the membranes of nearby U87 tumor cells, enabling propidium iodide dye to enter into these cells. High frame rate microscopy directly showed the formation of bubbles during ultrasound application to cells treated with GVs.
After showing the GVs can nucleate cavitation in free medium and when attached to cells, we investigated them as genetically encoded seeds for cellular inertial cavitation and payload release. We showed that GVs expressed by bacteria and mammalian cells could be used as a remote kill switch for engineered cells. Moreover, we showed that by detonating these GVs, we release co-expressed molecular payload from these cells with spatiotemporal control.
Finally, we performed three proof of concept experiments demonstrating GV-seeded cavitation and tissue disruption in vivo. GVs were directly administered into MC26 hind limb tumors under ultrasound imaging. The acoustic contrast of these GVs disappeared after exposure to focused ultrasound, while passive acoustic measurements showed significantly higher cavitation activity compared to control tumors. The ability of systemically administered GVs to damage surrounding tissue was demonstrated using the natural accumulation of GVs in the liver. Insonation in the presence of GVs was shown to produce selective tissue damage with a high number of hemorrhagic foci surrounded by necrotic regions. Finally, we endeavored to demonstrate the use of genetically encoded cavitation nuclei in the context of in vivo cell-based therapy. Mechanotherapy with GV-expressing tumor-homing cells enhanced cancer immunotherapy with checkpoint inhibitors.
This project demonstrated the power of GV seeded cavitation in vitro and in vivo, using tumor-homing probiotic bacteria. These bacteria are injected systemically and selectively multiply inside tumors. There, they produce GVs providing information about the structure of the tumor. Using the ability of tumor homing cells to express GV in situ, we have also tested the use of GVs cavitation in the context of immunotherapy. The insonation of GV expressing tumor cells in combination with systemic immune checkpoint inhibitors αCTLA-4 and αPD-L1 significantly reduced the tumor growth rate and extended the life span of these mice.
Another ongoing effort is improving the Imaging of GVs and their cavitation. Changes in tumor vasculature and microenvironment are important attributes associated with aggressive cancer phenotypes and reactions to various treatments. As part of this project, we developed methods that enable us to detect better GV expressing cells and interpret their location in the context of the tumor microenvironment and micro-vasculature. The cavitation activity seeded by GVs enabled ultrasensitive imaging of these reporters. This approach called burst ultrasound reconstructed with signal templates (BURST)— improves the cellular detection limit by more than 1,000-fold compared to conventional methods, enabling single-cell detection. Finally, we introduced a technique that resulted in a four-fold improvement in the resolution or non-contrast vascular ultrasound imaging. This approach could extend the clinical use of super-resolution ultrasound imaging and enable frequent imaging of awake and active animals.
This project and the technologies developed as part of it are expected to open a range of new therapeutic possibilities. This work introduces the first family of genetically encoded cavitation nuclei. The ability to produce GVs in many engineered cells enables us to combine focused ultrasound with emerging medical technologies such as synthetic biology, cell therapy, and immunotherapy. In addition to providing better solutions to critical medical problems, ultrasound-based imaging and therapy approaches could help deliver better healthcare to remote areas and low-income populations. Ultrasound is an excellent tool for tackling these problems due to its cost-efficiency, safety, and portability. These socio-economic issues are even more critical in the post-COVID-19 reality.
Another ongoing effort is improving the Imaging of GVs and their cavitation. Changes in tumor vasculature and microenvironment are important attributes associated with aggressive cancer phenotypes and reactions to various treatments. As part of this project, we developed methods that enable us to detect better GV expressing cells and interpret their location in the context of the tumor microenvironment and micro-vasculature. The cavitation activity seeded by GVs enabled ultrasensitive imaging of these reporters. This approach called burst ultrasound reconstructed with signal templates (BURST)— improves the cellular detection limit by more than 1,000-fold compared to conventional methods, enabling single-cell detection. Finally, we introduced a technique that resulted in a four-fold improvement in the resolution or non-contrast vascular ultrasound imaging. This approach could extend the clinical use of super-resolution ultrasound imaging and enable frequent imaging of awake and active animals.
This project and the technologies developed as part of it are expected to open a range of new therapeutic possibilities. This work introduces the first family of genetically encoded cavitation nuclei. The ability to produce GVs in many engineered cells enables us to combine focused ultrasound with emerging medical technologies such as synthetic biology, cell therapy, and immunotherapy. In addition to providing better solutions to critical medical problems, ultrasound-based imaging and therapy approaches could help deliver better healthcare to remote areas and low-income populations. Ultrasound is an excellent tool for tackling these problems due to its cost-efficiency, safety, and portability. These socio-economic issues are even more critical in the post-COVID-19 reality.