Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45−tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-β, producing 150 pg/106cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-β). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-β plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

Both adjuvants and focal ablation can alter the local innate immune system and trigger a highly effective systemic response. Our goal is to determine the impact of these treatments on directly treated and distant disease and the mechanisms for the enhanced response obtained by combinatorial treatments.Methods: We combined RNA-sequencing, flow cytometry and TCR-sequencing to dissect the impact of immunotherapy and of immunotherapy combined with ablation on local and systemic immune components.Results: With administration of a toll-like receptor agonist agonist (CpG) alone or CpG combined with same-site ablation, we found dramatic differences between the local and distant tumor environments, where the directly treated tumors were skewed to high expression of F4/80, Cd11b and Tnf and the distant tumors to enhanced Cd11c, Cd3 and Ifng. When ablation was added to immunotherapy, 100% (n=20/20) of directly treated tumors and 90% (n=18/20) of distant tumors were responsive. Comparing the combined ablation-immunotherapy treatment to immunotherapy alone, we find three major mechanistic differences. First, while ablation alone enhanced intratumoral antigen cross-presentation (up to ~8% of CD45+ cells), systemic cross-presentation of tumor antigen remained low. Combining same-site ablation with CpG amplified cross-presentation in the draining lymph node (~16% of CD45+ cells) compared to the ablation-only (~0.1% of CD45+ cells) and immunotherapy-only cohorts (~10% of CD45+ cells). Macrophages and DCs process and present this antigen to CD8+ T-cells, increasing the number of unique T-cell receptor rearrangements in distant tumors. Second, type I interferon (IFN) release from tumor cells increased with the ablation-immunotherapy treatment as compared with ablation or immunotherapy alone. Type I IFN release is synergistic with toll-like receptor activation in enhancing cytokine and chemokine expression. Expression of genes associated with T-cell activation and stimulation (Eomes, Prf1 and Icos) was 27, 56 and 89-fold higher with ablation-immunotherapy treatment as compared to the no-treatment controls (and 12, 32 and 60-fold higher for immunotherapy-only treatment as compared to the no-treatment controls). Third, we found that the ablation-immunotherapy treatment polarized macrophages and dendritic cells towards a CD169 subset systemically, where CD169+ macrophages are an IFN-enhanced subpopulation associated with dead-cell antigen presentation.Conclusion: While the local and distant responses are distinct, CpG combined with ablative focal therapy drives a highly effective systemic immune response.

Microbubble contrast agents are widely used in ultrasound imaging and therapy, typically with transmission center frequencies in the MHz range. Currently, an ultrasound center frequency near 250 kHz is proposed for clinical trials in which ultrasound combined with microbubble contrast agents is applied to open the blood brain barrier, since at this low frequency focusing through the human skull to a predetermined location can be performed with reduced distortion and attenuation compared to higher frequencies. However, the microbubble vibrational response has not yet been carefully evaluated at this low frequency (an order of magnitude below the resonance frequency of these contrast agents). In the past, it was assumed that encapsulated microbubble expansion is maximized near the resonance frequency and monotonically decreases with decreasing frequency. Our results indicated that microbubble expansion was enhanced for 250 kHz transmission as compared with the 1 MHz center frequency. Following 250 kHz insonation, microbubble expansion increased nonlinearly with increasing ultrasonic pressure, and was accurately predicted by either the modified Rayleigh–Plesset equation for a clean bubble or the Marmottant model of a lipid-shelled microbubble. The expansion ratio reached 30-fold with 250 kHz at a peak negative pressure of 400 kPa, as compared to a measured expansion ratio of 1.6 fold for 1 MHz transmission at a similar peak negative pressure. Further, the range of peak negative pressure yielding stable cavitation in vitro was narrow (~100 kPa) for the 250 kHz transmission frequency. Blood brain barrier opening using in vivo transcranial ultrasound in mice followed the same trend as the in vitro experiments, and the pressure range for safe and effective treatment was 75–150 kPa. For pressures above 150 kPa, inertial cavitation and hemorrhage occurred. Therefore, we conclude that (1) at this low frequency, and for the large oscillations, lipid-shelled microbubbles can be approximately modeled as clean gas microbubbles and (2) the development of safe and successful protocols for therapeutic delivery to the brain utilizing 250 kHz or a similar center frequency requires consideration of the narrow pressure window between stable and inertial cavitation.

High intensity focused ultrasound (HIFU) rapidly and non-invasively destroys tumor tissue. Here, we sought to assess the immunomodulatory effects of MR-guided HIFU and its combination with the innate immune agonist CpG and checkpoint inhibitor anti-PD-1. Mice with multi-focal breast cancer underwent ablation with a parameter set designed to achieve mechanical disruption with minimal thermal dose or a protocol in which tumor temperature reached 65 °C. Mice received either HIFU alone or were primed with the toll-like receptor 9 agonist CpG and the checkpoint modulator anti-PD-1. Both mechanical HIFU and thermal ablation induced a potent inflammatory response with increased expression of Nlrp3, Jun, Mefv, Il6 and Il1β and alterations in macrophage polarization compared to control. Furthermore, HIFU upregulated multiple innate immune receptors and immune pathways, including Nod1, Nlrp3, Aim2, Ctsb, Tlr1/2/4/7/8/9, Oas2, and RhoA. The inflammatory response was largely sterile and consistent with wound-healing. Priming with CpG attenuated Il6 and Nlrp3 expression, further upregulated expression of Nod2, Oas2, RhoA, Pycard, Tlr1/2 and Il12, and enhanced T-cell number and activation while polarizing macrophages to an anti-tumor phenotype. The tumor-specific antigen, cytokines and cell debris liberated by HIFU enhance response to innate immune agonists.

Non-invasive, quantitative methods to assess the properties of biological tissues are needed for many therapeutic and tissue engineering applications. Magnetic resonance elastography (MRE) has historically relied on external vibration to generate periodic shear waves. In order to focally assess a biomaterial or to monitor the response to ablative therapy, the interrogation of a specific region of interest by a focused beam is desirable and transient MRE (t-MRE) techniques have previously been developed to accomplish this goal. Also, strategies employing a series of discrete ultrasound pulses directed to increasing depths along a single line-of-sight have been designed to generate a quasi-planar shear wave. Such ‘supersonic’ excitations have been applied for ultrasound elasticity measurements. The resulting shear wave is higher in amplitude than that generated from a single excitation and the properties of the media are simply visualized and quantified due to the quasiplanar wave geometry and the opportunity to generate the wave at the site of interest. Here for the first time, we extend the application of supersonic methods by developing a protocol for supersonic transient magnetic resonance elastography (sst-MRE) using an MR-guided focused ultrasound system capable of therapeutic ablation. We apply the new protocol to quantify tissue elasticity in vitro using biologically-relevant inclusions and tissue-mimicking phantoms, compare the results with elasticity maps acquired with ultrasound shear wave elasticity imaging (US-SWEI), and validate both methods with mechanical testing. We found that a modified time-of-flight (TOF) method efficiently quantified shear modulus from sst-MRE data, and both the TOF and local inversion methods result in similar maps based on US-SWEI. With a three-pulse excitation, the proposed sst-MRE protocol was capable of visualizing quasi-planar shear waves propagating away from the excitation location and detecting differences in shear modulus of 1 kPa. The techniques demonstrated here have potential application in real-time in vivo lesion detection and monitoring, with particular significance for image-guided interventions.

Structured illumination microscopy is an optical method to increase the spatial resolution of wide-field fluorescence imaging beyond the diffraction limit by applying a spatially structured illumination light. Here, we extend this concept to facilitate super-resolution ultrasound imaging by manipulating the transmitted sound field to encode the high spatial frequencies into the observed image through aliasing. Post processing is applied to precisely shift the spectral components to their proper positions in k-space and effectively double the spatial resolution of the reconstructed image compared to one-way focusing. The method has broad application, including the detection of small lesions for early cancer diagnosis, improving the detection of the borders of organs and tumors, and enhancing visualization of vascular features. The method can be implemented with conventional ultrasound systems, without the need for additional components. The resulting image enhancement is demonstrated with both test objects and ex vivo rat metacarpals and phalanges.

Resiquimod is an immunopotent toll-like receptor 7/8 agonist with antitumor activity. Despite being potent against skin cancers, it is poorly tolerated systemically due to toxicity. Integrating resiquimod into nanoparticles presents an avenue to circumvent the toxicity problem. Herein, the preparation of degradable nanoparticles with covalently bound resiquimod and their systemic application in cancer immunotherapy is reported. Dispersion in water of amphiphilic constructs integrating resiquimod covalently bound via degradable amide or ester linkages yields immune-activating nanoparticles. The degradable agonist-nanoparticle bonds allow the release of resiquimod from the carrier nanoparticles. In vitro assays with antigen presenting cells demonstrate that the nanoparticles retain the immunostimulatory activity of resiquimod. Systemic administration of the nanoparticles and checkpoint blockade (aPD-1) to a breast cancer mouse model with multiple established tumors triggers antitumor activity evidenced by suppressed tumor growth and enhanced CD8 + T-cell infiltration. Nanoparticles with ester links, which hydrolyze more readily, yield a stronger immune response with 75% of tumors eliminated when combined with aPD-1. The reduced tumor growth and the presence of activated CD8 + T-cells across multiple tumors suggest the potential for treating metastatic cancer.

We propose the use of a new encoding mask in order to improve the performance of the conventional time multiplexing super resolution method. The resolution improvement is obtained using a 2D Barker-based array that is placed upon the object and shifted laterally. The Barker-based array is a 2D generalization of the standard 1D Barker code. The Barker-based array has stable autocorrelation sidelobes, making it ideal for the encoding process. A sequence of low resolution images are captured at different positions of the array, and are decoded properly using the same array. After removing the low resolution image from the resulting reconstruction, a high resolution image is established. The proposed method is presented analytically, demonstrated via numerical simulation, and validated by laboratory experiment.